International Journal of Biological Sciences

Physiological and Oncogenic Aurora-A Pathway

Toshiaki SAEKI, Mutsuko OUCHI, Toru OUCHI

Aurora family of protein kinases have emerged as crucial factors of, not only mitosis and cytokinesis, but also human carcinogenesis. Among these family members is Aurora-A that is frequently overexpressed in varieties of human cancer. Both in vitro and in vivo studies demonstrated that Aurora-A induces tumorigenesis through genome instability. These studies have further shown that cell signaling cross-talk between Aurora-A and other cellular proteins are essential for fully-transformed phenotypes. This review summarizes recent progress of Aurora-A-associated carcinogenesis.

A receptor and binding protein interplay in the detection of a distinct pheromone component in the silkmoth Antheraea polyphemus

Maike Forstner, Heinz Breer, Jürgen Krieger

Male moths respond to conspecific female-released pheromones with remarkable sensitivity and specificity, due to highly specialized chemosensory neurons in their antennae. In Antheraea silkmoths, three types of sensory neurons have been described, each responsive to one of three pheromone components. Since also three different pheromone binding proteins (PBPs) have been identified, the antenna of Antheraea seems to provide a unique model system for detailed analyzes of the interplay between the various elements underlying pheromone reception. Efforts to identify pheromone receptors of Antheraea polyphemus have led to the identification of a candidate pheromone receptor (ApolOR1). This receptor was found predominantly expressed in male antennae, specifically in neurons located beneath pheromone-sensitive sensilla trichodea. The ApolOR1-expressing cells were found to be surrounded by supporting cells co-expressing all three ApolPBPs. The response spectrum of ApolOR1 was assessed by means of calcium imaging using HEK293-cells stably expressing the receptor. It was found that at nanomolar concentrations ApolOR1-cells responded to all three pheromones when the compounds were solubilized by DMSO and also when DMSO was substituted by one of the three PBPs. However, at picomolar concentrations, cells responded only in the presence of the subtype ApolPBP2 and the pheromone (E,Z)-6,11-hexadecadienal. These results are indicative of a specific interplay of a distinct pheromone component with an appropriate binding protein and its related receptor subtype, which may be considered as basis for the remarkable sensitivity and specificity of the pheromone detection system.

Cloning, Expression and Purification of an Acetoacetyl CoA Thiolase from Sunflower Cotyledon

Thiolase I and II coexist as part of the glyoxysomal β-oxidation system in sunflower (Helianthus annuus L.) cotyledons, the only system shown to have both forms. The importance of thiolases can be underscored not only by their ubiquity, but also by their involvement in a wide variety of processes in plants, animals and bacteria. Here we describe the cloning, expression and purification of acetoacetyl CoA thiolase (AACT) in enzymatically active form. Use of the extensive amount of sequence information from the databases facilitated the efficient generation of the gene-specific primers used in the RACE protocols. The recombinant AACT (1233 bp) shares 75% similarity with other plant AACTs. Comparison of specific activity of this recombinant AACT to a previously reported enzyme purified from primary sunflower cotyledon tissue was very similar (263 nkat/mg protein vs 220 nkat/mg protein, respectively). Combining the most pure fractions from the affinity column, the enzyme was purified 88-fold with a 55% yield of the enzymatically active, 47 kDa AACT.

Identification of Anoectochilus based on rDNA ITS sequences alignment and SELDI-TOF-MS

Chuan Gao, Fusheng Zhang, Jun Zhang, Shunxing Guo, Hongbo Shao

The internal transcribed spacer (ITS) sequences alignment and proteomic difference of Anoectochilus interspecies have been studied by means of ITS molecular identification and surface enhanced laser desorption ionization time of flight mass spectrography. Results showed that variety certification on Anoectochilus by ITS sequences can not determine species, and there is proteomic difference among Anoectochilus interspecies. Moreover, proteomic finger printings of five Anoectochilus species have been established for identifying species, and genetic relationships of five species within Anoectochilus have been deduced according to proteomic differences among five species.

A Comparison of the Effects of Three GM Corn Varieties on Mammalian Health

We present for the first time a comparative analysis of blood and organ system data from trials with rats fed three main commercialized genetically modified (GM) maize (NK 603, MON 810, MON 863), which are present in food and feed in the world. NK 603 has been modified to be tolerant to the broad spectrum herbicide Roundup and thus contains residues of this formulation. MON 810 and MON 863 are engineered to synthesize two different Bt toxins used as insecticides. Approximately 60 different biochemical parameters were classified per organ and measured in serum and urine after 5 and 14 weeks of feeding. GM maize-fed rats were compared first to their respective isogenic or parental non-GM equivalent control groups. This was followed by comparison to six reference groups, which had consumed various other non-GM maize varieties. We applied nonparametric methods, including multiple pairwise comparisons with a False Discovery Rate approach. Principal Component Analysis allowed the investigation of scattering of different factors (sex, weeks of feeding, diet, dose and group). Our analysis clearly reveals for the 3 GMOs new side effects linked with GM maize consumption, which were sex- and often dose-dependent. Effects were mostly associated with the kidney and liver, the dietary detoxifying organs, although different between the 3 GMOs. Other effects were also noticed in the heart, adrenal glands, spleen and haematopoietic system. We conclude that these data highlight signs of hepatorenal toxicity, possibly due to the new pesticides specific to each GM corn. In addition, unintended direct or indirect metabolic consequences of the genetic modification cannot be excluded.

The presence of alpha-catenin in the VE-cadherin complex is required for efficient transendothelial migration of leukocytes

Jaap D. van Buul, Floris P. van Alphen, Peter L. Hordijk

The majority of the leukocytes cross the endothelial lining of the vessels through cell-cell junctions. The junctional protein Vascular Endothelial (VE)-cadherin is transiently re-distributed from sites of cell-cell contacts during passage of leukocytes. VE-cadherin is part of a protein complex comprising p120-catenin and beta-catenin as intracellular partners. Beta-catenin connects VE-cadherin to alpha-catenin. This VE-cadherin-catenin complex is believed to dynamically control endothelial cell-cell junctions and to regulate the passage of leukocytes, although not much is known about the role of alpha- and beta-catenin during the process of transendothelial migration (TEM). In order to study the importance of the interaction between alpha- and beta-catenin in TEM, we used a cell-permeable version of the peptide encoding the binding site of alpha-catenin for beta-catenin (S27D). The data show that S27D interferes with the interaction between alpha- and beta-catenin and induces a reversible decrease in electrical resistance of the endothelial monolayer. In addition, S27D co-localized with beta-catenin at cell-cell junctions. Surprisingly, transmigration of neutrophils across endothelial monolayers was blocked in the presence of S27D. In conclusion, our results show for the first time that the association of alpha-catenin with the cadherin-catenin complex is required for efficient leukocyte TEM.

Role of Ldb1 in Adult Intestinal Homeostasis

Ipsita Dey-Guha, Mahua Mukhopadhyay, Matthew Phillips, Heiner Westphal

Ldb1 is an essential co-regulator of transcription in embryonic development. It acts in conjunction with nuclear LIM-homeodomain and LIM-only proteins to control key events of organogenesis as precursor cells enter lineage specification. Here we ask whether Ldb1 exerts control over stem cell activation and differentiation throughout the life of the organism as required for tissue homeostasis. To help answer this question, we have generated conditional Ldb1 mouse mutants with an Ldb1 floxed/floxed;ROSA26CreER genotype. Tamoxifen treatment of 60 day-old mutant animals results in near-ubiquitous Cre-mediated Ldb1 inactivation. As a consequence, the stem cell microenvironment of intestinal crypts is drastically affected. Cells that normally express Ldb1 together with markers that identify them as lineage progenitors cease to retain bromodeoxyuridine and are gradually lost. Ldb1 inactivation in intestinal crypts and/or in neighboring mesenchymal cells also triggers activation of Wnt signaling in the stem cell niches of the small intestine. Cell proliferation is markedly increased in the epithelia of the small intestine, and Lgr5-expressing stem cells disappear from the base of the crypts. This perturbation of the normal process of tissue homeostasis causes apoptosis, and the animals do not survive. We conclude that Ldb1-mediated transcriptional regulation plays a major role in adult intestinal homeostasis.

Proteome Changes in Thai Indigenous Chicken Muscle during Growth Period

Tawatchai Teltathum, Supamit Mekchay

Proteomic profiling of the pectoralis muscle of Thai indigenous chickens during growth period was analyzed using two-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/MS). A total of 259, 161, 120 and 107 protein spots were found to be expressed in the chicken pectoralis muscles at 0, 3, 6 and 18 weeks of age, respectively. From these expressed proteins, five distinct protein spots were significantly associated with chicken age. These protein spots were characterized and showed homology with phosphoglycerate mutase 1 (PGAM1), apolipoprotein A1 (APOA1), triosephosphate isomerase 1 (TPI1), heat shock protein 25 kDa (HSP25) and fatty acid binding protein 3 (FABP3). These five protein spots were categorized as follows: (i) the expression levels of PGAM1 and TPI1 proteins were positively correlated with chicken aging (p<0.05), (ii) the expression levels of APOA1 and FABP3 proteins were negatively correlated with chicken aging (p<0.05) and (iii) the expression levels of the HSP25 protein were up- and down-regulated during growth period. Moreover, the mRNA expression levels of the FABP3 and HSP25 genes were significantly decreased in muscle during the growth period (p<0.05), whereas no significant changes of the PGAM1, TPI1 and APOA1 gene expression from the chicken muscle was observed. The identified proteins were classified as metabolic and stress proteins. This demonstrates a difference in energy metabolism and stress proteins between age groups and shows that proteomics is a useful tool to uncover the molecular basis of physiological differences in muscle during the growth period.

Azithromycin suppresses interleukin-12p40 expression in lipopolysaccharide and interferon-γ stimulated macrophages

Azithromycin (AZM), a 15-member macrolide antibiotic, possesses anti-inflammatory activity. Macrophages are important in innate and acquired immunity, and produce pro-inflammatory cytokines such as interleukin (IL)-12, which are composed of subunit p40 and p35. The key function of IL-12 is the induction and maintenance of T-helper-1 responses, which is associated with the pathogenesis of chronic inflammatory diseases. We investigated the effect of azithromycin on IL-12p40 production in macrophages after lipopolysaccharide (LPS)/interferon (IFN)-γ stimulation. RAW264.7 macrophage cell line was pre-treated with vehicle or AZM, followed by the stimulation with LPS/IFN-γ. We measured IL-12 production by RT-PCR and ELISA. IL-12 transcriptional regulation was assessed by electrophoretic mobility shift assay and reporter assay. Phosphorylation of activator protein (AP)-1 and interferon consensus sequence binding protein (ICSBP) was assessed by immunoprecipitation using phosphotyrosine antibody, and immunoblotting using specific antibodies against JunB and ICSBP. AZM reduced the induction of IL-12p40 by LPS/IFN-γ in a dose dependent manner. AZM inhibited the binding of AP-1, nuclear factor of activated T cells (NFAT), and ICSBP, to the DNA binding site in the IL-12p40 promoter. AZM also reduced LPS/IFN-γ-induced IL-12p40 promoter activity. Phosphorylation of JunB and ICSBP was inhibited by azithromycin-treatment in stimulated cells. In conclusion, AZM reduced IL-12p40 transcriptional activity by inhibiting the binding of AP-1, NFAT, and ICSBP to the promoter site. This may represent an important mechanism for regulating the anti-inflammatory effects of AZM in macrophages.

Karyology of eight species of bats (Mammalia: Chiroptera) from Hainan Island, China

Yi Wu, Masaharu Motokawa, Yu-Chun Li, Masashi Harada, Zhong Chen, Liang-Kong Lin

Karyotypes and chromosomal data are presented for eight bat species representing two families (Rhinolophidae and Vespertilionidae) from Hainan Island, China. The species investigated were Rhinolophus lepidus (2n = 62, FN = 60), R. pusillus (2n = 62, FN = 60), R. affinis (2n = 62, FN = 60), R. sinicus (2n = 36, FN = 60), Myotis horsfieldi (2n = 44, FN = 52), Pipistrellus abramus (2n = 26, FN = 44), Miniopterus australis (2n = 46, FN = 50) and M. schreibersii (2n = 46, FN = 50). The karyotype of Rhinolophus lepidus is reported for the first time.

Clinical diagnosis for discogenic low back pain

Yin-gang Zhang, Tuan-mao Guo, Xiong Guo, Shi-xun Wu

Discogenic lower back pain (DLBP) is the most common type of chronic lower back pain (LBP), accounting for 39% of cases, compared to 30% of cases due to disc herniation, and even lower prevalence rates for other causes, such as zygapophysial joint pain. Only a small proportion (approximately 20%) of LBP cases can be attributed with reasonable certainty to a pathologic or anatomical entity. Thus, diagnosing the cause of LBP represents the biggest challenge for doctors in this field. In this review, we summarize the process of obtaining a clinical diagnosis of DLBP and discuss the potential for serum-based diagnosis in the near future. The use of serum biomarkers to diagnose DLBP is likely to increase the ease of diagnosis as well as produce more accurate and reproducible results.

An apoA-I mimetic peptide facilitates off-loading cholesterol from HDL to liver cells through scavenger receptor BI

Xuelei Song, Paul Fischer, Xun Chen, Charlotte Burton, Jun Wang

Apolipoprotein A-I (apoA-I) mimetic peptides have been pursued as new therapeutic agents for the treatment of atherosclerosis, yet their precise mechanism responsible for atheroprotection remains unclear. Like apoA-I itself, most of these peptides are capable of stimulating cholesterol efflux from macrophages or foam cells, and some of them stimulate lecithin cholesterol acyltransferase (LCAT) activity in the reverse cholesterol transport (RCT) pathway. However, the ability of mimetic peptides to deliver cholesterol into hepatocytes (off-loading), the last step of the RCT pathway, has not been demonstrated. In this study, we compared a mimetic peptide D-4F to purified apoA-I, to address the role that mimetics play during the off-loading process. Both D-4F and apoA-I formed spherical nano-particles when reconstituted with cholesteryl ester and phospholipids. Compared to apoA-I, D-4F particles were 20 times more efficient in off-loading cholesterol to HepG2 hepatocytes with an apparent K_t (transport) of 0.74 μg/mL. Furthermore, D-4F also facilitated cholesteryl ester offloading from HDL particles into HepG2 cells when it was pre-incubated with these HDL particles. Using an inducible HEK293 cell line, we demonstrated that these nano-particles were able to be taken up through SR-BI, a HDL selective receptor. Cholesterol uptake by HepG2 cells was completely blocked by a neutralizing monoclonal antibody against SR-BI, demonstrating that D-4F particles, similar to HDL, specifically off-loaded cholesterol through SR-BI. Overall our data provides evidence that D-4F is capable of mimicking apoA-I to form HDL-like particles, and off-loads cholesterol for catabolism and excretion, thus completing RCT.

Krüppel-like family of transcription factors: an emerging new frontier in fat biology

In mammals, adipose tissue stores energy in the form of fat. The ability to regulate fat storage is essential for the growth, development and reproduction of most animals, thus any abnormalities caused by excess fat accumulation can result in pathological conditions which are linked to several interrelated diseases, such as cardiovascular diseases, diabetes, and obesity. In recent years significant effort has been applied to understand basic mechanism of fat accumulation in mammalian system. Work in mouse has shown that the family of Krüppel-like factors (KLFs), a conserved and important class of transcription factors, regulates adipocyte differentiation in mammals. However, how fat storage is coordinated in response to positive and negative feedback signals is still poorly understood. To address mechanisms underlying fat storage we have studied two Caenorhabditis elegans KLFs and demonstrate that both worm klfs are key regulators of fat metabolism in C. elegans. These results provide the first in vivo evidence supporting essential regulatory roles for KLFs in fat metabolism in C. elegans and shed light on the human counterpart in disease-gene association. This finding allows us to pursue a more comprehensive approach to understand fat biology and provides an opportunity to learn about the cascade of events that regulate KLF activation, repression and interaction with other factors in exerting its biological function at an organismal level. In this review, we provide an overview of the most current information on the key regulatory components in fat biology, synthesize the diverse literature, pose new questions, and propose a new model organism for understanding fat biology using KLFs as the central theme.

Erratum

Zhihua Jiang, Daniel S. Rokhsar, Richard M. Harland

Nucleotide excision repair and recombination are engaged in repair of trans-4-hydroxy-2-nonenal adducts to DNA bases in Escherichia coli

One of the major products of lipid peroxidation is trans-4-hydroxy-2-nonenal (HNE). HNE forms highly mutagenic and genotoxic adducts to all DNA bases. Using M13 phage lacZ system, we studied the mutagenesis and repair of HNE treated phage DNA in E. coli wild-type or uvrA, recA, and mutL mutants. These studies revealed that: (i) nucleotide excision and recombination, but not mismatch repair, are engaged in repair of HNE adducts when present in phage DNA replicating in E. coli strains; (ii) in the single uvrA mutant, phage survival was drastically decreased while mutation frequency increased, and recombination events constituted 48 % of all mutations; (iii) in the single recA mutant, the survival and mutation frequency of HNE-modified M13 phage was slightly elevated in comparison to that in the wild-type bacteria. The majority of mutations in recA^- strain were G:C → T:A transversions, occurring within the sequence which in recA⁺ strains underwent RecA-mediated recombination, and the entire sequence was deleted; (iv) in the double uvrA recA mutant, phage survival was the same as in the wild-type although the mutation frequency was higher than in the wild-type and recA single mutant, but lower than in the single uvrA mutant. The majority of mutations found in the latter strain were base substitutions, with G:C → A:T transitions prevailing. These transitions could have resulted from high reactivity of HNE with G and C, and induction of SOS-independent mutations.

Failure to detect active virus replication in mast cells at various tissue sites of HIV patients by immunohistochemistry

Ann Marie Nelson, Aaron Auerbach, Yan-gao Man

A recent report postulated that the mast cell population is a significant reservoir for persistent HIV infection. Our study attempted to validate this hypothesis by quantitatively comparing the distribution of mast cells and cells expressing the HIV protein p24 in HIV infected patients. Consecutive sections of paraffin-embedded human tissues from various tissue sites were subjected to immunohistochemistry with monoclonal antibodies to mast cell tryptase, viral protein p24, and other molecules. The sub-cellular distribution of these molecules was examined, to determine whether immunoreactivities to these molecules would be co-localized within the same cells. Our study revealed that, in two immediate adjacent sections immunostained for mast cell tryptase and p24, respectively, all or nearly all tryptase and p24 expressing cells were distributed at different areas. In the single section double immunostained for mast cell tryptase and p24, 5 (1.1%) of 460 large p24 expressing cell clusters encountered showed a single or few mast cells within or adjacent to p24 expressing cell clusters, but no distinct co-localization of these two proteins was observed. Similarly, no distinct co-localization was observed in any of over 500 isolated individual mast cells and p24 expressing cells. In contrast, macrophages were consistently intermixed with or adjacent to p24 expressing cells, and p24 immunostaining were seen in the cytoplasm of a subset of macrophages. These findings suggest that tissue mast cells do not show evidence for active virus replication by the techniques employed.

Bioinformatics Analysis the Complete Sequences of Cytochrome b of Takydromus sylvaticus and Modeling the Tertiary Structure of Encoded Protein

Qi-Long CHEN, Xin-Sheng TANG, Wen-Juan YAO, Shun-Qing LU

Cytochrome b (cyt b) gene complete sequences (1143bp) of Takydromus sylvaticus were sequenced. In order to clarify the phylogenetic position of the Takydromus sylvaticus, we investigated the phylogeny of 15 Takydromus spp. distributed in East-Asia by Maximum Parsimony (MP), Bayesian Inference (BI), and Maximum Likelihood (ML) methods using DNA fragments of cyt b genes. The results supported that the Platyplacopus merged into Takydromus and negated the validity of Platyplacopus. Furthermore, the prediction of tertiary structures of cyt b exhibited the CD loop region contain two short helices forming a hairpin arrangement, namely cd1 and cd2. Thermostability analysis shows that the CD-loop region is unstable thermodynamically and may provide mobility to amino acids located at the heme, and might provide high flexibility to the top of ISP (iron-sulfur protein) and the cavity region of Qo binding site. It suggested that the two short helices of CD loop region of cyt b was a dominating portion for ISP binding site.

Fungal Bioconversion of Lignocellulosic Residues; Opportunities & Perspectives

Mehdi Dashtban, Heidi Schraft, Wensheng Qin

The development of alternative energy technology is critically important because of the rising prices of crude oil, security issues regarding the oil supply, and environmental issues such as global warming and air pollution. Bioconversion of biomass has significant advantages over other alternative energy strategies because biomass is the most abundant and also the most renewable biomaterial on our planet. Bioconversion of lignocellulosic residues is initiated primarily by microorganisms such as fungi and bacteria which are capable of degrading lignocellulolytic materials. Fungi such as Trichoderma reesei and Aspergillus niger produce large amounts of extracellular cellulolytic enzymes, whereas bacterial and a few anaerobic fungal strains mostly produce cellulolytic enzymes in a complex called cellulosome, which is associated with the cell wall. In filamentous fungi, cellulolytic enzymes including endoglucanases, cellobiohydrolases (exoglucanases) and β-glucosidases work efficiently on cellulolytic residues in a synergistic manner. In addition to cellulolytic/hemicellulolytic activities, higher fungi such as basidiomycetes (e.g. Phanerochaete chrysosporium) have unique oxidative systems which together with ligninolytic enzymes are responsible for lignocellulose degradation. This review gives an overview of different fungal lignocellulolytic enzymatic systems including extracellular and cellulosome-associated in aerobic and anaerobic fungi, respectively. In addition, oxidative lignocellulose-degradation mechanisms of higher fungi are discussed. Moreover, this paper reviews the current status of the technology for bioconversion of biomass by fungi, with focus on mutagenesis, co-culturing and heterologous gene expression attempts to improve fungal lignocellulolytic activities to create robust fungal strains.

Disruption of the BMEI0066 gene attenuates the virulence of Brucella melitensis and decreases its stress tolerance

Xinglin Zhang, Jie Ren, Na Li, Wenjuan Liu, Qingmin Wu

Brucella melitensis is a facultative intracellular pathogen. An operon composed of BMEI0066, which encodes a two-component response regulator CenR, and BMEI0067, which encodes a cAMP-dependent protein kinase regulatory subunit, has been predicted to exist in many bacterial species. However, little is known about the function of this operon. In order to characterize this operon and assess its role in virulence, we constructed a marked deletion mutant of BMEI0066. The mutant was less able to withstand hyperosmotic conditions than wild-type (16M), but showed no significant difference with 16M when challenged by H₂O₂. The mutant also showed increased sensitivity to elevated temperature (42°C) and a reduced survival ratio under acidic conditions compared with 16M. The mutant failed to replicate in cultured murine macrophages and was rapidly cleared from the spleens of experimentally infected BALB/c mice. These findings suggest that these operon products make an important contribution to pathogenesis in mice, probably by allowing B. melitensis to adapt to the harsh environment encountered within host macrophages.

Comparative characterization of a temperature responsive gene (lactate dehydrogenase-B, ldh-b) in two congeneric tropical fish, Lates calcarifer and Lates niloticus

Richard C. Edmunds, Lynne van Herwerden, Carolyn Smith-Keune, Dean R. Jerry

The characterization of candidate loci is a critical step in obtaining insight into adaptation and acclimation of organisms. In this study of two non-model tropical (to sub-tropical) congeneric perciformes (Lates calcarifer and Lates niloticus) we characterized both coding and non-coding regions of lactate dehydrogenase-B (ldh-b), a locus which exhibits temperature-adaptive differences among temperate and sub-tropical populations of the North American killifish Fundulus heteroclitus. Ldh-b was 5,004 and 3,527 bp in length in L. calcarifer and L. niloticus, respectively, with coding regions comprising 1,005 bp in both species. A high level of sequence homology existed between species for both coding and non-coding regions of ldh-b (> 97% homology), corresponding to a 98.5% amino acid sequence homology. All six known functional sites within the encoded protein sequence (LDH-B) were conserved between the two Lates species. Ten simple sequence repeat (SSR) motifs (mono-, di-, tri- and tetranucleotide) and thirty putative microRNA elements (miRNAs) were identified within introns 1, 2, 5 and 6 of both Lates species. Five single nucleotide polymorphisms (SNPs) were also identified within miRNA containing intron regions. Such SNPs are implicated in several complex human conditions and/or diseases (as demonstrated by extensive genome-wide association studies). This novel characterization serves as a platform to further examine how non-model species may respond to changes in their native temperatures, which are expected to increase by up to 6°C over the next century.

Protective Effects of Garlic and Silymarin on NDEA-Induced Rats Hepatotoxicity

Background — The present study was conducted to investigate the chemopreventive effects of garlic extract and silymarin on N-nitrosodiethylamine (NDEA) and carbon tetrachloride (CCl₄)-induced hepatotoxicity in male albino rats. Methods and Results — Animals were pretreated with garlic, silymarin or both for one week prior to the injection of NDEA. Then animals received a single injection of NDEA followed by weekly subcutaneous injections of CCl₄ for 6 weeks. Oral administration was then continued along with the injection of CCl₄ for the duration of the experiment. Serum aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), hepatic lipid peroxidation (LPO), superoxide dismutase (SOD), reduced glutathione (GSH), glutathione-S-transferase (GST) and glutathione reductase (GSR) were measured. Injection of NDEA induced a significant elevation in serum AST, ALT and ALP. In the liver, NDEA increased oxidative stress through the increase in LPO and decrease in SOD, and GSH-dependent enzymes. Although administration of garlic or silymarin significantly reduced the liver toxicity, combined administration was more effective in preventing the development of hepatotoxicity. Conclusion — These novel findings suggest that silymarin and garlic have a synergistic effect, and could be used as hepatoprotective agents against hepatotoxicity.

A novel peptide inhibits the influenza virus replication by preventing the viral attachment to the host cells

Avian influenza viruses (AIV), the causative agent of avian flu or bird flu, cause widespread morbidity and mortality in poultry. The symptoms of the disease range from mild flu like symptoms to death. These viruses possess two important surface glycoproteins, namely hemagglutinin (HA) and neuraminidase (NA) against which neutralizing antibodies are produced. Due to the highly mutative nature of the genes which encode these proteins, the viruses often confer resistance to the current anti-viral drugs making the prevention and treatment of infection challenging. In our laboratory, we have recently identified a novel anti-viral peptide (P1) against the AIV H9N2 from a phage displayed peptide library. This peptide inhibits the replication of the virus in ovo and in vitro by its binding to the HA glycoprotein. In the current study, we demonstrate that the peptide inhibits the virus replication by preventing the attachment to the host cell but it does not have any effect on the viral fusion. The reduction in the viral nucleoprotein (NP) expression inside the host cell has also been observed during the peptide (P1) treatment. This novel peptide may have the potential to be developed as a therapeutic agent for the treatment and control of avian influenza virus H9N2 infections.

Discovery of novel genetic networks associated with 19 economically important traits in beef cattle

Quantitative or complex traits are determined by the combined effects of many loci, and are affected by genetic networks or molecular pathways. In the present study, we genotyped a total of 138 mutations, mainly single nucleotide polymorphisms derived from 71 functional genes on a Wagyu x Limousin reference population. Two hundred forty six F₂ animals were measured for 5 carcass, 6 eating quality and 8 fatty acid composition traits. A total of 2,280 single marker-trait association runs with 120 tagged mutations selected based on the HAPLOVIEW analysis revealed 144 significant associations (P < 0.05), but 50 of them were removed from the analysis due to the small number of animals (≤ 9) in one genotype group or absence of one genotype among three genotypes. The remaining 94 single-trait associations were then placed into three groups of quantitative trait modes (QTMs) with additive, dominant and overdominant effects. All significant markers and their QTMs associated with each of these 19 traits were involved in a linear regression model analysis, which confirmed single-gene associations for 4 traits, but revealed two-gene networks for 8 traits and three-gene networks for 5 traits. Such genetic networks involving both genotypes and QTMs resulted in high correlations between predicted and actual values of performance, thus providing evidence that the classical Mendelian principles of inheritance can be applied in understanding genetic complexity of complex phenotypes. Our present study also indicated that carcass, eating quality and fatty acid composition traits rarely share genetic networks. Therefore, marker-assisted selection for improvement of one category of these traits would not interfere with improvement of another.

Gene expression profiling of chromophobe renal cell carcinomas and renal oncocytomas by Affymetrix GeneChip using pooled and individual tumours

Maria V. Yusenko, Dmitry Zubakov, Gyula Kovacs

Due to overlapping morphology, malignant chromophobe renal cell carcinomas (RCC) and benign renal oncocytomas (RO) may pose a diagnostic problem. In the present study, we have applied different algorithms to evaluate the data sets obtained by hybridisation of pooled and also individual samples of renal cell tumours (RCT) onto two different gene expression platforms. The two approaches revealed high similarities in the gene expression profiles of chromophobe RCCs and ROs but also some differences. After identifying the differentially expressed genes by statistic analyses, the candidate genes were further selected by a real time and normal RT-PCR and their products were analysed by immunohistochemistry. We have identified CD82 and S100A1 as valuable markers for chromophobe RCC as well as AQP6 for ROs. However, these genes are expressed at the protein level in other types of RCTs as well albeit at a low frequency and low intensity. As none of the selected genes marks exclusively one type of RCTs, for the differential diagnosis of chromophobe RCCs and ROs, a set of markers such as CD82, S100A1 and AQP6 as well as some others would be an option in routine histological laboratories.

The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass

Miranda Maki, Kam Tin Leung, Wensheng Qin

Lignocellulosic biomass is a renewable and abundant resource with great potential for bioconversion to value-added bioproducts. However, the biorefining process remains economically unfeasible due to a lack of biocatalysts that can overcome costly hurdles such as cooling from high temperature, pumping of oxygen/stirring, and, neutralization from acidic or basic pH. The extreme environmental resistance of bacteria permits screening and isolation of novel cellulases to help overcome these challenges. Rapid, efficient cellulase screening techniques, using cellulase assays and metagenomic libraries, are a must. Rare cellulases with activities on soluble and crystalline cellulose have been isolated from strains of Paenibacillus and Bacillus and shown to have high thermostability and/or activity over a wide pH spectrum. While novel cellulases from strains like Cellulomonas flavigena and Terendinibacter turnerae, produce multifunctional cellulases with broader substrate utilization. These enzymes offer a framework for enhancement of cellulases including: specific activity, thermalstability, or end-product inhibition. In addition, anaerobic bacteria like the clostridia offer potential due to species capable of producing compound multienzyme complexes called cellulosomes. Cellulosomes provide synergy and close proximity of enzymes to substrate, increasing activity towards crystalline cellulose. This has lead to the construction of designer cellulosomes enhanced for specific substrate activity. Furthermore, cellulosome-producing Clostridium thermocellum and its ability to ferment sugars to ethanol; its amenability to co-culture and, recent advances in genetic engineering, offer a promising future in biofuels. The exploitation of bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a 'greener' technology.

An apoA-I mimetic peptide increases LCAT activity in mice through increasing HDL concentration

Lecithin cholesterol acyltransferase (LCAT) plays a key role in the reverse cholesterol transport (RCT) process by converting cholesterol to cholesteryl ester to form mature HDL particles, which in turn deliver cholesterol back to the liver for excretion and catabolism. HDL levels in human plasma are negatively correlated with cardiovascular risk and HDL functions are believed to be more important in atheroprotection. This study investigates whether and how D-4F, an apolipoprotein A-I (apoA-I) mimetic peptide, influences LCAT activity in the completion of the RCT process. We demonstrated that the apparent rate constant value of the LCAT enzyme reaction gives a measure of LCAT activity and determined the effects of free metals and a reducing agent on LCAT activity, showing an inhibition hierarchy of Zn²⁺>Mg²⁺>Ca²⁺ and no inhibition with β-mercaptoethanol up to 10 mM. We reconstituted nano-disc particles using apoA-I or D-4F with phospholipids. These particles elicited good activity in vitro in the stimulation of cholesterol efflux from macrophages through the ATP-binding cassette transporter A1 (ABCA1). With these particles we studied the LCAT activity and demonstrated that D-4F did not activate LCAT in vitro. Furthermore, we have done in vivo experiments with apoE-null mice and demonstrated that D-4F (20 mg/kg body weight, once daily subcutaneously) increased LCAT activity and HDL level as well as apoA-I concentration at 72 hours post initial dosing. Finally, we have established a correlation between HDL concentration and LCAT activity in the D-4F treated mice.

Lipoproteins, cholesterol homeostasis and cardiac health

Cholesterol is an essential substance involved in many functions, such as maintaining cell membranes, manufacturing vitamin D on surface of the skin, producing hormones, and possibly helping cell connections in the brain. When cholesterol levels rise in the blood, they can, however, have dangerous consequences. In particular, cholesterol has generated considerable notoriety for its causative role in atherosclerosis, the leading cause of death in developed countries around the world. Homeostasis of cholesterol is centered on the metabolism of lipoproteins, which mediate transport of the lipid to and from tissues. As a synopsis of the major events and proteins that manage lipoprotein homeostasis, this review contributes to the substantial attention that has recently been directed to this area. Despite intense scrutiny, the majority of phenotypic variation in total cholesterol and related traits eludes explanation by current genetic knowledge. This is somewhat disappointing considering heritability estimates have established these traits as highly genetic. Thus, the continued search for candidate genes, mutations, and mechanisms is vital to our understanding of heart disease at the molecular level. Furthermore, as marker development continues to predict risk of vascular illness, this knowledge has the potential to revolutionize treatment of this leading human disease.

Histopathological effects of cisplatin, doxorubicin and 5-flurouracil (5-FU) on the liver of male albino rats

Cisplatin, doxorubicin and fluorouracil (5-FU), drugs belonging to different chemical classes, have been extensively used for chemotherapy of various cancers. Despite extensive investigations into their hepatotoxicity, there is very limited information on their effects on the structure and ultra-structure of liver cells in vivo. Here, we demonstrate for the first time, the effects of these three anticancer drugs on rat liver toxicity using both light and electron microscopy. Light microscopic observations revealed that higher doses of cisplatin and doxorubicin caused massive hepatotoxicity compared to 5-FU treatment, including dissolution of hepatic cords, focal inflammation and necrotic tissues. Interestingly, low doses also exhibited abnormal changes, including periportal fibrosis, degeneration of hepatic cords and increased apoptosis. These changes were confirmed at ultrastructural level, including vesiculated rough endoplasmic reticulum and atrophied mitochondria with ill-differentiated cisternae, dense collection of macrophages and lymphocytes as well as fibrocytes with collagenous fibrils manifesting early sign of fibrosis, especially in response to cisplatin and doxorubicin -treatment. Our results provide in vivo evidence, at ultrastructural level, of direct hepatotoxicity caused by cisplatin, doxorubicin and 5-FU at both light and electron microscopi. These results can guide the design of appropriate treatment regimen to reduce the hepatotoxic effects of these anticancer drugs.

Site-Dependent Differences in Clinical, Pathohistological, and Molecular Parameters in Metastatic Colon Cancer

Christoph Wilmanns, Sandra Steinhauer, Joachim Grossmann, Günther Ruf

The purpose was to develop a metastatic score specific to the hepatic and peritoneal site in colorectal cancer patients from clinical, pathohistological and molecular markers potentially reflecting oncogenic activation (OA) or epithelial-mesenchymal transition (EMT), where OA may reflect an activation and EMT the functional loss of certain genes. The primary tumour stage (OA, EMT), lymphonodal stage (OA), the presence of a lymphangiosis carcinomatosa (OA), histological grade (OA, EMT), and immunoblot extraction of E-cadherin (OA, EMT) were differentially rated with zero to one or two points due to their potential contribution to each process and the resulting scores were validated in 27 colorectal cancer patients (three patients with pre-malignant adenomas, 16 with primaries and two with local recurrencies, three of which were metastatic to the peritoneum, six metastatic to the liver and two metastatic to both, the liver and the peritoneum, and five with hepatic secondaries, one of which at histology was metastatic to the peritoneum too). As a single parameter only the N-stage significantly contributed to OA (p<0.05). Median OA and EMT scores, however, were 3.5 and 2 in the case of primaries without further spread, 5 and 4 in those nodal positive, 5 and 4 in the case of peritoneal implants, 6 and 2 in the case of liver metastases, and 6.5 and 3 in the case of a simultaneous hepatic and peritoneal spread, respectively. These differences were significant when scores from patients with and without liver metastases (OA, p<0.002) or with peritoneal implants and isolated hepatic spread (EMT, p<0.01) were compared. The results suggest a site-specific contribution of OA and EMT to tumour progression in human colon cancer.

Construction of a full-length cDNA Library from Chinese oak silkworm pupa and identification of a KK-42-binding protein gene in relation to pupa-diapause termination

In this study we successfully constructed a full-length cDNA library from Chinese oak silkworm, Antheraea pernyi, the most well-known wild silkworm used for silk production and insect food. Total RNA was extracted from a single fresh female pupa at the diapause stage. The titer of the library was 5 × 10⁵ cfu/ml and the proportion of recombinant clones was approximately 95%. Expressed sequence tag (EST) analysis was used to characterize the library. A total of 175 clustered ESTs consisting of 24 contigs and 151 singlets were generated from 250 effective sequences. Of the 175 unigenes, 97 (55.4%) were known genes but only five from A. pernyi, 37 (21.2%) were known ESTs without function annotation, and 41 (23.4%) were novel ESTs. By EST sequencing, a gene coding KK-42-binding protein in A. pernyi (named as ApKK42-BP; GenBank accession no. FJ744151) was identified and characterized. Protein sequence analysis showed that ApKK42-BP was not a membrane protein but an extracellular protein with a signal peptide at position 1-18, and contained two putative conserved domains, abhydro_lipase and abhydrolase_1, suggesting it may be a member of lipase superfamily. Expression analysis based on number of ESTs showed that ApKK42-BP was an abundant gene in the period of diapause stage, suggesting it may also be involved in pupa-diapause termination.

Essential Roles of mTOR/Akt Pathway in Aurora-A Cell Transformation

Makoto Taga, Eiji Hirooka, Toru Ouchi

We have recently demonstrated that Aurora-A kinase is a potential oncogene to develop mammary gland tumors in mice, when expressed under MMTV promoter. These tumors contain phosphorylated forms of Akt and mTOR, suggesting that Akt-mTOR pathway is involved in transformed phenotype induced by Aurora-A. In the present studies, we discovered that stable cell lines expressing Aurora-A contain phosphorylation of Akt Ser473 after prolonged passages of cell culture, not in cells of the early period of cell culture. Levels of PTEN tumor suppressor are significantly reduced in these late passage cells at least in part due to increased poly ubiquitination of the protein. Akt-activated Aurora-A cells formed larger colonies in soft agar and are resistant to UV-induced apoptosis. Aurora-A inhibitor, VX-680, can cause cell death of Aurora-A cells in which Akt is not activated. siRNA-mediated depletion of mTOR in those cells resulted in decreased phosphorylation of Akt Ser473, suggesting that TORC2 complex phosphorylates Akt in Aurora-A cells. Treatment of late-passage Aurora-A cells with mTOR inhibitor reduced colony formation in soft agar. These results strongly suggest that commitment of cell transformation by Aurora-A is determined by at least co-activation of Akt/mTOR pathway.

How Subchronic and Chronic Health Effects can be Neglected for GMOs, Pesticides or Chemicals

Chronic health effects are increasing in the world such as cancers, hormonal, reproductive, nervous, or immune diseases, even in young people. During regulatory toxicological subchronic tests to prevent these on mammalian health, prior commercialization of chemicals, including pesticides and drugs, or GMOs, some statistically significant findings may be revealed. This discussion is about the need to investigate the relevant criteria to consider those as biologically significant. The sex differences and the non linear dose or time related effects should be considered in contrast to the claims of a Monsanto-supported expert panel about a GMO, the MON 863 Bt maize, but also for pesticides or drugs, in particular to reveal hormone-dependent diseases and first signs of toxicities.

Nuclear Localization of p38 MAPK in Response to DNA Damage

p38 MAP kinase (MAPK) is activated in response to environmental stress, cytokines and DNA damage, and mediates death, cell differentiation and cell cycle checkpoints. The intracellular localization of p38 MAPK upon activation remains unclear, and may depend on the stimulus. We show here that activation of p38 MAPK by stimuli that induce DNA double strand breaks (DSBs), but not other stimuli, leads to its nuclear translocation. In addition, naturally occurring DSBs generated through V(D)J recombination in immature thymocytes also promote nuclear accumulation of p38 MAPK. Nuclear translocation of p38 MAPK does not require its catalytic activity, but is induced by a conformational change of p38 MAPK triggered by phosphorylation within the active site. The selective nuclear accumulation of p38 MAPK in response to DNA damage could be a mechanism to facilitate the phosphorylation of p38 MAPK nuclear targets for the induction of a G2/M cell cycle checkpoint and DNA repair.

HCV Antibody Response and Genotype Distribution in Different Areas and Races of China

Hepatitis C virus (HCV) heterogeneity accounts for the failure of effective vaccine development and the lack of successful anti-viral therapy in some patients. Little is known about the immune response to HCV peptides and the region or race specific genotypes in China. The objective of this study was to characterize HCV antibody immune response to HCV peptides and HCV genotypes in different regions and races of China. A total of 363 serum samples were collected from HCV carriers in 6 regions in China. The immune response to HCV peptides was evaluated by ELISA. HCV genotypes were examined using nested RT-PCR. We found that the anti-HCV antibody neutralization rates were significantly different among the serum samples from different areas or from different races in the same area. For samples from Tibet and Sinkiang, the rates of neutralization by HCV peptides were only 3.2% and 30.8%, respectively. The genotypes of samples from Tibet and Sinkiang were apparently heterogeneic and included type I, II, III and multiple types (I/II/III, I/II, I/III, II/III). One specific sample with multiple-genotype (I/II/III) HCV infection was found to consist of type I, II, III, II/III and an unclassified genotype. These studies indicate that the anti-HCV antibody immune response to HCV peptides varied across regions and among races. The distribution of HCV genotypes among Tibetans in Tibet and Uighurs in Sinkiang was different from that in the inner areas of China. In addition, a “master” genotype, type II, was found to exist in HCV infection with multiple HCV genotypes.

In vivo measurement of protein functional changes

Aili Wang, Zhicheng Zhang, Qinyi Zhao

Conformational changes in proteins are fundamental to all biological functions. In protein science, the concept of protein flexibility is widely used to describe protein dynamics and thermodynamic properties that control protein conformational changes. In this study, we show that urea, which has strong sedative potency, can be administered to fish at high concentrations, and that protein functional changes related to anesthesia induction can be measured in vivo. Ctenopharyngodon idellus (the grass carp) has two different types of N-methyl d-aspartate (NMDA) receptors, urea-insensitive and urea-sensitive, which are responsible for the heat endurance of fish. The urea-sensitive NMDA receptor showed high protein flexibility, the gamma aminobutyric acid (GABA) receptor showed less flexibility, and the protein that is responsible for ethanol anesthesia showed the lowest flexibility. The results suggest that an increase in protein flexibility underlies the fundamental biophysical mechanisms of volatile general anesthetics.

Axial and appendicular skeletal transformations, ligament alterations, and motor neuron loss in Hoxc10 mutants

Sirkka Liisa Hostikka, Jun Gong, Ellen M. Carpenter

Vertebrate Hox genes regulate many aspects of embryonic body plan development and patterning. In particular, Hox genes have been shown to regulate regional patterning of the axial and appendicular skeleton and of the central nervous system. We have identified patterning defects resulting from the targeted mutation of Hoxc10, a member of the Hox10 paralogous family. Hoxc10 mutant mice have skeletal transformations in thoracic, lumbar, and sacral vertebrae and in the pelvis, along with alterations in the bones and ligaments of the hindlimbs. These results suggest that Hoxc10, along with other members of the Hox10 paralogous gene family, regulates vertebral identity at the transition from thoracic to lumbar and lumbar to sacral regions. Our results also suggest a general role for Hoxc10 in regulating chondrogenesis and osteogenesis in the hindlimb, along with a specific role in shaping femoral architecture. In addition, mutant mice have a reduction in lumbar motor neurons and a change in locomotor behavior. These results suggest a role for Hoxc10 in generating or maintaining the normal complement of lumbar motor neurons.

BMP13 Prevents the Effects of Annular Injury in an Ovine Model

Chronic back pain is a global health problem affecting millions of people worldwide and carries significant economic and social morbidities. Intervertebral disc damage and degeneration is a major cause of back pain, characterised by histological and biochemical changes that have been well documented in animal models. Recently there has been intense interest in early intervention in disc degeneration using growth factors or stem cell transplantation, to replenish the diseased tissues. Bone Morphogenetic Proteins (BMPs) have been approved for clinical use in augmenting spinal fusions, and may represent candidate molecules for intervertebral disc regeneration.

BMP13 has an important role in embryonic development and recent genetic evidence shows a role in the development of the human spine. This study explores the effect of BMP13 on a damaged intervertebral disc in an ovine model of discal degeneration. We found that, when injected at the time of injury, BMP13 reversed or arrested histological changes that occurred in the control discs such as loss of extracellular matrix proteins. In addition, BMP13 injected discs retained greater hydration after 4months, and possessed more cells in the NP.

Taken together, BMP13 may be a potent clinical therapeutic agent when used early in the degeneration cascade to promote healthy disc tissue.

Chemoprevention of rat liver toxicity and carcinogenesis by Spirulina

Spirulina platensis (SP) is a filamentous cyanobacterium microalgae with potent dietary phyto-antioxidant, anti-inflammatory and anti-cancerous properties. The present study aimed to investigate the chemopreventive effect of SP against rat liver toxicity and carcinogenesis induced by dibutyl nitrosamine (DBN) precursors, and further characterized its underlying mechanisms of action in HepG2 cell line. Investigation by light and electron microscopy showed that DBN treatment induced severe liver injury and histopathological abnormalities, which were prevented by SP supplementation. The incidence of liver tumors was significantly reduced from 80 to 20% by SP. Immunohistochemical results indicated that both PCNA and p53 were highly expressed in the liver of DBN-treated rats, but were significantly reduced by SP supplementation. Molecular analysis indicated that SP treatment inhibited cell proliferation, which was accompanied by increased p21 and decreased Rb expression levels at 48hrs post-treatment. In addition, SP increased Bax and decreased Bcl-2 expression, indicating induction of apoptosis by 48hrs. This is the first report of the in vivo chemopreventive effect of SP against DBN-induced rat liver cytotoxicity and carcinogenesis, suggesting its potential use in chemoprevention of cancer.

Targeting Promyelocytic Leukemia Protein: A Means to Regulating PML Nuclear Bodies

Erin L. Reineke, Hung-Ying Kao

The promyelocytic leukemia protein (PML) is involved in many cellular processes including cell cycle progression, DNA damage response, transcriptional regulation, viral infection, and apoptosis. These cellular activities often rely on the localization of PML to unique subnuclear structures known as PML nuclear bodies (NBs). More than 50 cellular proteins are known to traffic in and out of PML NBs, either transiently or constitutively. In order to understand the dynamics of these NBs, it is important to delineate the regulation of PML itself. PML is subject to extensive regulation at transcriptional, post-transcriptional, and post-translational levels. Many of these modes of regulation depend on the cellular context and the presence of extracellular signals. This review focuses on the current knowledge of regulation of PML under normal cellular conditions as well as the role for regulation of PML in viral infection and cancer.

Characterization of the complete mitochondrial genome of the giant silkworm moth, Eriogyna pyretorum (Lepidoptera: Saturniidae)

The complete mitochondrial genome (mitogenome) of Eriogyna pyretorum (Lepidoptera: Saturniidae) was determined as being composed of 15,327 base pairs (bp), including 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes, and a control region. The arrangement of the PCGs is the same as that found in the other sequenced lepidopteran. The AT skewness for the E. pyretorum mitogenome is slightly negative (-0.031), indicating the occurrence of more Ts than As. The nucleotide composition of the E. pyretorum mitogenome is also biased toward A + T nucleotides (80.82%). All PCGs are initiated by ATN codons, except for cytochrome c oxidase subunit 1 and 2 (cox1 and cox2). Two of the 13 PCGs harbor the incomplete termination codon by T. All tRNA genes have a typical clover-leaf structure of mitochondrial tRNA, with the exception of trnS1(AGN) and trnS2(UCN). Phylogenetic analysis among the available lepidopteran species supports the current morphology-based hypothesis that Bombycoidea, Geometroidea, Notodontidea, Papilionoidea and Pyraloidea are monophyletic. As has been previously suggested, Bombycidae (Bombyx mori and Bombyx mandarina), Sphingoidae (Manduca sexta) and Saturniidae (Antheraea pernyi, Antheraea yamamai, E. pyretorum and Caligula boisduvalii) formed a group.

THE EXPRESSION OF APELIN AND ITS RECEPTOR APJ DURING DIFFERENT PHYSIOLOGICAL STAGES IN THE BOVINE OVARY

Recent studies implicate that apelin and its receptor APJ may have important role for the modulation of angiogenesis. The aim of this study was to further characterise the regulation of apelin/APJ system in bovine ovary. Experiment 1: corpora lutea (CL) were assigned to the following stages: days 1-2, 3-4, 5-7, 8-12, 13-16, >18 (after regression) of oestrous cycle and of gravidity (month <3, 3-5, 6-7 and >8). Experiment 2: Follicles during maturation were divided into granulosa cells (GC) and theca interna (TI) and were examined separately. Classification of follicles occurred by follicle size and oestradiol-17β (E2) concentration in the follicular fluid (FF) (<0.5 ng/ml, 0.5-5 ng/ml; 5-40 ng/ml; 40-180 ng/ml; >180 ng/ml). Real-time RT-PCR (qPCR) was applied to investigate mRNA expression of examined factors. In general, the expression level of apelin during the oestrous cycle was significantly higher compared to the one during pregnancy. Apelin mRNA levels were always high during the cycle with a tendency of decrease after CL regression. The APJ mRNA in the CL was significantly up regulated on days 5-7 and 8-12 followed by a decrease on days 13-16, and further on days >18. The expression of APJ does not show any significant regulation in the CL throughout pregnancy. The expression of apelin and APJ was not statistically regulated in GC, but was significantly up regulated in follicles with an E2 concentration of more than 5 ng/ml and showed an increase according to growth and maturation of follicles. In conclusion, our data suggest that apelin/APJ system is involved in the mechanism regulating angiogenesis during follicle maturation as well as during CL formation and function in the bovine ovary.

Modulation of NKT cells and Th1/Th2 imbalance after α-GalCer treatment in progressive load-trained rats

Wang Ru, Chen Peijie

Purpose: The purpose of this study was to determine whether α-galactosylceramide (α-GalCer), a synthetic glycolipid agonist of natural killer T (NKT) cells, can ameliorate exercise-induced immune imbalance. Methods: Eight-week-old female Sprague-Dawley rats were trained with a progressively increasing load for 9 weeks. At 36 h and at 7 d after training, groups of rats were euthanized. The whole blood was used to detect hemoglobin(Hb), plasma was analyzed for hormones testosterone(T) and corticosterone(C), and spleen was harvested for detecting NKT cells and interferon-γ (IFN-γ) and interleukin (IL)-4 producing cells. Results: Two-way analysis of variance (ANOVA) showed significant differences between training and time in Series 1. The results showed, at 36h after training, that the decrease in Hb, T and C concentration reflected overtraining or excessive exercise. At 7 d after training, NKT cell populations decreased, and a T helper 1/T helper 2 (Th1/Th2) lymphocyte imbalance occurred. In Series 2, α-galactosylceramide (α-GalCer), an NKT cell activator was found to enhance NKT cell numbers by 69% and shift the Th1/Th2 lymphocyte imbalance by observably decreasing the frequency of IL-4 secreting cells. Conclusion: These data showed that, in addition to Th1/Th2 self-regulation, α-GalCer played an important modulatory role in the exercise-induced Th1/Th2 lymphocyte imbalance, which may be correlative with NKT immunoregulatory cells.

Identification of candidate genes for congenital splay leg in piglets by alternative analysis of DNA microarray data

The congenital splay leg syndrome in piglets is characterized by a temporarily impaired functionality of the hind leg muscles immediately after birth. Etiology and pathogenetic mechanisms for the disease are still not well understood. We compared genome wide gene expression of three hind leg muscles (M. adductores, M. gracilis and M. sartorius) between affected piglets and their healthy littermates with the GeneChip® Porcine Genome Array (Affymetrix) in order to identify candidate genes for the disease. Data analysis with standard algorithms revealed no significant differences between both groups. By application of an alternative approach, we identified 63 transcripts with differences in two muscles and 5 genes differing between the groups in three muscles. The expression of six selected genes (SQSTM1, SSRP1, DDIT4, ENAH, MAF, and PDK4) was investigated with SYBRGreen RT - Real time PCR. The differences obtained with the microarray analysis could be confirmed and demonstrate the validity of the alternative approach to microarray data analysis. Four genes with different expression levels in at least two muscles (SQSTM1, SSRP1, DDIT4, and MAF) are assigned to transcriptional cascades related to cell death and may thus indicate pathways for further investigations on congenital splay leg in piglets.

A male-specific odorant receptor conserved through the evolution of sex pheromones in Ostrinia moth species

In many moths, mate-finding communication is mediated by the female sex pheromones. Since differentiation of sex pheromones is often associated with speciation, it is intriguing to know how the changes in female sex pheromone have been tracked by the pheromone recognition system of the males. A male-specific odorant receptor was found to have been conserved through the evolution of sex pheromone communication systems in the genus Ostrinia (Lepidoptera: Crambidae). In an effort to characterize pheromone receptors of O. scapulalis, which uses a mixture of (E)-11- and (Z)-11-tetradecenyl acetates as a sex pheromone, we cloned a gene (OscaOR1) encoding a male-specific odorant receptor. In addition, we cloned a gene of the Or83b family (OscaOR2). Functional assays using Xenopus oocytes co-expressing OscaOR1 and OscaOR2 have shown that OscaOR1 is, unexpectedly, a receptor of (E)-11-tetradecenol (E11-14:OH), a single pheromone component of a congener O. latipennis. Subsequent studies on O. latipennis showed that this species indeed has a gene orthologous to OscaOR1 (OlatOR1), a functional assay of which confirmed it to be a gene encoding the receptor of E11-14:OH. Furthermore, investigations of six other Ostrinia species have revealed that all of them have a gene orthologous to OscaOR1, although none of these species, except O. ovalipennis, a species most closely related to O. latipennis, uses E11-14:OH as the pheromone component. The present findings suggest that the male-specific receptor of E11-14:OH was acquired before the divergence of the genus Ostrinia, and functionally retained through the evolution of this genus.

Body fat mass reduction and up-regulation of uncoupling protein by novel lipolysis-promoting plant extract

We have found natural products exhibiting lipolysis-promoting activity in subcutaneous adipocytes, which are less sensitive to hormones than visceral adipocytes. The activities and a action mechanisms of a novel plant extract of Cirsium oligophyllum (CE) were investigated in isolated adipocytes from rat subcutaneous fat, and its fat-reducing effects by peroral administration and topical application were evaluated in vivo. CE-induced lipolysis was synergistically enhanced by caffeine, a phosphodiesterase inhibitor, and was reduced by propranolol, a β adrenergic antagonist. The peroral administration of 10% CE solution to Wistar rats for 32 days reduced body weight gain, subcutaneous, and visceral fat weights by 6.6, 26.2, and 3.0%, respectively, as compared to the control group. By the topical application of 2% of this extract to rats for 7 days, weight of subcutaneous fat in the treated skin was reduced by 23.2%. This fat mass reduction was accompanied by the up-regulation of uncoupling protein 1 (UCP), a principal thermogenic mitochondrial molecule related to energy dissipating, in subcutaneous fat and UCP3 in skin except for the fat layer. These results indicate that CE promotes lipolysis via a mechanism involving the β adrenergic receptor, and affects the body fat mass. This fat reduction may be partially due to UCP up-regulation in the skin including subcutaneous fat. This is the first report showing that repeated lipolysis promotion through CE administration may be beneficial for the systematic suppression of body fat accumulation or the control of fat distribution in obesity.

Vesnarinone Represses the Fibrotic Changes in Murine Lung Injury Induced by Bleomycin

We investigated the potential usefulness of vesnarinone, a novel cytokine inhibitor, for the treatment of lung fibrosis using a murine model of bleomycin (BLM)-induced pulmonary fibrosis. Mice were fed a control diet (n=42), or a diet containing low (n=42) or high (n=42) dose of vesnarinone. Dietary intake of vesnarinone minimized the BLM toxicity as reflected by significant decreases in numbers of inflammatory cells, KC, and soluble TNF receptors in the bronchoalveolar lavage fluid. A quantitative evaluation of histology demonstrated significantly mild lung parenchymal lesions in BLM-treated mice fed with diet containing high dose of vesnarinone than in the control diet group. Consistent with the histopathology, hydroxyproline levels in lung tissue from BLM-treated mice fed with diet containing vesnarinone were significantly lower than that from mice fed with control diet. We concluded that vesnarinone inhibits BLM-induced pulmonary fibrosis, at least in part, by the inhibition of acute lung injuries in the early phase.

Old can be new again: HAPPY whole genome sequencing, mapping and assembly

Zhihua Jiang, Daniel S. Rokhsar, Richard M. Harland

During the last three decades, both genome mapping and sequencing methods have advanced significantly to provide a foundation for scientists to understand genome structures and functions in many species. Generally speaking, genome mapping relies on genome sequencing to provide basic materials, such as DNA probes and markers for their localizations, thus constructing the maps. On the other hand, genome sequencing often requires a high-resolution map as a skeleton for whole genome assembly. However, both genome mapping and sequencing have never come together in one pipeline. After reviewing mapping and next-generation sequencing methods, we would like to share our thoughts with the genome community on how to combine the HAPPY mapping technique with the new-generation sequencing, thus integrating two systems into one pipeline, called HAPPY pipeline. The pipeline starts with preparation of a HAPPY panel, followed by multiple displacement amplification for producing a relatively large quantity of DNA. Instead of conventional marker genotyping, the amplified panel DNA samples are subject to new-generation sequencing with barcode method, which allows us to determine the presence/absence of a sequence contig as a traditional marker in the HAPPY panel. Statistical analysis will then be performed to infer how close or how far away from each other these contigs are within a genome and order the whole genome sequence assembly as well. We believe that such a universal approach will play an important role in genome sequencing, mapping, and assembly of many species; thus advancing genome science and its applications in biomedicine and agriculture.

Characterization of persistent TTX-R Na+ currents in physiological concentration of sodium in rat visceral afferents

Guo-Fen Qiao, Bai-Yan Li, Yu-Hong Zhou, Yan-Jie Lu, John H. Schild

Persistent tetrodotoxin-resistant (TTX-R) Na⁺ (Na_v1.9/SCN11A) currents are not normally recorded in vagal afferent neurons (VANs) with 50 mM of extracellular Na⁺ although the functional expression of this current was observed in the presence of PGE₂ or forskolin. However, it is uncertain whether this current can be seen under physiological condition (150 mM Na⁺). Using the whole-cell patch-clamp technique, we showed that persistent TTX-R Na⁺ currents were expressed in 9 out of 38 VANs bathed in 150 mM Na⁺. The current density, but not the whole-cell capacitance, was significantly enhanced in the VANs expressing Nav1.9. Persistent TTX-R Na⁺ channels were activated at a more hyperpolarized membrane potential near -60 mV, compared with TTX-sensitive (TTX-S at -40 mV) and TTX-R Na⁺ channels (at -20 mV). This indicates that persistent TTX-R Na⁺ channels provide a wider activation window than TTX-S and TTX-R Na channels to up-regulate neuronal excitability. These results suggest that the persistent TTX-R Na⁺ currents may be involved in the neuronal excitability by setting a lower pressure-discharge threshold and higher discharge frequency of VANs, especially the unique subset and gender-specific distribution of myelinated Ah-type VANs, including Ah-type aortic baroreceptor neurons, identified in our previous study.

Responses of Plasma Acetate Metabolism to Hop (Humulus lupulus L.) in Sheep

Mohammad Al-Mamun, Kunio Goto, Sota Chiba, Hiroaki Sano

An isotope dilution method using [1-¹³C]sodium (Na) acetate was conducted to determine the effect of feeding hop (Humulus lupulus L.) residues on plasma acetate metabolism in six adult crossbred sheep. The sheep were fed 63 g/kg BW^0.75/d of either mixed hay (MH-diet) of orchardgrass (Dactylis glomerata L.) and reed canarygrass (Phalaris arundinacea L.) at a 60:40 ratio or MH-diet and hop-residues (Hop-diet) at 85:15 ratio with a crossover design for each of 3 week period. The isotope dilution method using single injection of [1-¹³C]Na acetate was performed thrice; before feeding (BF), 2 h after feeding (2F) and 4 h after feeding (4F), on the 21^st day of each dietary treatment. Plasma acetate concentration tended to increase (P= 0.06) and turnover rate was numerically higher (P= 0.16) for MH-diet than Hop-diet. Plasma glucose, NEFA, VFA and lactic acid concentrations were similar between dietary treatments. In both the diets, although plasma concentration of acetate did not change, turnover rate increased significantly (P= 0.02) 2F than BF. Hop-residues did not show any negative impacts on acetate metabolism as well as physiology of animals in the present experimental conditions, hence thereby it could be used as an alternative to MH-diet for rearing sheep.

Serum adipokine levels in the obese people

Correlated alterations in prostate basal cell layer and basement membrane

Aijun Liu, Lixin Wei, William A. Gardner, Chu-Xia Deng, Yan-Gao Man

Our recent studies revealed that focal basal cell layer disruption (FBCLD) induced auto-immunoreactions represented a contributing factor for human prostate tumor progression and invasion. As the basement membrane surrounds and attaches to the basal cell layer, our current study assessed whether FBCLD would impact the physical integrity of the associated basement membrane. Paraffin sections from 25-human prostate tumors were subjected to double immunohistochemistry to simultaneously elucidate the basal cell layer and the basement membrane with corresponding biomarkers. The physical integrity of the basement membrane overlying FBCLD was examined to determine the extent of correlated alterations. Of a total of 89 FBCLD encountered, 76 (85 %) showed correlated alterations in the overlying basement membrane, which included distinct focal disruptions or fragmentations. In the remaining 13 (15%) FBCLD, the overlying basement membrane showed significant attenuation or reduction of the immunostaining intensity. The basement membrane in all or nearly all ducts or acini with p63 positive basal cells was substantially thicker and more uniform than that in ducts or acini without p63 positive basal cells, and also, a vast majority of the focal disruptions occurred near basal cells that lack p63 expression. These findings suggest that focal disruptions in the basal cell layer and alterations in the basement membrane are correlated events and that the physical and functional status of the basal cells could significantly impact the physical integrity of the overlying basement membrane. As the degradation of both the basal cell layer and the basement membrane is a pre-requisite for prostate tumor invasion or progression, ducts or acini with focally disrupted basal cell layer and basement membrane are likely at greater risk to develop invasive lesions. Thus, further elucidation of the specific molecules and mechanism associated with these events may lead to the development of a more effective alternative for repeat biopsy to monitor tumor progression and invasion.

Osteoarthitis of Leptin-Deficient ob/ob Mice in Response to Biomechanical Loading in Micro-CT

Hansjoerg Heep, Gero Hilken, Sebastian Hofmeister, Christian Wedemeyer

Objective: Mechanotransduction is the mechanism that due to reacting chondrocytes on biomechanical loading of body mass. Higher biomechanical loading lead to increased degeneration of chondrocytes, whereas moderate loading is protecting. This suggests that body fat regulates bone metabolism first by means of hormonal factors and second that the effects of muscle and loading are signaling factors in mechanotransduction. Leptin, a peptide hormone produced predominantly by white fat cells, is one of these hormonal factors. The aim of this study was to investigate and measure the different effects of weight-bearing on trabecular bone formation in mice without the stimulation of leptin and with or without osteoarthritis. Materials and methods: 40 C57BL/ 6J ob/ob-mice in the age of 20 weeks have been devided into two groups with an ad-libitum-diet and with reduced diet. The hip- and knee-joints have been examinated in micro-CT-scan and histomorphologically. Results: Animals with an ad-libitum-diet were found to increase body weight significantly at the age of six weeks in comparison with lean mice. At the age of twenty weeks the obese mice were almost twice as heavy as the lean mice. Significant statistical differences are shown between the two groups for body weight and bone mineral density. Examination of trabecular bone in micro-CT revealed that the only statistically significant difference between the two groups was the trabecular number for the proximal femur. High weight-bearing insignificantly improved all trabecular bone parameters in the obese mice. Correlation was found between trabecular number and bone mineral density on the one hand and body weight on the other hand. The correlation between body weight and osteoarthritis shows a significant increase in grade of osteoarthritis as body weight increases in hip-joint and knee-joint but not in osteoarthritis-positive (OP) versus osteoarthritis-negative (ON) mices. The correlation of the hip-joint between micro-CT data and body weight shows an increase in these data as body weight increases in OP mices. The correlation of the hip-joint between micro-CT data and osteoarthritis shows a decrease in these data as osteoarthritis increases in OP mices. The correlation of the knee-joint between micro-CT data and body weight shows differencies between ON and OP mices. The correlation of the knee-joint between micro-CT data and osteoarthritis shows an increase in these data as osteoarthritis increases in OP mices. Conclusion: biomechanical loading led to decreased bone mineral density by a decrease in the number of trabeculae. Trabecular thickness was not increased by biomechanical loading in growing mice. Decreased body weight in leptin-deficient mice protects against bone loss. This finding is consistent with the principle of light-weight construction of bone. Differences in osteoarthritis-positive and osteoarthritis-negative mices show the eventual importance of diet in leptin-deficience. It is not possible to conclude that these results also apply to human beings.

The Role of SRC-1 in Murine Prostate Carcinogenesis Is Nonessential due to a Possible Compensation of SRC-3/AIB1 Overexpression

Jean Ching-Yi Tien, Suoling Zhou, Jianming Xu

The androgen and androgen receptor (AR)-regulated gene expression plays important roles in normal prostate and prostate cancer development, and AR transcriptional control of genes is mediated by transcriptional coactivators, including the three members of the steroid receptor coactivator (SRC) family, SRC-1 (NCOA1), SRC-2 (TIF2/GRIP1/NCOA2) and SRC-3 (AIB1, ACTR/RAC3/NCOA3). SRC-1 and SRC-3 are overexpressed in multiple human endocrine cancers and knockdown of either one of them in prostate cancer cell lines impedes cellular proliferation. Knockout of SRC-3 in mice suppresses the progression of spontaneous prostate carcinogenesis. In this study, we investigated SRC-1 contribution to prostate cancer in vivo by deleting the SRC-1 gene in TRAMP mice, which contain the probasin promoter-driven SV40 T/t antigen transgene. In assessing tumor mass of mice at various ages, we found that initiation and progression of prostate cancer induced by SV40 T/t antigens were unaltered in SRC-1^-/- mice versus WT mice. Primary tumor histology and metastasis to distant lymph nodes were also similar in these mice at all time points assessed. These results demonstrate that the role of SRC-1 in mouse prostate carcinogenesis is nonessential and different from the essential contribution of SRC-3 that is required for prostate cancer progression and metastasis in mice. Interestingly, we observed that during prostate tumorigenesis SRC-1 expression was relatively constant, while SRC-3 expression was significantly elevated. Therefore, the loss of SRC-1 function may be compensated by SRC-3 overexpression during prostate tumorigenesis in SRC-1^-/- mice.

In vivo evidence of hepato- and reno-protective effect of garlic oil against sodium nitrite-induced oxidative stress

Sodium nitrite (NaNO₂), a food color fixative and preservative, contributes to carcinogenesis. We investigated the protective role of garlic oil against NaNO₂-induced abnormalities in metabolic biochemical parameters and oxidative status in male albino rats. NaNO₂ treatment for a period of three months induced a significant increase in serum levels of glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), bilirubin, urea and creatinine as well as hepatic AST and ALT. However, significant decrease was recorded in liver ALP activity, glycogen content, and renal urea and creatinine levels. In parallel, a significant increase in lipid peroxidation, and a decrease in glutathione content and catalase activity were observed in the liver and the kidney. However, garlic oil supplementation showed a remarkable amelioration of these abnormalities. Our data indicate that garlic is a phytoantioxidant with powerful chemopreventive properties against chemically-induced oxidative stress.

Birth weight and coronary artery disease. The effect of gender and diabetes

Background: The developmental origin theory of coronary heart disease proposes that undernutrition in utero permanently changes body functions and metabolism leading to an increased risk of coronary artery diseases (CAD) in adult life. Some studies support this theory but others suggest that birth weight (BW) is not a major risk factor for cardiovascular diseases. Gender differences concerning the association between BW and risk factors for CAD have been reported in some studies but not in others.

In this paper we have analyzed the effect of gender and diabetes on the relationship between BW and CAD in the White population of Rome.

Material and Methods: 226 subjects admitted to the Hospital for non fatal CAD from the White population of Rome were studied. 395 consecutive newborn infants studied in the same population in the years 1968-1972 were considered for comparison.

Results: Among subjects with CAD, reliable information on BW was obtained in 127 subjects. The distribution of BW in CAD depends on gender (p=0.009). In females with CAD there is a tendency toward low BW, while in males with CAD there is a tendency toward high BW. These associations are very marked in non-diabetic subjects with CAD (p=.001), while no significant association is observed in diabetic subjects (p=0.557).

Conclusion: Our data confirm the association between BW and CAD and suggest that the association depends on gender and is influenced by diabetes.

A Curriculum Vitae of Teeth: Evolution, Generation, Regeneration

Despina S. Koussoulakou, Lukas H. Margaritis, Stauros L. Koussoulakos

The ancestor of recent vertebrate teeth was a tooth-like structure on the outer body surface of jawless fishes. Over the course of 500,000,000 years of evolution, many of those structures migrated into the mouth cavity. In addition, the total number of teeth per dentition generally decreased and teeth morphological complexity increased. Teeth form mainly on the jaws within the mouth cavity through mutual, delicate interactions between dental epithelium and oral ectomesenchyme. These interactions involve spatially restricted expression of several, teeth-related genes and the secretion of various transcription and signaling factors. Congenital disturbances in tooth formation, acquired dental diseases and odontogenic tumors affect millions of people and rank human oral pathology as the second most frequent clinical problem. On the basis of substantial experimental evidence and advances in bioengineering, many scientists strongly believe that a deep knowledge of the evolutionary relationships and the cellular and molecular mechanisms regulating the morphogenesis of a given tooth in its natural position, in vivo, will be useful in the near future to prevent and treat teeth pathologies and malformations and for in vitro and in vivo teeth tissue regeneration.

Diverse protein regulations on PHA formation in Ralstonia eutropha on short chain organic acids

Sung-Eun Lee, Qing X. Li, Jian Yu

Organic acids are considered as potential substrates for biosynthesis of polyhydroxyalkaonates. The acids may also be the metabolic inhibitors at moderate concentration levels. In this study, Ralstonia eutropha was used to elucidate the protein regulations when the bacterial cells pre-cultivated on glucose were exposed to three representative short chain organic acids, acetic, propionic and levulinic acids. The research compared and examined the proteins that might participate in PHA metabolism, primary metabolism, and cell's defense systems. A number of proteins were found to be induced in R. eutropha by using 1D-PAGE and nano-liquid chromatography tandem MS/MS. With the proteins being up-regulated, a dramatic change occurred in the induction of PHA metabolism, including fatty acid biosynthesis for acetate, β-oxidation for propionate and both for levulinic acid. Acetate kinase was induced in response to the presence of acetate or levulinic acid. The organic acids induced several proteins involved in amino acid biosynthesis, purine and pyrimidine biosynthesis, and cofactor biosynthesis in R. eutropha, but the regulations had a great variation. R. eutropha might employ different regulation mechanisms to maintain cell growth and PHA formation when the cells are exposed to the organic acids as sole source of carbon and energy.

Identifications of SUMO-1 cDNA and Its Expression Patterns in Pacific White Shrimp Litopeanaeus vannamei

Yanisa Laoong-u-thai, Baoping Zhao, Amornrat Phongdara, Harry Ako, Jinzeng Yang

Small ubiquitin-like modifiers (SUMO) work in a similar way as ubiquitin to alter the biological properties of a target protein by conjugation. A shrimp SUMO cDNA named LvSUMO-1 was identified in Litopenaeus vannamei. LvSUMO-1 cDNA contains a coding sequence of 282 nucleotides with untranslated regions of 37 bp at 5'-end and 347 bp at 3'-end, respectively. The deduced 93 amino acids exhibit 83% identity with the Western Honeybee SUMO-1, and more than 65% homologies with human and mouse SUMO-1. LvSUMO-1 mRNA is expressed in most L. vannamei tissues with the highest level in hepatopancrease. The mRNA expression of LvSUMO-1 over development stages in L. Vammamei is distinguished by a low level in nauplius stage and relatively high level in postlarva stage with continuous expression until juvenile stage. The LvSUMO-1 protein and its conjugated proteins are detected in both cytoplasm and nucleus in several tissues. Interestingly, LvSUMO-1 mRNA levels are high in abdominal muscle during the premolt stage, wherein it has significant activities of protein degradation, suggesting its possible role in the regulation of shrimp muscle protein degradation.

Is sodium current present in human sinoatrial node cells?

Arie O. Verkerk, Ronald Wilders, Marcel M.G.J. van Borren, Hanno L. Tan

Pacemaker activity of the sinoatrial node has been studied extensively in various animal species, but is virtually unexplored in man. As such, it is unknown whether the fast sodium current (I_Na) plays a role in the pacemaker activity of the human sinoatrial node. Recently, we had the unique opportunity to perform patch-clamp experiments on single pacemaker cells isolated from a human sinoatrial node. In 2 out of the 3 cells measured, we observed large inward currents with characteristics of I_Na. Although we were unable to analyze the current in detail, our findings provide strong evidence that I_Na is present in human sinoatrial node pacemaker cells, and that this I_Na is functionally available at potentials negative to -60 mV.

BMP-13 Emerges as a Potential Inhibitor of Bone Formation

Bone morphogenetic protein-13 (BMP-13) plays an important role in skeletal development. In the light of a recent report that mutations in the BMP-13 gene are associated with spine vertebral fusion in Klippel-Feil syndrome, we hypothesized that BMP-13 signaling is crucial for regulating embryonic endochondral ossification. In this study, we found that BMP-13 inhibited the osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro. The endogenous BMP-13 gene expression in MSCs was examined under expansion conditions. The MSCs were then induced to differentiate into osteoblasts in osteo-inductive medium containing exogenous BMP-13. Gene expression was analysed by real-time PCR. Alkaline phosphatase (ALP) expression and activity, proteoglycan (PG) synthesis and matrix mineralization were assessed by cytological staining or ALP assay. Results showed that endogenous BMP-13 mRNA expression was higher than BMP-2 or -7 during MSC growth. BMP-13 supplementation strongly inhibited matrix mineralization and ALP activity of osteogenic differentiated MSCs, yet increased PG synthesis under the same conditions. In conclusion, BMP-13 inhibited osteogenic differentiation of MSCs, implying that functional mutations or deficiency of BMP-13 may allow excess bone formation. Our finding provides an insight into the molecular mechanisms and the therapeutic potential of BMP-13 in restricting pathological bone formation.

Association between Apoptotis and CD4+/CD8+ T-Lymphocyte Ratio in Aseptic Loosening after Total Hip Replacement

Particle-induced osteolysis is a major cause of aseptic loosening after total joint replacement. While the osteolytic cascade initiated by cytokine release from macrophages has been studied extensively, the involvement of T-lymphocytes in this context is controversial and has been addressed by only a few authors. In a former study we detected that the quantity of T-lymphocytes may be influenced by apoptosis in patients with aseptic loosening. In this study we intended to find out more details about the apoptosis-induced shifting of the T-cell number. We focused our interest on the CD4⁺ and CD8⁺ T-cells and their relative ratio. Caspase-3 cleaved was evaluated immunohistochemically to detect apoptotic T-cells in capsules and interface membranes from patients with aseptic hip implant loosening and a varying degree of caspase-3 cleaved expression in CD4⁺ and CD8⁺ T-lymphocytes was detected. Moreover, a relationship between the intensity of the apoptotic reactions and the radiological extent of osteolysis was observed. The number of CD4⁺ cells was decreased in the presence of strong apoptotic reactions, respectively extensive osteolysis, while CD8⁺ cells were affected to a much lower degree. Thus, the CD4⁺/CD8⁺ ratio changed from 1.0 in cases with only small areas of periprosthetic osteolysis and minimally intense apoptosis to 0.33 in cases with large areas of osteolysis. This may suggest a causal relationship between the apoptosis-induced shift in the CD4⁺/CD8⁺ ratio and the osteolysis respectively aseptic loosening. It is possible that these findings may lead to a new understanding of particle-induced osteolysis.

Developing tTA Transgenic Rats for Inducible and Reversible Gene Expression

To develop transgenic lines for conditional expression of desired genes in rats, we generated several lines of the transgenic rats carrying the tetracycline-controlled transactivator (tTA) gene. Using a vigorous, ubiquitous promoter to drive the tTA transgene, we obtained widespread expression of tTA in various tissues. Expression of tTA was sufficient to strongly activate its reporter gene, but was below the toxicity threshold. We examined the dynamics of Doxycycline (Dox)-regulated gene expression in transgenic rats. In the two transmittable lines, tTA-mediated activation of the reporter gene was fully subject to regulation by Dox. Dox dose-dependently suppressed tTA-activated gene expression. The washout time for the effects of Dox was dose-dependent. We tested a complex regime of Dox administration to determine the optimal effectiveness and washout duration. Dox was administered at a high dose (500 μg/ml in drinking water) for two days to reach the effective concentration, and then was given at a low dose (20 μg/ml) to maintain effectiveness. This regimen of Dox administration can achieve a quick switch between ON and OFF statuses of tTA-activated gene expression. In addition, administration of Dox to pregnant rats fully suppressed postnatal tTA-activated gene expression in their offspring. Sufficient levels of Dox are present in mother's milk to produce maximal efficacy in nursing neonates. Administration of Dox to pregnant or nursing rats can provide a continual suppression of tTA-dependent gene expression during embryonic and postnatal development. The tTA transgenic rat allows for inducible and reversible gene expression in the rat; this important tool will be valuable in the development of genetic rat models of human diseases.

Understanding Quantitative Genetics in the Systems Biology Era

Mengjin Zhu, Mei Yu, Shuhong Zhao

Biology is now entering the new era of systems biology and exerting a growing influence on the future development of various disciplines within life sciences. In early classical and molecular periods of Biology, the theoretical frames of classical and molecular quantitative genetics have been systematically established, respectively. With the new advent of systems biology, there is occurring a paradigm shift in the field of quantitative genetics. Where and how the quantitative genetics would develop after having undergone its classical and molecular periods? This is a difficult question to answer exactly. In this perspective article, the major effort was made to discuss the possible development of quantitative genetics in the systems biology era, and for which there is a high potentiality to develop towards "systems quantitative genetics". In our opinion, the systems quantitative genetics can be defined as a new discipline to address the generalized genetic laws of bioalleles controlling the heritable phenotypes of complex traits following a new dynamic network model. Other issues from quantitative genetic perspective relating to the genetical genomics, the updates of network model, and the future research prospects were also discussed.

Activity of Chitosans in combination with antibiotics in Pseudomonas aeruginosa

San Tin, Kishore R. Sakharkar, Chu Sing Lim, Meena K. Sakharkar

Chitosan and its derivative water soluble Chitosan oligosaccharide are used in a variety of applications in pharmaceutical preparations. In this study, 2 wild (ATCC 15729 and PAO1) and 2 mutant strains (PT121 and PT149) of P. aeruginosa are investigated for drug-drug interactions in vitro. 10 antimicrobial agents (antibiotics) are combined with different degree of deacetylated Chitosans and Chitosan oligosaccharide. All the chitosans show synergistic activity with sulfamethoxazole, a sulfonamide antimicrobial agent. It is interesting to observe that the MIC value for the MexEF-OprN overexpressing mutant strain of P. aeruginosa is 5 fold higher than the other strains under investigation suggesting a possible role of this efflux pump in Sulfamethoxazole efflux. The findings suggest on the use of chitosans as enhancing agent in combination with antibiotics in pharmaceutical preparations.

SIRT1, Is It a Tumor Promoter or Tumor Suppressor?

Chu-Xia Deng

SIRT1 has been considered as a tumor promoter because of its increased expression in some types of cancers and its role in inactivating proteins that are involved in tumor suppression and DNA damage repair. However, recent studies demonstrated that SIRT1 levels are reduced in some other types of cancers, and that SIRT1 deficiency results in genetic instability and tumorigenesis, while overexpression of SIRT1 attenuates cancer formation in mice heterozygous for tumor suppressor p53 or APC. Here, I review these recent findings and discuss the possibility that activation of SIRT1 both extends lifespan and inhibits cancer formation.

Antitumor Activity of T Cells Generated from Lymph Nodes Draining the SEA-expressing Murine B16 Melanoma and Secondarily Activated with Dendritic Cells

The successful use of tumor-draining lymph nodes (TDLN) as a source of effector cells for cancer immunotherapy depends largely on the immunogenicity of the tumor drained by the lymph nodes as well as the methods for secondary in vitro T cell activation and expansion. We transferred the bacterial superantigen staphylococcal enterotoxin A (SEA) gene into B16 murine melanoma tumor cells, and used them to induce TDLN (SEA TDLN) in syngeneic hosts. Wild-type (wt) TDLN induced by parental B16 tumor was used as a control. In vitro, SEA TDLN cells proliferated more vigorously, produced more IFNγ and demonstrated higher CTL activity than wt TDLN cells when activated with anti-CD3/anti-CD28/IL-2. In vivo, SEA TDLN cells mediated tumor eradication more effectively than similarly activated wt TDLN cells (p<0.01). Furthermore, use of dendritic cells (DC) plus tumor antigen in vitro in addition to anti-CD3/anti-CD28/IL-2 stimulation further amplified the immune function and therapeutic efficacy of SEA TDLN cells. DC-stimulated SEA TDLN cells eliminated nearly 90% of the pulmonary metastasis in mice bearing established B16 melanoma micrometastases. These results indicate that enforced expression of superantigen SEA in poorly immunogenic tumor cells can enhance their immunogenicity as a vaccine in vivo. The combined use of genetically modified tumor cells as vaccine to induce TDLN followed by secondary stimulation using antigen-presenting cells and tumor antigen in a sequential immunization/activation procedure may represent a unique method to generate more potent effector T cells for adoptive immunotherapy of cancer.

Assay Validation for KIM-1: human urinary renal dysfunction biomarker

Shalini Chaturvedi, Takeisha Farmer, Gordon F. Kapke

Urinary kidney injury molecule (KIM-1) is a sensitive quantitative biomarker for early detection of kidney tubular injury. The objective of the present work was to analytically validate this urinary renal injury biomarker. Duo-set reagents from R&D were used to develop the ELISA and validate the assay's linearity, intra-run precision, inter-run precision, lower limit of quantification, recovery, dilutional verification, reference range, stability, and length of run. The reference range data suggests that the healthy population falls within the assay range (59 - 2146 pg/mL) and upper limit of quantitation for this assay is 17168 pg/mL for the patient population. This is a robust assay to detect urinary levels of KIM-1, which serves as a non-invasive sensitive, reproducible, and potentially high-throughput method to detect early kidney injury in drug development studies.

Differential expression of neurotrophins in postnatal C57BL/6 mice striatum

V. Zermeño, S. Espindola, E. Mendoza, E. Hernández-Echeagaray

Neurotrophin expression in early stages of development is crucial for brain assembly and function. In particular, postnatal expression of neurotrophins has not been well documented in the neostriatum and in general neurotrophins or their receptor mRNA's are normally reported, but not protein expression. In the present study, immunocytochemical expression of BDNF, NT-3 and NT-4/5 was characterized in striatal tissue of C57BL/6 mice at postnatal days 10^th(P10), 21^st (P21), 42^nd (P42) and 80^th (P80).

We found that the expression of BDNF diminished along the postnatal time course we evaluated, while staining for NT-4 increased up to age P42 and remained constant, thereafter in the cell's soma. In contrast, NT-3 was first expressed in the neostriatal bundles and later on, in neostriatal cell somas. These results provide information about differences in the spatial and temporal expression of each neurotrophin in the neostriatum during the first 80^th postnatal days.

RT-PCR procedures were also carried out to further determine whether protein levels of neurotrophins observed in the neostriatum were under control of gene expression. All neurotrophin mRNAs were expressed and only mRNA^BDNF was reduced during the postnatal evaluated days.

Differences in temporal expression of neurotrophins may be related to the heterochronic development of neostriatal cell populations, but also with the specificity of each neurotrophin modulating different neuronal targets.

The Fascinating World of RNA Interference

Micro- and short-interfering RNAs represent small RNA family that are recognized as critical regulatory species across the eukaryotes. Recent high-throughput sequencing have revealed two more hidden players of the cellular small RNA pool. Reported in mammals and Caenorhabditis elegans respectively, these new small RNAs are named piwi-interacting RNAs (piRNAs) and 21U-RNAs. Moreover, small RNAs including miRNAs have been identified in unicellular alga Chlamydomonas reinhardtii, redefining the earlier concept of multi-cellularity restricted presence of these molecules. The discovery of these species of small RNAs has allowed us to understand better the usage of genome and the number of genes present but also have complicated the situation in terms of biochemical attributes and functional genesis of these molecules. Nonetheless, these new pools of knowledge have opened up avenues for unraveling the finer details of the small RNA mediated pathways.

Aberrant p63 and WT-1 expression in myoepithelial cells of pregnancy-associated breast cancer: implications for tumor aggressiveness and invasiveness

Zheli Xu, Wan Wang, Chu-Xia Deng, Yan-gao Man

Our recent studies revealed that focal alterations in breast myoepithelial cell layers significantly impact the biological presentation of associated epithelial cells. As pregnancy-associated breast cancer (PABC) has a significantly more aggressive clinical course and mortality rate than other forms of breast malignancies, our current study compared tumor suppressor expression in myoepithelial cells of PABC and non-PABC, to determine whether myoepithelial cells of PABC may have aberrant expression of tumor suppressors. Tissue sections from 20 cases of PABC and 20 cases of stage, grade, and age matched non-PABC were subjected to immunohistochemistry, and the expression of tumor suppressor maspin, p63, and Wilms' tumor 1 (WT-1) in calponin positive myoepithelial cells were statistically compared. The expression profiles of maspin, p63, and WT-1 in myoepithelial cells of all ducts encountered were similar between PABC and non-PABC. PABC, however, displayed several unique alterations in terminal duct and lobular units (TDLU), acini, and associated tumor tissues that were not seen in those of non-PABC, which included the absence of p63 and WT-1 expression in a vast majority of the myoepithelial cells, cytoplasmic localization of p63 in the entire epithelial cell population of some lobules, and substantially increasing WT-1 expression in vascular structures of the invasive cancer component. All or nearly all epithelial cells with aberrant p63 and WT-1 expression lacked the expression of estrogen receptor and progesterone receptor, whereas they had a substantially higher proliferation index than their counterparts with p63 and WT-1 expression. Hyperplastic cells with cytoplasmic p63 expression often adjacent to, and share a similar immunohistochemical and cytological profile with, invasive cancer cells. To our best knowledge, our main finings have not been previously reported. Our findings suggest that the functional status of myoepithelial cells may be significantly associated with tumor aggressiveness and invasiveness.

STAT2*C related genotypes and allele but not TLR4 and CD40 gene polymorphisms are associated with higher susceptibility for asthma

Yao-Yuan Hsieh, Lei Wan, Chi-Chen Chang, Chang-Hai Tsai, Fuu-Jen Tsai

Objective: Asthma is caused by a complex interaction between multiple genes and environmental factors. Herein we aimed to investigate whether signal transducer and activator of transcription (STAT2), toll-like receptors 4 (TLRs4) and CD40-related polymorphisms are associated with asthma susceptibility.

Design: Children were divided: (1) asthma (n=117); (2) normal controls (n=60). The polymorphisms of STAT2, TLR4 and CD40 polymorphism were analyzed by PCR-RFLP genotyping. Genotypes, allelic frequencies and association of haplotypes in both groups were compared.

Results: STAT2*C related genotypes, but not TLR4 and CD40 polymorphism, are associated with higher susceptibility for asthma. Distributions of STAT2*CC/CG/GG and C/G allele in both groups are: (1) 0/11.1/88.9 % and 5.6/94.4%; (2) 0/1.7/98.3% and 0.8/99.2% (p<0.05). Proportions of TLR4*rs10983755 AA/AG/GG and rs1927914 CC/CT/TT homozygote are: (1) 35.1/8.5/56.4% and 9.4/56.4/34.2%; (2) 35/8.3/56.7% and 16.7/48.3/35% (non-difference). Proportions of CD40*rs1883832 CC/CT/TT, rs3765459 AA/AG/GG, and rs4810485 TT/GT/GG are: (1) 29.9/53/17.1%, 6.8/47.9/45.3 and 18.8/62.4/18.8%; (2) 36.7/41.7/21.6%, 1.6/46.7/ 51.7 and 15/51.7/33.3% (non-difference). Haplotype analyses for TLR4 and CD40 genes revealed their non-association and non-additional effect upon asthma susceptibilities.

Conclusion: STAT2*C related genotypes and alleles are associated with asthma susceptibilities and pathogenesis. There were non-association and non-additional effects of TLR4/CD40 gene polymorphisms and haplotypes upon asthma risk.

SpolvlgA is a DDX3/PL10-related DEAD-box RNA helicase expressed in blastomeres and embryonic cells in planarian embryonic development

Jordi Solana, Rafael Romero

Planarian flatworms have an impressive regenerative power. Although their embryonic development is still poorly studied and is highly derived it still displays some simple characteristics. We have identified SpolvlgA, a Schmidtea polychroa homolog of the DDX3/PL10 DEAD-box RNA helicase DjvlgA from the planarian species Dugesia japonica. This gene has been previously described as being expressed in planarian adult stem cells (neoblasts), as well as the germ line. Here we present the expression pattern of SpolvlgA in developing embryos of S. polychroa and show that it is expressed from the first cleavage rounds in blastomere cells and blastomere-derived embryonic cells. These cells are undifferentiated cells that engage in a massive wave of differentiation during stage 5 of development. SpolvlgA expression highlights this wave of differentiation, where nearly all previous structures are substituted by blastomere-derived embryonic cells. In late stages of development SpolvlgA is expressed in most proliferating and differentiating cells. Thus, SpolvlgA is a gene expressed in planarian embryos from the first stages of development and a good marker for the zygote-derived cell lineage in these embryos. Expression in adult worms is also monitored and is found in the planarian germ line, where it is showed to be expressed in spermatogonia, spermatocytes and differentiating spermatids.

Free tyrosine and tyrosine-rich peptide-dependent superoxide generation catalyzed by a copper-binding, threonine-rich neurotoxic peptide derived from prion protein

Ken Yokawa, Tomoko Kagenishi, Kaishi Goto, Tomonori Kawano

Previously, generation of superoxide anion (O₂^•-) catalyzed by Cu-binding peptides derived from human prion protein (model sequence for helical Cu-binding motif VNITKQHTVTTTT was most active) in the presence of catecholamines and related aromatic monoamines such as phenylethylamine and tyramine, has been reported [Kawano, T., Int J Biol Sci 2007; 3: 57-63]. The peptide sequence (corresponding to helix 2) tested here is known as threonine-rich neurotoxic peptide. In the present article, the redox behaviors of aromatic monoamines, 20 amino acids and prion-derived tyrosine-rich peptide sequences were compared as putative targets of the oxidative reactions mediated with the threonine-rich prion-peptide. For detection of O₂^•-, an O₂^•--specific chemiluminescence probe, Cypridina luciferin analog was used. We found that an aromatic amino acid, tyrosine (structurally similar to tyramine) behaves as one of the best substrates for the O₂^•- generating reaction (conversion from hydrogen peroxide) catalyzed by Cu-bound prion helical peptide. Data suggested that phenolic moiety is required to be an active substrate while the presence of neither carboxyl group nor amino group was necessarily required. In addition to the action of free tyrosine, effect of two tyrosine-rich peptide sequences YYR and DYEDRYYRENMHR found in human prion corresponding to the tyrosine-rich region was tested as putative substrates for the threonine-rich neurotoxic peptide. YYR motif (found twice in the Y-rich region) showed 2- to 3-fold higher activity compared to free tyrosine. Comparison of Y-rich sequence consisted of 13 amino acids and its Y-to-F substitution mutant sequence revealed that the tyrosine-residues on Y-rich peptide derived from prion may contribute to the higher production of O₂^•-. These data suggest that the tyrosine residues on prion molecules could be additional targets of the prion-mediated reactions through intra- or inter-molecular interactions. Lastly, possible mechanism of O₂^•- generation and the impacts of such self-redox events on the conformational changes in prion are discussed.

Non-Classical P38 Map Kinase Functions: Cell Cycle Checkpoints and Survival

Tina M. Thornton, Mercedes Rincon

The p38 MAPK kinase pathway is activated in response to a wide range of cellular stress stimuli and cytokines. Our understanding of the important functions of p38 MAPK in the process of differentiation and cell death has grown considerably in the recent years and is now relatively established. Here we discuss the role of p38 MAPK in the mediation of cell cycle checkpoints and cell survival, processes that have received less attention. We describe how p38 MAPK regulates both the G2/M as well as a G1/S cell cycle checkpoint in response to cellular stress such as DNA damage. While p38 MAPK has classically been associated with the induction of apoptosis, we discuss that p38 MAPK can also mediate cell survival in specific situations, such as in response to DNA damage. It is important to recognize these less appreciated functions of p38 MAPK when considering the potential use of pharmacological inhibitors of p38 MAPK in therapeutic treatments for disease.

Molecular cloning and characterization of pigeon (Columba liva) ubiquitin and ubiquitin-conjugating enzyme genes from pituitary gland library

In the study of the regulation of incubation, broodiness and laying performance in pigeons (Columba liva), a cDNA library, which was enriched with full-length brooding-related genes, was constructed by SMART LD-PCR techniques using the pituitary glands of incubating White King pigeons. The titers of optimal primary libraries were 1.54×10⁶ pfu/mL and 1.80×10⁶ pfu/mL and the titers of amplified libraries were 1.89×10⁸ pfu/mL and 2.32×10⁹pfu/mL. The percentages of recombinant clones of primary libraries and amplified libraries were all over 90%. A positive clone was sequenced and named ubiquitin based on the highly similar from other species. The fragment has the four initial codons of ATG, a termination codon of TAA and a signal sequence of AATAAA for adding the poly-A tail. The open reading frame of 918bp encodes 305 amino acids (NCBI accession number is EU981283). Recombinant pigeon ubiquitin protein was efficiently expressed with the form of insoluble inclusion bodies in E. coli BL21 transformed with a pET28a⁺ expression vector containing the DNA sequence encoding mature pigeon ubiquitin. The molecular weight of expressed protein is the same as predicted size of approximately 35kD. To improve the efficiency of cloning full-length cDNA, strategies of RACE combined with cDNA library were used. The length of pigeons ubiquitin-conjugating enzyme gene obtained was 1263 bp containing a complete open reading frame of 435 bp that encodes 144 aa (NCBI accession number is EU914824). The results of this study not only provide a starting point for further study of ubiquitin function in pigeon species, but also provide a starting point for investigating the brooding mechanisms of pigeons.

Differential display of expressed genes reveals a novel function of SFRS18 in regulation of intramuscular fat deposition

Intramuscular fat (IMF) content plays a key role in establishing pork quality. In the present study, differential-display reverse transcription-polymerase chain reaction (DDRT-PCR) was used to identify differentially expressed (DE) genes between longissimus dorsi (LD) muscles with extremely different IMF content. A major DE gene associated with IMF content was identified as splicing factor serine-arginine rich protein (SFRS18) gene, also known as SRrp130. The gene exhibited relatively higher expression levels in LD muscles with higher IMF content. A full-length cDNA sequence of pig SFRS18 gene was obtained by in silico comparative cloning coupled with PCR target sequencing, while the current EST (expressed sequence tag) database supported two transcript variants of the pig gene. Differential expression of the SFRS18 gene was further confirmed using quantitative PCR. The mRNA levels of SFRS18 gene showed significant and positive correlation with IMF content in LD muscle (r = 0.54, P < 0.01). Collectively, these results suggest that the SFRS18 gene is involved in the regulation of IMF deposition in pig and that it may be a useful tool in selecting animals for desired amounts of fatness for high quality pork.

BRCA1, Hormone, and Tissue-Specific Tumor Suppression

Yanfen Hu

Germline mutations of BRCA1 predispose women to breast and ovarian cancers. Elucidating molecular mechanism of tissue- and gender-specific phenomena in BRCA1-related tumors is a key to our understanding of BRCA1 function in tumor suppression. This review summarizes studies in recent years on the link between BRCA1 and estrogen/progesterone signaling pathways, as well as discusses various models underscoring a triangle relationship among BRCA1, estrogen and genome instability.

Using Profiles Based on Nucleotide Hydrophobicity to Define Essential Regions for Splicing

Galina Boldina, Anatoly Ivashchenko, Mireille Régnier

The splice-site sequences of U2-type introns are highly degenerate, so many different sequences can function as U2-type splice sites. Using our new profiles based on hydrophobicity properties we pointed out specific properties for regions surrounding splice sites. We built a set T of flanking regions of genes with 1-3 introns from 21st and 22nd chromosomes extracted from GenBank to define positions having conserved properties, namely hydrophobicity, that are potentially essential for recognition by spliceosome.

GT–AG introns exist in U2 and U12-types. Therefore, intron type cannot be simply determined by the dinucleotide termini. We attempted to distinguish U2 and U12-types introns with help of hydrophobicity profiles on sets of spice sites for U2 or U12-type introns extracted from SpliceRack database. The positions given by our method, which may be important for recognition by spliceosome, were compared to the nucleotide consensus provided by a classical method, Pictogram. We showed that there is a similarity of hydrophobicity profiles inside intron types. On one hand, GT–AG and GC–AG introns belonging to U2-type have resembling hydrophobicity profiles as well as AT–AC and GT–AG introns belonging to U12-type. On the other hand, hydrophobicity profiles of U2 and U12-types GT–AG introns are completely different. We suggest that hydrophobicity profiles facilitate definition of intron type, distinguishing U2 and U12 intron types and can be used to build programs to search splice site and to evaluate their strength.

Therefore, our study proves that hydrophobicity profiles are informative method providing insights into mechanisms of splice sites recognition.

Genome-wide Analysis of BP1 Transcriptional Targets in Breast Cancer Cell Line Hs578T

Homeobox genes are known to be critically important in tumor development and progression. The BP1 (Beta Protein 1) gene, an isoform of DLX4, belongs to the Distal-less (DLX) subfamily of homeobox genes and encodes a homeodomain-containing transcription factor. Our studies have shown that the BP1 gene was overexpressed in 81% of primary breast cancer and its expression was closely correlated with the progression of breast cancer. However, the exact role of BP1 in breast has yet to be elucidated. Therefore, it is important to explore the potential transcriptional targets of BP1 via whole genome-scale screening. In this study, we used the chromatin immunoprecipitation on chip (ChIP-on-chip) and gene expression microarray assays to identify candidate target genes and gene networks, which are directly regulated by BP1 in ER negative (ER-) breast cancer cells. After rigorous bioinformatic and statistical analysis for both ChIP-on-chip and expression microarray gene lists, 18 overlapping genes were noted and verified. Those potential target genes are involved in a variety of tumorigenic pathways, which sheds light on the functional mechanisms of BP1 in breast cancer development and progression.