International Journal of Biological Sciences

Concerted Transcriptional Regulation by BRCA1 and COBRA1 in Breast Cancer Cells

Sarah E. Aiyar, HyungJun Cho, Jae Lee, Rong Li

Cofactor of BRCA1 (COBRA1) was first identified as a protein that binds to the breast cancer susceptibility gene product BRCA1. COBRA1 modulates estrogen-dependent and independent transcription and suppresses the growth of breast cancer cells. Its expression is significantly reduced in metastatic and recurrent breast cancer, pointing to a tumor suppressor function in breast cancer development. In light of these initial implications of COBRA1 in human breast cancer, the current investigation sought to obtain more direct functional evidence that links COBRA1 with BRCA1 in transcriptional regulation in breast cancer cells. Small hairpin RNA (shRNA)-mediated gene knockdown and gene expression microarray were used to study the impact of COBRA1 and BRCA1 on global transcription in the same breast cancer cell background. The gene expression profiling study in tissue culture cells uncovers a significant overlap of COBRA1- and BRCA1-regulated genes, many of which have been previously implicated in breast cancer progression. The data shown herein support the notion that COBRA1 and BRCA1 may engage in common gene regulatory pathways to suppress breast cancer progression.

Smad3 -signalling and Th2 cytokines in normal mouse airways and in a mouse model of asthma

This study investigates the role of Smad3 signalling for the T-helper2 (Th2) cytokine homeostasis in normal lungs and in a mouse model of asthma.

We used mice deficient for Smad3, a central part of the major signal transduction pathway for TGF-β and other related cytokines, and a mouse model for allergic asthma with ovalbumin (OVA) as the antigen.

Compared to wild type mice, naive (unmanipulated) Smad3-/- mice exhibited significantly increased levels of proinflammatory cytokines and IL-4 as well as the Th2 associated transcription factor GATA-3 in the lung tissue and bronchoalveolar lavage (BAL). In the asthma model, mucin secretion and airway hyperresponsiveness (AHR) after allergen exposure was significantly increased in the Smad3-/- mice as compared to wild type (WT) mice. IL-4 levels in Smad3-/- were similar to those encountered in WT mice but IL-13 levels were decreased in the airways of OVA sensitized Smad3-/- mice compared to corresponding WT mice.

The results indicate that a lack of Smad3 dependent signalling in the normal state will lead to an increase in the GATA-3 levels and as a result of this the levels of IL-4 increase. However, the lack of Smad3 also seems to inhibit expression of some cytokines, especially IL-13. Our results also indicate that in the inflammatory state TGF-β or related cytokines functions to counterbalance the effects of IL-4 rather than to critically regulate its expression.

The Growth-hormone inducible transmembrane protein (Ghitm) belongs to the Bax inhibitory protein-like family

Kerstin Reimers, Claudia YU Choi, Vesna Bucan, Peter M Vogt

The conserved protein domain UPF0005 is a protein family signature distributed among many species including fungi and bacteria. Although of unknown functionality this motif has been found in newly identified antiapoptotic proteins comprising the BI-1 family, namely Bax-inhibitory Protein-1 (BI-1), Lifeguard (LFG), and h-GAAP. In a search for vertebrate proteins presumably belonging to the BI-1 family, we found that Growth-hormone inducible transmembrane protein (Ghitm) is another prospective member of the BI-1 family. Here we characterise Ghitm in a first analysis regarding its phylogeny, expression in cancer cell lines, and proteomical properties.

Rational Design of Analyte Channels of the Green Fluorescent Protein for Biosensor Applications

A novel solvent-exposed analyte channel, generated by F165G substitution, on the surface of green fluorescent protein (designated His₆GFPuv/F165G) was successfully discovered by the aid of molecular modeling software (PyMOL) in conjunction with site-directed mutagenesis. Regarding the high predictive performance of PyMOL, two pore-containing mutants namely His₆GFPuv/H148G and His₆GFPuv/H148G/F165G were also revealed. The pore sizes of F165G, H148G, and the double mutant H148G/F165G were in the order of 4, 4.5 and 5.5 Å, respectively. These mutants were subjected to further investigation on the effect of small analytes (e.g. metal ions and hydrogen peroxide) as elucidated by fluorescence quenching experiments. Results revealed that the F165G mutant exhibited the highest metal sensitivity at physiological pH. Meanwhile, the other 2 mutants lacking histidine at position 148 had lower sensitivity against Zn²⁺ and Cu²⁺ than those of the template protein (His₆GFPuv). Hence, a significant role of this histidine residue in mediating metal transfer toward the GFP chromophore was proposed and evidently demonstrated by testing in acidic condition. Results revealed that at pH 6.5 the order of metal sensitivity was found to be inverted whereby the H148G/F165G became the most sensitive mutant. The dissociation constants (K_d) to metal ions were in the order of 4.88×10^-6 M, 16.67×10^-6 M, 25×10^-6 M, and 33.33×10^-6 M for His₆GFPuv/F165G, His₆GFPuv, His₆GFPuv/H148G/F165G and His₆GFPuv/H148G, respectively. Sensitivity against hydrogen peroxide was in the order of H148G/F165G > H148G > F165G indicating the crucial role of pore diameters. However, it should be mentioned that H148G substitution caused a markedly decrease in pH- and thermo-stability. Taken together, our findings rendered the novel pore of GFP as formed by F165G substitution to be a high impact channel without adversely affecting the intrinsic fluorescent properties. This opens up a great potential of using F165G mutant in enhancing the sensitivity of GFP in future development of biosensors.

Adjuvant effect of anti-4-1BB mAb administration in adoptive T cell therapy of cancer

Qiao Li, Takekazu Iuchi, Maria N. Jure-Kunkel, Alfred E. Chang

Administration of anti-4-1BB mAb has been found to be a potent adjuvant when combined with other therapeutic approaches, e.g. chemotherapy, cytokine therapies, anti-OX40 therapy, and peptide or DC vaccines. However, the adjuvant effect of anti-4-1BB mAb administration in adoptive T cell therapy of cancer has not been fully evaluated. In this report, effector T cells were generated in vitro by anti-CD3/anti-CD28 activation of tumor-draining lymph node (TDLN) cells and used in an adoptive immunotherapy model. While T cells or anti-4-1BB alone showed no therapeutic efficacy in mice bearing macroscopic 10-day pulmonary metastases, T cells plus anti-4-1BB mediated significant tumor regression in an anti-4-1BB dose dependent manner. Mice bearing microscopic 3-day lung metastases treated with T cells alone demonstrated tumor regression which was significantly enhanced by anti-4-1BB administration. NK cell depletion abrogated the augmented therapeutic efficacy rendered by anti-4-1BB. Cell transfer between congenic hosts demonstrated that anti-4-1BB administration increased the survival of adoptively transferred TDLN cells. Using STAT4^-/- mice, we found that modulated IFNγ secretion in wt TDLN cells after anti-CD3/CD28/4-1BB activation in vitro was lost in similarly stimulated STAT4^-/- TDLN cells. Additionally, anti-4-1BB administration failed to augment the therapeutic efficacy of T cell therapy in STAT4^-/- mice. Together, these results indicate that administered anti-4-1BB mAb can serve as an effective adjuvant to augment the antitumor reactivity of adoptively transferred T cells by recruiting the host NK cells; increasing the persistence of infused effector T cells, and modulating the STAT4 molecular signaling pathway.

Immunoregulatory role of intestinal surfactant-like particles during Salmonella typhimurium infection

Surfactants like particles (SLP) are secreted by Intestinal epithelium. These particles have the ability to lower surface tension of intestinal epithelial cells and contain small amounts of surfactant specific proteins A, B, and D. In the intestinal lumen they are known to function as lubricants and/or as a vehicle to deliver digestive enzymes to the luminal fluid. These particles have been found to have the ability in binding of uropathogenic E.coli. But their immunological function is not known. The present study was designed to assess the role of the SLP in the regulation of immune response during Salmonella (S) typhimurium infection using a rat an enteric model. The animals were divided in four different groups including control (PBS), rats fed fat diet (corn oil), rats fed fat diet followed with S. typhimurium infection and rats with S. typhimurium infection alone. The Peyer's patches (PP), intraepithelial (IE) and lamina propria (LP) mononuclear cells were isolated from the above-mentioned groups. These mononuclear cells were then incubated in presence of S. typhimurium lysate alone, SLP alone and S. typhimurium lysate and SLP together. T cell markers CD4 and CD8, cytokines mainly pro-inflammatory ones including IFN-γ, TNF-α, IL-12 etc were studied under such conditions. In addition histological studies were also carried out under these conditions. We report in this study that SLP plays an important role in modulating the cytokine level during infection. The pro-inflammatory cytokines were found significantly reduced in SLP induced diet along with the infection group compared to the infection group alone. Histopathological studies revealed the breakdown of duodenal villi after infection while only broadening of villi was observed in rats given corn oil induced SLP along with infection. These results suggested an important immuno-modulatory role for SLP during Salmonella infection.

Phycobilisomes linker family in cyanobacterial genomes: divergence and evolution

Xiangyu Guan, Song Qin, Fangqing Zhao, Xiaowen Zhang, Xuexi Tang

Cyanobacteria are the oldest life form making important contributions to global CO₂ fixation on the Earth. Phycobilisomes (PBSs) are the major light harvesting systems of most cyanobacteria species. Recent availability of the whole genome database of cyanobacteria provides us a global and further view on the complex structural PBSs. A PBSs linker family is crucial in structure and function of major light-harvesting PBSs complexes. Linker polypeptides are considered to have the same ancestor with other phycobiliproteins (PBPs), and might have been diverged and evolved under particularly selective forces together. In this paper, a total of 192 putative linkers including 167 putative PBSs-associated linker genes and 25 Ferredoxin-NADP oxidoreductase (FNR) genes were detected through whole genome analysis of all 25 cyanobacterial genomes (20 finished and 5 in draft state). We compared the PBSs linker family of cyanobacteria in terms of gene structure, chromosome location, conservation domain, and polymorphic variants, and discussed the features and functions of the PBSs linker family. Most of PBSs-associated linkers in PBSs linker family are assembled into gene clusters with PBPs. A phylogenetic analysis based on protein data demonstrates a possibility of six classes of the linker family in cyanobacteria. Emergence, divergence, and disappearance of PBSs linkers among cyanobacterial species were due to speciation, gene duplication, gene transfer, or gene loss, and acclimation to various environmental selective pressures especially light.

cDNA Cloning and Overexpression of Acidic Ribosomal Phosphoprotein P1 Gene (RPLP1) from the Giant Panda

Yu-Jie Du, Xiao-Yan Luo, Yan-Zhe Hao, Tian Zhang, Wan-Ru Hou

RPLP1 is one of acidic ribosomal phosphoproteins encoded by RPLP1 gene, which plays an important role in the elongation step of protein synthesis. The cDNA of RPLP1 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology, which was also sequenced, analyzed preliminarily and expressed in E.coli. The cDNA fragment cloned is 449bp in size, containing an open reading frame of 344bp encoding 114 amino acids. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other five species studied, including Homo sapiens, Mus musculus, Rattus norvegicus, Bos Taurus and Sus scrofa. The homologies for nucleotide sequences of Giant Panda PPLP1 to that of these species are 92.4%, 89.8%, 89.0%, 91.3% and 87.5%, while the homologies for amino acid sequences are 96.5%, 94.7%, 95.6%, 96.5% and 88.6%. Topology prediction showed there are three Casein kinase II phosphorylation sites and two N-myristoylation sites in the RPLP1 protein of the Giant Panda (Ailuropoda melanoleuca). The RPLP1 gene was overexpressed in E. coli and the result indicated that RPLP1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 18kDa polypeptide, which was in accordance with the predicted protein and could also be used to purify the protein and study its function.

Candidate Gene Identification Approach: Progress and Challenges

Mengjin Zhu, Shuhong Zhao

Although it has been widely applied in identification of genes responsible for biomedically, economically, or even evolutionarily important complex and quantitative traits, traditional candidate gene approach is largely limited by its reliance on the priori knowledge about the physiological, biochemical or functional aspects of possible candidates. Such limitation results in a fatal information bottleneck, which has apparently become an obstacle for further applications of traditional candidate gene approach on many occasions. While the identification of candidate genes involved in genetic traits of specific interest remains a challenge, significant progress in this subject has been achieved in the last few years. Several strategies have been developed, or being developed, to break the barrier of information bottleneck. Recently, being a new developing method of candidate gene approach, digital candidate gene approach (DigiCGA) has emerged and been primarily applied to identify potential candidate genes in some studies. This review summarizes the progress, application software, online tools, and challenges related to this approach.

In Celebration of Dr. Mario R. Capecchi's Nobel Prize

Chuxia Deng

Charles River Sprague Dawley Rats Lack Early Age-Dependent Susceptibility to DMBA-Induced Mammary Carcinogenesis

R.B. Gear, M. Yan, J. Schneider, P. Succop, S.C. Heffelfinger, D.J. Clegg

Developmental stages of mammary glands influence their susceptibility to initiating events related to carcinogenesis. The “window of susceptibility” to mammary carcinogenesis is classically defined as the time in early puberty when the mammary gland morphology is most sensitive to initiation events. Administration of the polyaromatic hydrocarbon, 7,12-dimethylbenz(a)anthracene (DMBA), in a single oral dose yields maximal mammary tumor formation when administered in this “window”. We examined the DMBA treated mammary glands, precursor lesions, and morphology of the uninvolved mammary epithelium for the first 100 days of life for Charles River Sprague Dawley CD^R IGS. Our goal was to determine the DMBA dose at which 50% of the rats (IC50) developed carcinoma in situ (CIS) within three months of dosing. Here we demonstrate, rather than the classical U-shaped dose curve in which there is maximum sensitivity for DMBA at 50 days, there is an increasing degree of sensitivity with age in the CD^R IGS rat. Additionally, we report that vehicle-treated animals developed mammary CIS without any known initiator, and 100 day virgin animals demonstrated lactational changes, independent of DMBA exposure or dose. Lastly, we demonstrate this strain of virgin female rats has elevated pituitary prolactin immunoreactivity independent of the level of mammary differentiation. We conclude this strain of Charles River Sprague Dawley rats has prolactin-induced pituitary stimulation, and therefore, the window of susceptibility for mammary tumorigenesis is absent.

DNA damage repair is unaffected by mimicked heterozygous levels of BRCA2 in HT-29 cells

Brian Tannenbaum, Tobechukwu Mofunanya, Alan R. Schoenfeld

Functional loss of both alleles of the breast cancer susceptibility gene, BRCA2, facilitates tumorigenesis. However, the direct effects of BRCA2 heterozygosity remain unclear. Here, BRCA2 heterozygosity was mimicked in HT-29 colon cells by reducing levels of BRCA2 through stable RNA interference. No difference in RAD51 subcellular localization and focus formation was observed between control and mimicked heterozygous cell lines. DNA repair ability, as measured by colony survival following mitomycin C treatment and ultraviolet radiation exposure, was also unaffected by reduced levels of BRCA2. Interestingly, the growth rate of the mimicked BRCA2 heterozygous cell line was significantly lower than that of control cells. Increased expression of p53 in the mimicked heterozygous cells was observed, perhaps in response to BRCA2 deficiency. Levels of p27 were also found to be slightly increased in cells with reduced BRCA2, perhaps contributing to the slower growth rate. Overall, these results suggest that tumors are unlikely to arise directly from BRCA2 heterozygous cells without other genetic events such as loss of the wild-type BRCA2 allele and/or loss of p53 function or other cell cycle inhibitors.

Thermal injury-plus-sepsis contributes to a substantial deletion of intestinal mesenteric lymph node CD4+ T cell via apoptosis

Nadeem Fazal, Walid M Al-Ghoul

Thermal injury (TI) with septic complications continues to be a serious clinical problem. One of the main concerns in such patients is immunosuppression related to functional derangements in intestinal CD4⁺ T lymphocytes. Extensive previous studies in thermal injury/septic patients and animal models of thermal injury/sepsis have shown decreased responsiveness of intestinal CD4⁺ T cells to antigen/mitogen. This hyporesponsiveness could significantly contribute to increase injured host susceptibility to pathogens including those translocating from host's gut lumen. Our previous studies indicated that while thermal injury or sepsis alone lead to suppressed proliferation and IL-2 production of intestinal CD4⁺ T cells, this study showed a substantial deletion via apoptosis of the Mesenteric Lymph Nodes (MLN) CD4⁺ T cells. Hence, thermal injury-plus-sepsis contributes not only to suppressed CD4⁺ T proliferation/IL-2 production but also to a substantial modulation of CD4⁺ T cell survivability. These findings allow us to conclude that while thermal injury alone can produce attenuated cell mediated responses without an overt change in CD4⁺ T cell survival, thermal injury with septic complications causes CD4⁺ T cell death and an irreversible loss of cell-mediated responses. The latter happening could be responsible for high morbidity and mortality in the injured host afflicted with thermal injury plus a critical infection.

Reversal of Systemic Hypertension-Associated Cardiac Remodeling in Chronic Pressure Overload Myocardium by Ciglitazone

Elevated oxidative stress has been characterized in numerous disorders including systemic hypertension, arterial stiffness, left ventricular hypertrophy (LVH) and heart failure. The peroxisome proliferator activated receptor gamma (PPARγ) ameliorates oxidative stress and LVH. To test the hypothesis that PPARγ decreased LVH and cardiac fibrosis in chronic pressure overload, in part, by increasing SOD, eNOS and elastin and decreasing NOX4, MMP and collagen synthesis and degradation, chronic pressure overload analogous to systemic hypertension was created in C57BL/6J mice by occluding the abdominal aorta above the kidneys (aortic stenosis-AS). The sham surgery was used as controls. Ciglitazone (CZ, a PPARγ agonist, 4 µg/ml) was administered in drinking water. LV function was measured by M-Mode Echocardiography. We found that PPARγ protein levels were increased by CZ. NOX-4 expression was increased by pressure-overload and such an increase was attenuated by CZ. SOD expression was not affected by CZ. Expression of iNOS was induced by pressure-overload, and such an increase was inhibited by CZ. Protein levels for MMP2, MMP-9, MMP-13 were induced and TIMP levels were decreased by pressure-overload. The CZ mitigated these levels. Collagen synthesis was increased and elastin levels were decreased by pressure-overload and CZ ameliorated these changes. Histochemistry showed that CZ inhibited interstitial and perivascular fibrosis. Echocardiography showed that CZ attenuated the systolic and diastolic LV dysfunction induced by pressure-overload. These observations suggested that CZ inhibited pressure-overlaod-induced cardiac remodeling, and inhibition of an induction of NOX4, iNOS, MMP-2/MMP-13 expression and collagen synthesis/degradation may play a role in pressure-overload induced cardiac remodeling.

Versican Expression during Synovial Joint Morphogenesis

The extracellular matrix (ECM) plays a critical role in governing cell behavior and phenotype during limb skeletogenesis. Chondroitin sulfate proteoglycans (Cspgs) are highly expressed in the ECM of precartilage mesenchymal condensations and are important to limb chondrogenesis and cartilage structure, but little is known regarding their involvement in formation of synovial joints in the embryonic limb. Matrix versican Cspg expression has previously been reported in the epiphysis of developing long bones and presumptive joint; however, detailed analysis has not yet been conducted. In the present study we immunolocalized versican and aggrecan Cspgs during chick elbow joint morphogenesis between HH st25-41 of development. In this study we show that versican and aggrecan expression initially overlapped in the incipient cartilage model of long bones in the wing, but versican was also highly expressed in the perichondrium and presumptive joint interzone during early stages of morphogenesis (HH st25-34). By HH st36-41 versican localization was restricted to the future articular surfaces of the developing joint and surrounding joint capsule while aggrecan localized in an immediately adjacent and predominately non-overlapping region of chondrogenic cells at the epiphyses. These results suggest a potential role for versican proteoglycan in development and maintenance of the synovial joint interzone.

High level glucose increases mutagenesis in human lymphoblastoid cells

Ying Zhang, Junqing Zhou, Tieli Wang, Lu Cai

Epidemiological data have suggested an increased cancer rates in diabetic patients, for which the underlying mechanism is poorly understood. We studied whether high level of glucose (HG) treatment that mimic the hyperglycemic condition in diabetes mellitus is mutagenic. Mutagenesis studies were carried out at both hypoxanthine phosphoribosyltransferase (hprt) and thymidine kinase (tk) loci. Role of p53 in HG-induced mutagenesis was also investigated by using human lymphoblastoid cell lines derived from same donor but differs in p53 statuses; TK6 has wild-type p53, NH32 has null p53, and WTK1 has mutant p53 (ile237). In addition, we studied the influence of antioxidant treatment on HG-induced mutagenesis. Mutation fractions at both loci increased significantly in all three lines at 21 and 28 days after HG treatments. At tk locus, the increase of a class of mutants with normal growth rate is mainly responsible for the overall increased mutant fraction. Compared to TK6 cells, both NH32 and WTK1 cells showed an early onset of mutagenesis. Treatment of cells with antioxidant N-acetyl-L-cysteine partially reduced HG induced mutagenesis. This study is the first to indicate that HG is able to induce gene mutation which may be one of the important mechanisms of diabetes-associated carcinogenesis.

An mRNA-protein Fusion at N-terminus for Evolutionary Protein Engineering

Shingo Ueno, Hidenao Arai, Miho Suzuki, Yuzuru Husimi

A novel method to link a nascent protein (phenotype) to its mRNA (genotype) covalently through the N-terminus was developed. The mRNA harboring amber stop codon at just downstream of initiation site was hybridized with hydrazide-modified ssDNA at upstream of coding region and was ligated to the DNA. This construct was then modified with 4-acetyl-phenylalanyl amber suppressor tRNA. This modified construct was fused with the nascent protein via the phenylalanine derivative when the mRNA uses the amber suppressor tRNA to decode the amber stop codon. The obtained fusion molecule was used successfully in selective enrichment experiments. It will be applicable for high-through-put screening in evolutionary protein engineering. In contrast to fusion molecules generated by other methods in which the protein is linked to genotype molecule through the C- terminus, our fusion molecule will serve to select a protein for which the C-terminus is essential to be active.

The Amphioxus SoxB Family: Implications for the Evolution of Vertebrate Placodes

Daniel Meulemans, Marianne Bronner-Fraser

Cranial placodes are regions of thickened ectoderm that give rise to sense organs and ganglia in the vertebrate head. Homologous structures are proposed to exist in urochordates, but have not been found in cephalochordates, suggesting the first chordates lacked placodes. SoxB genes are expressed in discrete subsets of vertebrate placodes. To investigate how placodes arose and diversified in the vertebrate lineage we isolated the complete set of SoxB genes from amphioxus and analyzed their expression in embryos and larvae. We find that while amphioxus possesses a single SoxB2 gene, it has three SoxB1 paralogs. Like vertebrate SoxB1 genes, one of these paralogs is expressed in non-neural ectoderm destined to give rise to sensory cells. When considered in the context of other amphioxus placode marker orthologs, amphioxus SoxB1 expression suggests a diversity of sensory cell types utilizing distinct placode-type gene programs was present in the first chordates. Our data supports a model for placode evolution and diversification whereby the full complement of vertebrate placodes evolved by serial recruitment of distinct sensory cell specification programs to anterior pre-placodal ectoderm.

Superoxide Anion Radical Scavenging Activities of Herbs and Pastures in Northern Japan Determined Using Electron Spin Resonance Spectrometry

Free radicals are not only destructive to the living cells but also reduce the quality of animal products through oxidation. As a result the superoxide anion radical (O₂^・-), one of the most destructive reactive oxygen species, is a matter of concern for the animal scientists as well as feed manufacturers to ensure the quality of product to reach consumers demand. The superoxide anion radical scavenging activities (SOSA) of water and MeOH extracts of 2 herbs and 9 pasture samples collected from lowland and highland swards were determined against a 5,5-dimethyl-1-pyroline-N-oxide-O₂^・-spin adduct based on a hypoxanthine-xanthine oxidase reaction using electron spin resonance spectrometry. Both the water and MeOH extracted SOSA differed among the herbs and pastures. Species and altitudinal variations were observed between extraction methods. The herbs were higher in both water and MeOH extracted SOSA than the pastures except for water extracts of one pasture, white clover (Trifolium repens L.). Among the pastures, quackgrass (Agrophyron repens L.) showed higher SOSA in both the MeOH and water extracts, and timothy (Phleum pretense L.) showed higher MeOH extracted SOSA. It is apparent that the kind and amount of antioxidants differ among herbs and pastures. Animal health and quality of animal products could be improved by adequate selection and combining of herbs and pastures having higher SOSA.

Differences in Apoptosis and Cell Cycle Distribution between Human Melanoma Cell Lines UACC903 and UACC903(+6), before and after UV Irradiation

Qiuyang Zhang, Yuanbin Chen, Bi-Dar Wang, Ping He, Yan A. Su

Introduction of human chromosome 6 into malignant melanoma cell line UACC903 resulted in generation of the chromosome 6-mediated suppressed cell subline UACC903(+6) that displays attenuated growth rate, anchorage-dependency, and reduced tumorigenicity. We have showed that overexpression of a chromosome 6-encoded tumor suppressor gene led to partial suppression to UACC903 cell growth. We now describe the differences in apoptosis and cell cycle between UACC903 and UACC903(+6) before and after UV irradiation. MTT assay revealed 86.92±8.24% of UACC903 cells viable, significantly (p<0.01) higher than 48.76±5.31% of UACC903(+6), at 24 hr after 254-nm UV irradiation (40 J/M²). Before UV treatment, flow cytometry analysis revealed 6.06±0.20% apoptosis in UACC903, significantly (p=0.01) lower than 6.67±0.15% in UACC903(+6). The G0/G1, S and G2/M phase cells of UACC903 were, respectively, 54.10±0.59%, 22.31±0.50% and 16.85±0.25%, all significantly (p<0.01) different from the corresponding percentages (58.82±0.35%, 20.48±0.05%, and 13.17±0.45%) of UACC903(+6). After the UV treatment, UACC903 cells in apoptosis, G0/G1, S, and G2/M became 12.59±0.17%, 38.90±0.67%, 19.74±0.70%, and 27.01±0.66%, respectively, while UACC903(+6) cells were 24.16±0.48%, 37.97±0.62%, 19.20±0.52%, and 15.69±0.14%. TUNEL assay revealed 2.31±0.62% apoptosis in UACC903, significantly (p<0.01) lower than 9.60±1.14% of UACC903(+6), and a linear and exponential increase of apoptosis, respectively, in response to the UV treatment. These results indicate that UACC903(+6) cells have a greater tendency to undergo apoptosis and are thus much more sensitive to UV irradiation. Our findings further suggest a novel mechanism for chromosome 6-mediated suppression of tumorigenesis and metastasis, i.e., through increased cell death.

The responses of HT22 cells to the blockade of mitochondrial complexes and potential protective effect of selenium supplementation

Jun Panee, Wanyu Liu, Kyoko Nakamura, Marla J. Berry

Mitochondria are the major reactive oxygen species (ROS) – generating sites in mammalian cells. Blockade of complexes in the electron transport chain (ETC) increases the leakage of single electrons to O₂ and therefore increases ROS levels. Complexes I and III have been reported to be the major ROS-generating sites in mitochondria. In this study, using mouse hippocampal HT22 cells as in vitro model, we monitored the change of intracellular ROS level in response to the blockade of ETC at different complex, and measured changes of gene expression of antioxidant enzymes and phase II enzymes, also evaluated potential protective effect of selenium (Se) supplementation to the cells under this oxidative stress. In summary, our results showed that complex I was the major ROS-generating site in HT22 cells. Complex I blockade upregulated the mRNA levels of glutamylcysteine synthetase heavy and light chains, glutathione-S-transferases omega1 and alpha 2, hemoxygenase 1, thioredoxin reductase 1, and selenoprotein H. Unexpectedly, the expression of the enzymes that directly scavenge ROS decreased, including superoxide dismutases 1 and 2, glutathione peroxidase 1, and catalase. Se supplementation increased glutathione levels and glutathione peroxidase activity, indicating a potential protective role in oxidative stress caused by ETC blockade.

Reduced Risk of Human Lung Cancer by Selective Cyclooxygenase 2 (Cox-2) Blockade: Results of a Case Control Study

Randall E. Harris, Joanne Beebe-Donk, Galal A. Alshafie

We conducted a case control study of selective cyclooxygenase-2 (COX-2) blocking agents and lung cancer. A total of 492 newly diagnosed lung cancer cases were ascertained during January 1, 2002 to September 30, 2004, at The Ohio State University Medical Center, Columbus, Ohio. All cases were confirmed by examination of the pathology report. Healthy population controls without cancer were ascertained during the same time period. Controls were frequency matched at a rate of 2:1 to the cases by age, gender, and county of residence. We collected information on type, frequency, and duration of use of selective COX-2 inhibitors (primarily celecoxib or rofecoxib) and nonselective NSAIDs such as ibuprofen and aspirin. Estimates of odds ratios (OR) were obtained with adjustment for cigarette smoking, age and other potential confounders using logistic regression analysis. Odds Ratios for selective COX-2 inhibitors were adjusted for past use of other NSAIDs. Use of any selective COX-2 inhibitor for more than one year produced a significant (60%) reduction in the risk of lung cancer (OR=0.40, 95% CI=0.19-0.81). Observed risk reductions were consistent for men (OR=0.26, 95% CI=0.10-0.62) and women (OR=0.52, 95% CI=0.24-1.13) and for individual COX-2 inhibitors (OR=0.28, 95% CI=-0.12-0.67, for celecoxib and OR=0.55, 95% CI=0.19-1.56, for rofecoxib). Intake of ibuprofen or aspirin also produced significant risk reductions (OR=0.40, 95% CI=0.23-0.73 and OR=0.53, 95% CI=0.34-0.82, respectively), whereas acetaminophen, an analgesic with negligible COX-2 activity, had no effect on the risk (OR=1.36, 95% CI=0.53-3.37). This investigation demonstrates for the first time that selective COX-2 blocking agents have strong potential for the chemoprevention of human lung cancer.

In search of a function for the most frequent naturally-occurring length polymorphism (MFNLP) of the HIV-1 LTR: Retaining functional coupling, of Nef and RBF-2, at RBEIII?

Mario Clemente Estable

Although the prototypical HIV-1 LTR sequences were determined 22 years ago from the initial isolate, elucidating which transcription factors are critical to replication in vivo, has been difficult. One approach has been to examine HIV-1 LTRs that have gone through the gamut of in vivo mutation and selection, in search of absolutely conserved sequences. In this vein, RBEIII sequences are virtually 100% conserved in naturally occurring HIV-1 LTRs. This is because when they are mutated, the MFNLP recreates an RBEIII site. Here, I enumerate some retroviral mutation mechanisms, which could generate the MFNLP. I then review the literature corresponding to the MFNLP, highlighting the discovery in 1999, that RBEIII and MFNLP sequences, bind USF and TFII-I cooperatively, within the context of earlier and later work that suggests a role in HIV-1 activation, through T-cell receptor engagement and the MAPK cascade. One exception to the nearly absolute conservation of RBEIII, has been a group of long term non progressors (LTNP). These patients harbor deletions to the Nef gene. However, the Nef gene overlaps with the LTR, and the LTNP deletions abrogate RBEIII, in the absence of an MFNLP. I suggest that the MFNLP retains functional coupling between the MAPK-mediated effects of Nef and the HIV-1 LTR, through RBEIII. I propose that difficult-to-revert-mutations, to either Nef or RBEIII, result in the convergent LTNP Nef/LTR deletions recently observed. The potential exploitation of this highly conserved protein-binding site, for chimeric transcription factor repression (CTFR) of HIV-1, functionally striving to emulate the LTNP deletions, is further discussed.

The Forces Behind Cell Movement

Revathi Ananthakrishnan, Allen Ehrlicher

Cell movement is a complex phenomenon primarily driven by the actin network beneath the cell membrane, and can be divided into three general components: protrusion of the leading edge of the cell, adhesion of the leading edge and deadhesion at the cell body and rear, and cytoskeletal contraction to pull the cell forward. Each of these steps is driven by physical forces generated by unique segments of the cytoskeleton. This review examines the specific physics underlying these phases of cell movement and the origins of the forces that drive locomotion.

Human Maf1 negatively regulates RNA Polymerase III transcription via the TFIIB family members Brf1 and Brf2

Janet Rollins, Ingrid Veras, Stephanie Cabarcas, Ian Willis, Laura Schramm

RNA polymerase III (RNA pol III) transcribes many of the small structural RNA molecules involved in processing and translation, thereby regulating the growth rate of a cell. Initiation of pol III transcription requires the evolutionarily conserved pol III initiation factor TFIIIB. TFIIIB is the molecular target of regulation by tumor suppressors, including p53, RB and the RB-related pocket proteins. However, our understanding of negative regulation of human TFIIIB-mediated transcription by other proteins is limited. In this study we characterize a RNA pol III luciferase assay and further demonstrate in vivo that a human homolog of yeast Maf1 represses RNA pol III transcription. Additionally, we show that Maf1 repression of RNA pol III transcription occurs via TFIIIB, specifically through the TFIIB family members Brf1 and Brf2.

Effects of BRCA1 Transgene Expression on Murine Mammary Gland Development and Mutagen-Induced Mammary Neoplasia

To characterize the role of BRCA1 in mammary gland development and tumor suppression, a transgenic mouse model of BRCA1 overexpression was developed. Using the mouse mammary tumor virus (MMTV) promoter/enhancer, transgenic mice expressing human BRCA1 or select mutant controls were generated. Transgenic animals examined during adolescence were shown to express the human transgene in their mammary glands. The mammary glands of 13-week-old virgin homozygous MMTV-BRCA1 mice presented the morphology of moderately increased lobulo-alveolar development. The mammary ductal trees of both hemizygous and homozygous MMTV-BRCA1t340 were similar to those of control non-transgenic littermates. Interestingly, both hemi- and homozygous mice expressing a splice variant of BRCA1 lacking the N-terminal RING finger domain (MMTV-BRCA1sv) exhibited marked mammary lobulo-alveolar development, particularly terminal end bud proliferation. Morphometric analyses of mammary gland whole mount preparations were used to measure epithelial staining indices of ~35% for homozygous MMTV-BRCA1 mice and ~60% for both hemizygous and homozygous MMTV-BRCA1sv mice versus ~25% for non-transgenic mice. Homozygous MMTV-BRCA1 mice showed delayed development of tumors when challenged with 7,12 dimethylbenzanthracene (DMBA), relative to non-transgenic and homozygous BRCA1t340 expressing mice. In contrast, homozygous MMTV-BRCA1sv transgenic animals were sensitized to DMBA treatment and exhibited a very rapid onset of mammary tumor development and accelerated mortality. MMTV-BRCA1 effects on mortality were restricted to DMBA-induced tumors of the mammary gland. These results demonstrate in vivo roles for BRCA1 in both mammary gland development and in tumor suppression against mutagen-induced mammary gland neoplasia.

Development of a highly sensitive and selective microplate chemiluminescence enzyme immunoassay for the determination of free thyroxine in human serum

Xu Wang, Hui Chen, Jin-Ming Lin, Xitang Ying

A microplate chemiluminescence enzyme immunoassay (CLEIA) with high sensitivity, selectivity and reproducibility was developed for the determination of free thyroxine (FT4) in human serum. A competitive assay has been utilized with horseradish peroxidase (HRP) labeled thyroxine analog in the chemiluminescence (CL) detection. The CL signal produced by the emission of photons from luminol was directly proportional to the amount of analyte. The linear range was 0.45-7.5 ng dL^-1and the detection limit was 0.09 ng dL^-1. Experimental conditions, such as temperature, pH, incubation time, titration level and other relevant variables upon the CL signal have been examined and optimized. A coefficient of variance of less than 16% was obtained for intra- and inter-assay precision. The present method has been successfully applied to the analysis of FT4 in human serum. The positive and negative coincidence ratios are satisfactory. Good correlations were obtained between the results by the proposed method and radioimmunoassay (RIA), as well as a Bayer ACS-180SE detection system.

Proteomics Analysis of the Expression of Neurogranin in Murine Neuroblastoma (Neuro-2a) Cells Reveals Its Involvement for Cell Differentiation

Neurogranin (Ng) is a neural-specific, calmodulin (CaM)-binding protein that is phosphorylated by protein kinase C (PKC). Although its biochemical property has been well characterized, the physiological function of Ng needs to be elucidated. In the present study, we performed proteomics analysis of the induced compositional changes due to the expression of Ng in murine neuroblastoma (Neuro-2a) cells using isotope coded affinity tags (ICAT) combined with 2-dimensional liquid chromatography/tandem mass spectrometry (2D-LC/MS/MS). We found that 40% of identified proteins were down-regulated and most of these proteins are microtubule components and associated proteins that mediated neurite outgrowth. Western blot experiments confirmed the expression of α-tubulin and microtubule- associated protein 1B (MAP 1B) was dramatically reduced in Neuro-2a-Ng cells compared to control. Cell morphology of Neuro-2a-Ng showed far less neurites than the control. Serum deprivation induced the extension of only one or two long neurites per cell in Neuro-2a-Ng, contrasting to the extension of multiple neurites per control cell. Ng may be linked to neurite formation by affecting expression of several microtubule related proteins. Furthermore, the PKC activator (PMA) induced an enhanced ERK1/2 activity in the cells that expressed Ng. The mutation of Ng at S36A caused sustained increase of ERK1/2 activity, whereas the ERK1/2 activity in mutation at I33Q showed no difference compared to wild type Ng, suggesting the phosphorylation of Ng but not the CaM /Ng interaction plays an important role in ERK activation. Ng may be involved in neuronal growth and differentiation via PKC and ERK1/2 signaling pathways.

Enrichment of coenzyme Q10 in plasma and blood cells: defense against oxidative damage

Petra Niklowitz, Anka Sonnenschein, Bernd Janetzky, Werner Andler, Thomas Menke

Coenzyme Q10 (CoQ10) concentration in blood cells was analyzed by HPLC and compared to plasma concentration before, during, and after CoQ10 (3 mg/kg/day) supplementation to human probands. Lymphocyte DNA 8-hydroxydeoxy-guanosine (8-OHdG), a marker of oxidative stress, was analyzed by Comet assay.

Subjects supplemented with CoQ10 showed a distinct response in plasma concentrations after 14 and 28 days. Plasma levels returned to baseline values 12 weeks after treatment stopped. The plasma concentration increase did not affect erythrocyte levels. However, after CoQ10 supplementation, the platelet level increased; after supplementation stopped, the platelet level showed a delayed decrease. A positive correlation was shown between the plasma CoQ10 level and platelet and white blood cell CoQ10 levels. During CoQ10 supplementation, delayed formation of 8-OHdG in lymphocyte DNA was observed; this effect was long-lasting and could be observed even 12 weeks after supplementation stopped. Intracellular enrichment may support anti-oxidative defense mechanisms.

A polychromator-based microspectrophotometer

A microspectrophotometer is a digital microscope used to measure absorption and fluorescence spectra. In this paper we describe a polychromator-based microspectrophotometer that performs in vivo absorption or emission measurements at the same time on different subcellular compartments such as photoreceptive and photosynthetic structures of algal cells. In this system, a flat field imaging concave grating polychromator is connected to the slit-shaped exit pupil of a light-guide probe mounted onto a microscope equipped with an epifluorescence module.

The subcellular components, on which the spectra will be measured, are placed in the microscope field and finely adjusted. The outer bundle of the probe is used for centering the objects, while the central bundle of the probe, containing 19 light guides, is used for acquiring either transmitted or emitted light (i.e. fluorescence). The light transmitted or emitted by the subcellular components is collected by the probe mounted in the back focal plane of the ocular. The exit pupil of this probe, connected to a flat field imaging concave grating polychromator, produces a dispersion image that in turn is focused onto a digital slow scan cooled CCD camera. Absorption and emission spectra of algal subcellular compartments are presented

Silencing of the Pink1 Gene Expression by Conditional RNAi Does Not Induce Dopaminergic Neuron Death in Mice

Transgenic RNAi, an alternative to the gene knockout approach, can induce hypomorphic phenotypes that resemble those of the gene knockout in mice. Conditional transgenic RNAi is an attractive choice of method for reverse genetics in vivo because it can achieve temporal and spatial silencing of targeted genes. Pol III promoters such as U6 are widely used to drive the expression of RNAi transgenes in animals. Tested in transgenic mice, a Cre-loxP inducible U6 promoter drove the broad expression of an shRNA against the Pink1 gene whose loss-of-functional mutations cause one form of familial Parkinson's disease. The expression of the shRNA was tightly regulated and, when induced, silenced the Pink1 gene product by more than 95% in mouse brain. However, these mice did not develop dopaminergic neurodegeneration, suggesting that silencing of the Pink1 gene expression from embryo in mice is insufficient to cause similar biochemical or morphological changes that are observed in Parkinson's disease. The results demonstrate that silencing of the PINK1 gene does not induce a reliable mouse model for Parkinson's disease, but that technically the inducible U6 promoter is useful for conditional RNAi in vivo.

Tyrosine Sulfation of Statherin

C. Kasinathan, N. Gandhi, P. Ramaprasad, P. Sundaram, N. Ramasubbu

Tyrosylprotein sulfotransferase (TPST), responsible for the sulfation of a variety of secretory and membrane proteins, has been identified and characterized in submandibular salivary glands (William et al. Arch Biochem Biophys 1997; 338: 90-96). In the present study we demonstrate the sulfation of a salivary secretory protein, statherin, by the tyrosylprotein sulfotransferase present in human saliva. Optimum statherin sulfation was observed at pH 6.5 and at 20 mm MnCl₂. Increase in the level of total sulfation was observed with increasing statherin concentration. The K_m value of tyrosylprotein sulfotransferase for statherin was 40 μM. Analysis of the sulfated statherin product on SDS-polyacrylamide gel electrophoresis followed by autoradiography revealed ³⁵S-labelling of a 5 kDa statherin. Further analysis of the sulfated statherin revealed the sulfation on tyrosyl residue. This study is the first report demonstrating tyrosine sulfation of a salivary secretory protein. The implications of this sulfation of statherin in hydroxyapatite binding and Actinomyces viscosus interactions are discussed.

MCEF is localized to the nucleus by protein sequences encoded within three distinct exons, where it represses HIV-1 Tat-transactivation of LTR-directed transcription

Maksymilian F. Niedzielski, Robert Hopewell, Zohra Ismail, Mario C. Estable

Translocations between the human Mixed Lineage Leukemia (MLL) and AF4 Family (AFF) member genes, are implicated in leukemia. Mutations to AFFs can disrupt lymphopoesis, CNS development and spermatogenesis. However, despite the growing list of pathologies linked to AFF members, their evolutionary relationship and the structure/function of individual members, remain to be elucidated. Here, we first report that database mining and phylogenetic analysis with AFF proteins from multiple species, revealed two monophyletic sister clades, suggesting a common Bilateria ancestor. We then examined the structure/function of the most recently discovered AFF member, MCEF (also known as AF5q31 or AFF4). In silico, the human MCEF gene was found to have 21 exons, and code for a protein with seven nuclear localization sequences (NLS). In HeLa cells, an MCEF-EGFP fusion protein, localized exclusively to the nucleus. Consequently, we made twenty constructs, expressing MCEF deletion mutants fused to EGFP and/or DsRed fluorescent proteins. Three distinct protein sequences, encoded by three separate MCEF exons, were found to mediate nuclear localization, only two of which were predicted in silico. Importantly, we also found that ectopic expression of MCEF, repressed HIV-1 LTR-directed RNA Polymerase II transcription, at the level of Tat-transactivation. We suggest that portions of MCEF could be exploited for chimeric transcription factor repression (CTFR) of HIV-1.

Characterization of transcriptional regulation of neurogranin by nitric oxide and the role of neurogranin in SNP-induced cell death: implication of neurogranin in an increased neuronal susceptibility to oxidative stress

Jingang Gui, Yan Song, Nian-Lin Reena Han, Fwu-Shan Sheu

Neurogranin (Ng), a calmodulin (CaM)-binding protein kinase C (PKC) substrate, regulates the availability of Ca²⁺/CaM complex and modulates the homeostasis of intracellular calcium in neurons. Previous work showed Ng oxidation by NO donor induces increase in [Ca²⁺]_i. The current study demonstrated that the gene transcription of Ng could be up-regulated by various nitric oxide (NO) donors via a NO-soluble guanylyl cyclase (sGC)-mediated pathway. Furthermore, ectopic expression of neuronal nitric oxide synthase (nNOS) in human embryonic kidney 293 cells (HEK 293) exhibited a nNOS-concentration-dependent biphasic regulatory effect on Ng gene transcription. One of the NO donors, sodium nitroprusside (SNP), however, induced cell death of neuroblastoma Neuro-2a cells. The potency of SNP-induced cell death was shown to be higher in Neuro-2a cells expressing recombinant Ng, as compared with Neuro-2a control cells without Ng expression in cell viability and apoptosis assays. Single-cell fluorescence imaging and site-directed mutagenesis studies suggest that Ng promotes SNP-induced cell death through an amplification of calcium-mediated signaling, which requires the interaction between CaM and IQ motif of Ng. Increased neuronal susceptibility rendered by Ng in response to pathophysiological NO production is suggested to be involved in the selective vulnerability of neurons to oxidative insults in the CNS.

Structural stability and endonuclease activity of a PI-SceI GFP-fusion protein

Alireza G. Senejani, J. Peter Gogarten

Homing endonucleases are site-specific and rare cutting endonucleases often encoded by intron or intein containing genes. They lead to the rapid spread of the genetic element that hosts them by a process termed 'homing'; and ultimately the allele containing the element will be fixed in the population.

PI-SceI, an endonuclease encoded as a protein insert or intein within the yeast V-ATPase catalytic subunit encoding gene (vma1), is among the best characterized homing endonucleases. The structures of the Sce VMA1 intein and of the intein bound to its target site are known. Extensive biochemical studies performed on the PI-SceI enzyme provide information useful to recognize critical amino acids involved in self-splicing and endonuclease functions of the protein. Here we describe an insertion of the Green Fluorescence Protein (GFP) into a loop which is located between the endonuclease and splicing domains of the Sce VMA1 intein. The GFP is functional and the additional GFP domain does not prevent intein excision and endonuclease activity. However, the endonuclease activity of the newly engineered protein was different from the wild-type protein in that it required the presence of Mn²⁺ and not Mg²⁺ metal cations for activity.

Overexpression of Selenoprotein H Reduces Ht22 Neuronal Cell Death after UVB Irradiation by Preventing Superoxide Formation

Kamel E. Ben Jilani, Jun Panee, Qingping He, Marla J. Berry, Ping-An Li

Selenoproteins have been shown to exhibit a variety of biological functions, including antioxidant functions, maintaining cellular redox balance, and heavy metal detoxification. UV irradiation-induced damage is partially mediated by increased oxygen radical production. The present study is designed to examine the antioxidative effects of human selenoprotein H (hSelH) after brief period of UVB irradiation on the murine hippocampal neuronal cell line Ht22. Ht22 cells were stably transfected with the hSelH gene or with MSCV empty vector and exposed to UVB irradiation with or without the presence of serum. The results showed that cell viability was significantly higher in hSelH-transfected cells compared to the MSCV vector-transfected cells after 24 h of recovery with or without the presence of serum in the media. Further studies revealed that while the number of superoxide anion (O2˙-) positive cells was increased following a 7 mJ/cm² of UVB irradiation and 5 h of recovery, overexpression of hSelH significantly reduced superoxide production. These results suggest that hSelH overexpression protects cells from UVB irradiation-induced cell death by reducing the O2˙- formation.

Advances in QTL Mapping in Pigs

Max F. Rothschild, Zhi-liang Hu, Zhihua Jiang

Over the past 15 years advances in the porcine genetic linkage map and discovery of useful candidate genes have led to valuable gene and trait information being discovered. Early use of exotic breed crosses and now commercial breed crosses for quantitative trait loci (QTL) scans and candidate gene analyses have led to 110 publications which have identified 1,675 QTL. Additionally, these studies continue to identify genes associated with economically important traits such as growth rate, leanness, feed intake, meat quality, litter size, and disease resistance. A well developed QTL database called PigQTLdb is now as a valuable tool for summarizing and pinpointing in silico regions of interest to researchers. The commercial pig industry is actively incorporating these markers in marker-assisted selection along with traditional performance information to improve traits of economic performance. The long awaited sequencing efforts are also now beginning to provide sequence available for both comparative genomics and large scale single nucleotide polymorphism (SNP) association studies. While these advances are all positive, development of useful new trait families and measurement of new or underlying traits still limits future discoveries. A review of these developments is presented.

Identifying the Future Needs for Long-Term USDA Efforts in Agricultural Animal Genomics

R. D. Green, M. A. Qureshi, J. A. Long, P.J. Burfening, D.L. Hamernik

Agricultural animal research has been immensely successful over the past century in developing technology and methodologies that have dramatically enhanced production efficiency of the beef, dairy, swine, poultry, sheep, and aquaculture industries. In the past two decades, molecular biology has changed the face of agricultural animal research, primarily in the arena of genomics and the relatively new offshoot areas of functional genomics, proteomics, transcriptomics, metabolomics and metagenomics. Publication of genetic and physical genome maps in the past 15 years has given rise to the possibility of being able finally to understand the molecular nature of the genetic component of phenotypic variation. While quantitative geneticists have been remarkably successful in improving production traits, genomic technology holds potential for being able to lead to more accurate and rapid animal improvement, especially for phenotypic traits that are difficult to measure.

Recently, the agricultural research community has been able to capitalize on the infrastructure built by the human genome project by sequencing two of the major livestock genomes (Gallus domesticus and Bos Taurus). The 2005 calendar year is truly unprecedented in the history of agricultural animal research since draft genome sequences were completed for chickens and cattle. In addition, sequencing the swine and equine genome was initiated in early 2006. We now have in place a powerful toolbox for understanding the genetic variation underlying economically important and complex phenotypes.

Over the past few years, new challenges have emerged for animal agriculture. Enhancements in production efficiency have not come without some negative side effects on animal well-being and longevity in production environments, including losses in reproductive efficiency, increased stress susceptibility, increased animal waste issues, and increased susceptibility to animal metabolic and infectious diseases. When considered in concert with societal concerns in the areas of natural resource conservation and protection, animal welfare, and food safety, it is clear that publicly supported agricultural research must be focused on enhancing the functionality and well-being of livestock and poultry in environmentally neutral production systems in the future.

Realizing the great potential for animal genomics to address these and other issues, a workshop was convened by the U. S. Department of Agriculture (USDA) in Washington, DC in September of 2004. The workshop was entitled “Charting the Road Map for Long Term USDA Efforts in Agricultural Animal Genomics”. This paper summarizes the proceedings of the workshop and the resulting recommendations. The need for a cohesive, comprehensive long-term plan for all of USDA's research efforts in animal genomics was evident at the workshop, requiring further integration of the efforts of the USDA's Cooperative State Research, Education, and Extension Service (CSREES) and the USDA's Agricultural Research Service (ARS) to achieve the greatest return on investment.

Advances in Swine Biomedical Model Genomics

Joan K. Lunney

This review is a short update on the diversity of swine biomedical models and the importance of genomics in their continued development. The swine has been used as a major mammalian model for human studies because of the similarity in size and physiology, and in organ development and disease progression. The pig model allows for deliberately timed studies, imaging of internal vessels and organs using standard human technologies, and collection of repeated peripheral samples and, at kill, detailed mucosal tissues. The ability to use pigs from the same litter, or cloned or transgenic pigs, facilitates comparative analyses and genetic mapping. The availability of numerous well defined cell lines, representing a broad range of tissues, further facilitates testing of gene expression, drug susceptibility, etc. Thus the pig is an excellent biomedical model for humans. For genomic applications it is an asset that the pig genome has high sequence and chromosome structure homology with humans. With the swine genome sequence now well advanced there are improving genetic and proteomic tools for these comparative analyses. The review will discuss some of the genomic approaches used to probe these models. The review will highlight genomic studies of melanoma and of infectious disease resistance, discussing issues to consider in designing such studies. It will end with a short discussion of the potential for genomic approaches to develop new alternatives for control of the most economically important disease of pigs, porcine reproductive and respiratory syndrome (PRRS), and the potential for applying knowledge gained with this virus for human viral infectious disease studies.

Characterizing Linkage Disequilibrium in Pig Populations

Feng-Xing Du, Archie C. Clutter, Michael M. Lohuis

Knowledge of the extent and range of linkage disequilibrium (LD), defined as non-random association of alleles at two or more loci, in animal populations is extremely valuable in localizing genes affecting quantitative traits, identifying chromosomal regions under selection, studying population history, and characterizing/managing genetic resources and diversity. Two commonly used LD measures, r² and D', and their permutation based adjustments, were evaluated using genotypes of more than 6,000 pigs from six commercial lines (two terminal sire lines and four maternal lines) at ~4,500 autosomal SNPs (single nucleotide polymorphisms). The results indicated that permutation only partially removed the dependency of D' on allele frequency and that r² is a considerably more robust LD measure. The maximum r² was derived as a function of allele frequency. Using the same genotype dataset, the extent of LD in these pig populations was estimated for all possible syntenic SNP pairs using r² and the ratio of r² over its theoretical maximum. As expected, the extent of LD highest for SNP pairs was found in tightest linkage and decreased as their map distance increased. The level of LD found in these pig populations appears to be lower than previously implied in several other studies using microsatellite genotype data. For all pairs of SNPs approximately 3 centiMorgan (cM) apart, the average r² was equal to 0.1. Based on the average population-wise LD found in these six commercial pig lines, we recommend a spacing of 0.1 to 1 cM for a whole genome association study in pig populations.

Genetic Resources, Genome Mapping and Evolutionary Genomics of the Pig (Sus scrofa)

Kefei Chen, Tara Baxter, William M. Muir, Martien A. Groenen, Lawrence B. Schook

The pig, a representative of the artiodactyla clade, is one of the first animals domesticated, and has become an important agriculture animal as one of the major human nutritional sources of animal based protein. The pig is also a valuable biomedical model organism for human health. The pig's importance to human health and nutrition is reflected in the decision to sequence its genome (3X). As an animal species with its wild ancestors present in the world, the pig provides a unique opportunity for tracing mammalian evolutionary history and defining signatures of selection resulting from both domestication and natural selection. Completion of the pig genome sequencing project will have significant impacts on both agriculture and human health. Following the pig whole genome sequence drafts, along with large-scale polymorphism data, it will be possible to conduct genome sweeps using association mapping, and identify signatures of selection. Here, we provide a description of the pig genome sequencing project and perspectives on utilizing genomic technologies to exploit pig genome evolution and the molecular basis for phenotypic traits for improving pig production and health.

Advances in Swine Transcriptomics

Christopher K. Tuggle, Yanfang Wang, Oliver Couture

The past five years have seen a tremendous rise in porcine transcriptomic data. Available porcine Expressed Sequence Tags (ESTs) have expanded greatly, with over 623,000 ESTs deposited in Genbank. ESTs have been used to expand the pig-human comparative maps, but such data has also been used in many ways to understand pig gene expression. Several methods have been used to identify genes differentially expressed (DE) in specific tissues or cell types under different treatments. These include open screening methods such as suppression subtractive hybridization, differential display, serial analysis of gene expression, and EST sequence frequency, as well as closed methods that measure expression of a defined set of sequences such as hybridization to membrane arrays and microarrays. The use of microarrays to begin large-scale transcriptome analysis has been recently reported, using either specialized or broad-coverage arrays. This review covers published results using the above techniques in the pig, as well as unpublished data provided by the research community, and reports on unpublished Affymetrix data from our group. Published and unpublished bioinformatics efforts are discussed, including recent work by our group to integrate two broad-coverage microarray platforms. We conclude by predicting experiments that will become possible with new anticipated tools and data, including the porcine genome sequence. We emphasize that the need for bioinformatics infrastructure to efficiently store and analyze the expanding amounts of gene expression data is critical, and that this deficit has emerged as a limiting factor for acceleration of genomic understanding in the pig.

Swine Genome Science Comes of Age

Zhihua Jiang, Max F. Rothschild

Pigs were among the first animals to be domesticated and pork is one of the most widely eaten meats in the world today. The pig has also been an excellent biomedical model for understanding a variety of human health issues such as obesity, diabetes, cancer, female reproductive health, cardiovascular disease, and infectious diseases. Genome sequencing, mapping, expression and functional analyses have significantly advanced our ability to unravel the secrets of the pig. Therefore, this edition, with six reviews from leading scientists, offers the opportunity for all interested researchers and readers to see the big picture of porcine genomics.

Depletion of ceramides with very long chain fatty acids causes defective skin permeability barrier function, and neonatal lethality in ELOVL4 deficient mice

Very long chain fatty acids (VLCFA), either free or as components of glycerolipids and sphingolipids, are present in many organs. Elongation of very long chain fatty acids-4 (ELOVL4) belongs to a family of 6 members of putative fatty acid elongases that are involved in the formation of VLCFA. Mutations in ELOVL4 were found to be responsible for an autosomal dominant form of Stargardt's-like macular dystrophy (STGD3) in human. We have previously disrupted the mouse Elovl4 gene, and found that Elovl4^+/- mice were developmentally normal, suggesting that haploinsufficiency of ELOVL4 is not a cause for the juvenile retinal degeneration in STGD3 patients. However, Elovl4^-/- mice died within several hours of birth for unknown reason(s). To study functions of ELOVL4 further, we have explored the causes for the postnatal lethality in Elovl4^-/- mice. Our data indicated that the mutant mice exhibited reduced thickness of the dermis, delayed differentiation of keratinocytes, and abnormal structure of the stratum corneum. We showed that all Elovl4^-/- mice exhibited defective skin water permeability barrier function, leading to the early postnatal death. We further showed that the absence of ELOVL4 results in depletion in the epidermis of ceramides with ω-hydroxy very long chain fatty acids (≥C28) and accumulation of ceramides with non ω-hydroxy fatty acids of C26, implicating C26 fatty acids as possible substrates of ELOVL4. These data demonstrate that ELOVL4 is required for VLCFA synthesis that is essential for water permeability barrier function of skin.

Essential role of Elovl4 in very long chain fatty acid synthesis, skin permeability barrier function, and neonatal survival

Mutations in the gene ELOVL4 have been shown to cause stargardt-like macular dystrophy. ELOVL4 is part of a family of fatty acid elongases and is yet to have a specific elongase activity assigned to it. We generated Elovl4 Y270X mutant mice and characterized the homozygous mutant as well as homozygous Elovl4 knockout mice in order to better understand the function or role of Elovl4. We found that mice lacking a functional Elovl4 protein died perinatally. The cause of death appears to be from dehydration due to faulty permeability barrier formation in the skin. Further biochemical analysis revealed a significant reduction in free fatty acids longer than C26 in homozygous mutant and knockout mouse skin. These results implicate the importance of these long chain fatty acids in skin barrier development. Furthermore, we suggest that Elovl4 is likely involved in the elongation of C26 and longer fatty acids.

Application of Nanotechnology in Cancer Research: Review of Progress in the National Cancer Institute's Alliance for Nanotechnology

Beeta Ehdaie

Mouse 24p3 Protein Has an Effect on L929 Cell Viability

Pei-Tzu Li, Ying-Chu Lee, Namasivayam Elangovan, Sin-Tak Chu

It is well known that mouse uterine 24p3 protein, is an acute phase protein, secreted from the L929 cell line, and that it will be induced by the dexamethasone stimulation of the cell. We investigated the possible effects of 24p3 protein on the L929 cell line, by observing its morphological change, ROS increase and viability decrease, by the process of culturing in a 24p3 protein-supplemented medium. Following the L929 cells′ exposure to the 24p3 protein supplement for a period of 72 hours, S-phase cells accumulated to a significant degree, suggesting that the entry into the G2/M phase from the S phase, in the cell cycle progression, was blocked. There was a significant decrease in cell numbers and increased DNA damage within the cells in the presence of 24p3 protein within the medium for 96 hours, implying that they have undergone pathway of cell death. After 96h incubation in low concentration of 24p3 protein, the result of PI/annexin V double staining showed cell death obviously. These results suggest that 24p3 protein-induced S phase arrest in the cell cycle, would cause DNA damage, followed by cell death in the L929 cells.

RNAi-based conditional gene knockdown in mice using a U6 promoter driven vector

Vivek Shukla, Xavier Coumoul, Chu-Xia Deng

RNA interference (RNAi) is a powerful tool widely used for studying gene function in a number of species. We have previously developed an approach that allows conditional expression of a polymerase III promoter based small hairpin RNA (shRNA) in mice using the Cre-LoxP system. This approach uses a U6 promoter, which is inactive due to the presence of a ploxPneo cassette in the promoter; this promoter can be activated after excision of the neo gene in transgenic mice that express a Cre recombinase transgene. As a proof of principle, we have previously knocked down over 95% of Fgfr2 transcripts in mouse germlines, leading to embryonic lethality, while restricting the knockdown to the progress zone of the limb results in live animals with malformation of digits of both the forelimbs and hindlimbs. We now provide a detailed protocol, including a simplified single-step cloning procedure for vector construction. This method provides a fast yet efficient way to decipher gene functions in vivo in a tissue specific manner.

A complete mitochondrial genome sequence of Asian black bear Sichuan subspecies (Ursus thibetanus mupinensis)

We obtained the complete mitochondrial genome of U.thibetanus mupinensis by DNA sequencing based on the PCR fragments of 18 primers we designed. The results indicate that the mtDNA is 16 868 bp in size, encodes 13 protein genes, 22 tRNA genes, and 2 rRNA genes, with an overall H-strand base composition of 31.2% A, 25.4% C, 15.5% G and 27.9% T. The sequence of the control region (CR) located between tRNA-Pro and tRNA-Phe is 1422 bp in size, consists of 8.43% of the whole genome, GC content is 51.9% and has a 6bp tandem repeat and two 10bp tandem repeats identified by using the Tandem Repeats Finder. U. thibetanus mupinensis mitochondrial genome shares high similarity with those of three other Ursidae: U. americanus (91.46%), U. arctos (89.25%) and U. maritimus (87.66%).

The Environmental-Endocrine Basis of Gynandromorphism (Intersex) in a Crustacean

Allen W. Olmstead, Gerald A. LeBlanc

Commensurate with the decline in many crustacean populations has been an accumulation in reports of sexually ambiguous individuals within these populations. The cause of gynandromorphism or intersex among crustaceans is unknown. We show that gynandromorphism in the branchiopod crustacean Daphnia magna is initiated by the sex-determining hormone methyl farnesoate when levels of the hormone are intermediate between low levels that stimulate the production of broods containing all female offspring and high levels that stimulate the production of broods of all male offspring. The incidence of hormonally-induced gynandromorphism was low (0.14% at the maximum stimulatory hormone concentrations) but was significantly increased (46-fold) when the animals were hormone-treated at 30^oC. Some environmental chemicals also can stimulate the gynandromorphic phenotype as we demonstrated with the insecticide pyriproxyfen. Gynandromorphism occurs due to inadequate signaling of male-sex determination since: a) gynandromorphs did not occur in a population that was producing only female offspring; and, b) conditions that stimulated gynandromorphism also reduced the incidence of male offspring. We suggest that male sex determination normally occurs prior to the first embryonic cleavage. Elevated temperature may alter the timing of sex determination such that methyl farnesoate signaling occurs after the first embryonic cleavage and bilateral gynandromorphism occurs as a consequence of signaling to only one of the daughter cells. These results demonstrate that environmental factors can cause aberrant sex determination via perturbations in methyl farnesoate signaling.

Role of TAB1 in nitric oxide-induced p38 activation in insulin-producing cells

Natalia Makeeva, Godfried M. Roomans, Nils Welsh

The aim of present study was to elucidate the role of TAB1 in nitric oxide-induced activation of p38 MAPK. For this purpose we over-expressed TAB1 in insulin-producing β-TC6 cells. We observed in cells transiently over-expressing TAB1 that p38 activation was enhanced in response to DETA/NONOate. A lowering of TAB1 levels, using the siRNA technique, resulted in the opposite effect. The DETA/NONOate-induced cell death rate was increased in cells transiently overexpressing TAB1. In stable β-TC6 cell clones with very high TAB1 levels p38 phosphorylation was enhanced also at basal conditions. DETA/NONOate increased also the phosphorylation of JNK and ERK in β-TC6 cells, but these events were not affected by TAB1. Interestingly, the inhibitory effect of SB203580 on p38 phosphorylation was paralleled by a stimulatory effect on JNK phosphorylation and an inhibitory effect on ERK phosphorylation. In summary, we propose that TAB1 promotes nitric oxide-induced p38 autophosphorylation. In addition, nitric oxide-induced p38 activation seems to promote JNK inhibition and ERK activation, but this effect appears to not require TAB1. A better understanding of how the TAB1/p38 pathway promotes β-cell death in response to nitric oxide might help in the development of novel pharmacological approaches in the treatment of diabetes.

Urokinase Separation from Cell Culture Broth of a Human Kidney Cell Line

Vibha Bansal, Pradip K. Roychoudhury, Ashok Kumar

A single step ion-exchange chromatography on a sulfo-propyl (SP)- Sepharose column was performed to separate both the high molecular weight (HMW)- and low molecular weight (LMW)- forms of enzymatically active urokinase type plasminogen activator from human kidney (HT1080) cell culture media. The level of urokinase secreted by the cell line reached to about 145 Plough units/ml culture broth within 48 h of cultivation. The conditioned cell culture media was applied directly to the column without any prior concentration steps. Polyacrylamide gel electrophoresis of the column eluates in the presence of sodium dodecyl sulphate showed that the cell line secretes three forms of two-chain high molecular weight (HMW) urokinase of molecular weights (M_r) 64,000, 60,900 and 55,000. In addition, two low molecular weight (LMW) forms of M_r 22,000 and 20,000; proteolytic cleavage products of HMW, were also found. The HMW and LMW forms had intrinsic plasminogen dependent proteolytic activity as judged by zymographic analysis. The specific activity of the pooled peak fractions increased (approximately 93-fold) to values as high as 1481 Plough units/ mg protein. Both HMW as well as LMW forms were obtained in significantly high yields.

Prion-derived copper-binding peptide fragments catalyze the generation of superoxide anion in the presence of aromatic monoamines

Tomonori Kawano

Objectives: Studies have proposed two opposing roles for copper-bound forms of prion protein (PrP) as an anti-oxidant supporting the neuronal functions and as a pro-oxidant leading to neurodegenerative process involving the generation of reactive oxygen species. The aim of this study is to test the hypothesis in which putative copper-binding peptides derived from PrP function as possible catalysts for monoamine-dependent conversion of hydrogen peroxide to superoxide in vitro.

Materials and methods: Four peptides corresponding to the copper (II)-binding motifs in PrP were synthesized and used for analysis of peptide-catalyzed generation of superoxide in the presence of Cu (II) and other factors naturally present in the neuronal tissues.

Results: Among the Cu-binding peptides tested, the amino acid sequence corresponding to the Cu-binding site in the helical region was shown to be the most active for superoxide generation in the presence of Cu(II), hydrogen peroxide and aromatic monoamines, known precursors or intermediates of neurotransmitters. Among monoamines tested, three compounds namely phenylethylamine, tyramine and benzylamine were shown to be good substrates for superoxide-generating reactions by the Cu-bound helical peptide.

Conclusions: Possible roles for these reactions in development of prion disease were suggested.

Distinct domain-dependent effect of syntaxin1A on amiloride-sensitive sodium channel (ENaC) currents in HT-29 colonic epithelial cells

Sunil K Saxena, Madhurima Singh, Simarna Kaur, Constantine George

The amiloride-sensitive epithelial sodium channel (ENaC), a plasma membrane protein mediates sodium reabsorption in epithelial tissues, including the distal nephron and colon. Syntaxin1A, a trafficking protein of the t-SNARE family has been reported to inhibit ENaC in the Xenopus oocyte expression and artificial lipid bilayer systems. The present report describes the regulation of the epithelial sodium channel by syntaxin1A in a human cell line that is physiologically relevant as it expresses both components and also responds to aldosterone stimulation. In order to evaluate the physiological significance of syntaxin1A interaction with natively expressed ENaC, we over-expressed HT-29 with syntaxin1A constructs comprising various motifs. Unexpectedly, we observed the augmentation of amiloride-sensitive currents with wild-type syntaxin1A full-length construct (1-288) in this cell line. Both γENaC and neutralizing syntaxin1A antibodies blocked native expression as amiloride-sensitive sodium currents were inhibited while munc18-1 antibody reversed this effect. The coiled-coiled domain H3 (194-266) of syntaxin1A inhibited, however the inclusion of the transmembrane domain to this motif (194-288) augmented amiloride sensitive currents. More so, data suggest that ENaC interacts with multiple syntaxin1A domains, which differentially regulate channel function. This functional modulation is the consequence of the physical enhancement of ENaC at the cell surface in cells over-expressed with syntaxin(s). Our data further suggest that syntaxin1A up-regulates ENaC function by multiple mechanisms that include PKA, PLC, PI3 and MAP Kinase (p42/44) signaling systems. We propose that syntaxin1A possesses distinct inhibitory and stimulatory domains that interact with ENaC subunits, which critically determines the overall ENaC functionality/regulation under distinct physiological conditions.

Intrarenal Oxidative Stress and Augmented Angiotensinogen are Precedent to Renal Injury in Zucker Diabetic Fatty Rats

Yuki Suzaki, Yuri Ozawa, Hiroyuki Kobori

The Zucker diabetic fatty (ZDF) rat is a model of type II diabetes and metabolic syndrome based on impaired glucose tolerance caused by the inherited insulin-resistance gene. The ZDF rat exhibits progressive nephropathy; however, the detailed mechanisms have remained unclear. This study was performed to examine the possible involvement of enhanced intrarenal angiotensinogen in the development of renal injury in ZDF rats. Genetic pairs of male ZDF rats and control lean rats (N=6 each) were maintained from 12 to 17 weeks of age. At 17 weeks of age, fasting blood glucose and urinary 8-isoprostane levels were significantly higher in ZDF rats compared with the controls. Systolic blood pressure progressively increased in ZDF rats from 120+/-1 to 137+/-1 mmHg during this period. In contrast, systolic blood pressure did not increase in the controls. Kidney angiotensinogen protein levels were significantly increased in ZDF rats compared with the controls (1.83+/-0.34 vs. 1.00+/-0.17, relative ratio). Expression of angiotensin II type 1a receptor mRNA was similar between these groups. The measured indices of renal damage in the present study (glomerular sclerosis, interstitial expansion, glomerular macrophage infiltration, and renal arterial proliferation) were not significantly increased at this stage in ZDF rats. However, we previously showed that the increased reactive oxygen species-related angiotensinogen enhancement plays an important role in the development of renal injury in a genetic salt-sensitive hypertension. Thus, the present data suggest that elevated reactive oxygen species and reactive oxygen species-associated augmentation of intrarenal angiotensinogen may initiate the development of renal injury in ZDF rats.

Proteomic Identification of 14-3-3ζ as an Adapter for IGF-1 and Akt/GSK-3β Signaling and Survival of Renal Mesangial Cells

Lalit P. Singh, Yan Jiang, Davis W. Cheng

Recently we demonstrated that IGF-1 expression is increased in the diabetic kidney and that it may involve in renal hypertrophy and extracellular matrix protein (ECM) accumulation in mesangial cells as seen in diabetic glomerulopathy. The present study investigates the molecular mechanism(s) of IGF-1 and Akt/glycogen synthase kinase-3beta (GSK-3β) signaling pathway in the regulation of fibronectin and cyclin D1 expression and survival of renal mesangial cells. A proteomic approach is also employed to identify protein targets of IGF-1 signaling via GSK-3β inhibition in mesangial cells. We show that IGF-1 (100 ng/ml) significantly increases the protein kinase Akt/PKB activity (1.5-2-fold, p<0.05) within 1-5 minutes, which is completely blocked by the presence of 100 nM Wortmannin (phosphatidyl-inositol 3-kinase inhibitor). Akt activation is coupled with Ser9 phosphorylation and inactivation of its down-stream target GSK-3β. IGF-1 increases the cyclic AMP-responsive element (CRE) binding transcription factor CREB phosphorylation at Ser 133 and CRE-binding activity in mesangial cells, which parallels cyclin D1 and fibronectin expressions. Both proteins are known to have CRE-sequences in their promoter regions upstream of the transcription start site. Suppression of GSK-3β by SB216763 (100 nM) increases CREB phosphorylation, cyclin D1 and fibronectin levels. Two dimensional gel electrophoresis followed by MALDI-TOF mass spectrometric analysis of mesangial proteins reveals that IGF-1 treatment or an inhibition of GSK-3β increases the expression of the phosphorylated Ser/Thr binding signal adapter protein 14-3-3ζ. Immuno-precipitation of 14-3-3ζ followed by Western blotting validates the association of phosphorylated GSK-3β with 14-3-3ζ in renal mesangial cells. Stable expression of a constitutively active GSK-3β(Ser9Ala) induces cell death while overexpression of HA-tagged 14-3-3ζ increases cell viability as measured by MTT assays. These results indicate that the Akt/GSK-3β pathway and the adapter protein 14-3-3ζ may play an important role in IGF-1 signaling and survival of mesangial cells in diabetic nephropathy.

Are heat shock proteins therapeutic target for Parkinson's disease?

Guang-Rui Luo, Sheng Chen, Wei-Dong Le

Heat shock proteins (HSPs), known as molecular chaperone to assist protein folding, have recently become a research focus in Parkinson's disease (PD) because the pathogenesis of this disease is highlighted by the intracellular protein misfolding and inclusion body formation. The present review will focus on the functions of different HSPs and their protective roles in PD. It is postulated that HSPs may serve as protein folding machinery and work together with ubiquitin-proteasome system (UPS) to assist in decomposing aberrant proteins. Failure of UPS is thought to play a key role in the pathogenesis of PD. In addition, HSPs may possess anti-apoptotic effects and keep the homeostasis of dopaminergic neurons against stress conditions. The critical role of HSPs and recent discovery of some novel HSPs inducers suggest that HSPs may be potential therapeutic targets for PD and other neurodegenerative disorders.

Increased Susceptibility to Metabolic Alterations in Young Adult Females Exposed to Early Malnutrition

María del Carmen Miñana-Solis, Carolina Escobar

Early malnutrition during gestation and lactation modifies growth and metabolism permanently. Follow up studies using a nutritional rehabilitation protocol have reported that early malnourished rats exhibit hyperglycemia and/or hyperinsulinemia, suggesting that the effects of early malnutrition are permanent and produce a “programming” effect on metabolism. Deleterious effects have mainly been observed when early-malnutrition is followed by a high-carbohydrate or a high-fat diet.

The aim of this study was to evaluate whether following a balanced diet subsequent to malnutrition can deter the expression of metabolic disease and lead rats to exhibit metabolic responses, similar to those of well-nourished controls.

Young rats, born from dams malnourished during gestation and lactation with a low protein diet, were provided with a regular balanced chow diet upon weaning. At 90 days of age, the effects of rehabilitation were determined under three different feeding conditions: ad libitum, fasting or fasting-reefed satiated.

Early-malnourished rats showed an increased rate of body weight gain. Males under ad libitum conditions showed an elevated concentration of hepatic glycogen and low values of insulin. In the fasting-reefed satiated condition, only early-malnourished females showed an alteration in glucose response and glucagon level, compared with their well-nourished controls.

Data indicate that a balanced diet along life after early malnutrition can mask the expression of metabolic disorders and that a metabolic challenges due to a prolonged fasting and reefed state unmask metabolic deficiencies in early-malnourished females.

Gamma Protocadherin Expression in the Embryonic Chick Nervous System

Kenneth D. Cronin, Anthony A. Capehart

Protocadherin γ (pcdh-γ) family expression was examined in the embryonic chick central nervous system by in situ hybridization. Transcripts were visualized in discrete regions of fore-, mid-, and hindbrain at stages 23 and 25 and in spinal cord and optic lobe at stages 27 and 43, respectively. Results suggest that pcdh-γ may function cooperatively with other cell adhesion molecules in neuronal differentiation and establishment of neural networks in several areas of the developing brain, particularly regions involved in visual processing.

Effect of Reparation of Repeat Sequences in the Human α-Synuclein on Fibrillation Ability

Koji Sode, Sayaka Ochiai, Natsuki Kobayashi, Eri Usuzaka

The aggregation and fibrillation of α-synuclein has been implicated as a causative factor in the Parkinson's disease. The hexamer motif KTKEGV is found in each of the seven imperfect repeat sequences in the N-terminal half of α-synuclein. The motif is not fully conserved in the sixth and seventh repeats. We created mutants in which the motif was repaired to be fully conserved in either (Rep6 and Rep7) or both (Rep67) of these two repeats. The Rep6 and Rep67 mutants showed a greatly reduced propensity to aggregate and fibrillate while all three mutants showed greater resistance to HFIP-induced formation of the α-helix intermediate. Resistance to formation in the partially folded intermediate may repress the folding of α-synuclein, consequently interfering with the aggregation and fibril formation. These results demonstrated that KTKEGV repeats may have a significant role in keeping native unfolded status of α-synuclein.