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A formulation with stabilizers replacing albumin was developed for lyophilization of recombinant factor VIII (FVIII), GreenGene F (WHO INN: beroctocog alfa), to achieve stability and eliminate safety issues of blood-derived albumin. L-Arginine (hydrophilic amino acid, positively charged side chain), L-glutamic acid (hydrophilic amino acid, negatively charged side chain), and L-isoleucine (hydrophobic amino acid, nonpolar) were selected as stabilizers, and the mixture of the three amino acids were optimized. The mixture had results comparative with albumin and other commonly used stabilizers showing good preservation of recombinant FVIII during lyophilization, robust stability with consistently high recovery of FVIIII, very low aggregate formation, and good storage stability without alterations in protein characteristics. In vivo test results showed that the efficacy was maintained and had no signs of toxicity. The study demonstrated that the three amino acid mixture acts as a good stabilizer for lyophilization of recombinant FVIII and as a safe excipient., (Copyright © 2012 American Institute of Chemical Engineers (AIChE).)

The Rpd3S histone deacetylase complex represses cryptic transcription initiation within coding regions by maintaining the hypo-acetylated state of transcribed chromatin. Rpd3S recognizes methylation of histone H3 at lysine 36 (H3K36me), which is required for its deacetylation activity. Rpd3S is able to function over a wide range of H3K36me levels, making this a unique system to examine how chromatin regulators tolerate the reduction of their recognition signal. Here, we demonstrated that Rpd3S makes histone modification-independent contacts with nucleosomes, and that Rpd3S prefers di-nucleosome templates since two binding surfaces can be readily accessed simultaneously. Importantly, this multivalent mode of interaction across two linked nucleosomes allows Rpd3S to tolerate a two-fold intramolecular reduction of H3K36me. Our data suggest that chromatin regulators utilize an intrinsic di-nucleosome-recognition mechanism to prevent compromised function when their primary recognition modifications are diluted.

Objective: Malignant middle cerebral artery (MCA) infarction occurs in 10% of all ischemic strokes and these severe strokes are associated with high mortality rates. Recent clinical trials demonstrated that early decompressive craniectomy reduce mortality rates and improves functional outcomes in healthy young patients (less than 61 years of age) with a malignant infarction. The purpose of this study was to assess the efficacy of decompressive craniectomy in elderly patients (older than 70 years of age) with a malignant MCA infarction., Methods: Between February 2008 and October 2011, 131 patients were diagnosed with malignant MCA infarctions. We divided these patients into two groups: patients who underwent decompressive craniectomy (n = 58) and those who underwent conservative care (n = 73). A cut-off point of 70 years of age was set, and the study population was segregated into those who fell above or below this point. Mortality rates and functional outcome scores were assessed, and a modified Rankin Scale (mRS) score of > 3 was considered to represent a poor outcome., Results: Mortality rates were significantly lower at 29.3% (one-month mortality rate) and 48.3% (six-month mortality rate) in the craniectomy group as compared to 58.9% and 71.2%, respectively, in the conservative care group (p < 0.001, p = 0.007). Age (≥70 years vs. < 70 years) did not statistically differ between groups for the six-month mortality rate (p = 0.137). However, the pre-operative National Institutes of Health Stroke Scale (NIHSS) score did contribute to the six-month mortality rate (p = 0.047)., Conclusion: Decompressive craniectomy is effective for patients with a malignant MCA infarction regardless of their age. Therefore, factors other than age should be considered and the treatment should be individualized in elderly patients with malignant infarctions.

Background: As a human replacement, the crab-eating macaque (Macaca fascicularis) is an invaluable non-human primate model for biomedical research, but the lack of genetic information on this primate has represented a significant obstacle for its broader use., Results: Here, we sequenced the transcriptome of 16 tissues originated from two individuals of crab-eating macaque (male and female), and identified genes to resolve the main obstacles for understanding the biological response of the crab-eating macaque. From 4 million reads with 1.4 billion base sequences, 31,786 isotigs containing genes similar to those of humans, 12,672 novel isotigs, and 348,160 singletons were identified using the GS FLX sequencing method. Approximately 86% of human genes were represented among the genes sequenced in this study. Additionally, 175 tissue-specific transcripts were identified, 81 of which were experimentally validated. In total, 4,314 alternative splicing (AS) events were identified and analyzed. Intriguingly, 10.4% of AS events were associated with transposable element (TE) insertions. Finally, investigation of TE exonization events and evolutionary analysis were conducted, revealing interesting phenomena of human-specific amplified trends in TE exonization events., Conclusions: This report represents the first large-scale transcriptome sequencing and genetic analyses of M. fascicularis and could contribute to its utility for biomedical research and basic biology.

Increasing evidence supports an association between Alzheimer's disease (AD) and diabetes. In this context, anti-diabetic agents such as rosiglitazone and glucagon-like peptide (GLP)-1 have been reported to reduce pathologies associated with AD, including tau hyperphosphorylation, suggesting that such agents might be used to treat AD. One such anti-diabetic agent is sitagliptin, which acts through inhibition of dipeptidyl peptidase (DPP)-IV to increase GLP-1 levels. Given this action, sitagliptin would be predicted to reduce AD pathology. Accordingly, we investigated whether sitagliptin is effective in attenuating AD pathologies, focusing on tau phosphorylation in the OLETF type 2 diabetic rat model. Unexpectedly, we found that sitagliptin was not effective against pathological tau phosphorylation in the hippocampus of OLETF type 2 diabetes rats, and instead aggravated it. This paradoxically increased tau phosphorylation was attributed to activation of the tau kinase, GSK3β (glycogen synthase kinase 3β). Sitagliptin also increased ser-616 phosphorylation of the insulin receptor substrate (IRS)-1, suggesting increased insulin resistance in the brain. These phenomena were recapitulated in primary rat cortical neurons treated with sitagliptin, further confirming sitagliptin's effects on AD-related pathologies in neurons. These results highlight the need for caution in considering the use of sitagliptin in AD therapy., (Copyright © 2012 Elsevier Inc. All rights reserved.)

To study the functions of histone modifications in the context of chromatin, it is necessary to be able to prepare nucleosomal templates that carry specific posttranslational modifications in a defined biochemical system. Here, we describe two sets of protocols for reconstituting designer nucleosomes that contain specifically modified histones. The resulting nucleosomes are suitable for electromobility shift assays, chromatin remodeling assays, and other functional and structural studies.

The Zinc finger, RAN-binding domain-containing protein 2 (ZRANB2), contains arginine/serine-rich (RS) domains that mediate its function in the regulation of alternative splicing. The ZRANB2 gene contains 2 LINE elements (L3b, Plat&#95;L3) between the 9th and 10th exons. We identified the exonization event of a LINE element (Plat&#95;L3). Using genomic PCR, RT-PCR amplification, and sequencing of primate DNA and RNA samples, we analyzed the evolutionary features of ZRANB2 transcripts. The results indicated that 2 of the LINE elements were integrated in human and all of the tested primate samples (hominoids: 3 species; Old World monkey: 8 species; New World monkey: 6 species; prosimian: 1 species). Human, rhesus monkey, crab-eating monkey, African-green monkey, and marmoset harbor the exon derived from LINE element (Plat&#95;L3). RT-PCR amplification revealed the long transcripts and their differential expression patterns. Intriguingly, these long transcripts were abundantly expressed in Old World monkey lineages (rhesus, crab-eating, and African-green monkeys) and were expressed via intron retention (IR). Thus, the ZRANB2 gene produces 3 transcript variants in which the Cterminus varies by transposable elements (TEs) exonization and IR mechanisms. Therefore, ZRANB2 is valuable for investigating the evolutionary mechanisms of TE exonization and IR during primate evolution.

Choroideremia (CHM) is essential for the posttranslational activation of retina-specific Rab protein. Transcript variants (a and b) of the CHM gene were detected in human cancer cells and tissues. Sequence analysis of the both variants found that isoform b is caused by an LTR12C element offering an alternative splicing site within the CHM gene. We performed real-time RT-PCR analysis to study expression levels of the CHM transcript variants in tumor and adjacent normal tissue samples. Our results showed that CHM isoform b was highly expressed in tumor tissues but its expression levels were relatively low in those of adjacent normal tissues including colon, testis, and lung. In addition, high expression levels of CHM isoform b were detected in the cancer cell lines of colon and lung, and colon cancer patient tissues. Thus, we suggest that expression levels of alternative transcripts of the CHM gene could be used as a molecular marker system to identify human cancers., (2011. Published by Elsevier B.V. All rights reserved.)

Shiga toxin (STx) belongs to the AB(5) toxin family and is transiently localized in the periplasm before secretion into the extracellular milieu. While producing outer membrane vesicles (OMVs) containing only A subunit of the toxin (STxA), we created specific STx1B- and STx2B-deficient mutants of E. coli O157:H7. Surprisingly, STxA subunit was absent in the OMVs and periplasm of the STxB-deficient mutants. In parallel, the A subunit of heat-labile toxin (LT) of enterotoxigenic E. coli (ETEC) was absent in the periplasm of the LT-B-deficient mutant, suggesting that instability of toxin A subunit in the absence of the B subunit is a common phenomenon in the AB(5) bacterial toxins. Moreover, STx2A was barely detectable in the periplasm of E. coli JM109 when stx2A was overexpressed alone, while it was stably present when stxB was co-expressed. Compared with STx2 holotoxin, purified STx2A was degraded rapidly by periplasmic proteases when assessed for in vitro proteolytic susceptibility, suggesting that the B subunit contributes to stability of the toxin A subunit in the periplasm. We propose a novel role for toxin B subunits of AB(5) toxins in protection of the A subunit from proteolysis during holotoxin assembly in the periplasm., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Formyl peptide receptor 3 (FPR3) is a potential player in innate immunity and appears with FPR2 as a FPR cluster during primate evolution. Comparative genome analyses indicate that a segmental duplication (SD) event upstream of the FPR3 gene after the divergence of New and Old World monkeys led to the emergence of an alternative promoter. In this study we combined computational and experimental approaches to identify a FPR3 gene that is controlled by an alternative promoter derived during a SD event. Its transcriptional activity was detected by quantitative reverse transcription polymerase chain reaction. Human alternative transcripts (FPR3-1 and FPR3-2) showed tissue-specific patterns with strong expressions in lung or uterus, while the FPR3-1 transcript of rhesus macaque is broadly expressed in various tissues. Overall, transcriptional variations of FPR3 occur by an alternative promoter during primate evolution.

Objective: The purpose of this study was to analyze the clinical and anatomic features involved in determining treatment modalities for anterior communicating artery (AcoA) aneurysms., Methods: The authors retrospectively evaluated 112 AcoA aneurysms with pretreatment clinical features including age, Hunt and Hess grade, medical or neurological comorbidity, and anatomical features including aneurysm size, neck size, dome-to-neck ratio, vessel incorporation, multiple lobulation, and morphologic scoring system. Post-treatment clinical results were classified according to the Glasgow Outcome Scale, and anatomic results in coiled patients were classified according to the modified Raymond scale. Using multivariate logistic regression, the probabilities for decision making between surgical clipping and coil embolization were calculated., Results: Sixty-seven patients (60%) were treated with surgical clipping and 45 patients (40%) with endovascular coil embolization. The clinical factor significantly associated with treatment decision was age (≥65 vs. <65 years) and anatomical factors including aneurysm size (small or large vs. medium), dome-to-neck ratio (<2 vs. ≥2), presence of vessel incorporation, multiple lobulation, and morphologic score (≥2 vs. <2). In multivariate analysis, older patients (age, >65 years) had significantly higher odds of being treated with coil embolization relative to clipping (adjusted OR=3.78; 95% CI, 1.39-10.3; p=0.0093) and higher morphological score patients (≥2) had a higher tendency toward surgical clipping than endovascular coil embolization (OR=0.23; 95% CI, 0.16-0.93; p=0.0039)., Conclusion: The optimal decision for treating AcoA aneurysms cannot be determined by any single clinical or anatomic characteristics. All clinical and morphological features need to be considered, and a collaborative neurovascular team approach to AcoA aneurysms is essential.

Centromeres nucleate the formation of kinetochores and are vital for chromosome segregation during mitosis. The SNF2 family helicase PICH (Plk1-interacting checkpoint helicase) and the BLM (the Bloom's syndrome protein) helicase decorate ultrafine histone-negative DNA threads that link the segregating sister centromeres during anaphase. The functions of PICH and BLM at these threads are not understood, however. Here, we show that PICH binds to BLM and enables BLM localization to anaphase centromeric threads. PICH- or BLM-RNAi cells fail to resolve these threads in anaphase. The fragmented threads form centromeric-chromatin-containing micronuclei in daughter cells. Anaphase threads in PICH- and BLM-RNAi cells contain histones and centromere markers. Recombinant purified PICH has nucleosome remodelling activities in vitro. We propose that PICH and BLM unravel centromeric chromatin and keep anaphase DNA threads mostly free of nucleosomes, thus allowing these threads to span long distances between rapidly segregating centromeres without breakage and providing a spatiotemporal window for their resolution.

The retromer complex is a heteropentameric protein unit associated with retrograde transport of cargo proteins from endosomes to the trans-Golgi network. Functional silencing study of the Vps26a gene indicated the important role of the retromer complex during early developmental stages in the mouse. However, individual expression patterns and quantitative analysis of individual members of the retromer complex during the early developmental stages has not been investigated. In this study, we conducted quantitative expression analysis of six retromer complex genes (Vps26a, Vps26b, Vps29, Vps35, Snx1, and Snx2) and one related receptor gene (Ci-mpr) during the eleven embryonic stages with normal MEF (mouse embryonic fibroblast) and Vps26a(-/-) MEF cells. Remarkably, except for Vps26a (maternal expression pattern), all tested genes showed maternal-zygotic expression patterns. And five genes (Vps26b, Vps29, Vps35, Snx2, and Ci-mpr) showed a pattern of decreased expression in Vps26a(-/-) MEF cells by comparative analysis between normal MEF and Vps26a(-/-) MEF cells. However, the Snx1 gene showed a pattern of increased expression in Vps26a(-/-) MEF cells. From our results, we could assume that retromer complex-related genes have important roles during oocyte development. However, in the preimplantation stage, they did not have significant roles.

IGFBP-5 is known to be involved in various cell phenomena such as proliferation, differentiation, and apoptosis. However, the exact mechanisms by which IGFBP-5 exerts its functions are unclear. In this study, we demonstrate for the first time that IGFBP-5 is a TNFR1-interacting protein. We found that ectopic expression of IGFBP-5 induced TNFR1 gene expression, and that IGFBP-5 interacted with TNFR1 in both an in vivo and an in vitro system. Secreted IGFBP-5 interacted with GST-TNFR1 and this interaction was blocked by TNF-α, demonstrating that IGFBP-5 might be a TNFR1 ligand. Furthermore, conditioned media containing secreted IGFBP-5 inhibited PMA-induced NF-κB activity and IL-6 expression in U-937 cells. Coimmunoprecipitation assays of TNFR1 and IGFBP-5 wild-type and truncation mutants revealed that IGFBP-5 interacts with TNFR1 through its N- and L-domains. However, only the interaction between the L-domain of IGFBP-5 and TNFR1 was blocked by TNF-α in a dose-dependent manner, suggesting that the L-domain of IGFBP-5 can function as a TNFR1 ligand. Competition between the L-domain of IGFBP-5 and TNF-α resulted in inhibition of TNF-α-induced NF-κΒ activity. Taken together, our results suggest that the L-domain of IGFBP-5 is a novel TNFR1 ligand that functions as a competitive TNF-α inhibitor., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We introduce a novel three-step procedure for precise niobium (Nb)-etching on the nanometer-scale, including the design of high contrast resist patterning and sacrifice layer formation under high radio frequency (RF) power. We present the results of precise slit fabrication using this technique and discuss its application for the production of superconducting devices, such as superconductor-semiconductor-superconductor (S-Sm-S) Josephson junctions. For the reactive ion etching (RIE) of Nb, we selected CF(4) as etchant gas and a positive tone resist to form the etching mask. We found that the combination of resist usage and RIE process allows for etching of thicker Nb layers when utilizing the opposite dependence of the etching rate (ER) on the CF(4) pressure in the case of Nb as compared to the resist. Precise slit-width control of 80 and 200 nm thick Nb apertures was performed with three kinds of ER control, for the resist, the Nb, and the underlying layer. S-Sm-S Josephson junctions were fabricated with lengths as small as 80 nm, which can be considered clean and short and thus exhibit critical currents as high as 50 µA. Moreover, possible further applications, such as for apertures of superconducting light emitting diodes (SC LEDs), are addressed.

Somatic cell nuclear transfer (SCNT) is a powerful tool, not only for producing cloned animals, but also in revealing various early developmental events. However, relatively little is known regarding the biological events and underlying mechanism(s) directly associated with early development of SCNT embryos. Here, we show that production of high-quality bovine SCNT blastocysts is dependent on the method used for fusion and the associated reduction in endoplasmic reticulum (ER) stress. During fusion between the donor cell and the enucleated oocyte, electrofusion triggers spontaneous oocyte activation, accompanied by an increase in intracellular Ca(2+) and improper nuclear remodelling. These events can be greatly reduced by the use of Sendai virus (SV)-mediated fusion. Moreover, SV-SCNT improves the blastulation rate and blastocyst quality, defined by the number and ratio of inner cell mass and trophectoderm cells in each blastocyst, in comparison with electrofusion-mediated SCNT (E-SCNT). Interestingly, expression of ER-stress-associated genes and blastomere apoptosis were significantly increased in E-SCNT embryos. These increases could be reversed by inhibition of ER stress or by using the SV-mediated fusion method. Collectively, these results indicate that SV-mediated fusion improves the developmental competence and quality of SCNT blastocysts, by reducing ER-stress-associated apoptosis., (© CSIRO 2011 Open Access)

Recent research has suggested that a perfusion-weighted image (PWI) relative cerebral blood volume (rCBV) map after acute ischemic stroke (AIS) provides information about the collateral circulation in the ischemic region. In this study, we demonstrate the usefulness of the rCBV ratio in PWI in predicting poor outcome after using IV t-PA in AIS. We recruited 58 stroke patients who were treated with IV t-PA after diagnostic magnetic resonance imaging (MRI). Poor outcome was defined as a Modified Rankin Scale (mRS) score >2 measured 90 days after ischemic insult. In total, 21 patients (36.2%) demonstrated poor outcome (i.e. mRS score 3-6). Poor outcome after t-PA correlated with age (p = 0.03), serum glucose level (p = 0.01), NIHSS (p = 0.05), and the presence of T-occlusion (p = 0.05). Poor outcome also correlated with diffusion-weighted MR images of the lesion volume (p < 0.01), lower rCBV ratio on PWI (p < 0.01), and non-recanalization (p < 0.01). Among these, non-recanalization (p < 0.01), reduced rCBV ratio on PWI (p < 0.01), age (p = 0.04), and serum glucose level (p = 0.01) had an independent significance for predicting it. This suggests that the rCBV ratio on PWI may be used to determine prognosis after thrombolysis in AIS., (Copyright © 2011 S. Karger AG, Basel.)

DYX1C1 is a candidate gene for developmental dyslexia and has three alternative pre-mRNA spliced forms in the human genome. One of the transcripts contains an HERV-H LTR that could affect the expression level of DYX1C1. We speculate that the HERV-H LTR integrated into the DYX1C1 locus in the catarrhine lineage after its divergence from the platyrrhine lineage. Reverse transcription-PCR of the HERV-H LTR-related transcript produced four alternative forms from several human tissues. All of alternative forms were also identified in various rhesus macaque tissues. Through sequencing analysis of various primate DNA samples, we found that a part of the HERV-H LTR sequence was duplicated within the DYX1C1 exon 9 only in catarrhines. However, the duplication event did not cause frameshift mutation of the DYX1C1 transcript. Taken together, this HERV-H LTR insertion into DYX1C1 has contributed to transcript diversification of DYX1C1 during primate evolution.

Recent studies indicate major roles for transposable elements (TEs) in alternative splicing. In this study, we conducted genome-wide alternative splicing analyses focusing on new internal exon birth derived from TEs in human, mouse, and zebrafish genomes. We identified two different exon sets, TE-spliced exons and non-TE-spliced exons. The proportion of TE-spliced exons was nearly twice as high as the proportion of non-TE-spliced exons in the coding sequence (CDS) region. Detailed analysis of various families of TEs in three different species of TE-spliced exons revealed a different pattern in zebrafish. In our analysis, we could identify the functional role of TE insertions in the vertebrate genome affecting mRNA splicing machinery. Their effects can be directly linked to the shift from constitutive to alternative splicing during primate evolution. Our results indicate that TEs have a significant effect on shaping new internal exons in human, mouse, and zebrafish transcriptomes., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The pig genome contains the gamma 1 family of porcine endogenous retroviruses (PERVs), which are a major obstacle to the development of successful xenotransplantation from pig to human. Long terminal repeats (LTRs) found in PERVs are known to be essential elements for the control of the transcriptional activity of single virus by different transcription factors (TFs). To identify transcribed PERV LTR elements, RT-PCR and DNA sequencing analyses were performed. Twenty-nine actively transcribed LTR elements were identified in the kidney tissues of the NIH-Miniature pig. These elements were divided into two major groups (I and II), and four minor groups (I-1, I-2, I-3, and II-1), by the presence of insertion and deletion (INDEL) sequences. Group I elements showed strong transcriptional activity compared to group II elements. Four different LTR elements (PL1, PL2, PL3, and PL4) as representative of the groups were analyzed by using a transient transfection assay. The regulation of their promoter activity was investigated by treatment with M.SssI (CpG DNA methyltransferase) and garcinol (histone acetyltransferase inhibitor). The transcriptional activity of PERV LTR elements was significantly reduced by treatment with M.SssI. These data indicate that transcribed PERV LTR elements harbor sufficient promoter activity to regulate the transcription of a single virus, and the transcriptional activity of PERV LTRs may be controlled by DNA methylation events.

Background and Purpose: There is little information available about the effects of Emergency Medical Service (EMS) hospital notification on transfer and intrahospital processing times in cases of acute ischemic stroke., Methods: This study retrospectively investigated the real transfer and imaging processing times for cases of suspected acute stroke (AS) with EMS notification of a requirement for intravenous (IV) tissue-type plasminogen activator (t-PA) and for cases without notification. Also we compared the intra-hospital processing times for receiving t-PA between patients with and without EMS prehospital notification., Results: Between December 2008 and August 2009, the EMS transported 102 patients with suspected AS to our stroke center. During the same period, 33 patients received IV t-PA without prehospital notification from the EMS. The mean real transfer time after the EMS call was 56.0±32.0 min. Patients with a transfer distance of more than 40 km could not be transported to our center within 60 min. Among the 102 patients, 55 were transferred via the EMS to our emergency room for IV t-PA. The positive predictive value for stroke (90.9% vs. 68.1%, p=0.005) was much higher and the real transfer time was much faster in patients with an EMS t-PA call (47.7±23.1 min, p=0.004) than in those without one (56.3±32.4 min). The door-to-imaging time (17.8±11.0 min vs. 26.9±11.5 min, p=0.01) and door-to-needle time (29.7±9.6 min vs. 42.1±18.1 min, p=0.01) were significantly shorter in the 18 patients for whom there was prehospital notification and who ultimately received t-PA than in those for whom there was no prehospital notification., Conclusions: Our results indicate that prehospital notification could enable the rapid dispatch of AS patients needing IV t-PA to a stroke centre. In addition, it could reduce intrahospital delays, particularly, imaging processing times.

The leptin receptor (LEPR) is a crucial regulatory protein that interacts with Leptin. In our analysis of LEPR, novel AluJb-derived alternative transcripts were identified in the genome of the rhesus monkey. In order to investigate the occurrence of AluJb-derived alternative transcripts and the mechanism underlying exonization events, we conducted analyses using a number of primate genomic DNAs and adipose RNAs of tissue and primary cells derived from the crab-eating monkey. Our results demonstrate that the AluJb element has been integrated into our common ancestor genome prior to the divergence of simians and prosimians. The lineage-specific exonization event of the LEPR gene in chimpanzees, orangutans, and Old World monkeys appear to have been accomplished via transition mutations of the 5' splicing site (second position of C to T). However, in New World monkeys and prosimians, the AluJb-related LEPR transcript should be silenced by the additional transversion mutation (fourth position of T to G). The AluJb-related transcript of human LEPR should also be silenced by a mutation of the 5' splicing site (first position of G to A) and the insertion of one nucleotide sequence (minus fourth position of A). Our data suggests that lineage-specific exonization events should be determined by the combination event of the formation of splicing sites and protection against site-specific mutation pressures. These evolutionary mechanisms could be major sources for primate diversification.

Background: Rhesus monkeys (Macaca mulatta) are widely-used as experimental animals in biomedical research and are closely related to other laboratory macaques, such as cynomolgus monkeys (Macaca fascicularis), and to humans, sharing a last common ancestor from about 25 million years ago. Although rhesus monkeys have been studied extensively under field and laboratory conditions, research has been limited by the lack of genetic resources. The present study generated placenta full-length cDNA libraries, characterized the resulting expressed sequence tags, and described their utility for comparative mapping with human RefSeq mRNA transcripts., Results: From rhesus monkey placenta full-length cDNA libraries, 2000 full-length cDNA sequences were determined and 1835 rhesus placenta cDNA sequences longer than 100 bp were collected. These sequences were annotated based on homology to human genes. Homology search against human RefSeq mRNAs revealed that our collection included the sequences of 1462 putative rhesus monkey genes. Moreover, we identified 207 genes containing exon alterations in the coding region and the untranslated region of rhesus monkey transcripts, despite the highly conserved structure of the coding regions. Approximately 10% (187) of all full-length cDNA sequences did not represent any public human RefSeq mRNAs. Intriguingly, two rhesus monkey specific exons derived from the transposable elements of AluYRa2 (SINE family) and MER11B (LTR family) were also identified., Conclusion: The 1835 rhesus monkey placenta full-length cDNA sequences described here could expand genomic resources and information of rhesus monkeys. This increased genomic information will greatly contribute to the development of evolutionary biology and biomedical research.

A dual coding event, which is the translation of different isoforms from a single gene, is one of the special patterns among the alternative splicing events. This is an important mechanism for the regulation of protein diversity in human and mouse genomes. Although the regulation for dual coding events has been characterized in a few genes, the individual mechanism remains unclear. Numerous studies have described the exonization of transposable elements, which is the splicing mediated insertion of transposable element sequence fragments into mature mRNAs. Therefore, in this study, we investigated the number of transposable element (TE)-derived dual coding genes in human, chimpanzee and mouse genomes. TE fusion exons appeared in the dual coding regions of 309 human genes. Functional protein domain alterations by TE-derived dual coding events were observed in 129 human genes. Comparative TE-derived dual coding events were also analyzed in chimpanzee and mouse orthologs. Seventy chimpanzee orthologs had TE-derived dual coding events, but mouse orthologs did not have any TE-derived dual coding events. Taken together, our analyses listed the number of TE-derived dual coding genes which could be investigated by experimental analysis and suggested that TE-derived dual coding events were major sources for the functional diversity of human genes, but not mouse genes.

The Protocadherin 11X/Y (PCDH11X/Y) gene pair has been proposed as a carrier of the variation relating to cerebral asymmetry and psychosis on the ground that the Y gene was generated by duplication at 6 million years (close to the chimpanzee-human separation) and there is a case for an X/Y determinant of cerebral asymmetry. The present article investigated the patterns of alternative splicing and expression of the PCDH11X/Y genes. Twelve alternative transcripts of PCDH11X/Y genes were presently identified by in silico analysis. To investigate the biological roles of alternative transcripts of PCDH11X/Y genes, the transcripts were analyzed by real-time reverse transcription-polymerase chain reaction amplification. A total of 31 normal tissues including 11 different regions of human brain were used to investigate a wide spectrum of expression profiles. Dominant expression patterns were identified in several tissues (Tx1-fetal liver; Tx3-adult brain; Tx4-adult brain and kidney; Tx5-bone marrow; Ty1-fetal brain; Ty2-adrenal gland). Tx4 transcripts showed specific expression patterns in olfactory tissues. The findings can guide functional investigation of neuropsychiatric disorders.

The ADRA1A (Alpha-1-adrenergic receptor) gene is one of the members of G protein-coupled receptor superfamily. Alternative splicing of this gene was known to generate four transcript variants which code four isoforms with various C-terminal regions. In this study, we conducted expression analysis and evolutionary characterization of alternative transcripts of the ADRA1A gene. In total, 10 alternative transcripts were identified using experimental approaches and in silico analysis. Among them, 6 alternative transcripts (T1, T2, T3, T4, T4-1, and T8) were validated by RT-PCR approaches and sequencing procedures. From the alternative splicing analysis, it could be assumed that there were three different alternative transcripts generation mechanisms and unknown mechanism. First one is the integration event of three different TEs (AluSc, L1MC5, and MIR3) as seen on the last exons of T3, T4, T4-1, T5, T6, and T7 transcripts. The second mechanism is a differential promoter usage on T8. The third one is a substitution event of the 3' splicing site during the primate evolution on T3. The last one is an unknown mechanism which was identified in the T4-1 transcript. Therefore, alternative transcripts of human ADRA1A gene occurred by four different ways, such as integration of TEs, differential promoter usage, substitution of splicing sites, and unknown mechanism.

Primary cardiac angiosarcoma is a very rare disease with a poor prognosis. We report a case of a patient with a primary cardiac angiosarcoma who presented with cardiac tamponade; the angiosarcoma was successfully resected surgically.

The CHRM3 gene is a member of the muscarinic acetylcholine receptor family that plays important roles in the regulation of fundamental physiological functions. The evolutionary mechanism of exon-acquisition and alternative splicing of the CHRM3 gene in relation to transposable elements (TEs) were analyzed using experimental approaches and in silico analysis. Five different transcript variants (T1, T2, T3, T3-1, and T4) derived from three distinct promoter regions (T1: L1HS, T2, T4: original, T3, T3-1: THE1C) were identified. A placenta (T1) and testis (T3 and T3-1)-dominated expression pattern appeared to be controlled by different TEs (L1HS and THE1C) that were integrated into the common ancestor genome during primate evolution. Remarkably, the T1 transcript was formed by the integration event of the human specific L1HS element. Among the 12 different brain regions, the brain stem, olfactory region, and cerebellum showed decreased expression patterns. Evolutionary analysis of splicing sites and alternative splicing suggested that the exon-acquisition event was determined by a selection and conservation mechanism. Furthermore, continuous integration events of transposable elements could produce lineage specific alternative transcripts by providing novel promoters and splicing sites. Taken together, exon-acquisition and alternative splicing events of CHRM3 genes were shown to have occurred through the continuous integration of transposable elements following conservation.

Objective: To obtain prospective outcomes data on patients (pts) undergoing palliative operative or endoscopic procedures for malignant bowel obstruction due to recurrent ovarian cancer., Methods: An institutional study was conducted from July 2002 to July 2003 to prospectively identify pts who underwent an operative or endoscopic procedure to palliate the symptoms of advanced cancer. This report focuses on pts with malignant bowel obstruction due to recurrent ovarian cancer. Procedures performed with an upper or lower gastrointestinal (GI) endoscope were considered "endoscopic." All other cases were classified as "operative." Following the procedure, the presence or absence of symptoms was determined and followed over time. All pts were followed until death., Results: Palliative interventions were performed on 74 gynecologic oncology pts during the study period, of which 26 (35%) were for malignant GI obstruction due to recurrent ovarian cancer. The site of obstruction was small bowel in 14 (54%) cases and large bowel in 12 (46%) cases. Palliative procedures were operative in 14 (54%) pts and endoscopic in the other 12 (46%). Overall, symptomatic improvement or resolution within 30 days was achieved in 23 (88%) of 26 patients, with 1 (4%) postprocedure mortality. At 60 days, 10 (71%) of 14 pts who underwent operative procedures and 6 (50%) of 12 pts who had endoscopic procedures had symptom control. Median survival from the time of the palliative procedure was 191 days (range, 33-902) for those undergoing an operative procedure and 78 days (range, 18-284) for those undergoing an endoscopic procedure., Conclusion: Patients with malignant bowel obstructions due to recurrent ovarian cancer have a high likelihood of experiencing relief of symptoms with palliative procedures. Although recurrence of symptoms is common, durable palliation and extended survival are possible, especially in those patients selected for operative intervention.

SFTPB gene encoding pulmonary surfactant protein has been investigated to have transcript variants related with transposable elements (TEs). To investigate the alternative splicing event on SFTPB gene, various primate samples (tissue RNA and genomic DNA) were used. Two different transcript variants (T1-2 and T2-2) were newly identified by RT-PCR analysis. T1-1,-2 and T2-1,-2 are TE-related and original transcripts, respectively. T1-1 transcript was investigated to be a lung and human specific alternative transcript. T2-1 transcript shows dominant expression pattern in lung tissues. T1-related TEs (LTR7B and AluSx) were investigated by genomic PCR and sequencing procedure of different primate genomic DNAs. Sequencing analysis indicated that they had been integrated into our common ancestor genome before the divergence of simian and prosimian and Alu recombination-mediated deletion (ARMD) event had been occurred on our common ancestor genome after the divergence of New World monkeys and Old World monkeys in SFTPB gene 3' UTR region. Taken together, integration event of LTR7B and AluSx on SFTPB gene seem to cause lineage specific events via general exonization event (New World monkeys and prosimian), ARMD event (Old World monkeys and hominoids), and alternative splicing (human) during the primate evolution.

Purpose: DYX1C1 has three alternatively spliced transcripts. Therefore, we expect that alternative transcripts of DYX1C1 are used as a biomarker to detect specific cancer., Methods: RT-PCR analysis is conducted in order to detect expression of the DYX1C1 gene and the PCR products were analyzed using the Image J program to compare the expression levels of each transcript., Results: We found one of the transcripts was directly associated with an HERV-H LTR element that could be translated into protein sequence. Four new alternative transcripts were identified by RT-PCR analysis with various human tissue samples including 10 normal and adjacent tumor tissue sets. Semi-quantitative RT-PCR analysis showed the transcriptional activity of V3 and V2 was higher in tumor than in normal tissue samples, especially in the colorectal tissue samples., Conclusion: Our results indicated that alternatively spliced transcript variants of the DYX1C1 gene could be used as cancer biomarkers to detect colorectal cancer.

Porcine endogenous retroviruses (PERVs) gamma1 in the pig genome have the potential to act as harmful factors in xenotransplantation (pig-to-human). Long terminal repeats (LTRs) are known to be strong promoter elements that could control the transcription activity of PERV elements and the adjacent functional genes. To investigate the transcribed PERV gamma1 LTR elements in pig tissues, bioin-formatic and experimental approaches were conducted. Using RT-PCR amplification and sequencing approaches, 69 different transcribed LTR elements were identified. And 69 LTR elements could be divided into six groups (15 subgroups) by internal variation including tandem repeated sequences, insertion and deletion (INDEL). Remarkably, all internal variations were indentified in U3 region of LTR elements. Taken together, the identification and characterization of various PERV LTR transcripts allow us to extend our knowledge of PERV and its transcriptional study.

The RNF19 protein, which contains RING-finger and IBR motifs, acts as an E3 ubiquitin ligase localized to Lewy bodies. RNF19 is located on human chromosome 8q22.2, has a 4.4-kb transcript, and is ubiquitously expressed in various tissues. Here, we identified an alternative RNF19 promoter region and alternative RNF19 transcripts derived from MaLR (mammalian apparent LTR-retrotransposon) and AluJo elements. Comparative analyses indicated human-specific expression of the MaLR- and AluJo-related transcripts. From the expression analysis of 72 tissue samples including human normal, tumor, and primate tissues, three different isoforms (V1, V2, and V3) of MaLR-derived transcripts were identified. Quantitative RT-PCR analysis showed a dominant expression pattern of the V2 MaLR-derived transcript. A reporter gene assay for MaLR element promoter activity indicated that pGL2-RNF19/MaLR in the forward orientation is capable of driving luciferase gene expression in Cos7 and HCT116 cells. These findings suggest that RNF19 has acquired a new promoter and alternative exons via continuous retrotransposition events of MaLR and AluJo elements during mammalian and primate evolution, respectively.

We have examined polyphenols as potential inhibitors of UDP-glucose dehydrogenase (UGDH) activity. Gallic acid and quercetin decreased specific activities of UGDH and inhibited the proliferation of MCF-7 human breast cancer cells. Western blot analysis showed that gallic acid and quercetin did not affect UGDH protein expression, suggesting that UGDH activity is inhibited by polyphenols at the post-translational level. Kinetics studies using human UGDH revealed that gallic acid was a non-competitive inhibitor with respect to UDP-glucose and NAD+. In contrast, quercetin showed a competitive inhibition and a mixed-type inhibition with respect to UDP-glucose and NAD+, respectively. These results indicate that gallic acid and quercetin are effective inhibitors of UGDH that exert strong antiproliferative activity in breast cancer cells.

Background: The rhesus monkey (Macaca mulatta) is a valuable and widely used model animal for biomedical research. However, quantitative analyses of rhesus gene expression profiles under diverse experimental conditions are limited by a shortage of suitable internal controls for the normalization of mRNA levels. In this study, we used a systematic approach for the selection of potential reference genes in the rhesus monkey and compared their suitability to that of the corresponding genes in humans., Results: Eight housekeeping genes (HKGs) (GAPDH, SDHA, ACTB, RPL13A, RPL32, UBA52, PGK1Y, and YWHAZ) from rhesus monkeys and humans were selected to test for normalization of expression levels in six different tissue types (brain, colon, kidney, liver, lung, and stomach). Their stability and suitability as reference genes were validated by geNorm, NormFinder and BestKeeper programs. Intriguingly, RPL13A and RPL32 were selected as ideal reference genes only in rhesus monkeys., Conclusion: The results clearly indicated the necessity of using different reference genes for normalization of expression levels between rhesus monkeys and humans in various tissues.

We characterized the human endogenous retrovirus (HERV-W) family in humans and primates. In silico expression data indicated that 22 complete HERV-W families from human chromosomes 1-3, 5-8, 10-12, 15, 19, and X are randomly expressed in various tissues. Quantitative real-time RT-PCR analysis of the HERV-W env gene derived from human chromosome 7q21.2 indicated predominant expression in the human placenta. Several copies of repeat sequences (SINE, LINE, LTR, simple repeat) were detected within the complete or processed pseudo HERV-W of the human, chimpanzee, and rhesus monkey. Compared to other regions (5'LTR, Gag, Gag-Pol, Env, 3'LTR), the repeat family has been mainly integrated into the region spanning the 5'LTRs of Gag (1398 bp) and Pol (3242 bp). FISH detected the HERV-W probe (fosWE1) derived from a gorilla fosmid library in the metaphase chromosomes of all primates (five hominoids, three Old World monkeys, two New World monkeys, and one prosimian), but not in Tupaia. This finding was supported by molecular clock and phylogeny data using the divergence values of the complete HERV-W LTR elements. The data suggested that the HERV-W family was integrated into the primate genome approximately 63 million years (Myr) ago, and evolved independently during the course of primate radiation.

We introduce a nonconforming finite-element method for second order elliptic interface problems. Our approach applies to problems in which discontinuous coefficients and singular sources on the interface may give rise to jump discontinuities in either the solution or its normal derivative. Given a standard background mesh and an interface that passes between elements, the key idea is to construct a singular correction function that satisfies the prescribed jump conditions, providing accurate subgrid resolution of the discontinuities. Utilizing the closest point extension and an implicit interface representation by the signed distance function, an algorithm is established to construct the correction function. The result is a function that is supported only on the interface elements, represented by the regular basis functions, and bounded independently of the interface location with respect to the background mesh. In the particular case of a constant second-order coefficient, our regularization by a singular function is straightforward, and the resulting left-hand side is identical to that of a regular problem without introducing any instability. The influence of the regularization appears solely on the right-hand side, which simplifies the implementation. In the more general case of discontinuous second-order coefficients, a normalization is invoked which introduces a constraint equation on the interface. This results in a problem statement similar to that of a saddle-point problem. We employ two-level iteration as the solution strategy, which exhibits aspects similar to those of iterative preconditioning strategies.

A 345-bp gene that encodes human bone morphogenetic protein-2 (hBMP-2) has been synthesized. The codon usage of the resulting gene was modified to include those triplets that are utilized in highly expressed Escherichia coli genes. The hBMP-2 gene was efficiently expressed in E. coli as a soluble and active protein. Since the recombinant hBMP-2 was readily solublized, no further solublization steps were required throughout purification. No additional tagging residues were introduced into the synthetic hBMP-2 gene product. The developed synthetic gene is a promising approach for scaling-up the soluble expression of hBMP-2.

Objective: Hydrocephalus is a common sequelae of aneurysmal subarachnoid hemorrhage (SAH) and patients who develop hydrocephalus after SAH typically have a worse prognosis than those who do not. This study was designed to identify factors predictive of shunt-dependent chronic hydrocephalus among patients with aneurysmal SAH, and patients who require permanent cerebrospinal fluid diversion., Methods: Seven-hundred-and-thirty-four patients with aneurysmal SAH who were treated surgically between 1990 and 2006 were retrospectively studied. Three stages of hydrocephalus have been categorized in this paper, i.e., acute (0-3 days after SAH), subacute (4-13 days after SAH), chronic (>/=14 days after SAH). Criteria indicating the occurrence of hydrocephalus were the presence of significantly enlarged temporal horns or ratio of frontal horn to maximal biparietal diameter more than 30% in computerized tomography., Results: Overall, 66 of the 734 patients (8.9%) underwent shunting procedures for the treatment of chronic hydrocephalus. Statistically significant associations among the following factors and shunt-dependent chronic hydrocephalus were observed. (1) Increased age (p < 0.05), (2) poor Hunt and Hess grade at admission (p < 0.05), (3) intraventricular hemorrhage (p < 0.05), (4) Fisher grade III, IV at admission (p < 0.05), (5) radiological hydrocephalus at admission (p < 0.05), and (6) post surgery meningitis (p < 0.05) did affect development of chronic hydrocephalus. However the presence of intracerebral hemorrhage, multiple aneurysms, vasospasm, and gender did not influence on the development of shunt-dependent chronic hydrocephalus. In addition, the location of the ruptured aneurysms in posterior cerebral circulation did not correlate with the development of shunt-dependent chronic hydrocephalus., Conclusion: Hydrocephalus after aneurysmal SAH seems to have a multifactorial etiology. Understanding predisposing factors related to the shunt-dependent chronic hydrocephalus may help to guide neurosurgeons for better treatment outcomes.

Objective: Rapid increase in aged population and westernization of lifestyle have modified epidemiological status of stroke. The purpose of this study is to analyze changing trends of stroke epidemiology in South Korea., Methods: We reviewed retrospectively medical records of 1,124 cases diagnosed as stroke among 54,534 patients who visited the Emergency Medical Center at our hospital from January 1994 to December 1996 (Group A). Also, we evaluated 1,705 cases diagnosed as stroke among 55,381 patients who visited to the same hospital from January 2003 to December 2005 (Group B). The variable features of stroke, such as age, sex, seasonal variation and distribution of stroke subtypes were studied by comparing group A with B., Results: In group A, hemorrhagic stroke (67.9%) was more prevalent than ischemic stroke (32.1%). However, group B showed that the ratio of hemorrhagic stroke (40.3%) to ischemic stroke (59.6%) has been reversed. The highest incidence of stroke was noted in their sixties and seventies of age in group B, which was older than that of group A. In group A, male ischemic stroke (IS) patients outnumbered female patients (1.26:1). Moreover, this gender disproportion became higher in group B (1.53:1). In group A, the number of male intracerebral hermorrhage (ICH) patients were similar to that of female patients (0.97:1). However, male ICH patients outnumbered female patients in group B (1.23:1). As for subarachnoid hemorrhage (SAH), female patients outnumbered male patients more than two-fold in both groups. Both groups showed that the occurrence of ischemic stroke was highest in summer, but that of hemorrhagic stroke was the highest in winter., Conclusion: This study showed the changing trends of stroke in its distribution of subtypes. Multicenter prospective study using stroke registry would be required for the determination of national epidemiologic trends.

There are conflicting views for the polymerization process of human UDP-glucose dehydrogenase (UGDH) and no clear evidence has been reported yet. Based on crystal coordinates for Streptococcus pyogenes UGDH, we made double mutant A222Q/S233G. The double mutagenesis had no effects on expression, stability, and secondary structure. Interestingly, A222Q/S233G was a dimeric form and showed an UGDH activity, although it showed increased Km values for substrates. These results suggest that Ala222 and Ser233 play an important role in maintaining the hexameric structure and the reduced binding affinities for substrates are attributable to its altered subunit communication although quaternary structure may not be critical for catalysis.

Porcine endogenous retroviruses (PERVs) in the pig genome represent a potential risk of infection in pig-to-human transplantation and are transmitted vertically. The solitary long terminal repeat (LTR) elements of the PERVs affect the replication properties of the individual viruses via their repeat sequences and by encoding a set of specific transcription factors. We examined the promoter activities of solitary LTR elements belonging to the PERV-A and -B families of the Korean domestic pig (KDP) using luciferase reporters. Three of the LTR structures (of PERV-A5-KDP, PERV-A7-KDP, PERV-A8-KDP) had different promoter activities in human HCT116 cells and monkey Cos7 cells, and potential negatively and positively acting regions affecting transcription were identified by deletion analysis. These data suggest that specific sequences in the U3 region of a given LTR element can affect the activities of promoter or enhancer elements in the PERV.

Background: Hybrid genes are candidate risk factors for human tumors by inducing mutation, translocation, inversion, or rearrangement of genes. The occurrence of hybrid genes may also have given rise to new transcripts during hominid evolution., Description: HYBRIDdb is a database of hybrid genes in humans. This system encompasses the bioinformatics analysis of mRNA, EST, cDNA, and genomic DNA sequences in the INDC databases, and can be used to identify hybrid genes. We searched for hybrid genes among the 28,171 genes listed in the NCBI database, and analyzed their structural patterns in the human genome. The 2,344 gene pairs were detected as hybrid forms of transcriptional products. We classified the hybrid genes into two groups: chromosomal-mediated translocation fusion transcripts and transcription-mediated fusion transcripts., Conclusion: The HYBRIDdb database will provide genome scientists with insight into potential roles for hybrid genes in human evolution and disease.

Human endogenous retroviruses (HERVs) contribute to various kinds of genomic instability via rearrangement and retrotransposition events. In the present study the formation of a new human-specific solo-LTR belonging to the HERV-H family (AP001667; chromosome 21q21) was detected by a comparative analysis of human chromosome 21 and chimpanzee chromosome 22. The solo-LTR was formed as a result of an equal homologous recombination excision event. Several evolutionary processes have occurred at this locus during primate evolution, indicating that mammalian-wide interspersed repeat (MIR) and full-length HERV-H elements integrated into hominoid genomes after the divergence of Old World monkeys and hominoids, and that the solo-LTR element was created by recombination excision of the HERV-H only in the human genome.

Objective: The objective of this study was to identify independent prognostic factors for survival in patients with epithelial ovarian cancer who had persistent disease identified at second-look surgery., Methods: We performed a retrospective chart review of all patients with epithelial ovarian cancer who had positive findings at second-look surgery between June 1991 and June 2002. All patients achieved a complete clinical remission after a prescribed course of primary therapy. Survival was determined from the time of second-look surgery until last follow-up or death., Results: The study included a total of 262 patients, with a median age of 54 years (range, 22-80). Of the 262 patients, 166 (63%) had died of disease. Records of initial (salvage) treatment after the positive second-look surgery were available for 243 patients. Therapies included the following: intraperitoneal (IP) cisplatin, 71 (29%); IP cisplatin combined with a second drug, 53 (22%); IP therapy other than cisplatin, 29 (12%); intravenous (IV) chemotherapy, 50 (21%); IP and IV therapy, 35 (14%); and oral chemotherapy, 5 (2%). Of the 13 potential prognostic factors analyzed, only 2 factors emerged that, when combined, were significant--residual disease after primary surgery and size of persistent disease found at second-look surgery. Patients with

UDP-glucose dehydrogenase (UGDH) catalyzes the synthesis of UDP-glucuronic acid from UDP-glucose resulting in the formation of proteoglycans that are involved in promoting normal cellular growth and migration. Overproduction of proteoglycans has been implicated in the progression of certain epithelial cancers. Here, human UGDH (hUGDH) was purified and crystallized from a solution of 0.2 M ammonium sulfate, 0.1 M Na cacodylate, pH 6.5, and 21% PEG 8000. Diffraction data were collected to a resolution of 2.8 A. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1) with unit-cell parameters a = 173.25, b = 191.16, c = 225.94 A, and alpha = beta = gamma = 90.0 degrees. Based on preliminary analysis of the diffraction data, we propose that the biological unit of hUGDH is a tetramer.

The role of residue C323 in catalysis by human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) was examined by substituting Arg, Gly, Leu, Met, or Tyr at C323 by cassette mutagenesis using synthetic human GDH isozyme genes. As a result, the Km of the enzyme for NADH and alpha-ketoglutarate increased up to 1.6-fold and 1.1-fold, respectively. It seems likely that C323 is not responsible for substrate-binding or coenzyme-binding. The efficiency (kcat/Km) of the mutant enzymes was only 11-14% of that of the wild-type isozymes, mainly due to a decrease in kcat values. There was a linear relationship between incorporation of [14C]p-chloromercuribenzoic acid and loss of enzyme activity that extrapolated to a stoichiometry of one mol of [14C] incorporated per mol of monomer for wild type hGDHs. No incorporation of [14C]p-chloromer-curibenzoic acid was observed with the C323 mutants. ADP and GTP had no effect on the binding of p-chloromercuribenzoic acid, suggesting that C323 is not directly involved in allosteric regulation. There were no differences between the two hGDH isozymes in sensitivities to mutagenesis at C323. Our results suggest that C323 plays an important role in catalysis by human GDH isozymes.

The families of human endogenous retroviruses (HERVs) are widely distributed in the human genome. Here we examined their distribution and expression. Approximately forty thousand HERV elements including truncated and solitary long terminal repeats (LTRs) were identified. These elements were most dense on chromosomes 4, 20, X, and Y. From an analysis of genomic stability during primate evolution, the 5 cent -LTR of the HERV genome (5 cent LTR - internal HERV - 3 cent LTR) appeared to be more often truncated than the 3 cent -LTR. ESTs derived from normal placenta, skeletal muscle, hypothalamus, and testis gave frequent matches to HERV elements. We present a classification of genes associated with HERV elements according to the hierarchical structure of gene ontology.

Hyperinsulinism-hyperammonemia syndrome is due either to hyperactivity of GDH or impaired inhibition of GDH by GTP. We have investigated the effect of Cimicifuga heracleifolia extract on the activities of glutamate dehydrogenase (GDH) in cultured rat islets. When the extract was present in the culture medium for 24 h prior to cell harvest, the Vmax of GDH was decreased by 45% with no significant change in Km. In addition, the concentration of alpha-ketoglutarate increased by approximately 39%, and glutamate decreased by 48%. Perfusion of islets with C. heracleifolia extract reduced insulin release by up to 47%. Although the relation between GDH activity and insulin release remains to be clarified, our results suggest that C. heracleifolia extract regulates insulin release by altering GDH activity in primary cultured islets and that this natural compound may be used to modulate GDH activity in patients with hyperinsulinism-hyperammonemia syndrome.