A BAC-based contig map of the cynomolgus macaque (Macaca fascicularis) major histocompatibility complex genomic region

The construction of a cynomolgus macaque (Macaca fascicularis, Mafa) BAC library for genomic comparison between rhesus and cynomolgus macaques is necessary to promote the cynomolgus macaque as one of the important experimental animals for future medical and biological research. In this paper, we constructed a cynomolgus macaque BAC library and a map of the MHC (Mafa) genomic region for comparison of the genomic organization and nucleotide similarities between the human, the chimpanzee, and the rhesus macaque. The BAC library consists of 221,184 clones with an average insert size of 83 kb, providing a sixfold coverage of the haploid genome. A total of 114 BAC clones and 54 PCR primer sets were used to construct a 4.3-Mb contig of the MHC region. Diversity analysis of genomic sequence from selected subregions of the MHC revealed that the cynomolgus sequence varied compared to rhesus macaque, human, and chimpanzee sequences by 0.48, 4.15, and 4.10%, respectively. From these findings, we conclude that the BAC library and Mafa genomic map are useful tools for genome analysis and will have important applications for comparative genomics and identifying regions of consequence in medical research.     

In vitro neuroprotective action of recombinant rat erythropoietin produced by astrocyte cell lines and comparative studies with erythropoietin produced by Chinese hamster ovary cells

In the central nervous system, astrocytes produce erythropoietin (Epo) and neurons express its receptor. To examine whether or not the brain Epo protects the in vitro cultured neurons from glutamate-induced cell death, we established rat astrocyte cell lines containing the plasmid for production of recombinant rat Epo. Epo partially purified from the culture medium showed a neuroprotective effect similar to that of rat Epo produced by Chinese hamster ovary (CHO) cells. Comparison was made in some other properties between Epo produced by these astrocyte cell lines and that by CHO cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mutation patterns for two flaviviruses: hepatitis C virus and GB virus C/hepatitis G virus

We studied the mutation patterns of hepatitis C virus (HCV) and GB virus C/hepatitis G virus (HGV). Although the mutation patterns of the two viruses were similar to each other, they were quite different from that of HIV. In particular, the similarity of the patterns between HCV or HGV and human nuclear pseudogenes was statistically significant whereas there was no similarity between HIV and human nuclear pseudogenes. This finding suggests that the mutation patterns of HCV and HGV are similar to the patterns of spontaneous substitution mutations of human genes, implying that nucleotide analogues which are effective against HCV and HGV may have a side effect on the normal cells of humans.     

Sperm membrane incorporation into oolemma contributes to the oolemma block to sperm penetration: Evidence based on intracytoplasmic sperm injection experiments in the mouse

Mouse oocytes were fertilized by intracytoplasmic sperm injection (ICSI) and reinseminated after the removal of zonae pellucidae at pronuclear stage or at the 2-cell stage. Although these oocytes were activated normally by ICSI, as evidenced by resumption of meiosis and cortical granule exocytosis, they did not develop oolemma block to sperm penetration. They could be penetrated by spermatozoa at pronuclear stage and even at the 2-cell stage. This supports the notion that incorporation of sperm plasma membrane into oolemma contributes to the changes in oolemma that block sperm penetration. © 1996 Wiley-Liss, Inc.     

BMP4 induces primitive endoderm but not trophectoderm in monkey embryonic stem cells

Monkey embryonic stem (ES) cells share similar characteristics to human ES cells and provide a primate model of allotransplantation, which allows to validate efficacy and safety of cell transplantation therapy in regenerative medicine. Bone morphogenetic protein 4 (BMP4) is known to promote trophoblast differentiation in human ES cells in contrast to mouse ES cells where BMP4 synergistically maintains self-renewal with leukemia inhibitory factor (LIF), which represents a significant difference in signal transduction of self-renewal and differentiation between murine and human ES cells. As the similarity of the differentiation mechanism between monkey and human ES cells is of critical importance for their use as a primate model system, we investigated whether BMP4 induces trophoblast differentiation in monkey ES cells. Interestingly, BMP4 did not induce trophoblast differentiation, but instead induced primitive endoderm differentiation. Prominent downregulation of Sox2, which plays a pivotal role not only in pluripotency but also placenta development, was observed in cells treated with BMP4. In addition, upregulation of Hand1, Cdx2, and chorionic gonadotropin beta (CG-beta), which are markers of trophoblast, was not observed. In contrast, BMP4 induced significant upregulation of Gata6, Gata4, and LamininB1, suggesting differentiation into the primitive endoderm, visceral endoderm, and parietal endoderm, respectively. The threshold of BMP4 activity was estimated as about 10 ng/mL. These findings suggest that BMP4 induced differentiation into the primitive endoderm lineage but not into trophoblast in monkey ES cells.     

Simulation of C. elegans thermotactic behavior in a linear thermal gradient using a simple phenomenological motility model

The nematode Caenorhabditis elegans has been reported to exhibit thermotaxis, a sophisticated behavioral response to temperature. However, there appears to be some inconsistency among previous reports. The results of population-level thermotaxis investigations suggest that C. elegans can navigate to the region of its cultivation temperature from nearby regions of higher or lower temperature. However, individual C. elegans nematodes appear to show only cryophilic tendencies above their cultivation temperature. A Monte-Carlo style simulation using a simple individual model of C. elegans provides insight into clarifying apparent inconsistencies among previous findings. The simulation using the thermotaxis model that includes the cryophilic tendencies, isothermal tracking and thermal adaptation was conducted. As a result of the random walk property of locomotion of C. elegans, only cryophilic tendencies above the cultivation temperature result in population-level thermophilic tendencies. Isothermal tracking, a period of active pursuit of an isotherm around regions of temperature near prior cultivation temperature, can strengthen the tendencies of these worms to gather around near-cultivation-temperature regions. A statistical index, the thermotaxis (TTX) L-skewness, was introduced and was useful in analyzing the population-level thermotaxis of model worms.     

Development and application of a monoclonal antibody-based sandwich ELISA for quantification of Japanese medaka (Oryzias latipes) vitellogenin

Vitellogenin (Vtg) was purified from ascitic fluid of a 17beta-estradiol (E2)-treated female Japanese medaka by anion-exchange chromatography. The molecular mass of medaka Vtg by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), corresponding to the Vtg monomer, was 200 kDa. BALB/c mice were immunized with purified-Vtg and two hybridoma clones producing specific antibodies against medaka Vtg were selected. The specificity of these monoclonal antibodies (mAbs) was evaluated by Western blot analysis of the plasma proteins separated on SDS-PAGE, and no cross-reactivity was observed with plasma proteins from control males. A sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of medaka Vtg was developed using these mAbs. The assay range was between 1 and 100 ng/ml, and the intra- and inter-assay variations determined from plasma samples were within 7.7 and 8.5%, respectively. Recovery of medaka Vtg added to plasma was 92-111%. In a plasma dilution test, plots of Vtg concentration gave a straight line. After exposure of male medaka to E2 (10 ng/l), Vtg appeared in liver and plasma on the first day and reached a maximum on the 3rd to 5th day. The sandwich ELISA could be useful for the detection of estrogenic properties, and the medaka Vtg bioassay could be a very sensitive and good tool for screening of endocrine disrupting compounds.     

Effects of dietary di(2-ethylhexyl)phthalate, a putative endocrine disrupter, on enzyme activities involved in the metabolism of tryptophan to niacin in rats

We have reported that the conversion ratio of tryptophan to niacin increased with increasing dietary concentration of di(2-ethylhexyl)phthalate (DEHP); the conversion ratio was about 2.0% in the control rat, which increased by about 30% in the rat fed with 3.0% DEHP diet. In this study, we investigated whether this abnormal increase in the conversion ratio by DEHP occurred through the alteration of the enzyme activities involved in the metabolism of tryptophan to niacin. Rats were fed with a diet containing 0%, 0.1%, 0.5%, or 1.0% DEHP for 21 days. The nine kinds of enzyme activities involved in the biosynthesis and catabolism in the liver and kidney were measured. Based on previous findings that the formation of quinolinic acid and its' metabolites significantly increased with DEHP administration, we proposed that the activity of 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase would be inhibited by DEHP intake. However, we found that the activities in the liver and kidney did not decrease in the rat fed with DEHP-containing diet. We discuss the discrepancy between the metabolite results and the enzyme activities.     

Insulin and nateglinide reduce monocyte adhesion to endothelial cells in Goto-Kakizaki rats exhibiting repetitive blood glucose fluctuation

Epidemiological studies demonstrated the importance of postprandial hyperglycemia on the progression of atherosclerosis. However, whether treatment of postprandial hyperglycemia by insulin or insulin secretagogues has a beneficial effect on atherosclerosis has not been elucidated. To elucidate the effects of reduction of postprandial rise of blood glucose by insulin and nateglinide on monocyte adhesion to endothelial cells, we used non-obese type 2 diabetic Goto-Kakizaki (GK) rats fed twice daily, as a model of repetitive postprandial hyperglycemia. We investigated the effects of insulin injection and nateglinide administration just before each meal for 12 weeks on monocyte adhesion to endothelial cells. By setting the doses of insulin and nateglinide, both treatment significantly reduced postprandial hyperglycemia without significant reduction of HbA1c. Nateglinide also reduced serum insulin level just after 1 h meal. Both nateglinide and insulin therapy reduced the number of monocytes adherent to the aortic endothelial layer. Nateglinide, but not insulin, reduced intimal thickness of the thoracic aorta. While increased serum insulin level might be regarded as a factor responsible for the progression of atherosclerosis, our data showed that treatment with pre-meal insulin or nateglinide, which reduces postprandial hyperglycemia, reduced monocyte adhesion to endothelial cells.