Serine hydroxymethyltransferase from the silkworm Bombyx mori: Identification,distribution, and biochemical characterization

Serine hydroxymethyltransferase (SHMT) catalyzes the interconversion of serine and tetrahydrofolate (THF) to glycine and methylenetetrahydrofolate. cDNA encoding Bombyx mori SHMT (bmSHMT) was cloned and sequenced. The deduced amino acid sequence consisted of 465 amino acids and was found to share homology with other SHMTs. Recombinant bmSHMT was overexpressed in Escherichia coli and purified to homogeneity. The enzyme showed optimum activity at pH 3.0 and 30°C and was stable under acidic conditions. The K_m and k_cat/K_m values for THF in the presence of Nicotinamide adenine dinucleotide phosphate (NADP⁺) were 0.055 mM and 0.081 mM^?1 s^?1, respectively, whereas those toward NADP⁺ were 0.16 mM and 0.018 mM^?1 s^?1 and toward l ‐serine were 1.8 mM and 0.0022 mM^?1 s^?1, respectively. Mutagenesis experiments revealed that His119, His132, and His135 are important for enzymatic activity. Our results provide insight into the roles and regulation mechanism of one‐carbon metabolism in the silkworm B. mori.     

Association of APOE, GCPII and MMP9 polymorphisms with common diseases and lipid levels in an older adult/elderly cohort

Background and aims

The characterization of candidate gene polymorphisms in elderly populations is an important tool for the identification of risk factors for age-related diseases and conditions. We aimed to genotype the APOE polymorphisms (rs429358 and rs7412), rs61886492 (1561C>T) and rs202720 of GCPII gene and rs3918242 (− 1562C>T) of MMP9 gene in an older-adult/elderly cohort from Cuiabá city, Mato Grosso Brazil as well as to characterize risk factors for morbidities and conditions affecting this cohort.

Methods

The studied population consisted of 570 subjects from Cuiabá city, Brazil, who were subjected to clinical interviews and blood collection for laboratory examinations and DNA extraction. Restriction Fragment Length Polymorphism Polymerase Chain Reaction (RFLP-PCR), sequence-specific primer PCR (SSP-PCR) and TaqMan® allelic discrimination assay were used for genotyping.

Results

The frequencies of APOE ε2 and ε4 were 6.6% and 14.8%, respectively, and the frequencies of GCPII rs61886492 T allele, GCPII rs202720 C allele and MMP9 rs3918242 T allele were, respectively, 3.0%, 26.6% and 10.1%. Significant associations between APOE ε2 allele with lower total cholesterol and LDL-cholesterol were found. In addition, MMP9 rs3918242 T allele was associated with higher LDL-cholesterol levels, suggesting a link between lipid metabolism alteration and cardiovascular disease.

Conclusions

The present findings contributed to characterize risk factors specific for the studied population and to better understand the molecular physiopathology of common morbidities and conditions affecting older-adult/elderly people.     

Devising Assisted Reproductive Technologies for Wild-Derived Strains of Mice: 37 Strains from Five Subspecies of Mus musculus

Wild-derived mice have long offered invaluable experimental models for mouse genetics because of their high evolutionary divergence from laboratory mice. A number of wild-derived strains are available from the RIKEN BioResource Center (BRC), but they have been maintained as living stocks because of the unavailability of assisted reproductive technology (ART). In this study, we sought to devise ART for 37 wild-derived strains from five subspecies of Mus musculus maintained at the BRC. Superovulation of females was effective (more than 15 oocytes per female) for 34 out of 37 strains by treatment with either equine chorionic gonadotropin or anti-inhibin serum, depending on their genetic background (subspecies). The collected oocytes could be fertilized in vitro at mean rates of 79.0% and 54.6% by the optimized protocol using fresh or frozen-thawed spermatozoa, respectively. They were cryopreserved at the 2-cell stage by vitrification with an ethylene glycol-based solution. In total, 94.6% of cryopreserved embryos survived the vitrification procedure and restored their normal morphology after warming. A conventional embryo transfer protocol could be applied to 25 out of the 35 strains tested. In the remaining 10 strains, live offspring could be obtained by a modified embryo transfer protocol using cyclosporin A treatment and co-transfer of ICR (laboratory mouse strain) embryos. Thus, ART for 37 wild-derived strains was devised successfully and is now routinely used for their preservation and transportation. The information provided here might facilitate broader use and wider distribution of wild-derived mice for biomedical research.     

Isolation and characterization of CRF-related diuretic hormones from the whitelined sphinx moth Hyles lineata

We have isolated and characterized two diuretic hormones (DH), Hylli-DH41 and Hylli-DH30, from extracts of whole heads of the lepidopteran Hyles lineata. We monitored the isolation by measuring the ability of fractions to affect levels of cyclic AMP production by Malpighian tubules of Manduca sexta maintained in vitro. These DH are related to a family of vertebrate neuropeptides which includes sauvagine, corticotropin-releasing factor (CRF), and urotensin I. Both Hylli-DH41 (RMPSLSIDLPMSVLRQKLSLE KERKVQALRAAANRNFLNDI-NH2) and Hylli-DH30 (SFSVNPAVEILQHRYMEKVAQNNRNFLNRV-NH2) show extremely high similarity with two DH from the tobacco hornworm M. sexta. This is not surprising because both H. lineata and M. sexta are sphingid moths. The discovery of these DH provides a third example of two CRF-related DH occurring in one insect species.     

Inhibition of male chick phenotypes and spermatogenesis by Bisphenol-A

Bisphenol-A (BPA) has been reported to bind to the estrogen receptor (ER) and also to act as a xenoestrogen on the reproductive system of many species. In our previous study, a high dose of BPA disturbed the growth of the comb and testes of male chickens. In this study, the exposure of relatively low doses of BPA on the growth of the male chicken phenotypes was investigated. White Leghorn male chicks were orally administered various doses of BPA (2 microg to 200 mg/kg) from 2 weeks of age, and thereafter the comb, wattle and testes were examined at 5, 10, 15, 20 and 25 weeks of age. Although the body weight showed no significant difference among the birds of all ages, the growth of above organs was significantly affected in the chicks even with a minimal dose of 2-microg BPA. These inhibitory effects appeared in a dose-dependent manner. Histologically, the growth of the testes was negatively affected by exposure to over 20-microg/kg BPA: namely, the development of seminiferous tubuli and spermatogenesis were severely inhibited. The mRNA expressions of ERalpha and the aromatase gene (p450arom) increased in the testes in a dose-dependent manner after BPA administration. Accordingly, even low doses of BPA delayed the growth of the male chicken phenotype either by a direct effect or by an indirect response resulting in an increase in both of the endogenous estrogen levels and hyper-sensitivity to estrogen.     

Na+/H+ exchange and pH regulation in the control of neutrophil chemokinesis and chemotaxis

Large proton fluxes accompany cell migration, but their precise role remains unclear. We studied pH regulation during the course of chemokinesis and chemotaxis in human neutrophils stimulated by attractant peptides. Activation of cell motility by chemoattractants was accompanied by a marked increase in metabolic acid generation, attributable to energy consumption by the contractile machinery and to stimulation of the NADPH oxidase and the ancillary hexose monophosphate shunt. Despite the increase in acid production, the cytosol underwent a sizable alkalinization, caused by acceleration of Na(+)/H(+) exchange. The development of the alkalinization mirrored the increase in the rate of cell migration, suggesting a causal relationship. However, elimination of Na(+)/H(+) exchange by omission of external Na(+) or by addition of potent inhibitors was without effect on either chemokinesis or chemotaxis, provided the cytosolic pH remained near neutrality. At more acidic levels, cell motility was progressively inhibited. These observations suggest that Na(+)/H(+) exchange plays a permissive role in cell motility but is not required for the initiation or development of the migratory response. Chemokinesis also was found to be exquisitely sensitive to extracellular acidification. This property may account for the inability of neutrophils to access abscesses and solid tumors that have been reported to have inordinately low pH.