New methods for species and sex determination in three sympatric Mustelids, Mustela itatsi, Mustela sibirica and Martes melampus

We developed novel species and sex determination methods for three Japanese mustelid species. We used DDX3Y to determine sex and generated a primer set to amplify both DDX3X and DDX3Y DNA in Mustela itatsi, M. sibirica and Martes melampus. To determine species and sex simultaneously, we generated fluorescence-labelled primers that give different fragment lengths at D-loop, DDX3X and DDX3Y of these three species using a DNA sequencer.     

A sex-specific size–number tradeoff in clonal broods

Polyembryonic parasitoids producing single-sex broods of clonal offspring provide an unusually clear window into the classic tradeoff between the number and size of offspring. We conducted a laboratory study of the encyrtid parasitoid Copidosoma bakeri parasitizing the noctuid Agrotis ipsilon to examine the way that size and number of offspring tradeoff in broods of each sex and to determine how the fit between host and parasitoid brood is achieved. We found that brood mass (wasp body mass ×brood size) was proportional to host mass, independent of brood sex, indicating a tight fit between brood and host and ensuring a size–number tradeoff. By correcting brood size and body mass of each brood for host mass, we demonstrated the expected inverse relationship between wasp variables. We postulated that the wasp brood might achieve the fit to the host by (1) adjusting brood size based on information available early in host development before and during division of the embryo, (2) manipulating host size late in host development after completion of embryo division, or (3) simply adjusting individual wasp mass to fill the host. We evaluated host responses to parasitism – and correlations between brood size and host growth early and late in development – for broods of each sex. The data are consistent with adjustment of brood size to the amount of host growth early in host development and with manipulation of host mass late in host development. The tight link between host mass and brood mass also suggests a final adjustment by parasitoid growth to achieve complete filling. Within the tight fit, female broods were smaller but contained larger individuals than male broods. The sex-specific balance point of the tradeoff and sex differences in balancing mechanisms and responses to host size suggest different selection pressures on each sex requiring future investigation.     

Effect of embryonic cell cycle of nuclear donor embryos on the efficiency of nuclear transfer in Japanese black cattle

In the present study, the development in vitro and in vivo of nuclear transfer (NT) embryos reconstructed with embryonic cells (blastomeres) at the 32- to 63-cell (sixth cell cycle) and 64- to 127-cell (seventh cell cycle) stages was investigated to determine the optimum range of embryonic cell cycles for yielding the highest number of identical calves in Japanese black cattle. Rates of development to the blastocyst stage (overall efficiency) were higher in the sixth cell-cycle stage (45%) than in the seventh cell-cycle stage (12%). After the transfer of the blastocysts reconstructed with blastomeres of the sixth and seventh cell cycle-stage embryos to recipient heifers, there were no differences in the pregnancy (14/35: 40% versus 3/13: 23%, respectively) or calving rates (11/39: 28% versus 3/13: 23%, respectively). These results indicate that the highest number of identical calves would be obtained by using sixth cell cycle (32- to 63-cell)-stage embryos as nuclear donors.     

Neurotoxic prion protein (PrP) fragment 106-126 requires the N-terminal half of the hydrophobic region of PrP in the PrP-deficient neuronal cell line

The cytotoxicity of aged PrP(106-126) was examined using an immortalized prion protein (PrP) gene-deficient neuronal cell line. The N-terminal half of the hydrophobic region (HR) but not the octapeptide repeat (OR) of PrP was required for aged PrP(106-126) neurotoxicity, suggesting that neurotoxic signals of aged PrP(106-126) are mediated by this region.     

Discovery of a new type inhibitor of human glyoxalase I by myricetin-based 4-point pharmacophore

The human glyoxalase I (hGLO I), which is a rate-limiting enzyme in the pathway for detoxification of apoptosis-inducible methylglyoxal (MG), has been expected as an attractive target for the development of new anti-cancer drugs. We have previously identified a natural compound myricetin as a substrate transition-state (Zn²⁺-bound MG-glutathione (GSH) hemithioacetal) mimetic inhibitor of hGLO I. Here, we constructed a hGLO I/inhibitor 4-point pharmacophore based on the binding mode of myricetin to hGLO I. Using this pharmacophore, in silico screening of chemical library was performed by docking study. Consequently, a new type of compound, which has a unique benzothiazole ring with a carboxyl group, named TLSC702, was found to inhibit hGLO I more effectively than S-p-bromobenzylglutathione (BBG), a well-known GSH analog inhibitor. The computational simulation of the binding mode indicates the contribution of Zn²⁺-chelating carboxyl group of TLSC702 to the hGLO I inhibitory activity. This implies an important scaffold-hopping of myricetin to TLSC702. Thus, TLSC702 may be a valuable seed compound for the generation of a new lead of anti-cancer pharmaceuticals targeting hGLO I.     

Gastric acidity in patients with follicular gastritis is significantly reduced, but can be normalized after eradication for Helicobacter pylori

BACKGROUND: Follicular gastritis is thought to be caused by Helicobacter pylori infection. However, the pathophysiology of it remains unclear. MATERIALS AND METHODS: We assessed gastric acidity in 15 patients with follicular gastritis, aged 20-37 years, using a 24-hour intragastric pH-metry, as well as by histologic and serologic evaluations; and compared it with that in other age-matched groups: 18 cases of H. pylori-positive antrum-predominant gastritis, 12 of pangastritis, and 24 H. pylori-negative normals. In eight cases with follicular gastritis, it was re-assessed 6 months after the eradication therapy for H. pylori. RESULTS: During nighttime, the percentage of time with intragastric pH above 3.0 in follicular gastritis was significantly higher than that in normals (p<.0001), and in antrum-predominant gastritis (p<.001), but was comparable with that in pangastritis. In the daytime period, this parameter in follicular gastritis was significantly higher than that in normal (p<.001), in antrum-predominant gastritis (p<.001), and in pangastritis (p<.05). Marked mononuclear cell and neutrophil infiltration but no apparent glandular atrophy were observed in both the antrum and corpus. Serum pepsinogen I/II ratio was significantly lower in follicular gastritis than that in normals (p<.0001) and in antrum-predominant gastritis (p<.001), whereas serum gastrin was significantly higher than that in normals (p<.0001), in antrum-predominant gastritis (p<.01) and in pangastritis (p<.05). After eradication for H. pylori, all of the parameters in follicular gastritis were altered to the same ranges as those in normals. CONCLUSIONS: In follicular gastritis, gastric acidity is significantly reduced, but can be normalized by eradication of H. pylori. It can thus be speculated that inflammatory cytokines or H. pylori-infection-induced prostaglandins might strongly inhibit gastric acid secretion in follicular gastritis.     

Link between the ubiquitin conjugation system and the ISG15 conjugation system: ISG15 conjugation to the UbcH6 ubiquitin E2 enzyme

ISG15 is a ubiquitin-like protein that is upregulated on treatment with interferon. ISG15 is considered to be covalently conjugated to cellular proteins through a sequential reaction similar to that of the ubiquitin conjugation system consisting of E1/E2/E3 enzymes: UBE1L and UbcH8 have been reported to function as E1 and E2 enzymes, respectively, for ISG15 conjugation. Several cellular proteins have been identified as targets for ISG15 conjugation, but the roles of ISG15 conjugation remain unclear. In this study, we found that UbcH6 and UbcH8, E2 enzymes for ubiquitin conjugation, are covalently modified by ISG15. We also found that UbcH6 is capable of forming a thioester intermediate with ISG15 through Cys131. We determined that the Lys136 residue near the catalytic site Cys131 is the ISG15 conjugation site in UbcH6. We isolated ISG15-modified and unmodified UbcH6 proteins, and analyzed their abilities to form thioester intermediates with ubiquitin. A ubiquitin thioester intermediate was detected in the case of unmodified UbcH6, but not in that of ISG15-modified UbcH6, strongly suggesting that ISG15 conjugation to UbcH6 suppresses its ubiquitin E2 enzyme activity. Thus, we provide evidence for a link between the ubiquitin conjugation system and the ISG15 conjugation system.