Selection of HLA-B57-associated Gag A146P mutant by HLA-B∗48:01-restricted Gag140-147-specific CTLs in chronically HIV-1-infected Japanese

We previously showed the possibility that Gag A146P, which is an escape mutant from HLA-B157-restricted CTLs, was selected by HLA-B148:01-restricted Gag138–147(LI10)-specific CTLs in a Japanese cohort in which HLA-B157 individuals were not detected. We herein demonstrated Gag140–147(GI8) to be the optimal epitope rather than LI10 and that GI8-specific T cells failed to recognize the A146P mutant virus-infected cells. The sequence analysis of Gag146 in 261 chronically HIV-1-infected Japanese showed the accumulation of the A146P mutation in HLA-B148:01⁺ individuals. These findings together indicate that the A146P mutant is accumulating in Japanese by selection by GI8-specific CTLs.     

Localization and secretion of inhibins in the equine fetal ovaries

To clarify the source of inhibins in equine female fetuses, concentrations of immunoreactive (ir-) inhibin, inhibin pro-alphaC, and inhibin A in both fetal and maternal circulation and in fetal ovaries were measured. In addition, the localization of inhibin alpha and inhibin/activin beta(A), and beta(B) subunits and the expression of inhibin alpha(A) and inhibin/activin beta(A) subunit mRNA in fetal ovaries were investigated using immunohistochemistry and in situ hybridization. Concentrations of circulating ir-inhibin, inhibin pro-alphaC, and inhibin A were remarkably more elevated in the fetal than in the maternal circulation between Days 100 and 250 of gestation. Fetal ovaries contained large amounts of ir-inhibin, inhibin pro-alphaC, and inhibin A. In contrast, these inhibin forms were undetectable in both the maternal ovaries and placenta. The inhibin alpha and inhibin/activin beta(A) and beta(B) subunit proteins were localized to enlarged interstitial cells of the equine fetal ovary. Expression of inhibin alpha and inhibin/activin beta(A) subunit mRNAs were also observed in the interstitial cells. We conclude that the main source of large amounts of inhibins in fetal circulation is interstitial cells of fetal ovary and is not of maternal origin. Furthermore, these inhibins may play some important physiological roles in the development of gonads in the equine fetus.     

Studies of the genetic mechanism of radiation-induced mitotic segregation in yeast

Summary Sectoring was induced with x-rays or ultraviolet in a diploid yeast strain heterozygous for seven genes located on one chromosome arm. The frequencies of sectoring of different genes were approximately linearly related to their distance from the centromere. If two or more adjacent genes sectored, the event could be explained by mitotic crossing over. Sectoring of single genes, however, was mostly nonreciprocal and resembled a conversion-type event. Approximately 80% of the sectored colonies could be explained single mitotic crossovers in one of the intergenic regions.     

Simultaneous determination of tetrahydrocortisol, allotetrahydrocortisol and tetrahydrocortisone in human urine by liquid chromatography-electrospray ionization tandem mass spectrometry

Simultaneous quantification method of three major metabolites of cortisone and cortisol, tetrahydrocortisol, allotetrahydrocortisol and tetrahydrocortisone by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was investigated in a positive mode using a recently developed picolinyl derivatization. Conversion of each steroid into the corresponding picolinyl derivatives (1b, 2b or 3b) was performed by mixed anhydride method using picolinic acids and 2-methyl-6-nitrobenzoic anhydride. Derivatization proceeded smoothly to afford the corresponding 3, 21-dipicolinyl derivatives. Positive ion-ESI mass spectra of the picolinyl derivatives were dominated by an appearance of [M+H](+) as base peaks in all cases. The picolinyl derivatives provided 15 to 80-fold higher ESI response in the LC-ESI-MS/MS (selected reaction monitoring: SRM) when compared to those of underivatized molecules in a positive LC-ESI mode. The use of the picolinyl ester, solid-phase extraction, and deuterium labeled internal standards enabled the concentrations of these metabolites in human urine to be determined simultaneously by LC-ESI-MS/MS (SRM) with a small sample volume of less than 1microl urine.     

Development of a highly selective EP2-receptor agonist. Part 1: identification of 16-hydroxy-17,17-trimethylene PGE2 derivatives

Design and synthesis of an EP2-receptor selective agonist began with the chemical modification of alpha- and omega-chains of butaprost 1a, which exhibits an affinity for the IP-receptor. Two series of prostaglandin (PG) analogues with a 16-hydroxy-17,17-trimethylene moiety as an omega-chain were identified. Among those tested, 4a,b,e,f,h and 6a,b,e,f,h were found to be highly selective EP2-receptor agonists. Structure-activity relationships are discussed.     

Direct comparison of in vivo antisense activity of ENA oligonucleotides targeting PTP1B mRNA with that of 2'-O-(2-methoxy)ethyl-modified oligonucleotides

Protein-tyrosine phosphatase 1B (PTP1B) inhibitory activity of the 2'-O-(2-methoxy)ethyl (2'- MOE)-modified gapmer antisense oligonucleotide, ISIS113715, was previously reported. This antisense oligonucleotide increases insulin sensitivity and normalizes plasma glucose levels in diabetic ob/ob and db/db mice. In the present study, the isosequential 2'-O,4'-C-ethylene-bridged nucleic acid (ENA)-modified oligonucleotide, ENA-1, was synthesized, and its ability to further improve the downregulation of PTP1B in db/db mice was examined. We demonstrated that, compared with ISIS113715, intraperitoneal and subcutaneous administration of ENA-1 more effectively decreased the plasma glucose levels in db/db mice. Moreover, ENA-1 decreased expression of PTP1B in the liver and fat of db/db mice more effectively than ISIS113715. We describe for the first time the functional comparison of 2'-MOE- and ENA-modified antisense oligonucleotides. Our data indicate that the enhancement of the efficacy of antisense oligonucleotides by ENA modifications is superior to that of second-generation 2'-MOE modifications in certain aspects.     

Ranking the selectivity of PubChem screening hits by activity-based protein profiling: MMP13 as a case study

High-throughput screening (HTS) has become an integral part of academic and industrial efforts aimed at developing new chemical probes and drugs. These screens typically generate several 'hits', or lead active compounds, that must be prioritized for follow-up medicinal chemistry studies. Among primary considerations for ranking lead compounds is selectivity for the intended target, especially among mechanistically related proteins. Here, we show how the chemical proteomic technology activity-based protein profiling (ABPP) can serve as a universal assay to rank HTS hits based on their selectivity across many members of an enzyme superfamily. As a case study, four metalloproteinase-13 (MMP13) inhibitors of similar potency originating from a publically supported HTS and reported in PubChem were tested by ABPP for selectivity against a panel of 27 diverse metalloproteases. The inhibitors could be readily separated into two groups: (1) those that were active against several metalloproteases and (2) those that showed high selectivity for MMP13. The latter set of inhibitors was thereby designated as more suitable for future medicinal chemistry optimization. We anticipate that ABPP will find general utility as a platform to rank the selectivity of lead compounds emerging from HTS assays for a wide variety of enzymes.     

Reduction in excess sludge production in a dairy wastewater treatment plant via nozzle-cavitation treatment: Case study of an on-farm wastewater treatment plant

Nozzle-cavitation treatment was used to reduce excess sludge production in a dairy wastewater treatment plant. During the 450-d pilot-scale membrane bioreactor (MBR) operation, when 300 l of the sludge mixed liquor (1/10 of the MBR volume) was disintegrated per day by the nozzle-cavitation treatment with the addition of sodium hydrate (final concentration: 0.01% W/W) and returned to the MBR, the amount of excess sludge produced was reduced by 80% compared with that when sludge was not disintegrated.