Design, synthesis, and biological activity of non-basic compounds as factor Xa inhibitors: SAR study of S1 and aryl binding sites

Compound 7 was identified as the active metabolite of 6 by HPLC and mass spectral analysis. Modification of lead compound 7 by transformation of its N-oxide 6-6 biaryl ring system and fused aromatics produced a series of non-basic fXa inhibitors with excellent potency in anti-fXa and anticoagulant assays. The optimized compounds 73b and 75b showed sub to one digit micromolar anticoagulant activity (PTCT2). Particularly, anti-fXa activity was detected in plasma of rats orally administered with 1mg/kg of compound 75b.     

Expression and regulation of adrenomedullin in renal glomerular podocytes

Adrenomedullin (AM) is postulated to exert organ-protective effects. It is expressed in the renal glomeruli, but its roles in the glomerular podocytes have been poorly elucidated. In the present study, we investigated the expression and regulation of AM in recently established conditionally immortalized mouse podocyte cell line in vitro and podocyte injury model in vivo. The cultured differentiated podocytes expressed AM mRNA and secreted measurable amount of AM. AM secretion from the podocytes was increased by H(2)O(2), hypoxia, puromycin aminonucleoside (PAN), albumin overload, and TNF-alpha. Real-time RT-PCR analysis revealed that AM mRNA expression in the podocytes was enhanced by PAN and TNF-alpha, both of which were suppressed by mitochondrial antioxidants. Furthermore, AM expression was upregulated in the glomerular podocytes of PAN nephrosis rats. These results indicated that AM expression in the podocytes was upregulated by stimuli or condition relevant to podocyte injury, suggesting its potential role in podocyte pathophysiology.     

Roles of mTOR and JNK in serine phosphorylation, translocation, and degradation of IRS-1

In 3T3-L1 adipocytes, insulin or anisomycin stimulated phosphorylation of IRS-1 at Ser(307) and Ser(636/639), both of which were partially reduced by the mTOR inhibitor, rapamycin, or the JNK inhibitor, SP600125, and were further inhibited by a combination of them. Interestingly, anisomycin-induced p70(S6K) phosphorylation was reduced by SP600125, while insulin-induced p70(S6K) phosphorylation was not. Furthermore, unlike insulin, anisomycin failed to elicit translocation or degradation of IRS-1. These results indicate that mTOR and JNK play roles in phosphorylating IRS-1 serine residues, and that insulin and anisomycin are different in terms of the relationship of activation between mTOR and JNK, and the effects on IRS-1 localization and stability.     

Extrusion and removal of lipid from the cytoplasm of porcine oocytes at the germinal vesicle stage: centrifugation under hypertonic conditions influences vitrification

In the present study, we examined a novel lipid removal method, centrifugation in solutions made hypertonic by adding 0.27 M sugar. This allowed the lipid to be extruded and removed without the loss of active mitochondria or extra cytoplasm. The type of sugar influenced the proportion of oocytes that could be stratified by centrifugation. Glucose induced the highest extrusion rate of lipid droplets. After vitrification the rates of survival, germinal vesicle breakdown and metaphase II were 30, 26, and 7%, respectively, for lipid-removed GV oocytes; this was significantly higher (P<0.05) than for corresponding vitrified lipid-intact oocytes (2, 0, and 0%, respectively). These results indicated that this method is useful to remove whole lipid droplets without losing mitochondria and improves cryotolerance of porcine GV oocytes.     

Genetic transformation of <Emphasis Type="Italic">Coffea canephora</Emphasis> by particle bombardment

Stable transformation of Coffea canephora P. was obtained by particle bombardment of embryogenic tissue. Leaf explants were cultured on medium supplemented with 5 µM isopentenyl-adenosine to induce direct embryogenesis. Explants with somatic embryos were transferred to half strength MS medium with 9 µM 2,4 dichlorophenoxyacetic acid. After 2 weeks, the explants with somatic embryos and embryogenic tissue were bombarded with tungsten particles (M-25) carrying the plasmid pCambia3301 (containing the bar and uidA genes) using a high pressure helium microprojectile device. The bombarded explants were submitted to selection on medium containing 5 µM ammonium glufosinate herbicide as selective agent. After 6 months, putative transgenic embryos were transferred to a growth regulator-free medium for germination. The regenerated plantlets were β-glucuronidase (GUS) positive whereas no GUS activity was observed in non-transgenic controls. Incorporation of the bar gene into the genome was confirmed by PCR and Southern blot analysis of the regenerated transformed plants. Greenhouse grown transgenic coffee plants were found to withstand the recommended level of the herbicide Finale™ for weed control.This research was supported by the Consorcio Brasileiro de Pesquisa e Desenvolvimento do Cafe (CBP&D-Cafe).     

Green fluorescent protein labeling of primordial germ cells using a nontransgenic method and its application for germ cell transplantation in salmonidae

Transplanting primordial germ cells (PGCs) has a number of potential applications in fish bioengineering. Previously, we established a system to visualize live PGCs in the rainbow trout by introducing the green fluorescent protein (Gfp) gene driven by rainbow trout vasa gene regulatory regions. However, for PGC transplantation to be practically useful in aquaculture, visualization of PGCs using a nontransgenic technique is required. In this study, we demonstrate a method for labeling PGCs from various fish species by introducing chimeric RNAs composed of the Gfp coding region and vasa gene 3'-untranslated regions (UTRs); these sequences play a critical role in stabilizing mRNA in zebrafish PGCs. The GFP chimeric RNAs, including vasa 3'-UTR RNAs from rainbow trout, Nibe croaker, and zebrafish, were microinjected into the cytoplasm of fertilized eggs of several Salmonidae species. All the resulting embryos showed specific labeling in PGCs after the somatogenesis stage, which continued to be visible for at least 50 days. To apply this technique to PGC transplantation, PGCs labeled with chimeric RNA were microinjected into the peritoneal cavity of newly hatched salmonid embryos. The GFP labeling was sufficiently long-lived for the initial stage of donor PGC behavior to be followed in the recipient embryos. Importantly, donor PGCs from brown trout and masu salmon were incorporated into xenogeneic genital ridges in recipient rainbow trout. This nontransgenic method for labeling fish PGCs should be extremely useful for applications of PGC transplantation where the resulting progeny are to be released into the environment, such as PGC cryopreservation for fish stocks and surrogate brood stock technology.     

Feasibility of a nylon-mesh holder for vitrification of bovine germinal vesicle oocytes in subsequent production of viable blastocysts

To improve the feasibility of nylon-mesh holder for vitrification of bovine cumulus-oocytes complexes (GV-COCs) having germinal vesicle, this study was conducted to demonstrate effects of sugars and protocol of exposure in vitrification on subsequent in vitro maturation, ultrastructural changes, and in vitro development in bovine immature oocytes after cryopreservation using nylon mesh. Before vitrification, GV-COCs were exposed to the cryoprotectant, which was composed of 40% (v/v) ethylene glycol, 18% (w/v) Ficoll-70, and 0.3 M sucrose (EFS40) or 0.3 M trehalose (EFT40), either by single step or in a stepwise way. The maturation rates in the stepwise exposure with EFS40 or EFT40 were significantly higher (P < 0.05) compared with the corresponding rates in the single step. In the stepwise exposure, few abnormalities were observed compared with the single-step exposure, where most oocytes showed a highly vacuolated cytoplasm with many ruptured mitochondria. Cleavage rates in fertilized oocytes previously exposed stepwise to EFS40 or EFT40 were significantly higher than those exposed by the single-step procedure. The cleaved embryos derived from the stepwise exposure to EFS40 developed to blastocysts. After transfer of blastocysts derived from vitrified GV oocytes, a female calf was born. These results indicate that vitrification of large numbers of bovine GV-COCs using a nylon-mesh holder accompanied with stepwise exposure minimizes structural damage in organelles, resulting in yield of viable blastocysts following in vitro embryo production.     

Isolation and functional analysis of a chk2 homologue from Entamoeba histolytica

Mammalian Chk2 is a Ser/Thr kinase required for cell-division arrest induced by DNA damage. We found six new kinase genes of Entamoeba histolytica by analysis in silico. One of the kinase genes was a homologue of human chk2 gene. The chk2 homologue gene (Eh chk2) was expected to encode 398 amino acids and showed nearly 50% homology to human Chk2 in amino acid sequence. Eh chk2 had a catalytic domain of Ser/Thr kinase and a fork head-associated (FHA) domain that is highly conserved among Chk2 homologues in vertebrates. To examine the biological functions of Eh chk2, we synthesized Eh chk2 mRNA in vitro and injected it into immature frog eggs (Xenopus laevis oocytes) as a model system of cell division. Eh chk2 markedly delayed the cell division of frog eggs by disrupting transition of G2 phase to M phase. Eh chk2 also inhibited the activation of p42 MAPK and Cdc2 kinase which are representative events induced by cell division. These results suggest that Eh chk2 gene should be a cell-division regulator in E. histolytica.