Chorioallantoic placenta defects in cloned mice

Somatic cell nuclear transfer technology has been applied to produce live clones successfully in several mammalian species, but the success rates are very low. In mice, about half of the nuclear transfer embryos undergo implantation, but very few survive to term. We undertook detailed histological analyses of placentas from cloned mouse embryos generated from cumulus cells at 10.5 dpc of pregnancy, by which stage most clones have terminated their development. At 10.5 dpc, the extraembryonic tissues displayed several defined histological patterns, each reflecting their stage of developmental arrest. The most notable abnormality was the poor development of the spongiotrophoblast layer of diploid cells. This is in contrast to the placental hyperplasia frequently observed in somatic clones at 12.5 dpc or later stages. A variety of structural abnormalities were also observed in the embryos. Both placental and embryonic defects likely contribute to the low success rate of the mouse clones.     

Amino acid substitutions in the s2 region enhance severe acute respiratory syndrome coronavirus infectivity in rat angiotensin-converting enzyme 2-expressing cells

To clarify the molecular basis of severe acute respiratory syndrome coronavirus (SARS-CoV) adaptation to different host species, we serially passaged SARS-CoV in rat angiotensin-converting enzyme 2 (ACE2)-expressing cells. After 15 passages, the virus (Rat-P15) came to replicate effectively in rat ACE2-expressing cells. Two amino acid substitutions in the S2 region were found on the Rat-P15 S gene. Analyses of the infectivity of the pseudotype-bearing S protein indicated that the two substitutions in the S2 region, especially the S950F substitution, were responsible for efficient infection. Therefore, virus adaptation to different host species can be induced by amino acid substitutions in the S2 region.     

Expression of low density lipoprotein receptor gene in human placenta during pregnancy

Mammalian cells require cholesterol as a structural component of plasma membranes. It is also required for placental steroid synthesis. De novo synthesis of cholesterol is limited in human placenta and cholesterol is obtained mainly from plasma low density lipoprotein (LDL). Cholesterol delivery from LDL is mediated by receptor-mediated uptake and the receptor amount is the most important factor for cellular delivery. Thus, the regulation of receptor synthesis is important for placental development and function. Since the regulation of LDL receptor gene expression has not been studied in human placenta, LDL receptor mRNA was measured in placentae of 5-40 weeks of gestation by hybridization of RNA with 32P-labeled cDNA for human LDL receptor. Two mRNA species for LDL receptor were demonstrated by Northern blot analysis. The longer mRNA [5.3 kilobases (kb)] was much more abundant than the shorter mRNA (3.7 kb). The amount of 5.3 kb mRNA was highest early in gestation and decreased during pregnancy. However, the amount of 3.7 kb mRNA did not change appreciably during gestation. Dot blot analysis of 26 placental mRNAs obtained from various stages of gestation revealed a negative correlation between LDL receptor mRNA and gestation (r = -0.76, P less than 0.001). Considering the rapid growth of the trophoblast during gestation, especially in the first and the second trimester, increased expression of the LDL receptor gene and subsequent translation are expected for efficient cholesterol uptake to provide a sufficient substrate for cell growth. Possible mechanisms for the appearance of two mRNA species for LDL receptor are also discussed.     

Echosounding observations of coverage,height, PVI,and biomass of submerged macrophytes in the southern basin of Lake Biwa,Japan

We have developed a procedure to process echosounding data to map the distribution of submerged aquatic macrophytes in the southern basin of Lake Biwa, a water body that has a surface area of 52 km² and a mean depth of 4 m. Echosounding observations were made along 27 transect lines spaced at 500-m intervals on August 4 and September 2 and 30, 2003. Quantitative vegetation data including percent coverage, mean vegetation height, and percent vegetation infestation were directly determined using image data from the echosounder recorded digitally on videotape. Based on the image data from an echosounder, a regression model was developed for estimating biomass of submerged macrophytes. The regression model using the total echo strength as the explanatory variable could reliably estimate macrophyte biomass up to 300 g m⁻². Distribution maps of macrophyte height and biomass suggest that the recent summer decline of submerged macrophytes started earlier in shallow areas (<3 m of depth) than deep areas (>4 m) in the southern basin of Lake Biwa.     

Involvement of placental peptidases associated with renin-angiotensin systems in preeclampsia

Preeclampsia is characterized by pregnancy-induced hypertension accompanied with protein urea and generalized edema. Preeclampsia develops during the second half of pregnancy and resolves postpartum promptly, implicating the placenta as a primary cause in the disorder. Normal pregnancy is associated with reductions in arterial pressure and attenuated pressor response to exogenous infused angiotensin II (ANG II). In contrast, women with preeclampsia show the similar sensitivity to the pressor effect of ANG II as do non-pregnant women. To elucidate the involvement of placental peptidases associated with renin-angiotensin systems, we determined the localization of angiotensin-converting enzyme (ACE) and aminopeptidase A (AP-A), ANG II degrading enzyme, in the placenta and compared the expression of mRNA and protein in uncomplicated and preeclamptic placenta. In addition, AP-A expression in trophoblastic cells treated with ANG II and ACE expression in HUVECs under hypoxic condition were analyzed, respectively. The expression of both peptidases in the preeclamptic placenta was significantly higher than those from uncomplicated. ACE was primarily localized to venous endothelial cells of stem villous whereas AP-A expression was recognized in the trophoblast and pericytes of fetal arterioles and venules within stem villous. Hypoxia induced ACE expression in HUVECs while both hypoxia and ANG II evoked AP-A expression in trophoblast. These results suggested that hypoxic condition in preeclampsia induces ACE activation in feto-placental unit to maintain the fetal hemodynamics and placental AP-A plays a role as a component of the barrier of ANG II between mother and fetus.     

Selectivity improvement of an azole inhibitor of CYP707A by replacing the monosubstituted azole with a disubstituted azole

The plant growth-retardant uniconazole (UNI), a triazole inhibitor of gibberellin biosynthetic enzyme (CYP701A), inhibits multiple P450 enzymes including ABA 8′-hydroxylase (CYP707A), a key enzyme in ABA catabolism. Azole P450 inhibitors bind to a P450 active site by both coordinating to the heme-iron atom via sp² nitrogen and interacting with surrounding protein residues through a lipophilic region. We hypothesized that poor selectivity of UNI may result from adopting a distinct conformation and orientation for different active sites. Based on this hypothesis, we designed and synthesized novel UNI analogs with a disubstituted azole ring (DSI). These analogs were expected to have higher selectivity than UNI because the added functional group may interact with the active site to restrict orientation of the molecule in the active site. DSI-505ME and DSI-505MZ, which have an imidazolyl group with a methyl 5-acrylate, strongly inhibited recombinant CYP707A3, with no growth-retardant effect.     

Arabidopsis CYP90B1 catalyses the early C-22 hydroxylation of C27, C28 and C29 sterols

Arabidopsis dwf4 is a brassinosteroid (BR)-deficient mutant, and the DWF4 gene encodes a cytochrome P450, CYP90B1. We report the catalytic activity and substrate specificity of CYP90B1. Recombinant CYP90B1 was produced in Escherichia coli, and CYP90B1 activity was measured in an in vitro assay reconstituted with NADPH-cytochrome P450 reductase. CYP90B1 converted campestanol (CN) to 6-deoxocathasterone, confirming that CYP90B1 is a steroid C-22 hydroxylase. The substrate specificity of CYP90B1 indicated that sterols with a double bond at positions C-5 and C-6 are preferred substrates compared with stanols, which have no double bond at the position. In particular, the catalytic efficiency (k(cat)/K(m)) of CYP90B1 for campesterol (CR) was 325 times greater than that for CN. As CR is more abundant than CN in planta, the results suggest that C-22 hydroxylation of CR before C-5alpha reduction is the main route of BR biosynthetic pathway, which contrasts with the generally accepted route via CN. In addition, CYP90B1 showed C-22 hydroxylation activity toward various C(27-29) sterols. Cholesterol (C27 sterol) is the best substrate, followed by CR (C28 sterol), whereas sitosterol (C29 sterol) is a poor substrate, suggesting that the substrate preference of CYP90B1 may explain the discrepancy between the in planta abundance of C27/C28/C29 sterols and C27/C28/C29 BRs.