Altered vestibular function in fetal and newborn rats gestated in space

Researchers evaluated vestibular development and function in rat pups flown during gestation on the NASA-NIH R1 and R2 missions. Fetal and postnatal vestibular function were examined. Altered vestibular-mediated responses in the experimental fetal pups are attributed to either direct effect of gravity on the vestibular system or indirect effects of microgravity transduced through the mother. The postnatal tests confirmed the hypothesis that the vestibular system continually adapts and responds to tonic stimulation.     

Cardiac atrial natriuretic peptide (ANP) in rat dams and fetuses developed in space (NIH-R1 and NIH-R2 experiments).

NIH-R1 and R2 missions, conducted by NASA, allowed us to study the effects of the microgravitational environment 1) on cardiac ANP in pregnant rats, spaceflown for 11 days and dissected after a 2-day readaptation to Earth's gravity, after natural delivery, and 2) on maturation of cardiac ANP system in rat fetuses developed for 11 days in space and dissected on the day of landing, 2 days before birth. Immunocytochemical and electron microscopy analyses showed a typical formation of ANP-containing granules in atrial myocytes, in both dams and fetuses. Using competitive RT-PCR and radioimmunoassays, we observed that, after 2 days of readaptation to Earth's gravity, cardiac ANP biosynthesis of rat dams flown in space was increased by about twice, when compared to Synchronous and Vivarium Control rats. More obviously, rat fetuses developed in space and dissected on the day of landing displayed an altered maturation of cardiac ANP, evidenced by an increased mRNA biosynthesis (by about 6 fold, p<0.05), whereas the cardiac ANP storage was slightly reduced (by about twice, p<0.05) in both Flight and Synchronous Control groups, in comparison with Vivarium Control rats. These last results suggest that ANP metabolism during development is impacted by the microgravitational environment, but also by the housing conditions designed for space flight.     

Molecular analysis of X-linked ichthyosis in Japan

BACKGROUND: X-linked ichthyosis (XLI) is an inherited skin disorder caused by a deficiency of steroid sulfatase (STS). The gene and protein of STS were examined in 19 Japanese patients with XLI. RESULTS: In Western blotting analysis, no cross-reacting peptide was detected in the patients' placenta, although a single band (63 kD) corresponding to STS in a normal subject was observed. Southern blotting was performed using EcoRI digests of cellular DNA from 13 XLI patients and full-length human STS cDNA as a probe. Normal males had bands of 20, 15, 10, 9.0, 6.1, 4.2, 2.6, and 1.5 kb. Twelve of the 19 patients had only 20- and 1.5-kb bands. Only one patient had the same band pattern as that of normal males. The STS gene was analyzed by PCR in 6 of the 19 patients. PCR amplification products were sequenced to analyze the STS gene. Two cases with one-base change in the STS gene and variation in amino acids H444R and E560P were found. Mutant STS cDNA was transfected into COS-1 cells and the STS enzyme activity was assayed. The enzyme activities were less than the minimum detection value of the detection system. CONCLUSIONS: These results suggest that XLI is mainly caused by an extensive deletion of the STS gene and that the PCR method is useful for detection of STS point mutations.     

Identification and mapping of random amplified polymorphic DNA (RAPD) markers linked to resistance against beet necrotic yellow vein virus (BNYVV) in Beta accessions

Molecular markers linked to resistance genes are useful to facilitate the introgression of one or more of these genes in breeding materials. Following the approach of bulked segregant analysis, RAPD markers linked to resistance genes against beet necrotic yellow vein virus were identified in the four Beta accessions Holly-1-4, R104, R128 and WB42. Two primers were found which generate RAPD markers tightly linked to resistance in segregating families of Holly-1-4, R104 and R128, indicating that the resistance genes in these accessions might be situated at the same locus. Other, specific, primers were identified which generate RAPD markers linked to resistance in each of these accessions. Short-range maps were established around the resistance locus in these accessions. For WB42, RAPD markers were only identified at a relatively large distance from the resistance gene. Conversion of three RAPD primers of Holly-1-4, R104 and R128 into STS primers resulted in STS markers which can be readily used for marker-assisted selection in breeding programmes. Received: 8 January 1996 / Accepted: 14 June 1996     

Characterization of the Kallikrein-Kinin System Post Chemical Neuronal Injury: An In Vitro Biochemical and Neuroproteomics Assessment

Traumatic Brain Injury (TBI) is the result of a mechanical impact on the brain provoking mild, moderate or severe symptoms. It is acknowledged that TBI leads to apoptotic and necrotic cell death; however, the exact mechanism by which brain trauma leads to neural injury is not fully elucidated. Some studies have highlighted the pivotal role of the Kallikrein-Kinin System (KKS) in brain trauma but the results are still controversial and inconclusive. In this study, we investigated both the expression and the role of Bradykinin 1 and 2 receptors (B1R and B2R), in mediating neuronal injury under chemical neurotoxicity paradigm in PC12 cell lines. The neuronal cell line PC12 was treated with the apoptotic drug Staurosporine (STS) to induce cell death. Intracellular calcium release was evaluated by Fluo 4-AM staining and showed that inhibition of the B2R prevented calcium release following STS treatment. Differential analyses utilizing immunofluorescence, Western blot and Real-time Polymerase Chain Reaction revealed an upregulation of both bradykinin receptors occurring at 3h and 12h post-STS treatment, but with a higher induction of B2R compared to B1R. This implies that STS-mediated apoptosis in PC12 cells is mainly conducted through B2R and partly via B1R. Finally, a neuroproteomics approach was conducted to find relevant proteins associated to STS and KKS in PC12 cells. Neuroproteomics results confirmed the presence of an inflammatory response leading to cell death during apoptosis-mediated STS treatment; however, a “survival” capacity was shown following inhibition of B2R coupled with STS treatment. Our data suggest that B2R is a key player in the inflammatory pathway following STS-mediated apoptosis in PC12 cells and its inhibition may represent a potential therapeutic tool in TBI.     

Up-regulation of myometrial RHO effector proteins (PKN1 and DIAPH1) and CPI-17 (PPP1R14A) phosphorylation in human pregnancy is associated with increased GTP-RHOA in spontaneous preterm labor

RHO GTP-binding proteins are important regulators of actin-myosin interactions in uterine smooth muscle cells. Active (GTP-bound) RHOA binds to RHO-associated protein kinase (ROCK1), which inhibits the myosin-binding subunit (PPP1R12A) of myosin light chain phosphatase, leading to calcium-independent increases in myosin light chain phosphorylation and tension, which are termed "calcium sensitization." The RHO effector protein kinase N (PKN1) also increases calcium sensitization by phosphorylating the protein kinase C (PRKCB)-dependent protein CPI-17 (PPP1R14A) to inhibit the PPP1c subunit of myosin phosphatase. Moreover, other RHO proteins, such as RHOB, RHOD, and their effectors (DIAPH1 and DIAPH2), may modulate PKN1/ ROCK1 signaling to effect changes in myosin phosphatase activity and myosin light chain phosphorylation. The increases in contractile activity observed in term and preterm labor may be due to an increase in RHO activity and/or changes in RHO-related proteins. We found that the RHOA and RHOB mRNA levels in the myometrium were increased in pregnancy, although the expression levels of the RHOA and RHOB proteins did not change with pregnancy or labor. GTP-bound RHOA was increased in pregnancy, and this increase was significant in spontaneous preterm labor myometrium. PKN1 expression and PPP1R14A phosphorylation were dramatically increased in the pregnant myometrium. We also observed increases in DIAPH1 expression in spontaneous term and preterm labor myometrial tissues. The present study shows that human pregnancy is characterized by increases in PKN1 expression and PPP1R14A phosphorylation in the myometrium. Moreover, increases in GTP-bound RHOA and DIAPH1 expression may contribute to the increase in uterine activity in idiopathic preterm labor.     

Changes in plasma levels of PGF2 alpha and PGI2 metabolites at and after delivery at term

Plasma levels of 6-keto-PGF1 alpha and 13,14-dihydro-15-keto-PGF2 alpha (PGFM) were measured by high pressure liquid chromatography and radioimmunoassay during and up to 48 hours after term labor. PGFM levels increased during labor to reach values which at full dilatation, at delivery of the fetal head and at placental separation were each time higher than levels obtained earlier. In all women (n = 10) PGFM levels reached their maximum and started to decline within 10 min. after placental separation. Levels decreased to prelabor values within 2 to 3 hours after delivery and no temporary increases were observed within the first 2 days. Levels of 6-keto-PGF1 alpha on the other hand, showed no consistent trends throughout labor and the early puerperium. The observed changes are believed to be of relevance for ensuring adequate hemostasis after birth.     

县乡两级医院自控硬膜外分娩镇痛对产程及分娩结局的影响

目的观察县乡两级医院产妇行自控硬膜外分娩镇痛对产程及分娩结局的影响,评价县乡两级医院产妇行自控硬膜外分娩镇痛的安全性与可行性。方法选择足月单胎头位妊娠并经阴道试产的初产妇200例,均无硬膜外镇痛禁忌证,根据分娩镇痛要求,将其纳入镇痛组(P组,100例)及对照组(C组,100例)。记录两组第一、二产程及总产程时间,分娩方式,产后出血及催产素使用率,新生儿娩出后1、5min Apgar评分,分娩镇痛的不良反应等。结果 P组较C组的第二产程和总产程显著延长,催产素使用率P组明显高于C组;两组第一产程中转剖宫产率,器械助产率,羊水胎粪污染率,新生儿娩出后1、5min Apgar评分,分娩镇痛的不良反应均无统计学差异。结论硬膜外分娩镇痛可使第二产程及总产程延长、催产素使用率增加,但不增加急诊剖宫产率和经阴道器械助产率,明显降低了社会因素手术率,对分娩结局无不良影响,用于县乡两级医院产妇是安全可行的。     

徒手转胎纠正128 例头位难产的临床效果观察

目的:探讨应用徒手转胎术纠正头位难产的临床效果,为提高经阴道分娩的成功率提供参考。方法:选取2014年6月-2015年12月我院收治的128例头位难产患者作为研究对象,随机为研究组和对照组,每组64例。对照组在第二产程初期采用胎头吸引术,研究组患者通过徒手旋转胎头术调整胎头方位。观察并比较两组产后出血量、第二产程时间及新生儿神经行为测定(NBNA)评分。结果:研究组患者阴道自然分娩成功率(95.31%)高于对照组(73.44%),差异有统计学意义(P0.05);研究组产后出血量和第二产程时间均显著低于对照组,差异具有统计学意义(P0.05);研究组新生儿NBNA评分高于对照组,但两组之间比较,差异无统计学意义(P0.05)。结论:徒手转胎术与胎头吸引术两种干预方式对新生儿行为神经的影响无明显区别,但徒手转胎术可以有效缩短头位难产产妇的产程,并能显著提高阴道分娩率,有益于母婴的预后,值得临床推广应用。     

Apoptosis of mink lung epithelial cells by co-treatment of low-dose staurosporine and transforming growth factor-beta1 depends on the enhanced TGF-beta signaling and requires the decreased phosphorylation of PKB/Akt

We demonstrate how co-treatment of low-dose staurosporine (STS) and TGF-beta1, which alone have little effect on cell death, markedly induces apoptosis in Mv1Lu mink lung epithelial cells, but not in its clonal variant R1B cells lacking functional TGF-beta signaling. This process was associated with mitochondria-dependent apoptosis and the enhanced TGF-beta/Smad signaling in Mv1Lu cells. When R1B cells were infected with adenovirus carrying wild-type ALK5, a functional TGF-beta type I receptor gene, the apoptotic cell death was significantly restored in these cells following co-treatment of low-dose STS and TGF-beta1. Treatment of Mv1Lu cells with both low-dose STS and TGF-beta1 decreased the activity of phospho-Akt, which is involved in cell survival signal. In addition, pre-treatments of PI3 kinase inhibitors, LY294002 and wortmannin, further increased the apoptosis of MvlLu cells induced by co-treatment of low-dose STS and TGF-beta1. And overexpression of constitutively active Akt (myr-Akt) using adenoviral expression system inhibited the apoptotic cell death of Mv1Lu cells by about 50% upon co-treatment of low-dose STS and TGF-beta1. These results suggest that co-treatment of low-dose STS and TGF-beta1 induces apoptosis of mink lung epithelial cells by enhancing TGF-beta signaling and in part suppressing cytoprotective signaling.     

Estrone 3-sulfate mimics,inhibitors of estrone sulfatase activity: homology model construction and docking studies

Steroid sulfatase (STS) is a new target for the endocrine therapy of breast cancer. To ascertain some of the requirements for inhibition of estrone sulfatase activity, a number of novel analogues of estrone 3-O-sulfate possessing sulfate surrogates were synthesized and evaluated as inhibitors of estrone sulfatase (STS) in comparison to a lead inhibitor, estrone-3-O-methylthiophosphonate (E1-3-MTP). Using a selective enzyme digestion, one of the diastereoisomers of this compound, (R(p))-E1-3-MTP, could be prepared and evaluated. From structure-activity studies, we show that chirality at the phosphorus atom, hydrophobicity, basicity, size, and charge all influence the ability of a compound to inhibit estrone sulfatase activity. Of these, hydrophobicity seems to be the most important since simple, active nonsteroidal inhibitors, based on 5,6,7,8-tetrahydronaphth-2-ol (THN), can be prepared, provided that they are lipophilic enough to partition into a nonpolar environment. Also, a negatively charged group is favorable for optimal binding, although it appears that the presence of a potentially cleavable group can compensate for lack of charge in certain cases. A homology model of STS has been constructed from the STS sequence, and molecular docking studies of inhibitors have been performed to broaden the understanding of enzyme/inhibitor interactions. This model clearly shows the positions of the key amino acid residues His136, His290, Lys134, and Lys368 in the putative catalytic region of the formylglycine at position 75, with residues Asp35, Asp36, Asp342, and Gln343 as ligands in the coordination sphere of the magnesium ion. Docking studies using the substrate and estrone-3-sulfate mimics that are active inhibitors indicate they are positioned in the area of proposed catalysis, confirming the predictive power of the model.     

Use of RAPD- and STS-analysis for marking genes of a homeologic group from chromosome 5 of common wheat

The search for STS (sequence-tagged site) and RAPD (random amplified polymorphic DNA) markers tightly linked to some genes of homeologous group 5 chromosomes of common wheat Triticum aestivum L., more specifically, awns inhibitor genes (B1), vernalization response gene (Vrn1), and homeologous chromosome pairing gene (Ph1), was conducted. To estimate the linkage of the gene with the marker, wheat lines marked with recessive alleles b1 and vrn1 were used. RELP (restriction fragment length polymorphism) and SSR (simple sequence repeat) analyses of isogenic wheat lines were conducted to characterize the chromosomal region transferred to the isogenic line from the donor parent. In RAPD analysis of isogenic wheat lines marked with recessive alleles b1 and vrn1, 95 arbitrary primers were used. To develop STS markers, analysis of the primary structure of RELP markers Xpsr426 and Xcdo504, tightly linked to the Vrn1 gene, and the Xpsr1201 marker, located at the Ph1 locus, was carried out. Two markers that are tightly linked to the Vrn1 gene (5AL)--RAPD marker Xr405 and STS marker Xsts426--were obtained in this work. In addition, there is every reason to believe that Xsts426 can be used as a PCR marker of genes Vrn2 (5BL) and Vrn3 (5DL), while Xsts1201, of the gene Ph1 (5BL).     

Observations of parturition among captive patas monkeys (Erythrocebus patas)

Six cases of labor and delivery by laboratory-housed patas monkeys (Erythrocebus patas) are described. Five of the births occurred during the daytime, and the sixth occurred just after the usual “lights out” time. Although it was impossible to determine the actual onset of labor, the final 1.5–0.5 hr before parturition were observed in all cases. Throughout labor, females tended to begin a contraction every 2–4 min and contractions usually lasted 35–60 sec. Females did not show common patterns of change in contraction frequency or duration as birth neared. It was not possible to describe patas monkey labor and delivery in terms of “stages.” Postpartum, females typically began cleaning their infants within 90 sec and picked them up within 5 min. Placental delivery times varied among females, as did the extent of placental consumption. One “mother-only” - reared female proved to be an inadequate mother.     

Mitotic activity of the lymphocytes of the thymus cortex in hypokinesia during the period of readaptation

The changes in the weight and mitotic index were studied in the cortex of the thymus of Wistar rats during 10-day hypokinesia and 10-day readaptation (restoration). 24 hours after immobilization of the animals the mitotic index was 2 times as lower. No complete readaptation was attained during 10-day hypokinesia. No readaptation was attained during 10-day hypokinesia. In readaptation the stage of secondary stress was found (the mitotic index was 3.5 times as reduced), the stage of genuine restoration being revealed after 10 days.     

Marker assisted detection of gene (1Dx5) and translocation (1B/1R) in wheat genotypes

Detection of 1Dx5 gene and presence of 1B/1R wheat rye translocation were studied in nineteen elite Indian wheat genotypes using AS-PCR and STS markers, respectively. Fifteen genotypes had 1B/1R translocation whereas ten showed presence of 1Dx5 gene. More than 50 per cent of the genotypes tested were found positive for both 1Dx5 and 1B/1R translocation. The results are in conformity with HMW glutenin SDS-PAGE profile for 1Dx5 and cytological observations for 1B/1R translocation.     

Inactivation of recombinant human steroid sulfatase by KW-2581

Steroid sulfatase (STS) catalyses the hydrolysis of the sulfate esters of 3-hydroxy steroids, which are inactive transport or precursor forms of the active 3-hydroxy steroids. STS inhibitors are expected to block the local production and, consequently to reduce the active steroid levels; therefore, they are considered as potential new therapeutic agents for the treatment of estrogen- and androgen-dependent disorders such as breast and prostate cancers. KW-2581 is a novel steroidal STS inhibitor. In the present study, we found KW-2581 inhibited recombinant human STS (rhSTS) activity with an IC(50) of 2.9 nM when estrone sulfate was used as a substrate. The potency of KW-2581 was approximately 5-fold higher than that of a non-steroidal STS inhibitor, 667 COUMATE. KW-2581 was able to equally inhibit rhSTS activity when dehydroepiandrosterone sulfate was used as another substrate. KW-2581 inhibited rhSTS activity in a time- and concentration-dependent manner (k(inact), 0.439 min(-1); K(i, app), 15 nM), suggesting that it is an active site-directed irreversible inhibitor. Both decrease of KW-2581 concentration and increase of the des-sulfamoylated form's concentration were simultaneously observed during the reaction in a time-dependent manner with corresponding to the decrease of STS activity. Our findings for the first time demonstrated the production of des-sulfamoylated form of the compound as a consequence of STS inactivation.     

小麦品系5R618抗叶锈基因的初步定位

5R618是高抗叶锈病小麦品系。为了确定该品系所携带的抗叶锈基因,以5R618与感病小麦品种郑州5389杂交获得F1,自交获得F2分离群体以及F2∶3家系,用叶锈菌生理小种THJP对亲本、F2分离群体以及F2∶3家系进行叶锈抗性鉴定,然后进行分子标记分析。结果显示,5R618对生理小种THJP的抗病性由1对显性基因控制,该基因暂命名为Lr5R。经过亲本和抗感池间分子标记筛选以及F2∶3家系的标记检测,Lr5R定位于染色体3DL上,barc71和STS24-16是Lr5R最近的2个标记,遗传距离分别为0.9 c M和2.1 c M。     

Deletion pattern of the STS gene in X-linked ichthyosis in a Mexican population

BACKGROUND: X-linked ichthyosis (XLI) is an inherited disorder due to steroid sulfatase deficiency (STS). Most XLI patients (>90%) have complete deletion of the STS gene and flanking sequences. The presence of low copy number repeats (G1.3 and CRI-S232) on either side of the STS gene seems to play a role in the high frequency of these interstitial deletions. In the present study, we analyzed 80 Mexican patients with XLI and complete deletion of the STS gene. MATERIALS AND METHODS: STS activity was measured in the leukocytes using 7-[(3)H]-dehydroepiandrosterone sulfate as a substrate. Amplification of the regions telomeric-DXS89, DXS996, DXS1139, DXS1130, 5' STS, 3' STS, DXS1131, DXS1133, DXS237, DXS1132, DXF22S1, DXS278, DXS1134-centromeric was performed through PCR. RESULTS: No STS activity was detected in the XLI patients (0.00 pmoles/mg protein/h). We observed 3 different patterns of deletion. The first two groups included 25 and 32 patients, respectively, in which homologous sequences were involved. These subjects showed the 5' STS deletion at the sequence DXS1139, corresponding to the probe CRI-S232A2. The group of 32 patients presented the 3' STS rupture site at the sequence DXF22S1 (probe G1.3) and the remaining 25 patients had the 3' STS breakpoint at the sequence DXS278 (probe CRI-S232B2). The third group included 23 patients with the breakpoints at several regions on either side of the STS gene. No implication of the homologous sequences were observed in this group. CONCLUSION: These data indicate that more complex mechanisms, apart from homologous recombination, are occurring in the genesis of the breakpoints of the STS gene of XLI Mexican patients.