Rapid determination of parvalbumin amino acid sequence from Rana catesbeiana (pI 4.78) by combination of ESI mass spectrometry, protein sequencing, and amino acid analysis

The complete amino acid sequence of beta-type parvalbumin (PA) from bullfrog Rana catesbeiana (pI 4.78) was determined by tandem mass spectrometry in combination with amino acid analysis and peptide sequencing following Arg-C and V(8) protease digestion. The primary structure of the protein was compared with that of beta-type PA from R. esculenta (pI 4.50), with which it is highly homologous. Compared with R. esculenta beta-type PA4.50, R. catesbeiana beta-type parvalbumin (PA 4.78) differed in 15 out of 108 amino acid residues (14% displacement), PA4.78 had Cys at residue 64 and was acetylated at the amino terminus, but 25 residues of the carboxyl terminus were completely conserved. Several amino acid displacements were found between residues 51 and 80 (30% displacement), although the functionally important sequence of PA was completely conserved. The amino acids residues of putative calcium-binding sites were Asp-51, Asp-53, Ser-55, Phe-57, Glu-59, Glu-62, Asp-90, Asp-92, Asp-94, Lys-96, and Glu-101, which were conserved in all a and b-types of R. catesbeiana as well as other parvalbumins. In addition, Arg-75 and Glu-81, which are thought to form a salt bridge located in the interior of the molecule [Coffee, C.J. et al. (1976) Biochim. Biophys. Acta 453, 67-80], were also conserved in PA4.78.     

Scanning by DOVAM-S detects all unique sequence changes in blinded analyses: evidence that the scanning conditions are generic

The [detection of virtually all mutations]-SSCP (DOVAM-S) is a highly sensitive variant of single strand conformation polymorphism (SSCP). Mutations in the factor IX gene were used to find a set of five SSCP conditions that detects virtually all mutations. A blinded analysis of the factor IX gene in patients with hemophilia B detected 82 of 82 unique mutations. Since the method was developed and tested on the factor IX gene, it is possible that the conditions selected work more efficiently in the factor IX gene than in other genes. To test the general applicability of the conditions under which DOVAM-S detected all mutations in this gene, blinded analyses were performed in the human factor VIII and ataxia-telangiectasia (ATM) genes. Segments were amplified individually, combined into groups of 16 to 18 amplified segments and electrophoresed in five different nondenaturing conditions of varying matrices, buffers, temperatures and additives. Blinded analyses were performed in 92 samples from patients with hemophilia A (factor VIII gene) and 19 samples from A-T patients (ATM gene). Combined with an earlier blinded analysis in the factor IX gene, all of the 250 mutations and polymorphisms (180 of which are unique) were detected in both analyses. For two, three and four joint conditions, the average detection frequency ranged from 77%-97%, 91%-100% and 95%-100%, respectively. For each of the genes, one mutation may have been missed if only four conditions were used. With DOVAM-S, approximately 500 kb of autosomal sequence can be scanned in five gels with virtually 100% detection of mutations within the scanned region. The detection of 180 out of 180 unique sequence changes implies that DOVAM-S detects at least 96.5% (P = 0.03) of mutations. Blinded analyses that detect 400 unique sequence changes are required to determine that a scanning method detects at least 98.5% of mutations.     

Contribution of diet to the dosing time-dependent change of vitamin D3-induced hypercalcemia in rats

We have recently reported that the degree of hypercalcemia as an adverse effect induced by a single large-dose of active vitamin D3 varied with its dosing time without alteration in therapeutic effect for secondary hyperparathyroidism in patients with chronic renal failure. The present study was conducted to elucidate an effect of intestinal calcium (Ca) absorption on the chronopharmacological profiles of vitamin D3. 1, 25-dihydroxy-cholecalciferol (D3, 2 microg/kg) or vehicle alone was orally administered at two different times (2 and 14 hours after lights on; HALO) to male Wistar rats (n= 10) kept in rooms with a 12 h light-dark cycle. Blood samples for serum Ca concentration were taken before and 3, 6, 9, and 12 hours after the administration. Urine was collected for 6 hours after dosing. An identical protocol was repeated using the same animals after 16 hours fasting by a cross-over fashion. Under free-fed condition, basal concentration of serum Ca was higher at a resting period (lights on) than during an active period (lights off). Serum Ca reached its peak at 6 hours after dosing in both timings, while the value was significantly higher in the 2 HALO trial than in the 14 HALO trial. Area under the serum Ca concentration-time curve from 0 to 12 hours (AUC0-12h) and urinary excretion of Ca for 6 hours were also significantly higher in the 2 HALO trial than in the 14 HALO trial. When fasted, basal Ca concentration was reduced compared with the free-fed condition, while the daily variation was maintained. Serum Ca concentration profiles from 3 to 12 hours after dosing were not significantly different between the 2 HALO and 14 HALO trials. The AUC0-12h of serum Ca or its urinary excretion was not different between both trials. Serum concentrations of parathyroid hormone and total protein, measured before and 6 hours after the dosing were not affected by the dosing schedule. We have concluded that intestinal Ca absorption is a major factor for the chronopharmacological phenomenon of D3-induced hypercalcemia in intact rats, while intestinal and renal involvement may be relatively small in the mechanism of the intrinsic diurnal variation of serum Ca.     

Chronopharmacological study of furosemide; (V). Influence of pretreatment with 6-hydroxydopamine

Our previous indirect evidences suggested that the adrenergic nervous system is involved in the mechanisms responsible for the time-dependent changes in the effects of furosemide in Wistar rats. In the present study, the role of this system was examined more directly by means of 6-hydroxydopamine-induced sympathectomy. Thirty mg/kg of 6-hydroxydopamine hydrobromide (6-OH-DA) (n = 9) or its vehicle alone (n = 9) was injected intra-arterially (i.a.) twice in Wistar rats. Furosemide (5 mg/kg) was administered i.a. at 1000 hrs (03HALO*) or at 2200 hrs (15HALO). Urine was collected for 60 min after the drug and urinary excretion of sodium and furosemide were determined respectively. Urine volume and urinary excretion of sodium and furosemide were significantly greater at 1000 hrs (03HALO) than at 2200 hrs (15HALO) in the vehicle-injected rats as observed in the previous study. However these administration-time-dependent changes in the effects of furosemide disappeared in the rats with 6-OH-DA. Thus, the present study provides more direct evidence and supports our original hypothesis concerning the mechanisms of this chronopharmacological phenomenon of the agent. Since 6-OH-DA does not penetrate the central nervous system from the blood stream, the present data also indicate that the peripheral adrenergic system is involved in this event.     

多重聚合酶链反应与荚膜肿胀试验对肺炎链球菌血清分型的比较研究

为评估多重聚合酶链反应（PCR ）对肺炎链球菌血清分型的可行性,分别采用多重PCR和荚膜肿胀试验对568株肺炎链球菌进行血清分型,并对分型结果进行比较分析。结果显示,568株肺炎链球菌中,213株通过荚膜肿胀试验分出16个血清群,主要有血清群19（23．1％,131／568）、6（5．3％,30／568）、23（1．6％,9／568）、14（1．4％,8／568）、9（1．1％,6／568）、15（1．1％,6／568）等,分型率为37．5％（213／568）;356株通过多重PCR分出21个血清群,主要有血清群19（27．8％,158／568）、23（8．5％,48／568）、6（7．4％,42／568）、14（4．4％,25／568）、3（4．2％,24／568）、15（3．5％,20／568）等,分型率为62．7％（356／568）。荚膜肿胀试验鉴定出血清群4和18,但多重PCR未能鉴定;多重PCR鉴定出血清群5、12、35、16、17和22,但荚膜肿胀试验未能鉴定。多重PCR与荚膜肿胀试验对19F、19A血清型的鉴定无显著差异。结果提示,这2种方法对肺炎链球菌血清分型结果有差别,多重PCR的分型率高于荚膜肿胀试验。对来源复杂的标本进行肺炎链球菌血清分型,2种方法可相互补充,以提高分型率。     

红芽芋球茎片两步法离体快繁及其再生苗生理和光合特性研究

以江西铅山红芽芋(Colocasia esculenta L.Schott var.cormosus‘Hongyayu’)试管苗为材料,建立了芋球茎片两步法离体快繁体系,并对其再生苗的形态指标、染色体数目、生理和光合特性以及叶绿素荧光特性进行了检测。结果表明:(1)红芽芋球茎片单芽诱导的最佳培养基为MS+KT 2 mg/L+6-BA 1 mg/L+NAA0.1mg/L,诱导培养30d后将单芽从球茎片上分离,再接种到生根培养基(MS+KT 2mg/L+NAA 0.1mg/L)上培养30d即可形成完整植株,移栽成活率高达98%;(2)由球茎片单芽、丛生芽、不定芽离体快繁获得的红芽芋再生苗在形态指标、叶下表皮气孔参数、染色体数目、生理生化指标以及叶片光合特性参数和叶绿素荧光特性方面均无显著差异。说明红芽芋球茎片两步法离体培养的再生苗繁殖系数高、染色体数目稳定,该离体快繁体系可应用于江西铅山红芽芋的工厂化生产。     

Lens morphogenesis is dependent on Pax6‐mediated inhibition of the canonical Wnt/beta‐catenin signaling in the lens surface ectoderm

Lens formation in mouse is critically dependent on proper development of the retinal neuroectoderm that is located close beneath the head surface ectoderm. Signaling from the prospective retina triggers lens‐specific gene expression in the surface‐ectoderm. Supression of canonical Wnt/β‐catenin signaling in the surface ectoderm is one of the prerequisites for lens development because, as we show here, ectopic Wnt activation in the retina and lens abrogates lens formation. Wnt inhibiton is mediated by signals coming from the retina but its exact mechanism is unknown. We show that Pax6 directly controls expression of several Wnt inhibitors such as Sfrp1, Sfrp2, and Dkk1 in the presumptive lens. In accordance, absence of Pax6 function leads to aberrant canonical Wnt activity in the presumptive lens that subsequently impairs lens development. Thus Pax6 is required for down‐regulation of canonical Wnt signaling in the presumptive lens ectoderm. genesis 48:86–95, 2010. © 2009 Wiley‐Liss, Inc.     

A mouse line expressing Sall1‐driven inducible Cre recombinase in the kidney mesenchyme

Sall1 is expressed in the metanephric mesenchyme in the developing kidney, and mice deficient in Sall1 show kidney agenesis or dysgenesis. Sall1 is also expressed elsewhere, including in the limb buds, anus, heart, and central nervous system. Dominant‐negative mutations of Sall1 in mice and humans lead to developmental defects in these organs. Here, we generated a mouse line expressing tamoxifen‐inducible Cre recombinase (CreER^T2) under the control of the endogenous Sall1 promoter. Upon tamoxifen treatment, these mice showed genomic recombination in the tissues where endogenous Sall1 is expressed. When CreER^T2 mice were crossed with the floxed Sall1 allele, tamoxifen administration during gestation led to a significant decrease in Sall1 expression and small kidneys at birth, suggesting that Sall1 functions were disrupted. Furthermore, Sall1 expression in the kidney was significantly reduced by neonatal tamoxifen treatment. The Sall1CreER^T2 mouse is a valuable tool for in vivo time‐dependent and region‐specific knockout and overexpression studies. genesis 48:207–212, 2010. © 2010 Wiley‐Liss, Inc.     

不明原因男性不育人群中睾丸特异性乳酸脱氢酶基因突变的发现及其意义

为了研究睾丸特异性乳酸脱氢酶,即乳酸脱氢酶C4(LDH-C4)基因突变在男性不育发病中的作用,利用LDH-C4特异性底物对100名不明原因男性不育症患者的精子LDH-C4进行活性显色,用变性高效液相色谱(DHPLC)技术对LDH-C4活性低下的患者进行LDHC基因PCR产物的突变筛查,对DHPLC峰形异常的PCR产物进行序列测定.筛选到一组精子LDH-C4活性明显下降的患者,其中1名患者的LDHC基因PCR产物在DHPLC中呈异常洗脱峰.对这一PCR产物进行序列测定,发现患者LDHC基因第5外显子的115位碱基发生了T→A的杂合改变(GenBank登录号GU479375),该突变使LDHC基因的178位密码子由原来的TTG(编码亮氨酸)变为TAG(终止密码子),形成截短的C亚基.T克隆-测序进一步证实了该无义突变的杂合状态.这是在人类LDHC基因上发现的第一个突变,提示LDHC基因突变可能是男性不育发病的原因之一.