Calcium binding mode of gamma-carboxyglutamic acids in conantokins.

Conantokin-T (con-T) and conantokin-G (con-G) are two highly homologous peptide toxins found in Conus venom. The former is a 21-residue peptide with four gamma-carboxyglutamic acid (Gla) residues (at positions 3, 4, 10 and 14), while the latter is a 17-residue peptide with five gamma-carboxyglutamic acid residues (at positions 3, 4, 7, 10 and 14). Despite the apparent similarity in number and relative positions of the gamma-carboxyglutamic acid residues, (113)Cd-NMR studies indicated a distinct metal binding behavior for con-G and con-T. There appears to be four binding sites in con-G in contrast to one metal binding site in con-T. To elucidate the mode of calcium binding by the gamma-carboxyglutamic acid residues in these conantokins, we designed various analogous peptides with their gamma-carboxyglutamic acid replaced by other amino acid residues. (113)Cd-NMR experiments on conantokin analogues reveal that the major difference in the number of metal binding sites between con-G and con-T is due to the residue at position 7. We also performed molecular simulations to calculate the relative binding free energies of several potential binding sites. Based on our theoretical and experimental results, we propose a 'four-site' binding model for conantokin-G and a 'single-site' binding model for conantokin-T.     

Prenatal diagnosis of Pallister-Killian syndrome and literature review

Pallister-Killian syndrome (PKS) is a rare sporadic genetic disorder usually caused by mosaicism of an extra isochromosome of 12p (i(12p)). This retrospective study analysed the prenatal ultrasound manifestations and molecular and cytogenetic results of five PKS foetuses. Samples of amniotic fluid and/or cord blood, skin biopsy and placenta were collected. Conventional karyotyping and single nucleotide polymorphism array (SNP array) were performed on all the amniotic fluid or cord blood samples. Copy number variants sequencing (CNV-seq) and fluorescence in situ hybridization (FISH) were also used for the validation for one foetus. All the five foetuses were from pregnancies with advanced parental age. Two foetuses involved structural abnormalities and one foetus had only soft markers, all of which included increased nuchal translucency. The rest two foetuses had normal ultrasounds in the second trimester, which has rarely been reported before. The karyotype revealed typical i(12p) in four cases and a small supernumerary marker chromosome consisting of 12p and 20p in the remaining one case. The proportion of cells with i(12p) ranged from 0 to 100% in cultural cells, while SNP array results suggested 2−4 copies of 12p. For one foetus, metaphase FISH showed normal results, but the interphase FISH suggested cell lines with two, three and four copies of 12p in the amniotic fluid. Advanced parental age may be an important risk factor for PKS, and there were no typical ultrasound manifestations related to PKS. A combination of karyotype analysis and molecular diagnosis is an effective method for the diagnosis of PKS.     

Prevalence of Post-Stroke Cognitive Impairment in China: A Community-Based,Cross-Sectional Study

International hospital-based studies have indicated a high risk of cognitive impairment after stroke, evidence from community-based studies in China is scarce. To determine the prevalence of post-stroke cognitive impairment (PSCI) and its subtypes in stroke survivors residing in selected rural and urban Chinese communities, we conducted a community-based, cross-sectional study in 599 patients accounting for 48% of all stroke survivors registered in the 4 communities, who had suffered confirmed strokes and had undergone cognitive assessments via the Montreal Cognitive Assessment (MoCA), Mini-Mental State Examination (MMSE), and Hachinski Ischemia Scale (HIS). Detection of PSCI was based on scores in these neuropsychological scales. Factors potentially impacting on occurrence of PSCI were explored by comparing demographic characteristics, stroke features, and cardiovascular risk factors between patients with and without PSCI. The overall prevalence of PSCI was 80.97% (95%CI: 77.82%-84.11%), while that of non-dementia PSCI (PSCI-ND) and post-stroke vascular dementia (PSD) was 48.91% (95%CI: 44.91%-52.92%) and 32.05% (95%CI: 28.32%-35.79%), respectively. Prior stroke and complications during the acute phase were independent risk factors for PSCI. The risk of recurrent stroke survivors having PSCI was 2.7 times higher than for first-episode survivors, and it was 3 times higher for those with complications during the acute phase than for those without. The higher prevalence of PSCI in this study compared with previous Chinese studies was possibly due to the combined effects of including rural stroke survivors, a longer period from stroke onset, and different assessment methods. There is an urgent need to recognize and prevent PSCI in stroke patients, especially those with recurrent stroke and complications during the acute phase.     

Purification by affinity chromatography of the dicarboxylate carrier from bovine heart mitochondria

Submitochondrial particles were prepared from bovine heart mitochondria, solubilized with Triton X-114 in the presence of lipids and submitted to hydroxylapatite chromatography. The eluate obtained, containing a mixture of mitochondrial carriers, was processed further by affinity chromatography using as ligand p-aminophenylsuccinate coupled via a diazo bond to aminohexyl-Sepharose 4B. The activity of the dicarboxylate exchanger was measured after reconstitution into asolectin vesicles at each step of the purification procedure. All samples studied were found to display substrate and inhibitor specificity similar to those described for the dicarboxylate carrier in mitochondria. The specific activity of the final material eluted from the affinity column was found to be about 1000-times higher than that of the Triton X-114 extract of submitochondrial particles. SDS-polyacrylamide gel electrophoresis analysis of the affinity chromatography eluate showed the presence of only two polypeptides.     

Interaction of calf skin collagen with glycerol: linked function analysis

Glycerol stabilizes the triple-helical structure of solubilized calf skin collagen. The equilibrium melting temperature of the protein increased linearly from 38.0 degrees C in AS buffer (0.01 M NaOAc and 0.02 M NaCl, pH 4.0) to 43.0 degrees C in AS and 6 M glycerol buffer. To understand the thermodynamic basis of this effect on the equilibrium melting temperature and the glycerol inhibition of collagen self-association, the preferential interactions of native and denatured calf skin collagens in AS buffer containing 1.5, 3, and 4.5 M glycerol were measured with a precision densimeter. The results indicated that native collagen binds glycerol preferentially whereas denatured collagen neither binds nor repels glycerol. The preferential binding of glycerol by native collagen, when interpreted in terms of the three-component solution thermodynamics, suggests that the surface interaction of native collagen with glycerol is energetically more favorable than its interaction with water. By use of the Wyman linked function, the negative chemical potential change of collagen derived from its preferential binding of glycerol can account for both the glycerol stabilization of the triple-helical structure of collagen and the inhibition of in vitro self-association of monomers into fibrils.     

Transport of 2-oxoisocaproate in isolated hepatocytes and liver mitochondria

The transport of 2-oxoisocaproate into isolated hepatocytes and liver mitochondria of rat was studied using [U-14C]2-oxoisocaproate and the silicone oil filtration procedure. 2-Oxoisocaproate uptake by hepatocytes was composed of: rapid adsorption, unmediated diffusion and carrier-mediated transport. The carrier-mediated transport was strongly inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulphonic acid and p-chloromercuribenzoate, was less sensitive to alpha-cyano-4-hydroxycinnamate and insensitive to p-chloromercuriphenylsulphonate. Other 2-oxo acids: pyruvate, 2-oxoisovalerate and 2-oxo-3-methylvalerate, were also inhibitory. The kinetic parameters of the carrier-mediated transport were Km 30.6 mM and Vmax 23.4 nmol/min per mg wet wt, at 37 degrees C. It is concluded that at its low, physiological, concentration, 2-oxoisocaproate penetrates the hepatocyte membrane mainly by unmediated diffusion. The uptake of 2-oxoisocaproate by isolated liver mitochondria was partly inhibited by alpha-cyano-4-hydroxycinnamate, the inhibitor of mitochondrial monocarboxylate carrier. The remaining uptake was linearly dependent on 2-oxoisocaproate concentration and represented unmediated diffusion. The carrier-mediated transport exhibited the following kinetic parameters: Km 0.47 mM, Vmax 1.0 nmol/min per mg protein at 6 degrees C; and Km 0.075 mM and Vmax about 8 nmol/min per mg protein at 37 degrees C.     

碱性蛋白酶生产菌——地衣芽孢杆菌(B. licheniformis)的温和噬菌体

碱性蛋白酶生产菌——地衣芽孢杆菌经丝裂霉素C或紫外线处理,均可诱导释放噬菌体,电镜观察表明噬菌体头部外廓呈六边形,有不收缩的尾部(头部40nm×40nm,尾部107×5,7nm),噬菌体BLL1 DNA对限制酶Eco R Ⅰ,HindⅢ和Bam H Ⅰ敏感,分别切割成8,15,和9个片段,经电泳法测定。噬菌体DNA分子量约相当于23.4kb,根据解链温度计算出噬菌体的G+C含量约为31.2摩尔％,噬菌体提纯的外壳蛋白经SDS-聚丙烯酰胺凝胶电泳呈现5条主带,其分子量分别约为78000,72000,55000,39000,35000。     

Cytochromec oxidase fromParacoccus denitrificans in Triton X-100: Aggregation state and kinetics

Cytochromec oxidase fromParacoccus denitrificans was homogenously dispersed in Triton X-100. Using gel exclusion chromatography and sucrose gradient centrifugation analysis a molecular weight of the detergent-protein complex of 155,000 was determined. After subtraction of the bound detergent (111 mol/mol hemeaa ₃) a molecular weight of 85,000 resulted, which agreed well with the model of a monomer containing two subunits. This monomer showed high cytochromec oxidase activity when measured spectrophotometrically in the presence of Triton X-100 (V _max=85 s^–1). The molecular activity, plotted according to Eadie-Hofstee, was monophasic as a function of the cytochromec concentration. AK _m of 3.6×10^–6 M was evaluated, similar to theK _m observed in the presence of dodecyl maltoside [Naeczet al. (1985).Biochim. Biophys. Acta 808, 259–272].     

Effects of N,N′-dicyclohexylcarbodiimide on isolated and reconstituted cytochrome b-c1 complex from bovine heart mitochondria

N,N′-Dicyclohexylcarbodiimide (DCCD) inhibits the activity of ubiquinol-cytochrome c reductase in the isolated and reconstitued mitochondrial cytochrome b-c₁ complex. DCCD inhibits equally electron flow and proton translocation (i.e., the $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ ratio is not affected) catalysed by the enzyme reconstituted into phospholipid vesicles. The inhibitory effects are accompanied by structural alterations in the polypeptide pattern of both isolated and reconstituted enzyme. Cross-linking was observed between subunits V (iron-sulfur protein) and VII, indicating that these polypeptides are in close proximity. A clear correlation was found between the kinetics of inhibition of enzymic activity and the cross-linking, suggesting that the two phenomena may be coupled. Binding of [¹⁴C]DCCD was also observed, to all subunits with the isolated enzyme and preferentially to cytochrome b with the reconstituted vesicles; in both cases, however, it was not correlated kinetically with the inhibition of the enzymic activity.