Multiplication of a DNA fragment in Streptomyces antibioticus--producer of oleandomycin

Two strains of Str. antibioticus producing oleandomycin were studied. Strain 326 was obtained from the initial laboratory strain and strain 1607 from strain 326 as a result of multistage selection aimed at increasing the antibiotic production capacity. Extrachromosomal ring DNA could be identified in strain 1607. The identified DNA was designated as eSA1 or extrachromosomal element of Streptomyces antibioticus 1. The molecular weight of this DNA is 21.3 Md. It is represented by 1 copy per chromosome. No eSA1 was isolated from strain 326 at CsCl-EtBr gradient. Hybridization studies according to Southern with the use as a probe of 32-eSA1 DNA showed that strain 326 contained 1 copy of eSA1 per chromosome in the integrated state. The hybridization studies, electron microscopy and analysis of the total DNA in CsC1-EtBr gradient showed that eSA1 in strain 1607 was tandemly multireplicated in the chromosome content. In the autonomous state its number was approximately equal to 1 copy per chromosome. The presence of eSA1 in strain 1607 in the autonomous state probably results from its segregation during homologous recombination due to tandem multireplication. The data are indicative of multiplication in strain 1607 of the chromosome fragment 23.3 Md in size. It is suggested that an increase in the oleandomycin production capacity of strain 1607 is associated with multiplication of the DNA fragment (eSA1) containing the genes determining production of the antibiotic.     

Objectively Measured Physical Activity in European Adults: Cross-Sectional Findings from the Food4Me Study

Background

Comparisons of objectively measured physical activity (PA) between residents of European countries measured concurrently with the same protocol are lacking. We aimed to compare PA between the seven European countries involved in the Food4Me Study, using accelerometer data collected remotely via the Internet.

Methods

Of the 1607 participants recruited, 1287 (539 men and 748 women) provided at least 3 weekdays and 2 weekend days of valid accelerometer data (TracmorD) at baseline and were included in the present analyses.

Results

Men were significantly more active than women (physical activity level = 1.74 vs. 1.70, p < 0.001). Time spent in light PA and moderate PA differed significantly between countries but only for women. Adherence to the World Health Organization recommendation to accumulate at least 150 min of moderate-equivalent PA weekly was similar between countries for men (range: 54–65%) but differed significantly between countries for women (range: 26–49%). Prevalence estimates decreased substantially for men and women in all seven countries when PA guidelines were defined as achieving 30 min of moderate and vigorous PA per day.

Conclusions

We were able to obtain valid accelerometer data in real time via the Internet from 80% of participants. Although our estimates are higher compared with data from Sweden, Norway, Portugal and the US, there is room for improvement in PA for all countries involved in the Food4Me Study.     

Cdc14B: When a good kid turns bad

Comment on: Chiesa M, et al. Cell Cycle 2011; 10:1607-17.     

A phosphatase turns aggressive: The oncogenicity of Cdc14B

Comment on: Chiesa M, et al. Cell Cycle 2011; 10:1607-17.     

Cloning and nucleotide sequence analysis of the chloramphenicol resistance gene on conjugative R plasmids from the fish pathogen Photobacterium damselae subsp. piscicida

Transferable resistance to various drugs was investigated in Photobacterium damselae subsp. piscicida from Japan. Drug resistances were transferred via plasmids of 100, 50, and 40 kb. Resistance to chloramphenicol (Cmr) was transferred on plasmids of all 3 sizes. The Cmr gene (cat) was cloned from the 50 kb plasmids pPDP8511 and pPDP9106 transferred from P. damselae subsp. piscicida strains isolated in different years and places in Japan. Subcloning localized the cat to within 1.5 kb HindIII-HincII (or PstI) fragments. Nucleotide sequences of the coding and flanking region of the cat were determined as 1607 bp (HindIII-HincII fragment) in pPDP8511 and 1568 bp (HindIII-PstI fragment) in pPDP9106, which corresponded with the sequence from nucleotides 40 to 1607 in pPDP8511. The nucleotide sequences identified an open reading frame (ORF) encoding 213 amino acid residues with a calculated molecular mass of about 24.8 kDa, a size consistent with the molecular mass of known cat gene products, and the ORF had maximum homology (99.5%) with a Type II CAT variant from Haemophilus influenzae.     

Stability of fumarase activity of Brevibacterium flavum immobilized with ϰ-carrageenan and Chinese gallotannin

Summary In order to improve L-malic acid productivity by Brevibacterium flavum immobilized with -carrageenan, addition of Chinese gallotannin to the immobilization medium was investigated. As the results show, the optimal concentration of Chinese gallotannin was 0.1% (w/v). Fumarase activity and the stability of this improved preparation were higher than in one with only -carrageenan. Addition of Chinese gallotannin was more advantageous to stability towards ethanol than addition of polyethyleneimine. The L-malic acid productivity of the immobilized cells at 37°C was 42.2 kg/h per 1,000 l column, and increased threefold compared with that of B. flavum immobilized with only -carrageenan, and was 25 times that of B. ammoniagenes immobilized with polyacrylamide. Persimmon tannin also increased the stability of fumarase.     

Influence of Matrix Metalloproteinase-1 Gene −1607 (1G/2G) (rs1799750) Promoter Polymorphism on Circulating Levels of MMP-1 in Chronic Pancreatitis

This study investigated the role of ?1607 (1G/2G) (rs1799750) polymorphism of the MMP-1 gene in chronic pancreatitis. We genotyped 100 patients with chronic pancreatitis and 100 control subjects using tetra-primer ARMS-PCR followed by agarose gel electrophoresis. Serum levels of MMP-1 were determined by Elisa. Statistical analysis was applied to test the significance of the results. The genotypic and allelic distribution varied significantly between the disease group and the control subjects [OD = 1.981 (1.236–3.181), p = 0.004]. MMP-1 levels were higher in subjects homozygous for the 2G allele than in subjects with the 1G allele. The present study revealed a significant association of the MMP-1 ?1607 1G/2G (rs1799750) gene promoter polymorphism with chronic pancreatitis, and it can be considered a biological marker in the etiology of chronic pancreatitis.     

Moniliformin accumulation in kernels of oats used for food and feed purposes

Inoculation experiments with 12 genotypes of oats (9 lines: CHD 894, CHD 1095, CHD 1296, CHD 1607, CHD 1653, CHD 1692, STH 2293, STH 2393, STH 2494, STH 2694 and 2 cultivars: Farys, Slawko) were performed during the vegetation seasons of 1999 — 2000 in Zamosc region in the South — Eastern part of Poland. Panicles of oats were inoculated with a conidial suspension ofFusarium avenaceum, which caused the following average reductions of traits: yield by 37.8 % and weight of 1000 kernels by 40.2 % respectively. The highest susceptibility to scab development caused by theF. avenaceum inoculation was found particularly for two lines (CHD 894, and CHD 1296) while cultivar Farys and line CHD 1607 exhibited the highest resistance to the disease in terms of TKW and yield reductions after inoculation. During 2 consecutive years (1999 and 2000 respectively) the average level of moniliformin accumulation (mg/kg) in positive samples was respectively 0.51 and 0.12 the highest being 1.11 and 0.26, while the lowest was 0.30 and 0.01.     

Modeling ADAMTS13-von Willebrand factor interaction: Implications for oxidative stress-related cardiovascular diseases and type 2A von Willebrand disease

The haemostatic potential of von Willebrand factor, a glycoprotein expressed by endothelial cells as ultra-large polymers (UL-vWF)¹, increases with its length, which in turn is regulated proteolytically by ADAMTS13, a zinc-metalloprotease selectively cleaving vWF at the Tyr1605-Met1606 bond. We have recently shown that in vitro oxidation of Met1606, under conditions mimicking those found in diseases characterized by high oxidative stress, severely impairs proteolysis by ADAMTS13, with a resulting pro-thrombotic effect caused by the accumulation of UL-vWF species. Conversely, Val1607Asp mutation, found in vWF from patients with type 2A von Willebrand disease, accelerates proteolysis of vWF, with a final hemorrhagic effect. Considering the physio-pathological importance of ADAMTS13-vWF interaction and the absence of experimental structural data, here we produced by homology modeling techniques a three-dimensional model of ADAMTS13 metalloprotease domain (M13). Thereafter, the vWF(1604-1607) peptide, containing the cleavable Tyr1605-Met1606 bond, was manually docked into the protease active site and the resulting model complex provided us key information for interpreting on structural grounds the variable effects that chemical modifications/mutations in vWF have on proteolysis by ADAMTS13.     

Association between MMP-1 g.-1607dupG Polymorphism and Periodontitis Susceptibility: A Meta-Analysis

Background

Matrix metalloproteinase-1 (MMP-1) plays an important role during the destruction of periodontal tissue. Although multiple studies had focused on the association between MMP-1 g.-1607dupG and periodontitis susceptibility, the results remained inconclusive. The purpose of this meta-analysis was to explore its role in the development of periodontitis.

Methods

Retrieved studies from Pubmed, Web of Science, Medline and Google Scholar Search regarding MMP-1 g.-1607dupG and periodontitis susceptibility were included into the final analysis with definite selection and exclusion criteria. Overall and stratified analyses based on disease type, severity, ethnicity and smoking status were performed. Odds ratio (OR) and 95% confidence interval (CI) were used to evaluate the association between MMP-1 g.-1607dupG and periodontitis susceptibility, while Q test and Egger’s test were adopted respectively to assess heterogeneity among studies and publication bias.

Results

A total of 1580 periodontitis cases and 1386 controls in 11 case-control studies were included in the meta-analysis. The pooled results showed significant association between periodontitis susceptibility and MMP-1 g.-1607dupG polymorphism in homozygote (2G/2G versus 1G/1G, OR = 1.50, 95% CI = 1.02–2.20) and dominant model analysis (2G/2G+2G/1G versus 1G/1G, OR = 1.28, 95% CI = 1.04–1.57). For subgroups by type of periodontitis, increased risk of chronic periodontitis was observed on heterozygote (2G/1G versus 1G/1G, OR = 2.01, 95% CI = 1.58–2.56) and dominant model (OR = 1.27, 95% CI = 1.03–1.57). Furthermore, similar association was also detected in severe chronic periodontitis (2G/2G versus 1G/1G, OR = 2.15, 95% CI = 1.35–3.43; 2G/2G+2G/1G versus 1G/1G, OR = 1.64, 95% CI = 1.12–2.39; 2G/2G versus 2G/1G+1G/1G, OR = 1.86, 95% CI = 1.31–2.64).

Conclusions

Our meta-analysis demonstrated that MMP-1 g.-1607dupG polymorphism was associated with chronic periodontitis, especially the severity of the disease condition.     

Induction of mutations in Streptomyces antibioticus

Mutagenic action of UV-light, nitrosoguanidine and nitrosomethylbiuret was studied in Streptomyces antibioticus VNIIA 1607. Nitrosomethylbiuret appeared to be most effective inducer of auxotrophic mutations (mutation frequency reached 15%). By means of hybridological analysis, it was shown that heterokaryons predominated in the progeny of mixed cultures of multiply marked strains. The test for functional allelism using heterokaryons permitted us to divide 93 independently obtained mutations into 28 complementation groups.     

Matrix Metalloproteinase-9 -1562C/T Promoter Polymorphism Confers Risk for COPD: A Meta-Analysis

Background

The role of matrix metalloproteinase (MMP) gene polymorphisms in the development of chronic obstructive pulmonary disease (COPD) has been reported with inconsistent results. This meta-analysis was performed to assess the association of MMP-1 -1607G/GG and MMP-9 -1562C/T promoter polymorphisms with COPD susceptibility.

Methods

Published case-control studies from Pubmed and China National Knowledge Infrastructure (CNKI) databases were retrieved. Data were extracted and pooled odds ratios (OR) with 95% confidence intervals (CI) were calculated.

Results

A total of fourteen case-control studies were included in this meta-analysis. Pooled effect size showed an association of MMP-9 -1562 C/T with the risk of COPD (dominant model: TT+CT vs CC; OR: 1.46; 95% CI: 1.02–2.08; p = 0.04). However, no correlation with COPD was revealed in MMP-1 -1607G/GG polymorphism. When stratified by ethnicity, results indicated MMP-1 -1607G/GG (recessive model: G/G vs G/GG+GG/GG; OR: 1.20; 95% CI: 1.01–1.44; p = 0.04) and MMP-9 -1562 C/T (dominant model; OR: 1.66; 95% CI: 1.01–2.71; p = 0.04) were correlated with COPD susceptibility among Caucasians and Asians respectively. According to source of controls, signifiant association of MMP-9 -1562 C/T (additive model: T vs C; OR:1.71, 95% CI: 1.42–2.07; p<0.00001, and dominant model; OR: 1.92; 95% CI: 1.34–2.76; p = 0.0004) with COPD susceptibility was revealed in the subgroup with smoker-based controls. However, in the aforementioned risk estimates, only the association of MMP-9 -1562 C/T (additive and dominant models) with the risk of COPD in the subgroup with smoker-based controls persisted significantly after Bonferroni correction for multiple testing. Moreover, after excluding the studies without Hardy–Weinberg equilibrium and/or with small sample size, the pooled results were robust and no publication bias was found in this study.

Conclusion

This meta-analysis suggests, when using healthy smokers as controls, MMP-9 -1562 C/T, but not MMP-1 -1607 G/GG polymorphism is associated with the risk of COPD.     

Growth retardation in mice lacking the proteasome activator PA28gamma

The proteasome activator PA28 binds to both ends of the central catalytic machine, known as the 20 S proteasome, in opposite orientations to form the enzymatically active proteasome. The PA28 family is composed of three members designated alpha, beta, and gamma; PA28alpha and PA28beta form the heteropolymer mainly located in the cytoplasm, whereas PA28gamma forms a homopolymer that predominantly occurs in the nucleus. Available evidence indicates that the heteropolymer of PA28alpha and PA28beta is involved in the processing of intracellular antigens, but the function of PA28gamma remains elusive. To investigate the role of PA28gamma in vivo, we generated mice deficient in the PA28gamma gene. The PA28gamma-deficient mice were born without appreciable abnormalities in all tissues examined, but their growth after birth was retarded compared with that of PA28gamma(+/-) or PA28gamma(+/+) mice. We also investigated the effects of the PA28gamma deficiency using cultured embryonic fibroblasts; cells lacking PA28gamma were larger and displayed a lower saturation density than their wild-type counterparts. Neither the expression of PA28alpha/beta nor the subcellular localization of PA28alpha was affected in PA28gamma(-/-) cells. These results indicate that PA28gamma functions as a regulator of cell proliferation and body growth in mice and suggest that neither PA28alpha nor PA28beta compensates for the PA28gamma deficiency.     

尿激酶原变体Glu151-Glu154-mscu-PA的构建及其动力学性质

利用寡核苷酸介导的定点突变方法 ,将重组人尿激酶原 ( recombinant single chain uroki-nase- type plasminogen activator,rscu- PA)中 1 51位赖氨酸 ( Lys1 51 )突变为谷氨酸 ( Glu1 51 ) ,1 54位精氨酸 ( Arg1 54)突变为谷氨酸 ( Glu1 54) ,得到尿激酶原变体基因 ( mscu- PA) .尿激酶原变体和未突变的重组尿激酶原均在 E.coli中获得表达 ,超声后所得包涵体经体外变复性并得到纯化 .结果表明 ,尿激酶原变体对纤溶酶 ( plasmin)的敏感性比未突变的重组尿激酶原低约 40 % ,转变纤溶酶原 ( Glu- plasminogen)为纤溶酶的活性基本相同 .两种产物经纤溶酶活化后 ,分别得到了双链尿激酶 ( rtcu- PA)和双链尿激酶变体 ( mtcu- PA) .它们对人工合成的发色底物 S2 4 4 4反应的动力学基本一致 ,对 Glu- plasminogen的催化反应的米氏动力学常数 Km 基本一致 ,但 mtcu- PA的 Kcat仅为rtcu- PA的 80 % .酪蛋白降解系统 ( caseinolytic system)实验表明 ,在纤维蛋白和纤溶酶原存在的情况下 ,尿激酶原变体较未突变尿激酶原能加快酪蛋白的降解 ,说明 mtcu- PA对纤维蛋白有一定的亲和性     

Reconstitution of hybrid proteasomes from purified PA700-20 S complexes and PA28alphabeta activator: ultrastructure and peptidase activities.

The activity of the proteasome, the major non-lysosomal proteinase in eukaryotes, is stimulated by two activator complexes, PA700 and PA28. PA700-20 S-PA700 proteasome complexes, generally designated as 26 S proteasomes, degrade proteins, whereas complexes of the type PA28-20 S-PA28 degrade only peptides. We report, for the first time, the in vitro reconstitution of previously identified hybrid proteasomes (PA700-20 S-PA28) from purified PA700-20 S proteasome complexes and PA28 activator. In electron micrographs, the hybrid appears as a corkscrew-shaped particle with a PA700 and a PA28 activator each bound to a terminal alpha-disk of the 20 S core proteasome. The multiple peptidase activities of hybrid proteasomes are not different from those of PA28-20 S-PA28 or PA700-20 S-PA700 complexes.     

Preparation and characterization of two major isotypes of parvalbumins from skeletal muscle of bullfrog (Rana catesbeiana)

Two major isotypes of parvalbumins (PA1 and PA2) have been isolated from the skeletal muscle of bullfrog, Rana catesbeiana. The Mr values were estimated to be 10,100 (PA1) and 11,800 (PA2) by SDS/polyacrylamide gel electrophoresis, and the isoelectric points were determined to be 4.78 (PA1) and 4.97 (PA2) by polyacrylamide gel isoelectric focusing. The amino acid compositions and isoelectric points indicate that PA1 corresponds to Rana esculenta pI 4.50 and Rana temporaria pI 4.75 parvalbumins and PA2 to Rana esculenta pI 4.88 and Rana temporaria pI 4.97 parvalbumins, showing that PA1 is genetically a beta-parvalbumin and PA2 an alpha-parvalbumin. However, in terms of the amino acid compositions, PA1 and PA2 are distinctly different from the corresponding parvalbumins of Rana esculenta or Rana temporaria. The ultraviolet spectra of PA1 and PA2 are consistent with their amino acid compositions. An ultraviolet difference spectrum of the Ca2+-loaded form vs. metal-free form indicates that a Tyr and some Phe residues in PA1 are affected by a conformational change associated with the binding of Ca2+. On electrophoresis in polyacrylamide gel in 14 mM Tris and 90 mM glycine, the Ca2+-loaded form of PA1 migrated twice as fast as the Mg2+-loaded form. Both PA1 and PA2 show increased mobility in the Ca2+-loaded forms, like troponin C but different from calmodulin.