To elucidate further the pathogenesis of sporadic, idiopathic pulmonary arterial hypertension (IPAH) and identify potential therapeutic avenues, differential gene expression in IPAH was examined by ...suppression subtractive hybridisation (SSH).
Peripheral lung samples were obtained immediately after removal from patients undergoing lung transplant for IPAH without familial disease, and control tissues consisted of similarly sampled pieces of donor lungs not utilised during transplantation. Pools of lung mRNA from IPAH cases containing plexiform lesions and normal donor lungs were used to generate the tester and driver cDNA libraries, respectively. A subtracted IPAH cDNA library was made by SSH. Clones isolated from this subtracted library were examined for up regulated expression in IPAH using dot blot arrays of positive colony PCR products using both pooled cDNA libraries as probes. Clones verified as being upregulated were sequenced. For two genes the increase in expression was verified by northern blotting and data analysed using Student's unpaired two-tailed t-test.
We present preliminary findings concerning candidate genes upregulated in IPAH. Twenty-seven upregulated genes were identified out of 192 clones examined. Upregulation in individual cases of IPAH was shown by northern blot for tissue inhibitor of metalloproteinase-3 and decorin (P < 0.01) compared with the housekeeping gene glyceraldehydes-3-phosphate dehydrogenase.
Four of the up regulated genes, magic roundabout, hevin, thrombomodulin and sucrose non-fermenting protein-related kinase-1 are expressed specifically by endothelial cells and one, muscleblind-1, by muscle cells, suggesting that they may be associated with plexiform lesions and hypertrophic arterial wall remodelling, respectively.
The taxonomy of the "Aeromonas hydrophila" complex (comprising the species A. hydrophila, A. bestiarum, A. salmonicida, and A. popoffii) has been controversial, particularly the relationship between ...the two relevant fish pathogens A. salmonicida and A. bestiarum. In fact, none of the biochemical tests evaluated in the present study were able to separate these two species. One hundred and sixteen strains belonging to the four species of this complex were identified by 16S rDNA restriction fragment length polymorphism (RFLP). Sequencing of the 16S rDNA and cluster analysis of the 16S-23S intergenic spacer region (ISR)-RFLP in selected strains of A. salmonicida and A. bestiarum indicated that the two species may share extremely conserved ribosomal operons and demonstrated that, due to an extremely high degree of sequence conservation, 16S rDNA cannot be used to differentiate these two closely related species. Moreover, DNA-DNA hybridization similarity between the type strains of A. salmonicida subsp. salmonicida and A. bestiarum was 75.6 %, suggesting that they may represent a single taxon. However, a clear phylogenetic divergence between A. salmonicida and A. bestiarum was ascertained from an analysis based on gyrB and rpoD gene sequences, which provided evidence of a lack of congruence of the results obtained from 16S rDNA, 16S-23S ISR-RFLP, DNA-DNA pairing, and biochemical profiles.
Abstract The proinflammatory cytokine TNFα is a key player in insulin resistance (IR). The role of miRNAs in inflammation associated with IR is poorly understood. To investigate miR-181a-5p and ...miR-23a-3p expression profiles in obesity and to study their role in TNFα -induced IR in adipocytes. Two separate cohorts were used. Cohort 1 was used in adipose tissue (AT) expression studies and included 28 subjects with body mass index (BMI) <30 kg/m2 and 30 with BMI ≥30 kg/m2 . Cohort 2 was used in circulating serum miRNA studies and included 101 subjects with 4 years of follow-up (48 case subjects and 53 control subjects). miR-181a-5p and miR-23a-3p expression was assessed in subcutaneous and visceral AT. Functional analysis was performed in adipocytes, using miRNA mimics and inhibitors. Key molecules of the insulin pathway, AKT, PTEN, AS160, and S6K, were analyzed. Expression of miR-181a-5p and miR-23a-3p was reduced in adipose tissue from obese and diabetic subjects and was inversely correlated to adiposity and homeostasis model assessment of IR index. Overexpression of miR-181a-5p and miR-23a-3p in adipocytes upregulated insulin-stimulated AKT activation and reduced TNFα -induced IR, regulating PTEN and S6K expression. Serum levels of miR-181a-5p were reduced in case vs control subjects at baseline, suggesting a prognostic value. Variable importance in projection scores revealed miR-181a-5p had more effect on the model than insulin or glucose at 120 minutes. miR-181a-5p and miR-23a-3p may prevent TNFα -induced IR in adipocytes through modulation of PTEN and S6K expression. We have identified miR-181a and miR-23a as miRNAs downregulated by TNFα in human adipocytes and in human, obese, visceral adipose tissue, and we tested their functional mechanism in TNF-IR adipocytes.
Three typing methods, restriction fragment length polymorphism (RFLP) of the 16S-23S intergenic spacer region (ISR), PCR amplification of the enterobacterial repetitive intergenic consensus (ERIC) ...and of the repetitive extragenic palindromic units (REP), were evaluated for typing 26 isolates of Aeromonas popoffii from different geographical origins. When the methods were independently studied, ERIC showed the highest discriminatory power. When the methods were combined, the best combination of two methods was ERIC with REP since strains showed a tendency to cluster according to their geographical origin. However, this tendency was reinforced with the addition of ISR-RFLP.
We have isolated a full-length cDNA encoding an acetylcholinesterase secreted by the nematode parasite Nippostrongylus brasiliensis . The predicted protein is truncated in comparison with ...acetylcholinesterases from other organisms such that the carboxyl
terminus aligns closely to the end of the catalytic domain of the vertebrate enzymes. The residues in the catalytic triad
are conserved, as are the six cysteines which form the three intramolecular disulfide bonds. Three of the fourteen aromatic
residues which line the active site gorge in the Torpedo enzyme are substituted by nonaromatic residues, corresponding to
Tyr-70 (Thr), Trp-279 (Asn), and Phe-288 (Met).
High level expression was obtained via secretion from Pichia pastoris . The purified enzyme behaved as a monomeric hydrophilic species. Although of invertebrate origin and possessing the above
substitutions in the active site gorge residues, the enzyme efficiently hydrolyzed acetylthiocholine and showed minimal activity
against butyrylthiocholine. It displayed excess substrate inhibition with acetylthiocholine at concentrations over 2.5 m m and was highly sensitive to both active site and âperipheralâ site inhibitors. Northern blot analysis indicated a progressive
increase in mRNA for AChE B in parasites isolated from 6 days postinfection.
Obesity and HIV-1/HAART-associated lipodystrophy syndrome (HALS) share clinical, pathological and mechanistic features. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a ...multifunctional cytokine that plays an important role in obesity and related diseases. We sought to explore the relationship between HALS and circulating levels of soluble (s) TWEAK and its scavenger receptor sCD163. This was a cross-sectional multicenter study of 120 HIV-1-infected patients treated with a stable HAART regimen; 56 with overt HALS and 64 without HALS. Epidemiological and clinical variables were determined. Serum levels of sTWEAK and sCD163 levels were measured by ELISA. Results were analyzed with Student's t-test, Mann-Whitney U and X.sup.2 test. Pearson and Spearman correlation were used to estimate the strength of association between variables. Circulating sTWEAK was significantly decreased in HALS patients compared with non-HALS patients (2.81±0.2 vs. 2.94±0.28 pg/mL, p = 0.018). No changes were observed in sCD163 levels in the studied cohorts. On multivariate analysis, a lower log sTWEAK concentration was independently associated with the presence of HALS (OR 0.027, 95% CI 0.001-0.521, p = 0.027). HALS is associated with decreased sTWEAK levels.
Abstract The trafficking of glycerol from adipose and hepatic tissue is mainly mediated by 2 aquaporin channel proteins: AQP7 and AQP9 , respectively. In rodents, both aquaporins were found to act in ...a coordinated manner. The aim was to study the relationship between adipose AQP7 and hepatic AQP9 messenger RNA expression and the presence of glucose abnormalities simultaneously in morbid obesity. Adipose tissue (subcutaneous SAT and visceral VAT) and liver biopsies from the same patient were obtained during bariatric surgery in 30 (21 male and 9 female) morbidly obese subjects. Real-time quantification of AQP7 in SAT and VAT and hepatic AQP9 gene expression were performed. A 75-g oral glucose tolerance test was performed in all subjects. The homeostasis model assessment of insulin resistance and lipidic profile were also determined. Visceral adipose tissue AQP7 expression levels were significantly higher than SAT AQP7 ( P = .009). Subcutaneous adipose tissue AQP7 positively correlated with both VAT AQP7 and hepatic AQP9 messenger RNA expression ( r = 0.44, P = .013 and r = 0.45, P = .012, respectively). The correlation between SAT AQP7 and liver AQP9 was stronger in intolerant and type 2 diabetes mellitus subjects ( r = 0.602, P = .011). We have found no differences in compartmental AQP7 adipose tissue distribution or AQP9 hepatic gene expression according to glucose tolerance classification. The present study provides, for the first time, evidence of coordinated regulation between adipose aquaglyceroporins, with a greater expression found in visceral fat, and between subcutaneous adipose AQP7 and hepatic AQP9 gene expression within the context of human morbid obesity.
Abstract
Aeromonas popoffii is the most recent species within the genus Aeromonas described from freshwater. In our study this species was also recovered from this habitat and for the first time from ...seawater. Most of the virulence factors known in Aeromonas spp. (aerolysin/hemolysin, serine protease, lipases and DNases) were highly prevalent in this species. Third-generation cephalosporins and quinolones were the most active antimicrobial agents against A. popoffii.
The analysis of the complete nucleotide sequence of the African swine fever virus genome has revealed the existence of a number of genes potentially capable of modifying the host's response to the ...virus infection. In this report, we describe the results of the characterization of the A224L gene that encodes a novel member of the family of apoptosis inhibitors known as IAP proteins. A224L is expressed during the late phase of the infectious cycle, and its polypeptide product is assembled into virus particles.
Objectives
Treated HIV‐1‐infected patients with lipodystrophy often develop insulin resistance and proatherogenic dyslipidaemia. Zinc alpha‐2 glycoprotein (ZAG) is a recently characterized adipokine ...which has been shown to be involved in the development of obesity and metabolic syndrome in uninfected subjects. We assessed the relationship between circulating ZAG levels and metabolic derangements in HIV‐1‐infected patients receiving antiretroviral drugs.
Methods
Plasma ZAG levels were assessed in 222 individuals: 166 HIV‐1‐infected patients treated with antiretroviral drugs (77 with lipodystrophy and 89 without lipodystrophy) and 56 uninfected controls. Plasma ZAG levels were assessed by enzyme‐linked immunosorbent assay (ELISA) and were correlated with fat distribution abnormalities and metabolic parameters.
Results
HIV‐1‐infected patients had lower plasma ZAG levels compared with uninfected controls (P < 0.001). No differences were found in ZAG plasma levels according to the presence of lipodystrophy, components of the metabolic syndrome or type of antiretroviral treatment regimen. Circulating ZAG levels were strongly determined by high‐density lipoprotein cholesterol (HDLc) in men (B = 0.644; P < 0.001) and showed a positive correlation with total cholesterol (r = 0.312; P < 0.001) and HDLc (r = 0.216; P = 0.005).
Conclusions
HIV‐1‐infected patients have lower plasma ZAG levels than uninfected controls. In infected patients, plasma ZAG levels are in close relationship with total cholesterol and HDLc.
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