In silico and in vitro characterization of phospholipase A2 isoforms from soybean (Glycine max)

At the present, no secreted phospholipase A₂ (sPLA₂) from soybean (Glycine max) was investigated in detail. In this work we identified five sequences of putative secreted sPLA₂ from soybean after a BLAST search in G. max database. Sequence analysis showed a conserved PA2c domain bearing the Ca²⁺ binding loop and the active site motif. All the five mature proteins contain 12 cysteine residues, which are commonly conserved in plant sPLA₂s. We propose a phylogenetic tree based on sequence alignment of reported plant sPLA₂s including the novel enzymes from G. max. According to PLA₂ superfamily, two of G. max sPLA₂s are grouped as XIA and the rest of sequences as XIB, on the basis of differences found in their molecular weights and deviating sequences especially in the N- and C-terminal regions of the isoenzymes. Furthermore, we report the cloning, expression and purification of one of the putative isoenzyme denoted as GmsPLA₂-XIA-1. We demonstrate that this mature sPLA₂ of 114 residues had PLA₂ activity on Triton:phospholipid mixed micelles and determine the kinetic parameters for this system. We generate a model based on the known crystal structure of sPLA₂ from rice (isoform II), giving first insights into the three-dimensional structure of folded GmsPLA₂-XIA-1. Besides describing the spatial arrangement of highly conserved pair HIS-49/ASP-50 and the Ca⁺² loop domains, we propose the putative amino acids involved in the interfacial recognition surface. Additionally, molecular dynamics simulations indicate that calcium ion, besides its key function in the catalytic cycle, plays an important role in the overall stability of GmsPLA₂-XIA-1 structure.     

DNA binding and nucleolytic properties of Cu(ii) complexes of salicylaldehyde semicarbazones

The copper(ii) complexes of two salicylaldehyde semicarbazones, HOC(6)H(4)CH[double bond, length as m-dash]N-NHCONR(2) [H(2)Bnz(2) (R = CH(2)Ph) and H(2)Bu(2) (R = Bu)], were evaluated for their DNA binding and cleavage properties by spectrophotometric DNA titration, ethidium bromide displacement assay and electrophoretic mobility shift assay. Results showed that the Cu(ii) complexes can bind to DNA via a partial intercalation mode with binding constants of 1.1 × 10(4) and 9.5 × 10(3) M(-1) for [Cu(HBnz(2))Cl] and [Cu(HBu(2))Cl], respectively. These complexes also cleave DNA in the presence of ascorbic acid, most likely through hydroxyl radicals that are generated via the reduction of a Cu(ii) to a Cu(i) species. The complexes show similar DNA cleavage activity, which is reflected in the similarity of their frontier molecular orbital energies calculated by density functional theory. These results are discussed in relation to the anticancer properties of the complexes.     

Hemopoietic cell expression of the chemokine decoy receptor D6 is dynamic and regulated by GATA1

D6 scavenges inflammatory chemokines and is essential for the regulation of inflammatory and immune responses. Mechanisms explaining the cellular basis for D6 function have been based on D6 expression by lymphatic endothelial cells. In this study, we demonstrate that functional D6 is also expressed by murine and human hemopoietic cells and that this expression can be regulated by pro- and anti-inflammatory agents. D6 expression was highest in B cells and dendritic cells (DCs). In myeloid cells, LPS down-regulated expression, while TGF-beta up-regulated expression. Activation of T cells with anti-CD3 and soluble CD28 up-regulated mRNA expression 20-fold, while maturation of human macrophage and megakaryocyte precursors also up-regulated D6 expression. Competition assays demonstrated that chemokine uptake was D6 dependent in human leukocytes, whereas mouse D6-null cells failed to uptake and clear inflammatory chemokines. Furthermore, we present evidence indicating that D6 expression is GATA1 dependent, thus explaining D6 expression in myeloid progenitor cells, mast cells, megakaryocytes, and DCs. We propose a model for D6 function in which leukocytes, within inflamed sites, activate D6 expression and thus trigger resolution of inflammatory responses. Our data on D6 expression by circulating DCs and B cells also suggest alternative roles for D6, perhaps in the coordination of innate and adaptive immune responses. These data therefore alter our models of in vivo D6 function and suggest possible discrete, and novel, roles for D6 on lymphatic endothelial cells and leukocytes.     

Dilution-to-Extinction Culturing of Psychrotolerant Planktonic Bacteria from Permanently Ice-covered Lakes in the McMurdo Dry Valleys,Antarctica

Lakes in the McMurdo Dry Valleys of Antarctica are characterized by a permanent ice cover and little or no anthropogenic influence. Although bacterial cultures have been obtained from these habitats, recent culture-independent studies indicate that the most abundant microbes in these systems are not yet cultivated. By using dilution-to-extinction cultivation methods with sterilized and nutrient-amended lake water as media, we isolated 148 chemotrophic psychrotolerant bacterial cultures from fresh surface water of Lake Fryxell and the east lobe of Lake Bonney and the hypersaline, suboxic bottom water from the west lobes of Lake Bonney. Screening of the 16S ribosomal ribonucleic acid (rRNA) genes of the cultures by restriction fragment length polymorphism (RFLP) yielded 57 putatively pure psychrotolerant, slow growing cultures grouped into 18 clusters. The sequencing of 16S rRNA genes of randomly selected representatives of each RFLP cluster revealed that the corresponding isolates belong to the Alphaproteobacteria (six RFLP patterns), Betaproteobacteria (six RFLP patterns), Bacteroidetes (four RFLP patterns), and Actinobacteria (two RFLP patterns). Phylogenetic analysis of the sequences showed that the vast majority of the isolates were not closely related to previously described species. Thirteen of 18 RFLP patterns shared a 16S ribosomal deoxyribonucleic acid sequence similarity of 97% or less with the closest described species, and four isolates had a sequence similarity of 93% or less with the nearest described species. Phylogenetic analysis showed that these sequences were representatives of deeply branching organisms in the respective phylum. A comparison of the isolates with 16S rRNA clone libraries prepared from the same environments showed substantial overlap, indicating that dilution-to-extinction culturing in natural lake water media can help isolate some of the most abundant organisms in these perennially ice-covered lakes.     

High dose Losartan and ACE gene polymorphism in IgA nephritis

Background/aims Several studies have reported varying results of the influence of ACE gene on ACEI/ARB therapy. The efficacy of high dose ARB and its influence on ACE gene have not been explored. This is a 6 year randomised trial in IgA nephritis comparing high dose ARB (Losartan 200 mg/day) with normal dose ARB (Losartan 100 mg/day), normal dose ACEI (20 mg/day) and low dose ACEI (10 mg/day). Results Patients on high dose ARB had significantly lower proteinuria, 1.0 ± 0.8 gm/day compared to 1.7 ± 1.0 g/day in the other groups (P = 0.0005). The loss in eGFR was 0.7 ml min^?1year^?1 for high dose ARB compared to 3.2–3.5 ml min^?1year^?1 for the other three groups (P = 0.0005). There were more patients on high dose ARB with improvement in eGFR compared to other three groups (P < 0.001). Comparing patients with the three ACE genotypes DD, ID and II, all three groups responded well to therapy with decrease in proteinuria (P < 0.002). Only those on low dose ACEI (10 mg/day) with the I allele had increased in ESRF (P = 0.037). Conclusion High dose ARB is more efficacious in reducing proteinuria and preserving renal function when compared with normal dose ARB and ACEI, and also obviates the genomic influence of ACE gene polymorphism on renal survival.     

Association of Interleukin-1 gene polymorphisms with central obesity and metabolic syndrome in a coronary heart disease population

The objective of this study was to determine whether single nucleotide polymorphisms (SNPs) in the Interleukin-1 (IL-1) gene family are associated with central obesity and metabolic syndrome in a coronary heart disease population. The IL-1α C-889T (rs1800587) and IL-1β +3954 (rs1143634) SNPs were studied in a Western Australian coronary heart disease (CHD) population (N = 556). Subjects who were TT homozygous at either SNP had larger waist circumference (IL-1α: 1.8 cm greater, P = 0.04; IL-1β: 4 cm greater, P = 0.0004) compared with major allele homozygotes. Individuals with two copies of the IL-1α:IL-1β T:T haplotype had greater waist circumference (4.7 cm greater, P = 0.0001) compared to other haplotypes. There was a significant interaction between the IL-1β SNP and BMI level on waist circumference (P = 0.01). When the cohort was stratified by median BMI, TT carriers for IL-1β with above median BMI had greater waist circumference (6.1 cm greater, P = 0.007) compared to baseline carriers, whilst no significant association was seen in the below median group. Similarly, when the cohort was stratified by median fibrinogen level (IL-1α interaction P = 0.01; IL-1β interaction P = 0.04), TT carriers for both SNPs in the above median fibrinogen group had greater waist circumference (IL-1α 2.7 cm greater, P = 0.007; IL-1β 3.3 cm greater, P = 0.003) compared with major allele homozygotes. This association was not seen in the below median group. Also, we found a trend of increased metabolic syndrome for IL-1β TT homozygotes (P = 0.07). In conclusion, our findings suggest that in a CHD population IL-1 gene polymorphisms may be involved in increased central obesity, and the genetic influences are more evident among patients who have a higher level of obesity or inflammatory markers.     

Endogenous hydrogen sulphide mediates the cardioprotection induced by ischemic postconditioning

The present study aimed to investigate the role of hydrogen sulphide (H2S) in the cardioprotection induced by ischemic postconditioning and to examine the underlying mechanisms. Cardiodynamics and myocardial infarction were measured in isolated rat hearts. Postconditioning with six episodes of 10-s ischemia (IPostC) significantly improved cardiodynamic function, which was attenuated by the blockade of endogenous H2S production with d-l-propargylglycine. Moreover, IPostC significantly stimulated H2S synthesis enzyme activity during the early period of reperfusion. However, d-l-propargylglycine only attenuated the IPostC-induced activation of PKC-alpha and PKC-epsilon but not that of PKC-delta, Akt, and endothelial nitric oxide synthase (eNOS). These data suggest that endogenous H2S contributes partially to the cardioprotection of IPostC via stimulating PKC-alpha and PKC-epsilon. Postconditioning with six episodes of a 10-s infusion of NaHS (SPostC) or 2 min continuous NaHS infusion (SPostC2) stimulated activities of Akt and PKC, improved the cardiodynamic performances, and reduced myocardial infarct size. The blockade of Akt with LY-294002 (15 microM) or PKC with chelerythrine (10 microM) abolished the cardioprotection induced by H2S postconditioning. SPostC2, but not SPostC, also additionally stimulated eNOS. We conclude that endogenous H2S contributes to IPostC-induced cardioprotection. H2S postconditioning confers the protective effects against ischemia-reperfusion injury through the activation of Akt, PKC, and eNOS pathways.