The apolipoprotein A-I mimetic peptide 4F prevents defects in vascular function in endotoxemic rats

High density lipoprotein (HDL) and apolipoprotein A-I (apoA-I) reduce inflammatory responses to lipopolysaccharide (LPS). We tested the hypothesis that the apoA-I mimetic peptide 4F prevents LPS-induced defects in blood pressure and vascular reactivity. Systolic blood pressure (SBP) was measured in rats at baseline and 6 h after injection of LPS (10 mg/kg) or saline vehicle. Subgroups of LPS-treated rats also received 4F (10 mg/kg) or scrambled 4F (Sc-4F). LPS administration reduced SBP by 35% compared with baseline. 4F attenuated the reduction in SBP in LPS-treated rats (17% reduction), while Sc-4F was without effect. Ex vivo studies showed a reduced contractile response to phenylephrine (PE) in aortae of LPS-treated rats (ED₅₀ = 459 ± 83 nM) compared with controls (ED₅₀ = 57 ± 6 nM). This was associated with nitric oxide synthase 2 (NOS2) upregulation. 4F administration improved vascular contractility (ED₅₀ = 60 ± 9 nM), reduced aortic NOS2 protein, normalized plasma levels of NO metabolites, and reduced mortality in LPS-treated rats. These changes were associated with a reduction in plasma endotoxin activity. In vivo administration of ¹⁴C-4F and Bodipy-LPS resulted in their colocalization and retention in the HDL fraction. It is proposed that 4F promotes the localization of LPS to the HDL fraction, resulting in endotoxin neutralization. 4F may thus prevent LPS-induced hemodynamic changes associated with NOS2 induction.     

Biochemical alterations induced by nickel oxide nanoparticles in female Wistar albino rats after acute oral exposure

Context: Nickel oxide (NiO) nanoparticles (NPs) with appropriate surface chemistry have been widely used for their potential new applications in biomedical industry. Increased usage of these NPs enhances the chance of exposure of personnel involved in the work place.

Objective: This study was designed to assess the ability of NiO NPs to cause biochemical alterations post-acute oral exposure in female Wistar rats.

Materials and methods: Rats were administered with 125, 250, and 500?mg/kg doses of NiO NPs for haematological, biochemical, and histopathological studies. Biodistribution patterns of NiO NPs in female Wistar rats were also monitored.

Results: NiO NPs caused significant (p?Conclusions: This study revealed that exposure to nanosized NiO particles at acute doses may cause adverse changes in animal biochemical profiles. Further, the in vivo studies on toxicity evaluation help in biomonitoring of the potential contaminants.     

Structural consequences of replacement of an alpha-helical Pro residue in Escherichia coli thioredoxin

While it is well known that introduction of Pro residues into the interior of protein alpha-helices is destabilizing, there have been few studies that have examined the structural and thermodynamic effects of the replacement of a Pro residue in the interior of a protein alpha-helix. We have previously reported an increase in stability in the P40S mutant of Escherichia coli thioredoxin of 1-1.5 kcal/mol in the temperature range 280-330 K. This paper describes the structure of the P40S mutant at a resolution of 1.8 A. In wild-type thioredoxin, P40 is located in the interior of helix two, a long alpha-helix that extends from residues 32 to 49 with a kink at residue 40. Structural differences between the wild-type and P40S are largely localized to the above helix. In the P40S mutant, there is an expected additional hydrogen bond formed between the amide of S40 and the carbonyl of residue K36 and also additional hydrogen bonds between the side chain of S40 and the carbonyl of K36. The helix remains kinked. In the wild-type, main chain hydrogen bonds exist between the amide of 44 and carbonyl of 40 and between the amide of 43 and carbonyl of 39. However, these are absent in P40S. Instead, these main chain atoms are hydrogen bonded to water molecules. The increased stability of P40S is likely to be due to the net increase in the number of hydrogen bonds in helix two of E.coli thioredoxin.     

PARTAKE Survey of Public Knowledge and Perceptions of Clinical Research in India

Background

A public that is an informed partner in clinical research is important for ethical, methodological, and operational reasons. There are indications that the public is unaware or misinformed, and not sufficiently engaged in clinical research but studies on the topic are lacking. PARTAKE – Public Awareness of Research for Therapeutic Advancements through Knowledge and Empowerment is a program aimed at increasing public awareness and partnership in clinical research. The PARTAKE Survey is a component of the program.

Objective

To study public knowledge and perceptions of clinical research.

Methods

A 40-item questionnaire combining multiple-choice and open-ended questions was administered to 175 English- or Hindi-speaking individuals in 8 public locations representing various socioeconomic strata in New Delhi, India.

Results

Interviewees were 18–84 old (mean: 39.6, SD±16.6), 23.6% female, 68.6% employed, 7.3% illiterate, 26.3% had heard of research, 2.9% had participated and 58.9% expressed willingness to participate in clinical research. The following perceptions were reported (% true/% false/% not aware): ‘research benefits society’ (94.1%/3.5%/2.3%), ‘the government protects against unethical clinical research’ (56.7%/26.3%/16.9%), ‘research hospitals provide better care’ (67.2%/8.7%/23.9%), ‘confidentiality is adequately protected’ (54.1%/12.3%/33.5%), ‘participation in research is voluntary’ (85.3%/5.8%/8.7%); ‘participants treated like ‘guinea pigs’’ (20.7%/53.2%/26.0%), and ‘compensation for participation is adequate’ (24.7%/12.9%/62.3%).

Conclusions

Results suggest the Indian public is aware of some key features of clinical research (e.g., purpose, value, voluntary nature of participation), and supports clinical research in general but is unaware of other key features (e.g., compensation, confidentiality, protection of human participants) and exhibits some distrust in the conduct and reporting of clinical trials. Larger, cross-cultural surveys are required to inform educational programs addressing these issues.     

Combinations of Susceptibility Genes Are Associated with Higher Risk for Multiple Sclerosis and Imply Disease Course Specificity

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system that predominantly affects young adults. The genetic contributions to this multifactorial disease were underscored by a genome wide association study (GWAS) conducted by the International Multiple Sclerosis Genetic Consortium in a multinational cohort prompting the discovery of 57 non-MHC MS-associated common genetic variants. Hitherto, few of these newly reported variants have been replicated in larger independent patient cohorts. We genotyped a cohort of 1033 MS patients and 644 healthy controls with a consistent genetic background for the 57 non-MHC variants reported to be associated with MS by the first large GWAS as well as the HLA DRB1*1501 tagging SNP rs3135388. We robustly replicated three of the 57 non-MHC reported MS-associated single nucleotide polymorphisms (SNPs). In addition, our study revealed several genotype-genotype combinations with an evidently higher degree of disease association than the genotypes of the single SNPs. We further correlated well-defined clinical phenotypes, i.e. ataxia, visual impairment due to optic neuritis and paresis with single SNPs and genotype combinations, and identified several associations. The results may open new avenues for clinical implications of the MS associated genetic variants reported from large GWAS.     

Expression of functional TRPA1 receptor on human lung fibroblast and epithelial cells

The transient receptor potential subfamily A member 1 (TRPA1) is a non-selective cation channel implicated in the pathogenesis of several airway diseases like asthma and chronic obstructive pulmonary disease (COPD). Most of the research on TRPA1 focuses on its expression and function in neuronal context; studies investigating non-neuronal expression of TRPA1 are lacking. In the present study, we show functional expression of TRPA1 in human lung fibroblast cells (CCD19-Lu) and human pulmonary alveolar epithelial cell line (A549). We demonstrate TRPA1 expression at both mRNA and protein levels in these cell types. TRPA1 selective agonists like allyl isothiocyanate (AITC), 4-hydroxynonenal (4-HNE), crotonaldehyde and zinc, induced a concentration-dependent increase in Ca+2 influx in CCD19-Lu and A549 cells. AITC-induced Ca+2 influx was inhibited by Ruthenium red (RR), a TRP channel pore blocker, and by GRC 17536, a TRPA1 specific antagonist. Furthermore, we also provide evidence that activation of the TRPA1 receptor by TRPA1 selective agonists promotes release of the chemokine IL-8 in CCD19-Lu and A549 cells. The IL-8 release in response to TRPA1 agonists was attenuated by TRPA1 selective antagonists. In conclusion, we demonstrate here for the first time that TRPA1 is functionally expressed in cultured human lung fibroblast cells (CCD19-Lu) and human alveolar epithelial cell line (A549) and may have a potential role in modulating release of this important chemokine in inflamed airways.     

The gonococcal fur regulon: identification of additional genes involved in major catabolic,recombination, and secretory pathways

In this study, we have characterized the in vitro binding of Neisseria gonorrhoeae Fur to several well-defined iron transport genes, as well as to additional genes involved in major catabolic, secretory, and recombination pathways of gonococci. The gonococcal Fur protein was recombinantly expressed in Escherichia coli HBMV119. Fur was isolated from inclusion bodies and partially purified by ion-exchange chromatography. Gonococcal Fur was found to bind to the promoter/operator region of a gene encoding the previously identified Fur-regulated periplasmic binding protein (FbpA) in a metal ion-dependent fashion, demonstrating that purified Fur is functional. In silico analysis of the partially completed gonococcal genome (FA1090) identified Fur boxes in the promoters of several genes, including tonB, fur, recN, secY, sodB, hemO, hmbR, fumC, a hypothetical gene (Fe-S homolog), and the opa family of genes. By using purified gonococcal Fur, we demonstrate binding to the operator regions of tonB, fur, recN, secY, sodB, hemO, hmbR, fumC, the Fe-S homolog gene, and the opa gene family as determined by an electrophoretic mobility shift assay. While gonococcal Fur was demonstrated to bind to the promoter regions of all 11 opa genes (opaA through -K), we did not detect binding of purified E. coli Fur with 8 of the 11 opa members, indicating that target DNA sequence specificities between these two closely related proteins exist. Furthermore, we observed differences in the relative strengths of binding of gonococcal Fur for these different genes, which most likely reflect a difference in affinity between gonococcal Fur and its DNA targets. This is the first report that definitively demonstrates the binding of gonococcal Fur to its own promoter/operator region, as well as to the opa family of genes that encode surface proteins. Our results demonstrate that the gonococcal Fur protein binds to the regulatory regions of a broad array of genes and indicates that the gonococcal Fur regulon is larger than originally proposed.     

Meningococcal group W-135 and Y capsular polysaccharides paradoxically enhance activation of the alternative pathway of complement

Although capsular polysaccharide (CPS) is critical for meningococcal virulence, the molecular basis of alternative complement pathway (AP) regulation by meningococcal CPSs remains unclear. Using serum with only the AP active, the ability of strains to generate C3a (a measure of C3 activation) and subsequently deposit C3 fragments on bacteria was studied in encapsulated group A, B, C, W-135, and Y strains and their isogenic unencapsulated mutants. To eliminate confounding AP regulation by membrane-bound factor H (fH; AP inhibitor) and lipooligosaccharide sialic acid, the meningococcal fH ligands (fHbp and NspA) and lipooligosaccharide sialylation were deleted in all strains. Group A CPS expression did not affect C3a generation or C3 deposition. C3a generated by encapsulated and unencapsulated group B and C strains was similar, but CPS expression was associated with reduced C3 deposition, suggesting that these CPSs blocked C3 deposition on membrane targets. Paradoxically, encapsulated W-135 and Y strains (including the wild-type parent strains) enhanced C3 activation and showed marked C3 deposition as early as 10 min; at this time point C3 was barely activated by the unencapsulated mutants. W-135 and Y CPSs themselves served as a site for C3 deposition; this observation was confirmed using immobilized purified CPSs. Purified CPSs bound to unencapsulated meningococci, simulated findings with naturally encapsulated strains. These data highlight the heterogeneity of AP activation on the various meningococcal serogroups that may contribute to differences in their pathogenic mechanisms.     

Influence of nonhistone chromatin protein HMG-1 on the enzymatic digestion of purified DNA

The effect of chicken erythrocyte High Mobility Group protein 1 (HMG-1) on the enzymatic hydrolysis of purified double-stranded and single-stranded bacteriophage lambda DNA was studied. HMG-1 was found to inhibit the digestion of single- and double-stranded DNA by S1 nuclease and DNase I, respectively. HMG-I increased the rate of hydrolysis of double-stranded DNA by micrococcal nuclease, particularly at low HMG-1/DNA ratios, and had little effect on the hydrolysis of single-stranded DNA by micrococcal nucleases, even at high HMG-1 DNA ratios. We also present a semi-quantitative estimate that HMG-1 and HMG-2 occur in chromatin from rapidly dividing, cultured rat hepatoma cells at about 8 times the level that they occur in adult rat liver chromatin.     

Purification and characterization of Plasmodium yoelii adenosine deaminase

Plasmodium lacks the de novo pathway for purine biosynthesis and relies exclusively on the salvage pathway. Adenosine deaminase (ADA), first enzyme of the pathway, was purified and characterized from Plasmodium yoelii, a rodent malarial species, using ion exchange and gel exclusion chromatography. The purified enzyme is a 41 kDa monomer. The enzyme showed K_m values of 41 μM and 34 μM for adenosine and 2′-deoxyadenosine, respectively. Erythro-9-(2-hydroxy-3-nonyl) adenine competitively inhibited P. yoelii ADA with K_i value of 0.5 μM. The enzyme was inhibited by DEPC and protein denaturing agents, urea and GdmCl. Purine analogues significantly inhibited ADA activity. Inhibition by p-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM) indicated the presence of functional –SH groups. Tryptophan fluorescence maxima of ADA shifted from 339 nm to 357 nm in presence of GdmCl. Refolding studies showed that higher GdmCl concentration irreversibly denatured the purified ADA. Fluorescence quenchers (KI and acrylamide) quenched the ADA fluorescence intensity to the varied degree. The observed differences in kinetic properties of P. yoelii ADA as compared to the erythrocyte enzyme may facilitate in designing specific inhibitors against ADA.