Directed discovery of bivalent peptide ligands to an SH3 domain

The Caenorhabditis elegans SEM-5 SH3 domains recognize proline-rich peptide segments with modest affinity. We developed a bivalent peptide ligand that contains a naturally occurring proline-rich binding sequence, tethered by a glycine linker to a disulfide-closed loop segment containing six variable residues. The glycine linker allows the loop segment to explore regions of greatest diversity in sequence and structure of the SH3 domain: the RT and n-Src loops. The bivalent ligand was optimized using phage display, leading to a peptide (PP-G(4)-L) with 1000-fold increased affinity for the SEM-5 C-terminal SH3 domain over that of a natural ligand. NMR analysis of the complex confirms that the peptide loop segment is targeted to the RT and n-Src loops and parts of the beta-sheet scaffold of this SH3 domain. This binding region is comparable to that targeted by a natural non-PXXP peptide to the p67(phox) SH3 domain, a region not known to be targeted in the Grb2 SH3 domain family. PP-G(4)-L may aid in the discovery of additional binding partners of Grb2 family SH3 domains.     

Phosphorylation enhances mitochondrial targeting of GSTA4-4 through increased affinity for binding to cytoplasmic Hsp70

Recently we showed that three different isoforms of cytosolic glutathione S-transferases (GST), including GSTA4-4, are also localized in the mitochondrial compartment. In this study, we have investigated the mechanism of mouse GSTA4-4 targeting to mitochondria, using a combination of in vitro mitochondrial import assay and in vivo targeting in COS cells transfected with cDNA. Our results show that the mitochondrial GSTA4-4 is more heavily phosphorylated compared with its cytosolic counterpart. Protein kinase activators (cAMP, forskolin, or phorbol-12-myristate-13-acetate) markedly increased GSTA4-4 targeting to mitochondria, whereas kinase inhibitors caused its retention in the cytosol. Immunoinhibition and immunodepletion studies showed that the Hsp70 chaperone is required for the efficient translation of GSTA4-4 as well as its translocation to mitochondria. Co-immunoprecipitation studies showed that kinase inhibitors attenuate the affinity of GSTA4-4 for cytoplasmic Hsp70 suggesting the importance of phosphorylation for binding to the chaperone. Mutational analysis show that the putative mitochondrial targeting signal resides within the C-terminal 20 amino acid residues of the protein and that the targeting signal requires activation by phosphorylation at the C-terminal-most protein kinase A (PKA) site at Ser-189 or protein kinase C (PKC) site at Thr-193. We demonstrate for the first time that PKA and PKC modulate the cytoplasmic and mitochondrial pools of GSTA4-4.     

Progress in the development of a recombinant vaccine for human hookworm disease: the Human Hookworm Vaccine Initiative

Hookworm infection is one of the most important parasitic infections of humans, possibly outranked only by malaria as a cause of misery and suffering. An estimated 1.2 billion people are infected with hookworm in areas of rural poverty in the tropics and subtropics. Epidemiological data collected in China, Southeast Asia and Brazil indicate that, unlike other soil-transmitted helminth infections, the highest hookworm burdens typically occur in adult populations, including the elderly. Emerging data on the host cellular immune responses of chronically infected populations suggest that hookworms induce a state of host anergy and immune hyporesponsiveness. These features account for the high rates of hookworm reinfection following treatment with anthelminthic drugs and therefore, the failure of anthelminthics to control hookworm. Despite the inability of the human host to develop naturally acquired immune responses to hookworm, there is evidence for the feasibility of developing a vaccine based on the successes of immunising laboratory animals with either attenuated larval vaccines or antigens extracted from the alimentary canal of adult blood-feeding stages. The major antigens associated with each of these larval and adult hookworm vaccines have been cloned and expressed in prokaryotic and eukaryotic systems. However, only eukaryotic expression systems (e.g., yeast, baculovirus, and insect cells) produce recombinant proteins that immunologically resemble the corresponding native antigens. A challenge for vaccinologists is to formulate selected eukaryotic antigens with appropriate adjuvants in order to elicit high antibody titres. In some cases, antigen-specific IgE responses are required to mediate protection. Another challenge will be to produce anti-hookworm vaccine antigens at high yield low cost suitable for immunising large impoverished populations living in the developing nations of the tropics.     

Enantioselectivity in Candida antarctica lipase B: a molecular dynamics study

A major problem in predicting the enantioselectivity of an enzyme toward substrate molecules is that even high selectivity toward one substrate enantiomer over the other corresponds to a very small difference in free energy. However, total free energies in enzyme-substrate systems are very large and fluctuate significantly because of general protein motion. Candida antarctica lipase B (CALB), a serine hydrolase, displays enantioselectivity toward secondary alcohols. Here, we present a modeling study where the aim has been to develop a molecular dynamics-based methodology for the prediction of enantioselectivity in CALB. The substrates modeled (seven in total) were 3-methyl-2-butanol with various aliphatic carboxylic acids and also 2-butanol, as well as 3,3-dimethyl-2-butanol with octanoic acid. The tetrahedral reaction intermediate was used as a model of the transition state. Investigative analyses were performed on ensembles of nonminimized structures and focused on the potential energies of a number of subsets within the modeled systems to determine which specific regions are important for the prediction of enantioselectivity. One category of subset was based on atoms that make up the core structural elements of the transition state. We considered that a more favorable energetic conformation of such a subset should relate to a greater likelihood for catalysis to occur, thus reflecting higher selectivity. The results of this study conveyed that the use of this type of subset was viable for the analysis of structural ensembles and yielded good predictions of enantioselectivity.     

Effect of vaccinations with recombinant fusion proteins on Ancylostoma caninum habitat selection in the canine intestine

Laboratory dogs were vaccinated subcutaneously with 3 different recombinant fusion proteins, each precipitated with alum or calcium phosphate. The vaccinated dogs were then challenged orally with 400 third-stage infective larvae (L3) of the canine hookworm, Ancylostoma caninum. The 3 A. caninum antigens selected were Ac-TMP, an adult-specific secreted tissue inhibitor of metalloproteases; Ac-AP, an adult-specific secreted factor Xa serine protease inhibitor anticoagulant; and Ac-ARR-1, a cathepsin D-like aspartic protease. Each of the 3 groups comprised 6 male beagles (8 +/- 1 wk of age). A fourth group comprised control dogs injected with alum. All of the dogs vaccinated with Ac-TMP or Ac-APR-1 exhibited a vigorous antigen-specific antibody response, whereas only a single dog vaccinated with Ac-AP developed an antibody response. Dogs with circulating antibody responses exhibited 4.5-18% reduction in the numbers of adult hookworms recovered from the small intestines at necropsy, relative to alum-injected dogs. In contrast, there was a concomitant increase in the number of adult hookworms recovered from the colon. The increase in colonic hookworms was as high as 500%, relative to alum-injected dogs. Female adult hookworms were more likely to migrate into the colon than were males. Anti-enzyme and anti-enzyme inhibitor antibodies correlated with an alteration in adult hookworm habitat selection in the canine gastroinntestinal tract.     

The transmission dynamics of BSE and vCJD

The bovine spongiform encephalopathy (BSE) epidemic in cattle has had a huge economic impact on the agricultural industries across Europe. Furthermore, scientific evidence now strongly supporting a link between a new variant of Creutzfeldt-Jakob disease (vCJD) and consumption of BSE-infected animals has further heightened the need both to understand the transmission of these new diseases and to improve control measures to protect public health. In this paper we review work undertaken by our group using epidemiological models to understand the transmission dynamics of BSE and vCJD. We present new estimates of the future number of cases of BSE and the number of infected animals slaughtered for consumption for Great Britain, and summarise similar analyses undertaken for Northern Ireland, Ireland, Portugal and France. We also consider the epidemiological determinants of the future course of the vCJD epidemic, including the age and genetic characteristics of the confirmed cases, and present predictions of future case numbers.     

Effect of ninhydrin on the biochemical and histopathological changes induced by ethanol in gastric mucosa of rats

Studies on the effect of ninhydrin in the normal gastric mucosa and against the ethanol induced gastric injury were undertaken in rats in view of the presence of a carbonyl function as well as hydroxyl groups in its chemical structure. In spite of its potentials to generate hydroxyl radicals, it is deemed to possess antioxidant property by virtue of its electrophilic nature. Recent studies have shown gastro-protection to mediate through a reaction between the electrophilic compounds and sulfhydryl groups of the mucosa. Hence it was found worthwhile to evaluate the interaction between the oxidant and antioxidant functions in the structure of the same compound. The effects of ninhydrin pretreatment on gastric mucosal injuries caused by 80% ethanol, 25% NaCl and 0.2M NaOH were investigated in rats. The gastric tissue in ethanol-treated rats was analyzed for different histopathological lesions. In addition, the effects on ethanol-induced changes in the gastric levels of proteins, nucleic acids, non-protein sulfhydryl (NP-SH) and malondialdehyde (MDA) were also evaluated. Ninhydrin, as such, failed to induce any significant changes in normal gastric mucosa, while its pretreatment at oral doses of 5, 10 and 20 mg/kg was found to provide a dose-dependent protection against the ulcers induced by ethanol, NaOH and NaCl. The results of histopathological evaluation revealed a protective effect of ninhydrin on congestion, hemorrhage, edema, erosions and necrosis caused by ethanol. Furthermore, the pretreatment afforded a dose-dependent inhibition of the ethanol-induced depletion of proteins, nucleic acids, NP-SH and increase of MDA in the gastric tissue. The results obtained clearly demonstrate the anti-ulcerogenic activity of ninhydrin. The exact mechanism of action is not known. However, the carbonyl function in ninhydrin appears to achieve antioxidant balance and protect the gastric mucosa from the ethanol-induced gastric injury. Further studies are warranted to investigate the toxicity and detailed mechanism of action of this potent compound before any clinical trials, especially at the effective lower doses.     

Energetic Pathway Sampling in a Protein Interaction Domain

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Evidence for the role of glycoprotein G of respiratory syncytial virus in binding of Neisseria meningitidis to HEp-2 cells

Abstract Viral glycoproteins G and F are expressed on the surface of cells infected with respiratory syncytial virus (RSV). We investigated the role of these proteins in the previously reported enhanced binding of Neisseria meningitidis to RSV-infected HEp-2 cells. Virus particles attached to bacteria were detected by immunofluorescence with flow cytometry. Binding of FITC-labelled bacteria to RSV-infected cells was significantly inhibited by monoclonal antibody against glycoprotein G. Unlabelled bacteria interfered with binding of the anti-G monoclonal antibody to these cells. These interactions were not found with a monoclonal antibody against glycoprotein F. We propose that glycoprotein G of RSV expressed on the surface of infected cells might act as an additional receptor for meningococci.     

Psychosocial Factors of Antenatal Anxiety and Depression in Pakistan: Is Social Support a Mediator?

Introduction

Pregnancy is generally viewed as a time of fulfillment and joy; however, for many women it can be a stressful event. In South Asia it is associated with cultural stigmas revolving around gender discrimination, abnormal births and genetic abnormalities.

Methodology

This cross-sectional study was done at four teaching hospitals in Lahore from February, 2014 to June, 2014. A total of 500 pregnant women seen at hospital obstetrics and gynecology departments were interviewed with a questionnaire consisting of three sections: demographics, the Hospital Anxiety and Depression Scale (HADS) and the Social Provisions Scale (SPS). Pearson’s chi-squared test, bivariate correlations and multiple linear regression were used to analyze associations between the independent variables and scores on the HADS and SPS.

Results

Mean age among the 500 respondents was 27.41 years (5.65). Anxiety levels in participants were categorized as normal (145 women, 29%), borderline (110, 22%) or anxious (245, 49%). Depression levels were categorized as normal (218 women, 43.6%), borderline (123, 24.6%) or depressed (159, 31.8%). Inferential analysis revealed that higher HADS scores were significantly associated with lower scores on the SPS, rural background, history of harassment, abortion, cesarean delivery and unplanned pregnancies (P < .05). Social support (SPS score) mediated the relationship between the total number of children, gender of previous children and HADS score. Women with more daughters were significantly more likely to score higher on the HADS and lower on the SPS, whereas higher numbers of sons were associated with the opposite trends in the scores (P < .05).

Conclusion

Because of the predominantly patriarchal sociocultural context in Pakistan, the predictors of antenatal anxiety and depression may differ from those in developed countries. We therefore suggest that interventions designed and implemented to reduce antenatal anxiety and depression should take into account these unique factors.