Induction of an antigen-specific CTL response by a conformationally biased agonist of human C5a anaphylatoxin as a molecular adjuvant

A conformationally biased decapeptide agonist of human C5a anaphylatoxin (YSPKPMPLaR) was used as a molecular adjuvant in stimulating an Ag-specific CTL response against murine P815S target cells expressing an Ld-restricted CTL epitope of the hepatitis B surface Ag (HBsAg), Groups of BALB/c mice (H-2(d)) were immunized with aqueous solutions of the HBsAg CTL epitopes (IPQSLDSWWTSL and IPQSLDSTaVTSLRR); the C5a agonist (YSFKPMPLaR); the C5a agonist and HBsAg CTL epitopes admired (IPQSLDSWWTSL and IPQSLDSWWTSLRR + YSFKPMPLaR); the C5a-active, HBsAg CTL epitope-C5a agonist constructs (IPQSLDSWWTSLYSFKPMPLaR, IPQSLDSWWTSLRRYSFKPMPLaR, and IPQSLDSWWTSLRVRRYSFPMPLaR); a C5a-inactive, reverse-moiety construct (YSFKPMPLaRRRIPQSLDSWWTSL); and a C5a-attenuated, carboxyl-terminal-blocked construct (IPQSLDSWWTSLRRYSFKPMPLaRG). Ag-specific CD8(+) CTL responses were observed after the secondary boost in the absence of any added adjuvant only in mice that were immunized with C5a-active contructs, IPQSLDSWWTSLRRYSFKPMPLaR and IPQSLDSWWTSLRVRRYSFKPMPLaR. These two C5a-active immunogens contained potential subtilisin-sensitive linker sequences between the HBsAg CTL epitope and the C5a agonist; i.e., a double-Arg (RR) and a furin protease sensitive sequence (RVRR), The introduction of these potentially cleavable sequences may be a method of increasing the likelihood of liberating the CTL epitope from the C5a agonist by intracellular proteases, thereby facilitating entry of the epitope into Ag-processing pathways via an exogenous route.

MHC class II expression is regulated in dendritic cells independently of invariant chain degradation

We have investigated the mechanisms that control MHC class II (MHC II) expression in immature and activated dendritic cells (DC) grown from spleen and bone marrow precursors. Degradation of the MHC II chaperone invariant chain (li), acquisition of peptide cargo by MHC II, and delivery of MHC II-peptide complexes to the cell surface proceeded similarly in both immature and activated DC. However, immature DC reendocytosed and then degraded the MHC II-peptide complexes much faster than the activated DC. MHC II expression in DC is therefore not controlled by the activity of the protease(s) that degrade Ii, but by the rate of endocytosis of peptide-loaded MHC II. Late after activation, DC downregulated MHC II synthesis both in vitro and in vivo.

Removal of a cysteine ligand from rubredoxin: assembly of Fe2S2 and Fe(S-Cys)(3)(OH) centres

The electron transfer protein rubredoxin from Clostridium pasteurianum contains an Fe(S-Cys)(4) active site. Mutant proteins C9G, C9A, C42G and C42A, in which cysteine ligands are replaced by non-ligating Gly or Ala residues, have been expressed in Escherichia coli. The C42A protein expresses with a (Fe2S2)-S-III cluster in place. In contrast, the other proteins are isolated in colourless forms, although a (Fe2S2)-S-III cluster may be assembled in the C42G protein via incubation with Fe-III and sulfide. The four mutant proteins were isolated as stable mononuclear Hg-II forms which were converted to unstable mononuclear Fe-III preparations that contain both holo and apo protein. The Fe-III systems were characterized by metal analysis and mass spectrometry and by electronic, electron paramagnetic resonance, X-ray absorption and resonance Raman spectroscopies. The dominant Fe-III form in the C9A preparation is a Fe(S-Cys)(3)(OH) centre, similar to that observed previously in the C6S mutant protein. Related centres are present in the proteins NifU and IscU responsible for assembly and repair of iron-sulfur clusters in both prokaryotic and eukaryotic cells. In addition to Fe(S-Cys)(3)(OH) centres, the C9G, C42G and C42A preparations contain a second four-coordinate Fe-III form in which a ligand appears to be supplied by the protein chain. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-0020355-1.

Isolation and characterization of a novel venom protein from an endoparasitoid, Cotesia rubecula (Hym : Braconidae)

Insects are important vectors of diseases with remarkable immune defense capabilities. Hymenopteran endoparasitoids are adapted to overcome the host defense system and, therefore, are useful sources of immune-suppressing proteins. Not much is known about venom proteins in endoparasitoids, especially those that have a functional relationship with polydnaviruses (PDVs). Here, we describe the isolation and characterization of a small venom protein (Vn4.6) from an endoparositoid, Cotesia rubecula, which interferes with the activation of the host hemolymph prophenoloxidose. The coding region for Vn4.6 is located upstream in the opposite direction of a gene coding for a C rubecula PDV-protein (Crp32). Arch. Insect Biochem. Physiol. 53:92-100, 2003. (C) 2003 Wiley-Liss, Inc.

Proteasomal degradation of N-acetyltransferase 1 is prevented by acetylation of the active site cysteine - A mechanism for the slow acetylator phenotype and substrate-dependent down-regulation

Many drugs and chemicals found in the environment are either detoxified by N-acetyltransferase 1 (NAT1, EC 2.3.1.5) and eliminated from the body or bioactivated to metabolites that have the potential to cause toxicity and/or cancer. NAT1 activity in the body is regulated by genetic polymorphisms as well as environmental factors such as substrate-dependent down-regulation and oxidative stress. Here we report the molecular mechanism for the low protein expression from mutant NAT1 alleles that gives rise to the slow acetylator phenotype and show that a similar process accounts for enzyme down-regulation by NAT1 substrates. NAT1 allozymes NAT1 14, NAT1 15, NAT1 17, and NAT1 22 are devoid of enzyme activity and have short intracellular half-lives (similar to4 h) compared with wild-type NAT1 4 and the active allozyme NAT1 24. The inactive allozymes are unable to be acetylated by cofactor, resulting in ubiquitination and rapid degradation by the 26 S proteasome. This was confirmed by site-directed mutagenesis of the active site cysteine 68. The NAT1 substrate p-aminobenzoic acid induced ubiquitination of the usually stable NAT1 4, leading to its rapid degradation. From this study, we conclude that NAT1 exists in the cell in either a stable acetylated state or an unstable non-acetylated state and that mutations in the NAT1 gene that prevent protein acetylation produce a slow acetylator phenotype.

CysView: protein classification based on cysteine pairing patterns

CysView is a web-based application tool that identifies and classifies proteins according to their disulfide connectivity patterns. It accepts a dataset of annotated protein sequences in various formats and returns a graphical representation of cysteine pairing patterns. CysView displays cysteine patterns for those records in the data with disulfide annotations. It allows the viewing of records grouped by connectivity patterns. CysView's utility as an analysis tool was demonstrated by the rapid and correct classification of scorpion toxin entries from GenPept on the basis of their disulfide pairing patterns. It has proved useful for rapid detection of irrelevant and partial records, or those with incomplete annotations. CysView can be used to support distant homology between proteins. CysView is publicly available at http://research.i2r.a-star.edu.sg/CysView/.

The prevalence, intensities and risk factors associated with geohelminth infection in tea-growing communities of Assam, India

OBJECTIVE To determine the prevalence, intensity and associated risk factors for infection with Ascaris, hookworms and Trichuris in three tea-growing communities in Assam, India. METHODS Single faecal samples were collected from 328 individuals and subjected to centrifugal floatation and the Kato Katz quantitation technique and prevalence and intensities of infection with each parasite calculated. Associations between parasite prevalence, intensity and host and environmental factors were then made using both univariate and multivariate analysis. RESULTS The overall prevalence of Ascaris was 38% [95% confidence interval (CI): 33, 43], and the individual prevalence of hookworm and Trichuris was 43% (95% CI: 38, 49). The strongest predictors for the intensity of one or more geohelminths using multiple regression (P less than or equal to 0.10) were socioeconomic status, age, household crowding, level of education, religion, use of footwear when outdoors, defecation practices, pig ownership and water source. CONCLUSION A universal blanket treatment with broad-spectrum anthelmintics together with promotion of scholastic and health education and improvements in sanitation is recommended for helminth control in the communities under study.

Continuous reproduction and recruitment in the narrowback mud crab Panopeus americanus (Brachyura, Panopeidae) in a remnant human-impacted mangrove area

The population structure, reproductive period, and juvenile recruitment of Panopeus americanus were studied in order to enhance knowledge of its life cycle and reproductive strategy and promote the maintenance of its natural stocks in an impacted region. Specimens were collected in the remnant human-impacted mangrove at Araca, state of Sao Paulo, Brazil, at two-month intervals from January to November 2005, at low tide, with a capture effort by three people. The crabs were measured (carapace width, CW) and sexed. The total catch was 275 animals, including 132 males (48.0%); 127 females (46.2%), of which 39 were ovigerous (14.2% of total catch); and 16 individuals whose sex could not be identified (5.8%). No correlation was observed between water temperature and the number of collected individuals; however, there was a significant, positive correlation with salinity. Males were significantly larger than females. The size-frequency distribution was bimodal, reflecting the occurrende of more than one recruitment pulse and the differential abundance of adults during the period of study. The overall sex ratio was 1:0.97 favoring males, and was not significantly different from the expected value, i.e., this population fits the anomalous pattern of sex occurrence in size classes. Ovigerous females were captured in all sampled months, which explain the continuous recruitment observed. Expected low levels of individuals of different size classes in the population were not confirmed. All population aspects found here allowed us to infer that this population of P. americanus is well established in the impacted mangrove by virtue of its reproductive strategy.

Microdistribution of juveniles and adults of the mud crab Panopeus americanus (Brachyura, Panopeidae) in a remnant mangrove area in the southwest Atlantic

The distribution and density of a population of the narrowback mud crab Panopeus americanus were evaluated, with emphasis on the different developmental stages (juveniles and adults) and sexes. Specimens were collected in a remnant human-impacted mangrove at Araca on the northern coast of Sao Paulo, Brazil, at two-month intervals from January through July 2006, using 1m x 1m sample plots randomly distributed in the intertidal zone. The population was abundant, with a mean density of 14.3 +/- 9.3 individuals/m(2). Larger individuals were more abundant in the upper and middle intertidal zones, whereas smaller crabs were predominant in the lower intertidal and thus remained submerged longer. Males and juveniles were homogeneously distributed. Females preferred regions farther from the waterline. A clustered distribution pattern suggested gregarious behaviour and settling of juveniles in the vicinity of the adults` habitats, in an adaptive process as a function of tidal rhythms.

Non-ribosomal peptides produced by Brazilian cyanobacterial isolates with antimicrobial activity

Cyanobacterial strains isolated from terrestrial and freshwater habitats in Brazil were evaluated for their antimicrobial and siderophore activities. Metabolites of fifty isolates were extracted from the supernatant culture media and cells using ethyl acetate and methanol, respectively. The extracts of 24 isolates showed antimicrobial activity against several pathogenic bacteria and one yeast. These active extracts were characterized by Q-TOF/MS. The cyanobacterial strains Cylindrospermopsis raciborskii 339-T3, Synechococcus elongatus PCC7942, Microcystis aeruginosa NPCD-1, M. panniformis SCP702 and Fischerella sp. CENA19 provided the most active extracts. The 50 cyanobacterial strains were also screened for the presence of non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes and microcystin production. Putative fragment genes coding for NRPS adenylation domains and PKS keto-synthase domains were successfully PCR amplified from 92% and 80% of cyanobacterial strains, respectively. The potential therapeutical compounds siderophores were detected in five cyanobacterial isolates. Microcystin production was detected by ELISA test in 26% of the isolates. Further a protease inhibitor substance was detected by LC-MS/MS in the M. aeruginosa NPLJ-4 extract and the presence of aeruginosin and cyanopeptolin was confirmed by PCR amplification using specific primers, and sequenced. This screening study showed that Brazilian cyanobacterial isolates are a rich source of natural products with potential for pharmacological and biotechnological applications. (C) 2010 Elsevier GmbH. All rights reserved.

Dyslipidemia in a Cohort of HIV-infected Latin American Children Receiving Highly Active Antiretroviral Therapy*

In order to describe the prevalence of hypercholesterolemia and hypertriglyceridemia in a cohort of HIV-infected children and adolescents in Latin America and to determine associations with highly active antiretroviral therapy (HAART), we performed this cross-sectional analysis within the NICHD International Site Development Initiative pediatric cohort study. Eligible children had to be at least 2 years of age and be on HAART. Among the 477 eligible HIV-infected youth, 98 (20.5%) had hypercholesterolemia and 140 (29.4%) had hypertriglyceridemia. In multivariable analyses, children receiving protease inhibitor (PI)-containing HAART were at increased risk for hypercholesterolemia [adjusted odds ratio (AOR) = 2.7, 95% confidence interval (CI) 1.3-5.6] and hypertriglyceridemia (AOR = 3.5, 95% CI 1.9-6.4) compared with children receiving non-nucleoside reverse transcriptase inhibitor (NNRTI)-containing HAART. In conclusion, HIV-infected youth receiving PI-containing HAART in this Latin American cohort were at increased risk for hypercholesterolemia and hypertriglyceridemia compared with those receiving NNRTI-containing HAART.

Influence of Lactic Acid on Endogenous and Viral RNA-Induced Immune Mediator Production by Vaginal Epithelial Cells

OBJECTIVE: To evaluate the influence of lactic acid on immune mediator release from vaginal epithelial cells. METHODS: The human vaginal epithelial cell line, VK2/E6E7, was cultured in the presence or absence of physiological concentrations of lactic acid, and in the presence or absence of the viral Toll-like receptor 3 agonist, poly (inosinic acid: cytidylic acid). Supernatants were assayed by enzyme-linked immunosorbent assay (ELISA) for interleukin (IL)-1 beta, IL-6, IL-8, IL-23, transforming growth factor (TGF)-beta and secretory leukocyte protease inhibitor. RESULTS: Vaginal epithelial cells spontaneously released IL-1 beta (25.9 pg/mL), IL-8 (1.0 ng/mL), TGF-beta (175 pg/mL), and secretory leukocyte protease inhibitor (33.8 ng/mL). Only TGF-beta production was marginally enhanced (49%) by addition of lactic acid alone. Poly (inosinic acid: cytidylic acid) by itself stimulated the release of IL-6 (305 pg/mL) and enhanced IL-8 production (2.8 ng/mL). The combination of poly (inosinic acid: cytidylic acid) and lactic acid markedly increased IL-8 production (5.0 ng/mL) and induced the release of IL-1 beta (96.2 pg/mL). The poly (inosinic acid: cytidylic acid)-mediated lactic acid effect on IL-1 beta and IL-8 release was abrogated when the lactic acid was neutralized or if acetic acid was substituted for lactic acid. CONCLUSION: Lactic acid enhances the release of selective mediators from vaginal epithelial cells and stimulates antiviral immune responses. (Obstet Gynecol 2011;118:840-6) DOI: 10.1097/AOG.0b013e31822da9e9

Protein crystallography and examples of its applications in medicinal chemistry

The field of protein crystallography inspires and enthrals, whether it be for the beauty and symmetry of a perfectly formed protein crystal, the unlocked secrets of a novel protein fold, or the precise atomic-level detail yielded from a protein-ligand complex. Since 1958, when the first protein structure was solved, there have been tremendous advances in all aspects of protein crystallography, from protein preparation and crystallisation through to diffraction data measurement and structure refinement. These advances have significantly reduced the time required to solve protein crystal structures, while at the same time substantially improving the quality and resolution of the resulting structures. Moreover, the technological developments have induced researchers to tackle ever more complex systems, including ribosomes and intact membrane-bound proteins, with a reasonable expectation of success. In this review, the steps involved in determining a protein crystal structure are described and the impact of recent methodological advances identified. Protein crystal structures have proved to be extraordinarily useful in medicinal chemistry research, particularly with respect to inhibitor design. The precise interaction between a drug and its receptor can be visualised at the molecular level using protein crystal structures, and this information then used to improve the complementarity and thus increase the potency and selectivity of an inhibitor. The use of protein crystal structures in receptor-based drug design is highlighted by (i) HIV protease, (ii) influenza virus neuraminidase and (iii) prostaglandin H-2-synthetase. These represent, respectively, examples of protein crystal structures that (i) influenced the design of drugs currently approved for use in the treatment of HIV infection, (ii) led to the design of compounds currently in clinical trials for the treatment of influenza infection and (iii) could enable the design of highly specific non-steroidal anti-inflammatory drugs that lack the common side-effects of this drug class.

Acute Pancreatitis Hypertonic Saline Increases Heat Shock Proteins 70 and 90 and Reduces Neutrophil Infiltration in Lung Injury

Objectives: Acute pancreatitis (AP) protease release induces lung parenchymal destruction via matrix metalloproteinases (MMPs), a neutrophil (polymorphonuclear leukocyte)-dependent process. Recent studies in hemorrhagic shock revealed that hypertonic saline (HTS) has an anti-inflammatory effect and can inhibit a variety of neutrophil functions. The aim of this study was to determine whether HTS and its actions in the pathway of neutrophil migration, MMPs, and heat shock proteins (HSPs) are effective in protecting the lung from injury associated with AP. Methods: We determined neutrophil infiltration and expressions of MMPs and HSPs in the lung tissue after AP induced by retrograde infusion of 2.5% of sodium taurocholate. Results: Animals submitted to AP that received HTS compared with those who received normal saline presented with increased HSP70 and HSP90 expressions and reduced myeloperoxidase levels and MMP-9 expression and activity. Conclusions: Our data raised the hypothesis that a sequence of HTS lung protection events increases HSP70 and HSP90, inhibiting infiltration of neutrophils and their protease actions in the lung.