Resuscitation of Vibrio cholerae O1 strain TSI-4 from a viable but nonculturable state by heat shock

Abstract Vibrio cholerae strain TSI-4 was incubated in an M9 salt solution at 15 °C for more than 100 days. The plate counts showed no viable cells on day 30, but a broth culture from that day showed the growth of bacteria. However, after 35 days the bacteria entered the nonculturable state, based on the assessment of both the plate counts and broth culture. A portion of the culture was heated at 45 °C for 1 min in a water bath and subsequently plated onto a nutrient agar plate. More than 1000 colonies were recovered after this heat-shock treatment. The recovered cells showed the same chromosomal DNA pattern in the restriction map and the same outer membrane protein pattern in SDS-PAGE. Recovery of viable cells by heat-shock was achieved in cultures grown on M9 salt but not from cultures grown in phosphate-buffered saline. This suggests that the presence of NH₄Cl in the M9 salt solution may support the growth of the bacteria in a low nutrient medium, while also playing an important role in resuscitation.     

The Purification of a GroEL-Like Stress Protein from Aerobically Adapted Campylobacter jejuni

From plate cultures of Campylobacter jejuni grown in room air a particulate protein of 62 kDa was isolated by ion-exchange chromatography. The protein had a square shape from the side view but when viewed from the top it had a star-shaped structure. The molecular size of the whole particle determined by gel filtration was 850 kDa which suggested the presence of 14 subunits of 62 kDa in each particle. The N-terminal 37 amino residues showed more than 80% homology with the sequence of these heat shock protein (HSP) 60 homologs of Chlamydia trachomatis, Helicobacter pylori, and Escherichia coli (GroEL). This protein is immunologically cross-reactive with the antiserum for the 60-kDa HSP of Yersinia enterocolitica. Production of the 62-kDa protein increased under heat stress and growth in an aerobic atmospheric environment. From these observations we concluded that the 62-kDa protein is a Campylobacter stress protein (Cj62) which belongs to the HSP 60 family.     

A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Naturally-Acquired Immune Response against Plasmodium vivax Rhoptry-Associated Membrane Antigen

Rhoptry-associated membrane antigen (RAMA) is an abundant glycophosphatidylinositol (GPI)-anchored protein that is embedded within the lipid bilayer and is implicated in parasite invasion. Antibody responses against rhoptry proteins are produced by individuals living in a malaria-endemic area, suggesting the immunogenicity of Plasmodium vivax RAMA (PvRAMA) for induction of immune responses during P. vivax infection. To determine whether PvRAMA contributes to the acquisition of immunity to malaria and could be a rational candidate for a vaccine, the presence of memory T cells and the stability of the antibody response against PvRAMA were evaluated in P. vivax-exposed individuals. The immunogenicity of PvRAMA for the induction of T cell responses was evaluated by in vitro stimulation of peripheral blood mononuclear cells (PBMCs). High levels of interferon (IFN)-γ and interleukin (IL)-10 cytokines were detected in the culture supernatant of PBMCs, and the CD4⁺ T cells predominantly produced IL-10 cytokine. The levels of total anti-PvRAMA immunoglobulin G (IgG) antibody were significantly elevated, and these antibodies persisted over the 12 months of the study. Interestingly, IgG1, IgG2 and IgG3 were the major antibody subtypes in the response to PvRAMA. The frequency of IgG3 in specific to PvRAMA antigen maintained over 12 months. These data could explain the immunogenicity of PvRAMA antigen in induction of both cell-mediated and antibody-mediated immunity in natural P. vivax infection, in which IFN-γ helps antibody class switching toward the IgG1, IgG2 and IgG3 isotypes and IL-10 supports PvRAMA-specific antibody production.     

Trichothecenes and zearalenone production by fusarium species isolated from Argentinean black beans

Isolates of Fusarium species obtained from freshly harvested bean grains for human consumption collected from different Argentinean regions, were investigated for their ability to biosynthesise trichothecenes and zearalenone either on rice grains or beans. Low incidence of toxigenic fungi was observed. These mycotoxigenic species produced several toxins when grown on rice but none or little amount when cultured on beans. The results of this report suggest that contamination of Argentinean beans with Fusarium mycotoxins will not be common and therefore people would be at low mycotoxicosis risk through consumption of beans.     

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.     

Ligand-dependent differences in estrogen receptor beta-interacting proteins identified in lung adenocarcinoma cells corresponds to estrogenic responses

Background

A recent epidemiological study demonstrated a reduced risk of lung cancer mortality in breast cancer patients using antiestrogens. These and other data implicate a role for estrogens in lung cancer, particularly nonsmall cell lung cancer (NSCLC). Approximately 61% of human NSCLC tumors express nuclear estrogen receptor β (ERβ); however, the role of ERβ and estrogens in NSCLC is likely to be multifactorial. Here we tested the hypothesis that proteins interacting with ERβ in human lung adenocarcinoma cells that respond proliferatively to estradiol (E₂) are distinct from those in non-E₂-responsive cells.

Methods

FLAG affinity purification of FLAG-ERβ-interacting proteins was used to isolate ERβ-interacting proteins in whole cell extracts from E₂ proliferative H1793 and non-E₂-proliferative A549 lung adenocarcinoma cell lines. Following trypsin digestion, proteins were identified using liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). Proteomic data were analyzed using Ingenuity Pathway Analysis. Select results were confirmed by coimmunoprecipitation.

Results

LC-MS/MS identified 27 non-redundant ERβ-interacting proteins. ERβ-interacting proteins included hsp70, hsp60, vimentin, histones and calmodulin. Ingenuity Pathway Analysis of the ERβ-interacting proteins revealed differences in molecular and functional networks between H1793 and A549 lung adenocarcinoma cells. Coimmunoprecipitation experiments in these and other lung adenocarcinoma cells confirmed that ERβ and EGFR interact in a gender-dependent manner and in response to E₂ or EGF. BRCA1 interacted with ERβ in A549 cell lines and in human lung adenocarcinoma tumors, but not normal lung tissue.

Conclusion

Our results identify specific differences in ERβ-interacting proteins in lung adenocarcinoma cells corresponding to ligand-dependent differences in estrogenic responses.

     

The relation between cartilage biomarkers (C2C,C1,2C,CS846, and CPII) and the long-term outcome of rheumatoid arthritis patients within the CAMERA trial

Introduction  

The aim of this study was to investigate whether serum biomarker levels of C2C, C1,2C, CS846, and CPII can predict the long-term course of disease activity and radiographic progression early in the disease course of rheumatoid arthritis (RA).