Molecular characterization of the Bacillus anthracis main S-layer component: evidence that it is the major cell-associated antigen

Bacillus anthracis, the aetiological agent of anthrax, is a Gram-positive spore-forming bacterium. The cell wall of vegetative cells of B. anthracis is surrounded by an S-layer. An array remained when sap, a gene described as encoding an S-layer component, was deleted. The remaining S-layer component, termed EA1, is chromosomally encoded. The gene encoding EA1 (eag) was obtained on two overlapping fragments in Escherichia coli and shown to be contiguous to the sap gene. The EA1 amino acid sequence, deduced from the eag nucleotide sequence, shows classical S-layer protein features (no cysteine, only 0.1% methionine, 10% lysine, and a weakly acidic pi). Similar to Sap and other Gram-positive surface proteins, EA1 has three 'S-layer-homology’motifs immediately downstream from a signal peptide. Single- and double-disrupted mutants were constructed. EA1 and Sap were co-localized at the cell surface of the wild-type bacilli. However, EA1 was more tightly bound than Sap to the bacteria. Electron microscopy studies and in vivo experiments with the constructed mutants showed that EA1 constitutes the main lattice of the B. anthracis S-layer, and is the major cell-associated antigen.     

IL9 maps to mouse chromosome 13 and human chromosome 5

Mouse and human cDNA clones encoding the T-cell and mast cell growth factor P40, now designated IL-9, were used to identify DNA restriction fragment length polymorphisms (RFLPs) in sets of somatic cell hybrids and between inbred strains of mice and interspecific backcross progeny. Segregation of mouse and human chromosomes among somatic cell hybrids indicated a location on mouse chromosome 13 and human chromosome 5. RFLPs were identified among inbred strains of mice. Analysis of chromosome 13 alleles for Tcrg, Dhfr, and Il-9 in an interspecific cross between Mus musculus and NFS/N or C58/J mice indicates that IL-9 is distal to Tcrg and Proximal to Dhfr.     

Characterization of the effects of a polyunsaturated fatty acid (PUFA) on mitochondrial bioenergetics of chronologically aged yeast

Increased membrane unsaturation has been associated with shorter longevity due to higher sensitivity to lipid peroxidation (LP) leading to enhanced mitochondrial dysfunction and ROS overproduction. However, the role of LP during aging has been put in doubt along with the participation of electron leak at the electron transport chain (ETC) in ROS generation in aged organisms. Thus, to test these hypothesis and gain further information about how minimizing LP preserves ETC function during aging, we studied the effects of α-linolenic acid (C18:3) on in situ mitochondrial ETC function, ROS production and viability of chronologically aged cells of S. cerevisiae, whose membranes are intrinsically resistant to LP due to the lack of PUFA. Increased sensitivity to LP was observed in cells cultured with C18:3 at 6 days of aging. This was associated with higher viability loss, dissipated membrane potential, impaired respiration and increased ROS generation, being these effects more evident at 28 days. However, at this point, lower sensitivity to LP was observed without changes in the membrane content of C18:3, suggesting the activation of a mechanism counteracting LP. The cells without C18:3 display better viability and mitochondrial functionality with lower ROS generation even at 28 days of aging and this was attributed to full preservation of complex III activity. These results indicate that the presence of PUFA in membranes enhances ETC dysfunction and electron leak and suggest that complex III is crucial to preserve membrane potential and to maintain a low rate of ROS production during aging.     

A Prospective Study of Romanian Agriculture Workers for Zoonotic Influenza Infections

Background

In this prospective study we sought to examine seroepidemiological evidence for acute zoonotic influenza virus infection among Romanian agricultural workers.

Methods

Sera were drawn upon enrollment (2009) and again at 12 and 24 months from 312 adult agriculture workers and 51 age-group matched controls. Participants were contacted monthly for 24 months and queried regarding episodes of acute influenza-like illnesses (ILI). Cohort members meeting ILI criteria permitted respiratory swab collections as well as acute and convalescent serum collection. Serologic assays were performed against 9 avian, 3 swine, and 3 human influenza viruses.

Results

During the two-year follow-up, a total of 23 ILI events were reported. Two subjects'' specimens were identified as influenza A by rRT-PCR. During the follow-up period, three individuals experienced elevated microneutralization antibody titers ≥1∶80 against three (one each) avian influenza viruses: A/Teal/Hong Kong/w312/97(H6N1), A/Hong Kong/1073/1999(H9N2), or A/Duck/Alberta/60/1976(H12N5). However, none of these participants met the criteria for poultry exposure. A number of subjects demonstrated four-fold increases over time in hemagglutination inhibition (HI) assay titers for at least one of the three swine influenza viruses (SIVs); however, it seems likely that two of these three responses were due to cross-reacting antibody against human influenza. Only elevated antibody titers against A/Swine/Flanders/1/1998(H3N2) lacked evidence for such confounding. In examining risk factors for elevated antibody against this SIV with multiple logistic regression, swine exposure (adjusted OR = 1.8, 95% CI 1.1–2.8) and tobacco use (adjusted OR = 1.8; 95% CI 1.1–2.9) were important predictors.

Conclusions

While Romania has recently experienced multiple incursions of highly pathogenic avian influenza among domestic poultry, this cohort of Romanian agriculture workers had sparse evidence of avian influenza virus infections. In contrast, there was evidence, especially among the swine exposed participants, of infections with human and one swine H3N2 influenza virus.     

Biparental care in house sparrows: negotiation or sealed bid?

We explored the responses of monogamous house sparrow parentsto deviations in their mates' contributions to nestling provisioning.Following 1-2 days of baseline measurement of parental fooddelivery rates, we applied small lead fishing weights to thetail feathers of either male or female parents. Weighting hadmuch greater immediate impact on male parental care than on female care, but the handicapping had little long-term effecton either male or female provisioning behavior. When parentalperformance of handicapped males was most impaired, their matesdid not show significant increases in parental care as compensation,nor did females mated to handicapped males reduce their provisioningas their mates recovered from weighting. Similarly, males matedto handicapped females did not respond to their partners' recovery with declines in their own efforts; paradoxically, these malesshowed a sustained elevation of provisioning throughout thepost-treatment interval, despite no significant reduction inprovisioning by weighted females. The apparent insensitivityof both males and females to changes in their mates' parentalbehavior, and the ineffectiveness of current partner behaviorat predicting an individual's provisioning effort, fail toconform to assumptions of biparental care models that requirefacultative responses to partner deviations in effort. Instead,the remarkable consistency of each individual's behavior supportsthe notion of "sealed bids" and suggests that variation innestling provisioning is largely attributable to factors thatare independent of the mate's current behavior, such as differencesin individual quality.     

Structural and functional relationships between Pasteurella multocida and enterobacterial adenylate cyclases.

The Pasteurella multocida adenylate cyclase gene has been cloned and expressed in Escherichia coli. The primary structure of the protein (838 amino acids) deduced from the corresponding nucleotide sequence was compared with that of E. coli. The two enzymes have similar molecular sizes and, based on sequence conservation at the protein level, are likely to be organized in two functional domains: the amino-terminal catalytic domain and the carboxy-terminal regulatory domain. It was shown that P. multocida adenylate cyclase synthesizes increased levels of cyclic AMP in E. coli strains deficient in the catabolite gene activator protein compared with wild-type strains. This increase does not occur in strains deficient in both the catabolite gene activator protein and enzyme III-glucose, indicating that a protein similar to E. coli enzyme III-glucose is involved in the regulation of P. multocida adenylate cyclase. It also indicates that the underlying process leading to enterobacterial adenylate cyclase activation has been conserved through evolution.     

Exercise training decreases rat heart mitochondria free radical generation but does not prevent Ca2+-induced dysfunction.

Exercise provides cardioprotection against ischemia-reperfusion injury, a process involving mitochondrial reactive oxygen species (ROS) generation and calcium overload. This study tested the hypotheses that isolated mitochondria from hearts of endurance-trained rats have decreased ROS production and improved tolerance against Ca(2+)-induced dysfunction. Male Fischer 344 rats were either sedentary (Sed, n = 8) or endurance exercise trained (ET, n = 11) by running on a treadmill for 16 wk (5 days/wk, 60 min/day, 25 m/min, 6 degrees grade). Mitochondrial oxidative phosphorylation measures were determined with glutamate-malate or succinate as substrates, and H(2)O(2) production and permeability transition pore (PTP) opening were determined with succinate. All assays were carried out in the absence and presence of calcium. In response to 25 and 50 microM CaCl(2), Sed and ET displayed similar decreases in state 3 respiration, respiratory control ratio, and ADP:O ratio. Ca(2+)-induced PTP opening was also similar. However, H(2)O(2) production by ET was lower than Sed (P < 0.05) in the absence of calcium (323 +/- 12 vs. 362 +/- 11 pmol.min(-1).mg protein(-1)) and the presence of 50 microM CaCl(2) (154 +/- 3 vs. 197 +/- 7 pmol.min(-1).mg protein(-1)). Rotenone, which blocks electron flow from succinate to complex 1, reduced H(2)O(2) production and eliminated differences between ET and Sed. Mitochondrial superoxide dismutase and glutathione peroxidase were not affected by exercise. Catalase activity was extremely low but increased 49% in ET (P < 0.05). In conclusion, exercise reduces ROS production in myocardial mitochondria through adaptations specific to complex 1 but does not improve mitochondrial tolerance to calcium overload.     

The S-layer homology domain as a means for anchoring heterologous proteins on the cell surface of Bacillus anthracis

Bacillus anthracis synthesizes two S-layer proteins, each containing three S-layer homology (SLH) motifs towards their amino-terminus. In vitro experiments suggested that the three motifs of each protein were organized as a structural domain sufficient to bind purified cell walls. Chimeric genes encoding the SLH domains fused to the levansucrase of Bacillus subtilis were constructed and integrated on the chromosome. Cell fractionation and electron microscopy studies showed that both heterologous polypeptides were targeted to the cell surface. In addition, surface-exposed levansucrase retained its enzymatic and antigenic properties. Preliminary results concerning applications of this work are presented.     

Alterations in plasma concentrations of testosterone, LH, and prolactin associated with mating in the male rat.

The hormonal response of the male rat to sexual activity was investigated in two studies. In the first, no evidence of a chronic elevation in plasma levels of testosterone (T), LH, or prolactin (PRL) was observed in sexually experienced rats compared to naive controls. Both groups showed an acute increase in plasma levels of all three hormones following mating, but the increases shown by the experienced group were more pronounced. In the second study, plasma levels of T, LH and PRL rose in sexually experienced male rats following exposure to a mating arena whether it contained an estrous female, an anestrous female, or no other animal. However, the increases were considerably larger in the group exposed to estrous females. It is suggested that plasma hormones rise in anticipation of mating, although not to the same extent as following mating, and that the anticipatory rise may function to initiate or facilitate mating behavior.     

Bacterial SLH domain proteins are non-covalently anchored to the cell surface via a conserved mechanism involving wall polysaccharide pyruvylation

Several bacterial proteins are non-covalently anchored to the cell surface via an S-layer homology (SLH) domain. Previous studies have suggested that this cell surface display mechanism involves a non-covalent interaction between the SLH domain and peptidoglycan-associated polymers. Here we report the characterization of a two-gene operon, csaAB, for cell surface anchoring, in Bacillus anthracis. Its distal open reading frame (csaB) is required for the retention of SLH-containing proteins on the cell wall. Biochemical analysis of cell wall components showed that CsaB was involved in the addition of a pyruvyl group to a peptidoglycan-associated polysaccharide fraction, and that this modification was necessary for binding of the SLH domain. The csaAB operon is present in several bacterial species that synthesize SLH-containing proteins. This observation and the presence of pyruvate in the cell wall of the corresponding bacteria suggest that the mechanism described in this study is widespread among bacteria.