A misfolded form of 5S rRNA is complexed with the Ro and La autoantigens.

In both vertebrate and invertebrate cells, the 60-kDa Ro autoantigen is bound to small cytoplasmic RNAs known as Y RNAs. In Xenopus oocytes, the 60-kDa Ro protein is also complexed with a class of 5S rRNA precursors that contain internal mutations. Because these 5S rRNA precursors are processed inefficiently and degraded eventually, the Ro protein may function in a quality control pathway for 5S rRNA biosynthesis. We have investigated the sequence and secondary structure determinants in the mutant 5S rRNAs that confer binding by the 60-kDa Ro protein. The mutant 5S rRNAs fold to form an alternative helix that is required for recognition by the 60-kDa Ro protein. Mutations that disrupt the alternative helix eliminate Ro protein binding, whereas compensatory changes that restore the helix are bound efficiently by the Ro protein. When the structure of the mutant RNA was probed using dimethylsulfate and oligonucleotide-directed RNase H cleavage, the results were consistent with the formation of the alternative structure. The La protein, which is also complexed with the mutant 5S rRNA precursors, protects similar sequences from nuclease digestion as does the 60-kDa Ro protein. Thus, the binding sites for these two proteins are either nearby on the RNA, or the two proteins may be complexed through protein-protein interactions. When the human Ro protein is expressed in the yeast Saccharomyces cerevisiae, the protein binds wild-type 5S rRNA precursors, suggesting that a population of wild-type precursors also folds into the alternative structure.     

Methanogens and anaerobes in a colon segment isolated from the normal fecal stream.

Methanogens were in the isolated sigmoid colon but not in the colostomy bag of a woman with a left hemicolectomy with end colostomy and mucous fistula. The predominant methanogen was Methanobrevibacter smithii. An anaerobic bacterial community supported by nondietary substrates was present in the isolated sigmoid colon.     

Hydrogen peroxide elicits activation of bovine pulmonary arterial soluble guanylate cyclase by a mechanism associated with its metabolism by catalase

Guanylate cyclase activity in the soluble extract of bovine pulmonary arteries is activated by hydrogen peroxide generated by glucose oxidase only in the presence of catalase. This mechanism of guanylate cyclase activation is not blocked by scavengers for superoxide anion or hydroxyl radical, but is selectively inhibited by methylene blue, inactivation of catalase and ethanol. The time dependency of increases in guanylate cyclase activity in the presence of peroxides that are substrates for catalase are associated with the spectral detection of compound I, a species of catalase formed during the metabolism of peroxide. Thus, activation of soluble guanylate cyclase appears to be elicited by compound I of catalase or by a mediator generated by this species.     

Glutamine Supplementation Alleviates Vasculopathy and Corrects Metabolic Profile in an In Vivo Model of Endothelial Cell Dysfunction

Endothelial Cell Dysfunction (ECD) is a recognized harbinger of a host of chronic cardiovascular diseases. Using a mouse model of ECD triggered by treatment with L-Nω-methylarginine (L-NMMA), we previously demonstrated that renal microvasculature displays a perturbed protein profile, including diminished expression of two key enzymes of the Krebs cycle associated with a Warburg-type suppression of mitochondrial metabolism. We hypothesized that supplementation with L-glutamine (GLN), that can enter the Krebs cycle downstream this enzymatic bottleneck, would normalize vascular function and alleviate mitochondrial dysfunction. To test this hypothesis, mice with chronic L-NMMA-induced ECD were co-treated with GLN at different concentrations for 2 months. Results confirmed that L-NMMA led to a defect in acetylcholine-induced relaxation of aortic rings that was dose-dependently prevented by GLN. In caveolin-1 transgenic mice characterized by eNOS inactivation, L-NMMA further impaired vasorelaxation which was partially rescued by GLN co-treatment. Pro-inflammatory profile induced by L-NMMA was blunted in mice co-treated with GLN. Using an LC/MS platform for metabolite profiling, we sought to identify metabolic perturbations associated with ECD and offset by GLN supplementation. 3453 plasma molecules could be detected with 100% frequency in mice from at least one treatment group. Among these, 37 were found to be differentially expressed in a 4-way comparison of control vs. LNMMA both with and without GLN. One of such molecules, hippuric acid, an “uremic toxin” was found to be elevated in our non-uremic mice receiving L-NMMA, but normalized by treatment with GLN. Ex vivo analysis of hippuric acid effects on vasomotion demonstrated that it significantly reduced acetylcholine-induced vasorelaxation of vascular rings. In conclusion, functional and metabolic profiling of animals with early ECD revealed macrovasculopathy and that supplementation GLN is capable of improving vascular function. Metabolomic analyses reveal elevation of hippuric acid, which may further exacerbate vasculopathy even before the development of uremia.     

Influence of heme and vitamin B12 on growth and fermentations of Bacteroides species.

We examined the effects of heme on the growth and fermentations of Bacteroides species. Bacteroides fragilis ATCC 25285 required heme for growth and produced malate and lactate as major products of glucose fermentation when the concentration of heme was 1 ng/ml. With 1 microgram of heme per ml, malate was not formed, lactate production decreased, and succinate and acetate were the major fermentation products. B. eggerthii ATCC 27754 grew without heme, with the production of mainly malate and lactate from glucose. Its fermentation with 1 microgram of heme per ml was similar to that of B. fragilis grown with the same concentration of heme. B. splanchicus VPI 6842 grew without heme, with the production of mainly malate, acetate, and H2 from glucose. With 1 microgram of heme per ml, malate disappeared, H2 decreased significantly, and succinate, acetate, and butyrate were the major products. The addition of vitamin B12 to media containing 1 microgram of heme per ml caused all species to produce propionate at the expense of succinate and, with B. splanchnicus, also at the expense of butyrate. Thus, the concentration of heme and the presence of vitamin B12 significantly influenced the course of glucose fermentation by these bacteria.     

A Serum Bottle Modification of the Hungate Technique for Cultivating Obligate Anaerobes

The modification described utilizes a serum bottle closed with a butyl rubber stopper with a crimped metal seal for the growth of obligately anaerobic bacteria.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Safe To Walk? Neighborhood Safety and Physical Activity Among Public Housing Residents

Background

Despite its health benefits, physical inactivity is pervasive, particularly among those living in lower-income urban communities. In such settings, neighborhood safety may impact willingness to be regularly physically active. We examined the association of perceived neighborhood safety with pedometer-determined physical activity and physical activity self-efficacy.

Methods and Findings

Participants were 1,180 predominantly racial/ethnic minority adults recruited from 12 urban low-income housing complexes in metropolitan Boston. Participants completed a 5-d pedometer data-collection protocol and self-reported their perceptions of neighborhood safety and self-efficacy (i.e., confidence in the ability to be physically active). Gender-stratified bivariate and multivariable random effects models were estimated to account for within-site clustering. Most participants reported feeling safe during the day, while just over one-third (36%) felt safe at night. We found no association between daytime safety reports and physical activity among both men and women. There was also no association between night-time safety reports and physical activity among men (p = 0.23) but women who reported feeling unsafe (versus safe) at night showed significantly fewer steps per day (4,302 versus 5,178, p = 0.01). Perceiving one''s neighborhood as unsafe during the day was associated with significantly lower odds of having high physical activity self-efficacy among both men (OR 0.40, p = 0.01) and women (OR 0.68, p = 0.02).

Conclusions

Residing in a neighborhood that is perceived to be unsafe at night is a barrier to regular physical activity among individuals, especially women, living in urban low-income housing. Feeling unsafe may also diminish confidence in the ability to be more physically active. Both of these factors may limit the effectiveness of physical activity promotion strategies delivered in similar settings.     

Characterization of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> Beijing isolates from the Mediterranean area

Background  

The Beijing lineage of Mycobacterium tuberculosis is causing concern due to its global distribution and its involvement in severe outbreaks. Studies focused on this lineage are mainly restricted to geographical settings where its prevalence is high, whereas those in other areas are scarce. In this study, we analyze Beijing isolates in the Mediterranean area, where this lineage is not prevalent and is mainly associated with immigrant cases.