Bacterial cell wall synthesis: new insights from localization studies.

In order to maintain shape and withstand intracellular pressure, most bacteria are surrounded by a cell wall that consists mainly of the cross-linked polymer peptidoglycan (PG). The importance of PG for the maintenance of bacterial cell shape is underscored by the fact that, for various bacteria, several mutations affecting PG synthesis are associated with cell shape defects. In recent years, the application of fluorescence microscopy to the field of PG synthesis has led to an enormous increase in data on the relationship between cell wall synthesis and bacterial cell shape. First, a novel staining method enabled the visualization of PG precursor incorporation in live cells. Second, penicillin-binding proteins (PBPs), which mediate the final stages of PG synthesis, have been localized in various model organisms by means of immunofluorescence microscopy or green fluorescent protein fusions. In this review, we integrate the knowledge on the last stages of PG synthesis obtained in previous studies with the new data available on localization of PG synthesis and PBPs, in both rod-shaped and coccoid cells. We discuss a model in which, at least for a subset of PBPs, the presence of substrate is a major factor in determining PBP localization.     

Statistical properties of the variation at linked microsatellite loci: implications for the history of human Y chromosomes [published erratum appears in Mol Biol Evol 1997 Mar;14(3):354]

It has recently been suggested that observed levels of variation at microsatellite loci can be used to infer patterns of selection in genomes and to assess demographic history. In order to evaluate the feasibility of these suggestions it is necessary to know something about how levels of variation at microsatellite loci are expected to fluctuate due simply to stochasticity in the processes of mutation and inheritance (genetic sampling). Here we use recently derived properties of the stepwise mutation model to place confidence intervals around the variance in repeat score that is expected at mutation-drift equilibrium and outline a statistical test for whether an observed value differs significantly from expectation. We also develop confidence intervals for the time course of the buildup of variation following a complete elimination of variation, such as might be caused by a selective sweep or an extreme population bottleneck. We apply these methods to the variation observed at human Y-specific microsatellites. Although a number of authors have suggested the possibility of a very recent sweep, our analyses suggest that a sweep or extreme bottleneck is unlikely to have occurred anytime during the last approximately 74,000 years. To generate this result we use a recently estimated mutation rate for microsatellite loci of 5.6 x 10(-4) along with the variation observed at autosomal microsatellite loci to estimate the human effective population size. This estimate is 18,000, implying an effective number of 4,500 Y chromosomes. One important general conclusion to emerge from this study is that in order to reject mutation-drift equilibrium at a set of linked microsatellite loci it is necessary to have an unreasonably large number of loci unless the observed variance is far below that expected at mutation-drift equilibrium.      

Schistosoma mansoni egg-induced hepatic granulomas in mice deficient for the interferon-gamma receptor have altered populations of macrophages, lymphocytes and connective tissue cells

Systemic production and mobilization of inflammatory cells and formation of hepatic periovular granulomas were studied in Schistosoma mansoni-infected mice with deficient interferon gamma (IFN-gamma) receptor (IFN-gammaR(o/o)). The impaired IFN-gamma signaling did not cause a significant modification of the overall kinetics of inflammatory cells, but mutant mice developed smaller hepatic periovular granulomas with a two-fold reduction in all the cell lineages. In granulomas of normal mice, the fully differentiated macrophages were progressively predominant, whilst in IFN-gammaR(o/o) mice, the granulomas contained a higher percentage of immature and proliferating monocytes. Granulomas of IFN-gammaR(o/o) mice had an enhanced and accelerated fibrotic reaction, corresponding to an increased content of proliferative and activated connective tissue cells. Simultaneously, their granulomas had an increased ratio of T over B cells, with an increase in CD8(+) and a reduction in CD4(+) T cells. The functional IFN-gamma receptor was not required for initial recruitment of monocytes and lymphocytes into granulomas, but it was necessary for the maturation of macrophages, upregulation of major histocompatibility class 2 (MHC-II) expression and consequent stimulation of lymphocyte subpopulations depending upon the MHC-II-mediated antigen presentation.     

Photosynthesis of soybean under the action of a photosystem II-inhibiting herbicide

Photosynthesis, the fundamental physiological process of plant responsible for the growth and yield of crops, is strongly affected by environmental stresses. Several methods have been used to study changes in the physiological parameters of plants exposed to stresses. The work aimed to study physiological parameters related to photosynthesis in leaf discs of soybean plants exposed to a photosystem II-inhibiting herbicide. Soybean leaf discs obtained from mature leaves of plants in the vegetative stage immersed in bentazon herbicide solutions at concentrations of 0, 100, 250 or 500 μM were evaluated. In experiment I, the effect of the herbicide on chlorophyll a fluorescence transient was measured using a portable fluorometer. In the second experiment, the effect of the herbicide on modulated chlorophyll a fluorescence and gas exchange were evaluated, with the latter being measured with an infrared gas analyzer. The evaluations of transient and modulated fluorescence provided additional information on the photosynthetic activity of soybean leaf discs exposed to the action of bentazon. For the fluorescence transient analysis, performance indices were the parameters most sensitive to the action of bentazon, showing a decrease of approximately 70 % at a dose of 500 μM. For the modulated fluorescence analysis, the photochemical quenching coefficient, the electron transport rate, the photochemical efficiency of photosystem II and the net assimilation rate, decreased in response to herbicide application, with values that were almost equal to zero at a dose of 500 µM, which are the parameters that showed the greatest sensitivity to bentazon in soybean.     

Biomarkers in renal cell carcinoma: a metabolomics approach

Since Otto Warburg, many studies have explored the unique metabolic phenotype of cancer cells highlighting the value and applicability of metabolomics in the oncology field, particularly in the development of cancer biomarkers. With respect to renal cell carcinoma (RCC), a metabolomics approach would own a great potential since urinary system is intimately connected with urine and, this biofluid, offers some advantages allowing the development of an assay suitable for use in clinical practice. Moreover, the assessment of metabolic derangements characteristics of RCC might provide a complete health assessment of this pathology, enabling the development of novel targeted therapies and even the stratification of responsive patients to specific therapeutic options improving the effectiveness of therapy. Metabolomic studies performed so far showed that the RCC metabolic signature is characterized by alterations in metabolites related to energy metabolic pathways, particularly glycolysis, amino acid and fatty acid catabolism, known to be crucial to cell proliferation. Despite some of those alterations are common to carcinogenesis, the potential role of acylcarnitines, gentisate, α-ketoglutarate and quinolinate in RCC pathophysiology has been proposed recently. The ability of metabolomics to discriminate between RCC and normal samples shows convincing evidence of its applicability in RCC management. Furthermore, the studies already carried out have not only tried to unveil the metabolic profile of RCC but also to evaluate the impact of some factors, namely technical, in RCC-metabolomics research. This type of study is pivotal in the design of metabolomics studies, helping to increase the reliability of the results. The present review updates the current knowledge on the metabolic alterations recognized in the RCC, and provides insight to the characteristics, strengths, limitations, and recent advances in RCC-metabolomics studies, always keeping in mind its potential application in clinical/health areas as a biomarker discovery tool.     

Inactivation of the SauI Type I Restriction-Modification System Is Not Sufficient To Generate Staphylococcus aureus Strains Capable of Efficiently Accepting Foreign DNA

Genetic manipulation of Staphylococcus aureus is limited by the availability of only a single strain, RN4220, that is capable of easily accepting foreign DNA. Inactivation of the hsdR gene of the SauI type I restriction-modification system was shown previously to be responsible for the high transformation efficiency of RN4220 (D. E. Waldron and J. A. Lindsay, J Bacteriol. 188:5578-5585, 2006). However, deletion of this gene in three different S. aureus strains was not sufficient to make them readily transformable, which would be remarkably useful for genetic studies of this pathogenic organism. These results indicate that another unknown factor(s) is required for the transformable phenotype in S. aureus.Staphylococcus aureus is a major pathogen that causes both nosocomial and community-acquired infections, which range from superficial skin infections to severe systemic diseases. It is an extremely versatile pathogen which has developed resistance to virtually all known classes of antibiotics and which expresses various virulence factors that allow it to cause infection in different environments.Molecular genetic studies of S. aureus have resulted in a better understanding of both the virulence and antibiotic resistance mechanisms of this organism, which is crucial for discovery of new approaches to treat staphylococcal infections. One of the limitations in genetically manipulating S. aureus is the fact that there is only a single strain available which can easily accept plasmid DNA isolated from Escherichia coli. This strain, RN4220, is a chemical mutant obtained in the early 1980s by highly mutagenizing NCTC8325-4 (= RN450) with nitrosoguanidine and selecting for a mutant that was able to accept and maintain S. aureus plasmids (10; B. Kreiswirth, personal communication).One of the most important bacterial defenses against uptake of foreign DNA is restriction-modification (R-M) systems. These systems, comprising restriction endonucleases and methyltransferases, recognize and modify specific DNA sequences, protecting “self” DNA from restriction while eliminating potentially harmful foreign DNA which lacks appropriate modification (12). There are three distinct well-characterized types of classical R-M systems, including type II restriction enzymes, which cut DNA within specific recognition sequences and are therefore widely used as molecular biology tools (3). S. aureus strains may contain different R-M systems, including the Sau3AI and Sau96I type II systems (present in isolates of particular lytic groups) (19, 20) or the Sau42I BcgI-like R-M system expressed by S. aureus φ42 lysogens (5). However, the only chromosomal R-M system widely distributed in the sequenced S. aureus isolates is the SauI type I system (21). Type I R-M systems require the products of three genes, hsdR (restriction), hsdM (modification), and hsdS (sequence specificity), and cut DNA at sites remote from the recognition sequence (12). The staphylococcal SauI system includes a single hsdR gene and two copies of the hsdM and hsdS genes, and there is substantial variation between the hsdS genes from different isolates (21). It was recently shown that transformable RN4220 carries a stop mutation in the sauI hsdR gene and that complementation with a functional copy of this gene restores a nontransformable phenotype (21). An hsdR mutant does not cleave foreign unmodified DNA, but it does modify incoming DNA, which therefore is not cleaved when it is transferred to other S. aureus strains that contain an identical R-M system. For this reason, RN4220 has become an essential intermediate for laboratory manipulation of S. aureus, despite its limited clinical relevance. DNA first introduced into RN4220 by electroporation can then be transferred into other laboratory strains (for example, by phage transduction).The relevance of the SauI type I R-M system for horizontal transfer of foreign DNA in different S. aureus isolates in nature has been confirmed with bovine isolates that are hypersusceptible to gene transfer from enterococci and have stop mutations in each of the two SauI hsdS gene copies (18). These “hyperrecipient” strains may be ideal backgrounds for the acquisition of new antibiotic resistance markers, such as the vanA gene complex present in vancomycin-resistant S. aureus strains (18). However, the existence of a second pathway in S. aureus that blocks horizontal transfer of foreign DNA or of an unknown but necessary factor essential for SauI activity has also been proposed based on the fact that some strains that are hypersusceptible to gene transfer (such as B111 used by Noble et al. [13]) do not have a mutation in any of the five hsd genes (18).The availability of laboratory and clinical strains other than RN4220 that are capable of accepting foreign DNA would be remarkably useful for genetic studies of S. aureus, including studies of virulence and antibiotic resistance mechanisms present only in relevant clinical isolates. Therefore, we have tried to reproduce the hsdR mutation in RN4220 in different backgrounds of widely used laboratory strains in order to generate useful, easily transformable strains.     

Social Networks Shape the Transmission Dynamics of Hepatitis C Virus

Hepatitis C virus (HCV) infects 170 million people worldwide, and is a major public health problem in Brazil, where over 1% of the population may be infected and where multiple viral genotypes co-circulate. Chronically infected individuals are both the source of transmission to others and are at risk for HCV-related diseases, such as liver cancer and cirrhosis. Before the adoption of anti-HCV control measures in blood banks, this virus was mainly transmitted via blood transfusion. Today, needle sharing among injecting drug users is the most common form of HCV transmission. Of particular importance is that HCV prevalence is growing in non-risk groups. Since there is no vaccine against HCV, it is important to determine the factors that control viral transmission in order to develop more efficient control measures. However, despite the health costs associated with HCV, the factors that determine the spread of virus at the epidemiological scale are often poorly understood. Here, we sequenced partial NS5b gene sequences sampled from blood samples collected from 591 patients in São Paulo state, Brazil. We show that different viral genotypes entered São Paulo at different times, grew at different rates, and are associated with different age groups and risk behaviors. In particular, subtype 1b is older and grew more slowly than subtypes 1a and 3a, and is associated with multiple age classes. In contrast, subtypes 1a and 3b are associated with younger people infected more recently, possibly with higher rates of sexual transmission. The transmission dynamics of HCV in São Paulo therefore vary by subtype and are determined by a combination of age, risk exposure and underlying social network. We conclude that social factors may play a key role in determining the rate and pattern of HCV spread, and should influence future intervention policies.     

Oxidative protein folding by an endoplasmic reticulum-localized peroxiredoxin

Endoplasmic reticulum (ER) oxidation 1 (ERO1) transfers disulfides to protein disulfide isomerase (PDI) and is essential for oxidative protein folding in simple eukaryotes such as yeast and worms. Surprisingly, ERO1-deficient mammalian cells exhibit only a modest delay in disulfide bond formation. To identify ERO1-independent pathways to disulfide bond formation, we purified PDI oxidants with a trapping mutant of PDI. Peroxiredoxin IV (PRDX4) stood out in this list, as the related cytosolic peroxiredoxins are known to form disulfides in the presence of hydroperoxides. Mouse embryo fibroblasts lacking ERO1 were intolerant of PRDX4 knockdown. Introduction of wild-type mammalian PRDX4 into the ER rescued the temperature-sensitive phenotype of an ero1 yeast mutation. In the presence of an H(2)O(2)-generating system, purified PRDX4 oxidized PDI and reconstituted oxidative folding of RNase A. These observations implicate ER-localized PRDX4 in a previously unanticipated, parallel, ERO1-independent pathway that couples hydroperoxide production to oxidative protein folding in mammalian cells.