A Novel Pulse-Chase SILAC Strategy Measures Changes in Protein Decay and Synthesis Rates Induced by Perturbation of Proteostasis with an Hsp90 Inhibitor.

Standard proteomics methods allow the relative quantitation of levels of thousands of proteins in two or more samples. While such methods are invaluable for defining the variations in protein concentrations which follow the perturbation of a biological system, they do not offer information on the mechanisms underlying such changes. Expanding on previous work [1], we developed a pulse-chase (pc) variant of SILAC (stable isotope labeling by amino acids in cell culture). pcSILAC can quantitate in one experiment and for two conditions the relative levels of proteins newly synthesized in a given time as well as the relative levels of remaining preexisting proteins. We validated the method studying the drug-mediated inhibition of the Hsp90 molecular chaperone, which is known to lead to increased synthesis of stress response proteins as well as the increased decay of Hsp90 "clients". We showed that pcSILAC can give information on changes in global cellular proteostasis induced by treatment with the inhibitor, which are normally not captured by standard relative quantitation techniques. Furthermore, we have developed a mathematical model and computational framework that uses pcSILAC data to determine degradation constants kd and synthesis rates Vs for proteins in both control and drug-treated cells. The results show that Hsp90 inhibition induced a generalized slowdown of protein synthesis and an increase in protein decay. Treatment with the inhibitor also resulted in widespread protein-specific changes in relative synthesis rates, together with variations in protein decay rates. The latter were more restricted to individual proteins or protein families than the variations in synthesis. Our results establish pcSILAC as a viable workflow for the mechanistic dissection of changes in the proteome which follow perturbations. Data are available via ProteomeXchange with identifier PXD000538.

Application of biochemical and polymerase chain reaction assays for identification of Campylobacter isolates from non-human primates

A multiplex polymerase chain reaction (PCR) assay was performed on 167 thermophilic campylobacters isolated from non-human primates. Samples were first identified by phenotypic methods resulting in 64 Campylobacter jejuni and 103 C. coli strains. Four strains identified biochemically as C. coli, were then determined to be C. jejuni by PCR. Comparison of methodologies showed that the main discrepancies were attributed to the hippurate hydrolysis test and sensitivity to cephalothin and nalidixic acid. Analysis of data showed that the application of phenotypic methods should be supplemented by a molecular method to offer a more reliable Campylobacter identification.

Changes in antihypertensive drug treatment in the general population: the Colaus Study

Background and aims: there is little information regar ding changes in antihypertensive drug treatment in Switzerland. We aimed at assessing those changes in a population-based, prospective study. Methods: 768 hypertensive subjects (372 women, 397 men) followed for 5 years. Subjects were defined as continuers (no change), switchers (one antihypertensive class replace by another), combiners (one antihypertensive class added) and discontinuers (stopped treatment). Results: Analysis of all patients (mono or combination therapy) showed that 54.6% were continuers, 27.2% combiners, 12.9% switchers and 5.3 % discontinuers. Similar findings were obtained for participants on monotherapy only: 42.2% continuers, 36.7% combiners, 13.4% switchers and 7.7% discontinuers. Combiners had higher systolic and diastolic blood pressure values at baseline than the other groups (p<0.001), while no difference were found for personal and family history and other clinical and biological variables. Compared to continuers, combiners and switchers improved their blood pressure status at follow-up: 26.7% of combiners and 26.3% of switchers improved, versus 17.7% of continuers and 7.3% of discontinuers (p<0.001). Among participants on monotherapy at baseline, continuation was greatest for angiotensin II type 1 receptor blocking agents (ARBs, 53.1%), angiotensin-converting enzyme inhibitors (44.4%) and β-blockers (41.8%). Only one quarter of participants treated with diuretic or calcium channel blockers at baseline remained so at follow-up. Conclusion: Antihypertensivedrug treatment is very stable in Switzerland. There are no big differences in persistence between antihypertensive classes, even if ARBs had the most favorable utilization pattern. Changes are only due to blood pressure level and improve blood pressure status.

Changes induced in Biomphalaria glabrata (Say, 1818) following trials for artificial stimulation of its internal defense system

Biomphalaria glabrata can react through different pathways to Schistosoma mansoni miracidium penetration, according to the degree of resistance/susceptibility presented by different snail strains, which is a genetically determined character, resistance being the dominant feature. However, it has been observed that previous susceptible snail strain may change its reactive behavior along the course of infection, exhibiting later a pattern of cercarial shedding and histopatopathological picture compatible with high resistance. Such observation suggests the possibility of B. glabrata to develop a sort of adaptative immunity face a schistosome infection. To explore on this aspect, the present investigation looked for the behavior of S. mansoni infection in B. glabrata previously subjected to different means of artificial stimulation of its internal defense system. Snails previously inoculated with irradiated miracídia (Group I); treated with S. mansoni antigens (Group II) or with a non-related parasite antigen (Group III) were challenged with 20 viable S. mansoni miracidia, and later looked for cercarial shedding and histopathologic changes at different times from exposition. Nodules of hemocyte accumulations were found at the site of antigen injection. These nodules resembled solid granulomas, and were larger and more frequent in snails injected with S. mansoni products as compared to those injected with Capillaria hepatica. However, the presence of such granulomas did not avoid the S. mansoni challenge infection from developing in a similar way as that seen in controls. The data are indicative that hemocytes are able to proliferate locally when stimulated, such capacity also remaining localized, not being shared by the population of hemocytes located elsewhere within the snail body.

Remodeling of hepatic vascular changes after specific chemotherapy of schistosomal periportal fibrosis

Hepatosplenic schistosomiasis was the first human disease in which the possibility of extensive long standing hepatic fibrosis being degraded and removed has been demonstrated. When such changes occurred, the main signs of portal hypertension (splenomegaly, esophageal varices) progressively disappeared, implying that a profound vascular remodeling was concomitantly occurring. Hepatic vascular alterations associated with advanced schistosomiasis have already been investigated. Obstruction of the intrahepatic portal vein branches, plus marked angiogenesis and compensatory hyperplasia and hypertrophy of the arterial tree are the main changes present. However, there are no data revealing how these vascular changes behave during the process of fibrosis regression. Here the mouse model of pipestem fibrosis was used in an investigation about these vascular alterations during the course of the infection, and also after treatment and cure of the disease. Animals representing the two polar hepatic forms of the infection were included: (1) "isolated granulomas" characterized by isolated periovular granulomas sparsely distributed throughout the hepatica parenchyma; and (2) 'pipestem fibrosis' with periovular granulomas and fibrosis being concentrated within portal spaces, before and after treatment, were studied by means of histological and vascular injection-corrosion techniques. Instances of widespread portal vein obstruction of several types were commonly found in the livers of the untreated animals. These obstructive lesions were soon repaired, and completely disappeared four months following specific treatment of schistosomiasis. Treatment was accomplished by the simultaneous administration of praziquantel and oxamniquine. The most impressive results were revealed by the technique of injection of colored masses into the portal system, followed by corrosion in strong acid. The vascular lesions of non-treated pipestem fibrosis were represented in the plastic casts by considerable diminution of the fine peripheral portal vein radicles, plus dilatation of periportal collaterals. Four months after treatment, this last picture appeared replaced by tufts of newly interwoven vessels formed along the main portal vein branches, disclosing a strong angiomatoid reparative change. Understanding about the cellular elements at play during fibro-vascular repairing changes of hepatic schistosomiais represents a matter of considerable scientific and conceptual importance. At present time one may only speculate about the participation of some type of natural stem-cell capable of restoring the diseased liver back to normal once the cause of the disorder has been eliminated.

Carbon isotope excursions and microfacies changes in marine Permian-Triassic boundary sections in Hungary

Several Permian-Triassic boundary sections occur in various structural units within Hungary. These sections represent different facies zones of the western Palaeotethys margin. The Gardony core in the NE part of the Transdanubian Range typically represents the inner ramp, while the Balvany section in the Bukk Mountains of northern Hungary represents an outer ramp setting. The two sections have different patterns for their delta(13)C values. The Balvany section shows a continuous change towards more negative delta(13)C values starting at the first biotic decline, followed by a sharp, quasi-symmetric negative peak at the second decline. The appearance of the delta(13)C peak has no relationship to the lithology and occurs within a shale with low overall carbonate content, indicating that the peak is not related to diagenesis or other secondary influences. Instead, the shift and the peak reflect primary processes related to changes in environmental conditions. The continuous shift in delta(13)C values is most probably related to a decrease in bioproductivity, whereas the sharp peak can be attributed to an addition of C strongly depleted in (13)C to the ocean-atmosphere system. The most plausible model is a massive release of methane-hydrate. The quasi-symmetric pattern suggests a rapid warming-cooling cycle or physical unroofing of sediments through slope-failure and releasing methane-hydrate. The Gidony-1 core shows a continuous negative delta(13)C shift starting below the P-T boundary. However, the detailed analyses revealed a sharp delta(13)C peak in the boundary interval, just below the major biotic decline, although its magnitude doesn't reach that observed in the Balvany section. Based on careful textural examination and high-resolution stable isotope microanalyses we suggest that the suppression of the delta(13)C peak that is common in the oolitic boundary sections is due to combined effects of condensed sedimentation, sediment reworking and erosion, as well as perhaps diagenesis. (c) 2005 Elsevier B.V All rights reserved.

Breast awareness looking out for changes (English and 12 translations)

This leaflet is used to support the Northern Ireland breast screening programme and describes how women should check their breasts regularly for any changes that are new to themThis is also available in audio format by clicking here.�

Effects of exercise on the secondary blood markers commonly used to suspect erythropoietin doping.

Numerous trials have reported that some haematological and biochemical parameters could be put together and be used to detect and fight recombinant erythropoietin doping. Unfortunately, none of the studies mentioned the necessity of taking pre-analytical precautions to avoid possible suspicious results coming from major plasma volume changes caused notably by dehydration. Therefore we studied the behaviour of the most common secondary blood markers before and after a strenuous physical activity to find out how reliable these parameters were. The soluble transferrin receptor and the haemoglobin concentrations as well as the haematocrit level increased significantly after effort, whereas the plasma EPO concentration and the reticulocyte count remained constant. On the other hand, if the values were corrected for haemoconcentration, the soluble transferrin receptor concentration remained stable.

Causes of health expenditure growth : the predominance of changes in medical practices over population ageing

On the basis of French individual data, this paper compares the effects of demographic change, changes in morbidity and changes in practices on the growth in health expenditures that occurred between 1992 and 2000. Micro simulations show that the rise in expenditures due to ageing is relatively small and that the impact of changes in practices is times larger. Furthermore, changes in morbidity induce savings which more than offset the increase in spending due to population ageing. [Authors]

Urban and suburban malaria in Rondônia (Brazilian Western Amazon) II: perennial transmissions with high anopheline densities are associated with human environmental changes

Longitudinal entomological surveys were performed in Vila Candelária and adjacent rural locality of Bate Estaca concomitantly with a clinical epidemiologic malaria survey. Vila Candelária is a riverside periurban neighborhood of Porto Velho, capital of the state of Rondônia in the Brazilian Amazon. High anopheline densities were found accompanying the peak of rainfall, as reported in rural areas of the region. Moreover, several minor peaks of anophelines were recorded between the end of the dry season and the beginning of the next rainy season. These secondary peaks were related to permanent anopheline breeding sites resulting from human activities. Malaria transmission is, therefore, observed all over the year. In Vila Candelária, the risk of malaria infection both indoors and outdoors was calculated as being 2 and 10/infecting bites per year per inhabitant respectively. Urban malaria in riverside areas was associated with two factors: (1) high prevalence of asymptomatic carriers in a stable human population and (2) high anopheline densities related to human environmental changes. This association is probably found in other Amazonian urban and suburban communities. The implementation of control measures should include environmental sanitation and better characterization of the role of asymptomatic carriers in malaria transmission.

Epigenetic features of human mesenchymal stem cells determine their permissiveness for induction of relevant transcriptional changes by SYT-SSX1.

BACKGROUND: A characteristic SYT-SSX fusion gene resulting from the chromosomal translocation t(X;18)(p11;q11) is detectable in almost all synovial sarcomas, a malignant soft tissue tumor widely believed to originate from as yet unidentified pluripotent stem cells. The resulting fusion protein has no DNA binding motifs but possesses protein-protein interaction domains that are believed to mediate association with chromatin remodeling complexes. Despite recent advances in the identification of molecules that interact with SYT-SSX and with the corresponding wild type SYT and SSX proteins, the mechanisms whereby the SYT-SSX might contribute to neoplastic transformation remain unclear. Epigenetic deregulation has been suggested to be one possible mechanism. METHODOLOGY/PRINCIPAL FINDINGS: We addressed the effect of SYT/SSX expression on the transcriptome of four independent isolates of primary human bone marrow mesenchymal stem cells (hMSC). We observed transcriptional changes similar to the gene expression signature of synovial sarcoma, principally involving genes whose regulation is linked to epigenetic factors, including imprinted genes, genes with transcription start sites within a CpG island and chromatin related genes. Single population analysis revealed hMSC isolate-specific transcriptional changes involving genes that are important for biological functions of stem cells as well as genes that are considered to be molecular markers of synovial sarcoma including IGF2, EPHRINS, and BCL2. Methylation status analysis of sequences at the H19/IGF2 imprinted locus indicated that distinct epigenetic features characterize hMSC populations and condition the transcriptional effects of SYT-SSX expression. CONCLUSIONS/SIGNIFICANCE: Our observations suggest that epigenetic features may define the cellular microenvironment in which SYT-SSX displays its functional effects.

Purification and biochemical characterization of four iron superoxide dismutases in Trypanosoma cruzi

Four superoxide dismutase (SOD) activities (SOD I, II, III, and IV) have been characterized in the epimastigote form of Trypanosoma cruzi. The total extract was subjected to two successive ammonium sulphate additions between 35 and 85%, and the resulting fraction was purified using two continuous chromatography processes (ion exchange and filtration). Enzymes were insensitive to cyanide but sensitive to hydrogen peroxide, properties characteristic of iron-containing SODs. The molecular masses of the different SODs were 20 kDa (SOD I), 60 kDa (SOD II), 50 kDa (SOD III) and 25 kDa (SOD IV), whereas the isoelectric points were 6.9, 6.8, 5.2 and 3.8, respectively. Subcellular location and digitonin experiments have shown that these SODs are mainly cytosolic, with small amounts in the low-mass organelles (SOD II and SOD I) and the mitochondrion (SOD III), where these enzymes play an important role in minimizing oxidative damage.

Changes in the transcriptional profile of transporters in the intestine along the anterior-posterior and crypt-villus axes.

BACKGROUND: The purpose of this work was to characterize the expression of drug and nutrient carriers along the anterior-posterior and crypt-villus axes of the intestinal epithelium and to study the validity of utilizing whole gut tissue rather than purified epithelial cells to examine regional variations in gene expression. RESULTS: We have characterized the mRNA expression profiles of 76 % of all currently known transporters along the anterior-posterior axis of the gut. This is the first study to describe the expression profiles of the majority of all known transporters in the intestine. The expression profiles of transporters, as defined according to the Gene Ontology consortium, were measured in whole tissue of the murine duodenum, jejunum, ileum and colon using high-density microarrays. For nine transporters (Abca1, Abcc1, Abcc3, Abcg8, Slc10a2, Slc28a2, Slc2a1, Slc34a2 and Slc5a8), the mRNA profiles were further measured by RT-PCR in laser micro-dissected crypt and villus epithelial cells corresponding to the aforementioned intestinal regions. With respect to differentially regulated transporters, the colon had a distinct expression profile from small intestinal segments. The majority (59 % for p cutoff < or = 0.05) of transporter mRNA levels were constant across the intestinal sections studied. For the transporter subclass "carrier activity", which contains the majority of known carriers for biologically active compounds, a significant change (p < or = 0.05) along the anterior-posterior axis was observed. CONCLUSION: All nine transporters examined in laser-dissected material demonstrated good replication of the region-specific profiles revealed by microarray. Furthermore, we suggest that the distribution characteristics of Slc5a8 along the intestinal tract render it a suitable candidate carrier for monocarboxylate drugs in the posterior portion of the intestine. Our findings also predict that there is a significant difference in the absorption of carrier-mediated compounds in the different intestinal segments. The most pronounced differences can be expected between the adjoining segments ileum and colon, but the differences between the other adjoining segments are not negligible. Finally, for the examined genes, profiles measured in whole intestinal tissue extracts are representative of epithelial cell-only gene expression.

Evolution of C(4) phosphoenolpyruvate carboxykinase in grasses, from genotype to phenotype.

C(4) photosynthesis is an adaptation over the classical C(3) pathway that has evolved multiple times independently. These convergences are accompanied by strong variations among the independent C(4) lineages. The decarboxylating enzyme used to release CO(2) around Rubisco particularly differs between C(4) species, a criterion used to distinguish three distinct biochemical C(4) subtypes. The phosphoenolpyruvate carboxykinase (PCK) serves as a primary decarboxylase in a minority of C(4) species. This enzyme is also present in C(3) plants, where it is responsible for nonphotosynthetic functions. The genetic changes responsible for the evolution of C(4)-specific PCK are still unidentified. Using phylogenetic analyses on PCK sequences isolated from C(3) and C(4) grasses, this study aimed at resolving the evolutionary history of C(4)-specific PCK enzymes. Four independent evolutions of C(4)-PCK were shown to be driven by positive selection, and nine C(4)-adaptive sites underwent parallel genetic changes in different C(4) lineages. C(4)-adaptive residues were also observed in C(4) species from the nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME) subtype and particularly in all taxa where a PCK shuttle was previously suggested to complement the NADP-ME pathway. Acquisitions of C(4)-specific PCKs were mapped on a species tree, which revealed that the PCK subtype probably appeared at the base of the Chloridoideae subfamily and was then recurrently lost and secondarily reacquired at least three times. Linking the genotype to subtype phenotype shed new lights on the evolutionary transitions between the different C(4) subtypes.