The plasma membrane of Streptococcus cremoris: isolation and partial characterization

Plasma membrane was isolated from Streptococcus cremoris using mutanolysin from a streptomycete as the cell wall-degrading enzyme and phenylmethylsulfonyl fluoride as protease inhibitor. The specific activity of membrane-bound enzyme, adenosine triphosphatase (ATPase), was 4 μmol/mg protein per min, which was 5–10 times higher than the activity found in other fractions obtained during the isolation procedure. The number of polypeptides in the plasma membrane was approximately 50 with molecular weights 13 500–100 000, minor changes in the polypeptide pattern were observed when the plasma membrane was isolated without a protease inhibitor. The chemical composition of the membrane preparation was 49.7% protein, 21.9% lipid, 5.1% aminosugars, 17.3% RNA and 0.03% DNA. Electron microscopic examination confirmed the membrane to be practically devoid of cell wall components. Our results indicate that the membrane integrity is well retained and therefore the membrane preparation is suitable for detailed studies on vectorial metabolism and its enzymes, e.g. ATPase.     

Environmental predictability of taxonomic and functional community composition in high‐latitude streams

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Rapid identification of bacterial pathogens using a PCR- and microarray-based assay

Background  

During the course of a bacterial infection, the rapid identification of the causative agent(s) is necessary for the determination of effective treatment options. We have developed a method based on a modified broad-range PCR and an oligonucleotide microarray for the simultaneous detection and identification of 12 bacterial pathogens at the species level. The broad-range PCR primer mixture was designed using conserved regions of the bacterial topoisomerase genes gyrB and parE. The primer design allowed the use of a novel DNA amplification method, which produced labeled, single-stranded DNA suitable for microarray hybridization. The probes on the microarray were designed from the alignments of species- or genus-specific variable regions of the gyrB and parE genes flanked by the primers. We included mecA-specific primers and probes in the same assay to indicate the presence of methicillin resistance in the bacterial species. The feasibility of this assay in routine diagnostic testing was evaluated using 146 blood culture positive and 40 blood culture negative samples.     

Early Life Stress and Physical and Psychosocial Functioning in Late Adulthood

Background

Severe stress experienced in early life may have long-term effects on adult physiological and psychological health and well-being. We studied physical and psychosocial functioning in late adulthood in subjects separated temporarily from their parents in childhood during World War II.

Methods

The 1803 participants belong to the Helsinki Birth Cohort Study, born 1934–44. Of them, 267 (14.8%) had been evacuated abroad in childhood during WWII and the remaining subjects served as controls. Physical and psychosocial functioning was assessed with the Short Form 36 scale (SF-36) between 2001 and 2004. A test for trends was based on linear regression. All analyses were adjusted for age at clinical examination, social class in childhood and adulthood, smoking, alcohol intake, physical activity, body mass index, cardiovascular disease and diabetes.

Results

Physical functioning in late adulthood was lower among the separated men compared to non-separated men (b = −0.40, 95% confidence interval [95% CI]: −0.71 to −0.08). Those men separated in school age (>7 years) and who were separated for a duration over 2 years had the highest risk for lower physical functioning (b = −0.89, 95% CI: −1.58 to −0.20) and (b = −0.65, 95% CI: −1.25 to −0.05), respectively). Men separated for a duration over 2 years also had lower psychosocial functioning (b = −0.70, 95% CI: −1.35 to −0.06). These differences in physical and psychosocial functioning were not observed among women.

Conclusion

Early life stress may increase the risk for impaired physical functioning in late adulthood among men. Timing and duration of the separation influenced the physical and psychosocial functioning in late adulthood.     

A confirmatory analysis of malachite green residues in rainbow trout with liquid chromatography-electrospray tandem mass spectrometry

A quantitative liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has been developed for the determination of malachite green (MG) and its metabolite leucomalachite green (LMG) in fish. Residues were extracted with an acetonitrile-acetate buffer and purified using the automated solid-phase extraction (ASPEC). Residues were analyzed with a reversed-phase LC-MS/MS using a positive-ion electrospray ionisation (ESI). Isotope-labelled leucomalachite green (LMG-D5) was used as an internal standard for the quantification of LMG residues. The related dye, brilliant green (BG) was used as an instrumental standard. Identification and quantification of analytes were based on the ion transitions monitored by multiple reaction monitoring (MRM). The decision limit (CCalpha) for MG and LMG was 0.13 and 0.16 microgkg(-1). The respective detection capabilities (CCbeta) were 0.22 and 0.27 microgkg(-1). The absolute recovery (repeatability SD(r)) was in the range of 58-65% (7.8-11.2%) for MG and 59-68% (9.7-16.9%) for LMG. LMG was quantified also based on the internal standard, giving a recovery (repeatability SD(r)) of 103-110% (4.8-9.3%). The method was further evaluated by analyzing a total of 34 fish residue monitoring samples, of which eight samples were found to be non-compliant containing low residues of LMG.     

Endosomal escape of delivered mRNA from endosomal recycling tubules visualized at the nanoscale

Delivery of exogenous mRNA using lipid nanoparticles (LNPs) is a promising strategy for therapeutics. However, a bottleneck remains in the poor understanding of the parameters that correlate with endosomal escape versus cytotoxicity. To address this problem, we compared the endosomal distribution of six LNP-mRNA formulations of diverse chemical composition and efficacy, similar to those used in mRNA-based vaccines, in primary human adipocytes, fibroblasts, and HeLa cells. Surprisingly, we found that total uptake is not a sufficient predictor of delivery, and different LNPs vary considerably in endosomal distributions. Prolonged uptake impaired endosomal acidification, a sign of cytotoxicity, and caused mRNA to accumulate in compartments defective in cargo transport and unproductive for delivery. In contrast, early endocytic/recycling compartments have the highest probability for mRNA escape. By using super-resolution microscopy, we could resolve a single LNP-mRNA within subendosomal compartments and capture events of mRNA escape from endosomal recycling tubules. Our results change the view of the mechanisms of endosomal escape and define quantitative parameters to guide the development of mRNA formulations toward higher efficacy and lower cytotoxicity.     

Extracellular transglutaminase 2 has a role in cell adhesion, whereas intracellular transglutaminase 2 is involved in regulation of endothelial cell proliferation and apoptosis

Objective: Transglutaminase 2 (TG2) is a multifunctional protein with an important role in vascular biology, where it is involved in cell–matrix interaction, cell attachment and cell population expansion. In efforts to elucidate the role of TG2 in endothelial cell biology, in this study, we measured several endothelial cell characteristics in cells where TG2 was specifically knocked down by RNAi. Materials and methods: The effect of small interfering RNA (siRNA)‐TG2 on human umbilical vein endothelial cells was studied. Adhesion and cell viability were assessed by chemical reduction of MTT, and cell proliferation was analysed by flow cytometry. Apoptosis was evaluated by annexin V/PI dual staining and protein expression level was assayed by western blotting. Results: We found that siRNA‐TG2 reduced endothelial cell number, lead to cell adhesion deficiency, cell cycle arrest in G₁ phase and induction of apoptosis. Our results show that exogenously added TG2 could reverse loss of adhesion but did not overcome the defect in cell proliferation, nor could it inhibit siRNA‐TG2‐induced apoptosis. Conclusion: We conclude that TG2 loss in endothelial cells causes reduction in cell number as a result of cell cycle arrest, flaws in adhesion and induction of apoptosis. Our results imply that reduction in cell number and increased apoptosis in response to TG2 silencing is independent of the cell adhesion process. Altogether, our findings underline the significance of TG2 in endothelial cell cycle progression and cell survival, in vitro.