Bacterial fingerprinting by flow cytometry: bacterial species discrimination.

BACKGROUND: A flow cytometric measurement (FCM) technique has been developed to size DNA fragments. Individual fragments of a restriction digest of genomic DNA, stained with an intercalating dye, are passed through an ultrasensitive cytometer. The measured fluorescence intensity from each fragment is proportional to the fragment length. METHODS: The isolation of bacterial genomic DNA and digestion by restriction enzymes were performed inside an agarose plug. Rare cutting enzymes were employed to produce a manageable number of DNA fragments. Electroelution was used to move the DNA fragments from the agarose plug into a solution containing polyamines to protect the DNA from shear-induced breakage. The DNA was stained with the bisintercalating dye thiazole orange homodimer and introduced into our ultrasensitive flow cytometer. A histogram of the fluorescence intensities (fingerprint) was constructed. RESULTS: Gram-positive Bacillus globigii and gram-negative bacteria Escherichia coli and Erwinia herbicola were distinguished by the fingerprint pattern of restriction fragments of their genomic DNA. DNA sizes determined by FCM are in good agreement with pulsed-field gel electrophoresis (PFGE) analysis. Flow cytometry requires only picogram quantities of purified DNA and takes less than 10 min for data collection and analysis. When the total sample preparation time is included, the analysis times for PFGE and FCM are similar ( approximately 3 days). CONCLUSIONS: FCM is an attractive technique for the identification of bacterial species. It is more sensitive and potentially much faster than PFGE.     

Identification of MarvelD3 as a tight junction-associated transmembrane protein of the occludin family

Background

Protein translocation across the membrane of the Endoplasmic Reticulum (ER) is the first step in the biogenesis of secretory and membrane proteins. Proteins enter the ER by the Sec61 translocon, a proteinaceous channel composed of three subunits, α, β and γ. While it is known that Sec61α forms the actual channel, the function of the other two subunits remains to be characterized.

Results

In the present study we have investigated the function of Sec61β in Drosophila melanogaster. We describe its role in the plasma membrane traffic of Gurken, the ligand for the Epidermal Growth Factor (EGF) receptor in the oocyte. Germline clones of the mutant allele of Sec61β show normal translocation of Gurken into the ER and transport to the Golgi complex, but further traffic to the plasma membrane is impeded. The defect in plasma membrane traffic due to absence of Sec61β is specific for Gurken and is not due to a general trafficking defect.

Conclusion

Based on our study we conclude that Sec61β, which is part of the ER protein translocation channel affects a post-ER step during Gurken trafficking to the plasma membrane. We propose an additional role of Sec61β beyond protein translocation into the ER.     

Enhanced Expression of Endochitinase in Trichoderma harzianum with the cbh1 Promoter of Trichoderma reesei

Production of extracellular endochitinase could be increased 5-fold in the mycoparasite fungus Trichoderma harzianum by using the cellulase promoter cbh1 of Trichoderma reesei, whereas the total endochitinase activity increased 10-fold. The cbh1 promoter was not expressed on glucose and sucrose in T. harzianum and was induced by sophorose and on cellulase-inducing medium. The endogenous endochitinase gene was expressed at a low basal level on glucose and sucrose. No specific induction by crab shell chitin or sophorose was observed.     

Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.      

Drinking and driving: choosing the legal limits.

The legal limit for drinking and driving in Britain is 80 mg/dl (17.4 mmol/l) of alcohol in the blood. This was chosen 20 years ago on the basis of studies that have recently been reanalysed. Changes in public opinion, the results of recent research, and the evaluation of other countermeasures, such as random breath testing, show that there are good grounds for revising the legal limit downwards. It is suggested that the legal limit should be reduced from 80 mg/dl to 50 mg/dl (10.9 mmol/l) and random breath testing introduced as in most Nordic countries. A zero limit is proposed for learner and first year drivers, who are likely to have accidents even with low concentrations of alcohol in their blood.     

Studies on the modification of the cellular response to injury. IV. Protective effect of extracellular acidosis against anoxia,thermal, and p-chloromercuribenzene sulfonic acid treatment of isolated rat liver cells

Isolated rat liver cells were exposed $\text{in}$$\text{vitro}$ to ageing in an aerobic condition and to the effects of anoxia, thermal and $\text{p}$-chloromercuribenzene sulfonic (PCMBS) acid treatment, The survival of the cells was studied at normal and acidic pH using vital dye staining, intracellular concentration of potassium and ATP as indicators of viability. In aerobic conditions at pH 7.4, control cells lost their viability within approximately 6 hours. Extracellular acidosis not only prolonged the life span of isolated control hepatocytes against mechanical and other possible stress factors associated with the incubation and the absence of substrates in the medium, but also protected the cells significantly against various other superimposed injurious effects. These observations augment our previous observations on Ehrlich ascites tumore cells and lend further support to the hypothesis that extracellular acidosis, a common phenomenon associated with cell injury, is not harmful to cell survival $\text{in}$$\text{vitro}$ evidently prolongs it. The mechanism(s) behind this effect is not fully understood but it is suggested that extracellular acidosis stabilizes, and thereby protects cellular membrane systems making them more resistant to various deteriorating effects.     

Studies on the modification of the cellular response to injury

Extracellular acidosis (pH 6.5) was found to significantly retard the response of Ehrlich ascites tumor cells (EATC) to direct plasma membrane injury with the non-penetrating organic mercurial compound, p-chloromercuribenzene sulfonic acid (PCMBS). Treatment of cells with 1 mM PCMBS resulted in loss of viability of all cells by 45 minutes at pH 7.4, and by 90 minutes at pH 6.5. Pregression of cellular changes through the various stages of cell injury at the ultrastructural level was correspondingly slower at pH 6.5. The results support the concept that stage 3 of cell injury, associated with condensed mitochondria, dilated ER and swollen cell sap is compatible with cell survival, while stage 5 with high amplitude swelling of mitochondria, fragmentation of membrane systems, and beginning of karyolysis is characteristic of irreversible injury. All cells entered stage 3 at 7.5 minutes at pH 7.4, while essentially all cells entered stage 5 by 45 minutes. At pH 6.5, stage 3 was maintained for 45 minutes and 100% of the cells entered stage 5 by 90 minutes. Although the mechanism of the protection against PCMBS-induced injury is not known, the present electron microscopical results are compatible with the hypothesis that the extracellular acidosis acts to partially stabilize plasma membrane, perhaps by interaction with sulfhydryl (SH) groups.     

带有木糖还原酶基因和木糖醇脱氢酶基因的重组酿酒酵母的构建

采用双载体系统,将携带有瑞氏木霉木糖醇脱氢酶基因的表达质粒pAJ401－Xdh1转化已带有树干毕赤氏酵母木糖还原酶基因的重组酿酒酵母H475,构建了同时带有毕赤氏酵母木糖还原酶基因和瑞氏木霉木糖醇脱氢酶基因的重组酿酒酵母HX1。研究了重组酿酒酵母HX1对木糖的转化利用情况。     

Role of the reticulum in the stability and shape of the isolated human erythrocyte membrane

In order to examine the widely held hypothesis that the reticulum of proteins which covers the cytoplamsic surface of the human erythrocyte membrane controls cell stability and shape, we have assessed some of its properties. The reticulum, freed of the bilayer by extraction with Triton X-100, was found to be mechanically stable at physiological ionic strength but physically unstable at low ionic strength. The reticulum broke down after a characteristic lag period which decreased 500-fold between 0 degrees and 37 degrees C. The release of polypeptide band 4.1 from the reticulum preceded that of spectrin and actin, suggesting that band 4.1 might stabilize the ensemble but is not essential to its integrity. The time-course of breakdown was similar for ghosts, the reticulum inside of ghosts, and the isolated reticulum. However, at very low ionic strength, the reticulum was less stable within the ghost than when free; at higher ionic strength, the reverse was true. Over a wide range of conditions the membrane broke down to vesicles just as the reticulum disintegrated, presumably because the bilayer was mechanically stabilized by this network. The volume of both ghosts and naked reticula varied inversely and reversibly with ionic strength. The volume of the naked reticulum varied far more widely than the ghost, suggesting that its deformation was normally limited by the less extensible bilayer. The contour of the isolated reticulum was discoid and often dimpled or indented, as visualized in the fluorescence microscope after labeling of the ghosts with fluoroscein isothiocyanate. Reticula derived from ghosts which had lost the ability to crenate in isotonic saline were shriveled, even though the bilayer was smooth and expanded. Conversly, ghosts crenated by dinitrophenol yielded smooth, expanded reticula. We conclude that the reticulum is a durable, flexible, and elastic network which assumes and stabilizes the contour of the membrane but is not responsible for its crenation.