Total internal reflection with fluorescence correlation spectroscopy

Total internal reflection-fluorescence correlation spectroscopy (TIR-FCS) is an emerging technique that is used to measure events at or near an interface, including local fluorophore concentrations, local translational mobilities and the kinetic rate constants that describe the association and dissociation of fluorophores at the interface. TIR-FCS is also an extremely promising method for studying dynamics at or near the basal membranes of living cells. This protocol gives a general overview of the steps necessary to construct and test a TIR-FCS system using either through-prism or through-objective internal reflection geometry adapted for FCS. The expected forms of the autocorrelation function are discussed for the cases in which fluorescent molecules in solution diffuse through the depth of the evanescent field, but do not bind to the surface of interest, and in which reversible binding to the surface also occurs.     

A general method for the isolation of haptoglobin 1-1, 2-1, and 2-2 from human plasma

A general method for the isolation of haptoglobin 1-1, 2-1, and 2-2 human plasma is described. Plasma is fractionated by affinity chromatography on chicken hemoglobin-Sepharose using the full capacity of the column; then after washing the column thoroughly, haptoglobin is eluted with 8 M urea and the eluate is collected in fractions to separate active and denatured haptoglobin. The urea-free, active fractions of haptoglobin are fractionated by affinity chromatography on Affi-Gel Con A to remove nonglycoproteins, principally apolipoprotein A-I, and the haptoglobin is eluted with 0.5 M glucose. Then the haptoglobin-containing fractions are fractionated by negative immunoadsorption chromatography on anti-chicken hemoglobin-protein A-Sepharose to remove chicken hemoglobin-human haptoglobin complexes. Haptoglobin prepared by this three-step procedure is biologically active and nearly homogeneous. The recovery is approximately 70%, irrespective of phenotype. The procedure can be completed in 3 days. A partial purification of apolipoprotein A-I is obtained simultaneously by this method.     

The purine nucleotide cycle in the regulation of ammoniagenesis during induction and cessation of chronic acidosis in the rat kidney.

The effect of chronic acid feeding and its subsequent withdrawal was determined on the amounts of the metabolic intermediates and enzymic activities of the purine nucleotide cycle. Sprague-Dawley rats were given 1.5% (w/v) NH4Cl in their drinking water for 5 days. The renal excretion of NH3 rose 70-fold and the rats developed acidosis. The amount of renal IMP rose from a control value of 4.5 +/- 2.2 to 20.4 +/- 3.7nmol/g of kidney after 48h of acid feeding (P less than 0.001) and fell to normal within 48h of the recovery. Adenylosuccinate concentrations fell from a control value of 4.5 +/- 0.9nmol/g of kidney to 1.2 +/- 0.3nmol/g (P less than 0.005) by day 5 of acidosis and continued to fall to undetectable values by 48h after recovery. The amount of AMP remained constant through the acid-feeding and the recovery periods. The activity of adenylosuccinate synthetase, the rate-limiting enzyme of the purine nucleotide cycle, paralleled the rise and fall in NH3 excretion. The activities of phosphate-dependent glutaminase and glutamate dehydrogenase were elevated during the acid-feeding and the recovery period. Thus changes in the purine nucleotide cycle correlate with changes in NH3 excretion to a more parallel degree than does the activity of glutaminase or glutamate dehydrogenase.     

Regulatory T cells dampen pulmonary inflammation and lung injury in an animal model of pneumocystis pneumonia

CD4+CD25+FoxP3+ regulatory T cells are decreased in patients infected with HIV and have been shown to be critical in mediating Ag tolerance in the lung. Because a subset of Pneumocystis-infected individuals develop substantial lung injury, which can be modeled in immune reconstituted scid mice, we used mouse models of Pneumocystis carinii to investigate the role of regulatory T cells in opportunistic infection and immune reconstitution. In this study, we show that CD4+CD25+FoxP3+ cells are part of the host response to Pneumocystis in CD4+ T cell-intact mice. Moreover, lung injury and proinflammatory Th1 and Th2 cytokine levels in the bronchoalveolar lavage fluid and lung homogenate were increased following CD4+CD25- immune reconstitution in Pneumocystis-infected SCID mice but not in CD4+CD25+ T cell-reconstituted animals. The ability of CD4+CD25+ T cells to control inflammation and injury during the course of Pneumocystis was confirmed by treatment of wild-type C57BL/6 mice with anti-CD25 mAb. These data show that CD4+CD25+ T cells control pulmonary inflammation and lung injury associated with Pneumocystis infection both in the setting of immune reconstitution as well as new acquisition of infection.     

Comparative High-Density Microarray Analysis of Gene Expression during Growth of Lactobacillus helveticus in Milk versus Rich Culture Medium

Lactobacillus helveticus CNRZ32 is used by the dairy industry to modulate cheese flavor. The compilation of a draft genome sequence for this strain allowed us to identify and completely sequence 168 genes potentially important for the growth of this organism in milk or for cheese flavor development. The primary aim of this study was to investigate the expression of these genes during growth in milk and MRS medium by using microarrays. Oligonucleotide probes against each of the completely sequenced genes were compiled on maskless photolithography-based DNA microarrays. Additionally, the entire draft genome sequence was used to produce tiled microarrays in which noninterrupted sequence contigs were covered by consecutive 24-mer probes and associated mismatch probe sets. Total RNA isolated from cells grown in skim milk or in MRS to mid-log phase was used as a template to synthesize cDNA, followed by Cy3 labeling and hybridization. An analysis of data from annotated gene probes identified 42 genes that were upregulated during the growth of CNRZ32 in milk (P < 0.05), and 25 of these genes showed upregulation after applying Bonferroni's adjustment. The tiled microarrays identified numerous additional genes that were upregulated in milk versus MRS. Collectively, array data showed the growth of CNRZ32 in milk-induced genes encoding cell-envelope proteinases, oligopeptide transporters, and endopeptidases as well as enzymes for lactose and cysteine pathways, de novo synthesis, and/or salvage pathways for purines and pyrimidines and other functions. Genes for a hypothetical phosphoserine utilization pathway were also differentially expressed. Preliminary experiments indicate that cheese-derived, phosphoserine-containing peptides increase growth rates of CNRZ32 in a chemically defined medium. These results suggest that phosphoserine is used as an energy source during the growth of L. helveticus CNRZ32.     

DNA-DNA homology among lactose- and sucrose-fermenting transconjugants from Lactococcus lactis strains exhibiting reduced bacteriophage sensitivity.

DNA-DNA homology between a reduced bacteriophage sensitivity (Rbs+) probe and DNA from both Rbs+ and Rbs- Lactococcus lactis strains was examined. Homology was detected between the probe and five plasmids (pCI750, pCC34, pEB56, pNP2, and pJS88) isolated from lactose-positive Rbs+ transconjugants and between the probe and genomic DNA of a sucrose-positive Rbs+ transconjugant. Additionally, hybridizations conducted between the probe and plasmids reported to encode abortive bacteriophage infection indicated homology with pTR2030 but not with pBF61 and pGBK17. The results suggest that a common genetic determinant(s) may be present in a variety of lactococcal plasmids coding for Rbs+.     

Detection and characterization of erythromycin-resistant methylase genes in Gram-positive bacteria isolated from poultry litter

The epidemiology of four erythromycin-resistant methylase ( erm) genes, ermA, ermB, ermC and msrA, was determined in erythromycin-resistant staphylococci, enterococci and streptococci isolated from poultry litter. All isolates were resistant to multiple antibiotics. Southern hybridization indicated that 4 of the 20 staphylococci contained the ermC gene on plasmids: on a 2.2 kb plasmid in Staphylococcus hominis and S. sciuri, on a 6.0 kb plasmid in S. xylosus, and on a 7.0 kb plasmid in S. lentus. In 16 of the 20 staphylococci, the ermA gene was harbored exclusively on the chromosome, as a double chromosomal insert on 8.0 and 6.2 kb EcoRI fragments. None of the staphylococci harbored the msrA gene. Dot-blot analysis indicated that all enterococci and streptococci hybridized with a biotinylated ermB gene probe. Southern hybridization indicated that only 2 of the 19 erythromycin-resistant enterococci contained the ermB gene on plasmids. The gene was localized on 4.0 kb and 5.9 kb plasmids, respectively, in two Enterococcus faecium isolates. Results from our studies indicate that the patterns of occurrence of erm genes, the sizes of the plasmids and the copy numbers of the inserts were different from the existing information on the presence of erm genes in clinical strains of Staphylococcus spp.     

Immortalisation of human ovarian surface epithelium with telomerase and temperature-sensitive SV40 large T antigen

Epithelial ovarian cancer is the most common form of gynaecological malignancy. This lethal disease is thought to arise in ovarian surface epithelial (OSE) cells. The biology of these cells is not well understood, due to the limited amount of tissue that can be obtained from a single biopsy and their limited life span in culture. To overcome these problems, we have conditionally immortalised OSE cells with the catalytic subunit of telomerase (hTERT) and a temperature-sensitive form of SV40 Large T antigen (tsT). We have maintained these cells (designated OSE-C2) in culture for more than 100 population doublings after introduction of the immortalising genes. Early passage OSE-C2 cells have a near-tetraploid karyotype and exhibit a dual mesenchymal-epithelial phenotype, with consistent expression of vimentin and variable expression of cytokeratins and type III collagen, and absence of E cadherin expression. OSE-C2 cells proliferate steadily at the permissive temperature of 33 degrees C, but fail to increase in number at the nonpermissive temperature of 39 degrees C. Serum-deprived OSE-C2 cells are stimulated to grow at 33 degrees C by EGF, whereas they are growth inhibited at 33 degrees C by TGFbeta in the presence or the absence of serum. When temperature shifted to the nonpermissive temperature, OSE-C2 cells modulate to a more mesenchymal phenotype, and a proportion of the cells undergo senescence and/or apoptosis. Moreover, at the nonpermissive temperature, the levels of p53 and SV40 Large T antigen diminish, whilst the level of p21 increases, whereas the level of p16 and telomerase activity is unchanged. This experimental system shows that expression of telomerase alone only allows limited proliferative potential of OSE cells; expression of tsT is necessary to maintain these cells in culture for longer periods, perhaps by its ability to inactivate components of the p53/Rb pathway. OSE-C2 cells may be useful in studying the physiology and differentiation of human OSE cells and provide insight into the poorly understood earliest stages of epithelial ovarian cancer.