A simple technique for mass cultivation of Campylobacter fetus.
Studies using 86 media for maximum growth of Campylobacter fetus for antigen production showed that a diphasic medium (solid base with liquid overlay) was most suitable. The solid base was double strength cystine heart agar. The liquid overlay was thioglycollate medium of Brewer (135-C) without agar. This medium yielded maximum growth of C. fetus in six days with good motility, less clumping and less filament formation than all other media tried. (+info)
Stimulation of thymidine uptake and cell proliferation in mouse embryo fibroblasts by conditioned medium from mammary cells in culture.
Undialyzed conditioned medium from several cell culture sources did not stimulate thymidine incorporation or cell overgrowth in quiescent, density-inhibited mouse embryo fibroblast cells. However, dialyzed conditioned medium (DCM) from clonal mouse mammary cell lines MCG-V14, MCG-T14, MCG-T10; HeLa cells; primary mouse adenocarcinoma cells; and BALB/c normal mouse mammary epithelial cells promoted growth in quiescent fibroblasts. The amount of growth-promoting activity produced per cell varied from 24% (HeLa) to 213% (MCG-V14) of the activity produced by primary tumor cells. The production of growth-promoting activity was not unique to tumor-derived cells or cells of high tumorigenicity. The amount of growth-promoting activity produced per cell in the active cultures was not correlated with any of the following: tumorigenicity, growth rat, cell density achieved at saturation, cell type, or species of cell origin. It is concluded that transformed and non-transformed cells of diverse origin, cell type, and tumorigenicity can produce growth factors in culture. The growth-promoting potential of the active media from primary tumor cultures accumulated with time of contact with cells and was too great to be accounted for entirely by the removal of low-molecular-weight inhibitors by dialysis. The results are consistent with the hypothesis that conditioned medium from the active cultures contained a dialyzable, growth-promoting activity. Different cell lines exhibited differential sensitivity to tumor cell DCM and fetal bovine serum. Furthermore, quiescent fibroblasts were stimulated by primary tumor cell DCM in the presence of saturating concentrations of fetal bovine serum. These observations support the notion that the active growth-promoting principle in primary tumor cell DCM may not be a serum factor(s). (+info)
Improved medium for recovery and enumeration of Pseudomonas aeruginosa from water using membrane filters.
A modified mPA medium, designated mPA-C, was shown to recover Pseudomonas aeruginosa from a variety of water sources with results comparable to those with mPA-B and within the confidence limits of a most-probable-number technique. Enumeration of P. aeruginosa on mPA-C was possible after only 24 h of incubation at 41.5 degrees C, compared with 72 h of incubation required for mPA-B and 96 h of incubation for a presumptive most probable number. (+info)
Unsaturated fatty acid requirements for growth and survival of a rat mammary tumor cell line.
A cell line, the growth and survival of which is markedly affected by linoleic acid, has been established from a carcinogen-induced rat mammary tumor. The cells have been continuously passaged in 5% rat serum plus 10% fetal calf serum-supplemented medium. The rat serum component was found to be indispensalbe, for when it was omitted the growth rate rapidly declined and the cells died by 5 to 7 days. Removal of the rat serum from the growth medium also resulted in a dramatic loss of Oil Red O-positive droplets in the cells, suggesting that the lipid component of rat serum might be a major growth-promoting principle in rat serum. This is likely since the total lipid fraction, but not the delipidized protein fraction, could largely supplant requirement of the cells for rat serum. Pure linoleic acid was found to be effective in maintaining the cell growth in delipidized serum or in whole fetal calf serum-supplemented medium. Fatty acid analysis revealed a 19-fold higher amount of linoleic acid in rat serum than in fetal calf serum. (+info)
Nrg1 is a transcriptional repressor for glucose repression of STA1 gene expression in Saccharomyces cerevisiae.
Expression of genes encoding starch-degrading enzymes is regulated by glucose repression in the yeast Saccharomyces cerevisiae. We have identified a transcriptional repressor, Nrg1, in a genetic screen designed to reveal negative factors involved in the expression of STA1, which encodes a glucoamylase. The NRG1 gene encodes a 25-kDa C2H2 zinc finger protein which specifically binds to two regions in the upstream activation sequence of the STA1 gene, as judged by gel retardation and DNase I footprinting analyses. Disruption of the NRG1 gene causes a fivefold increase in the level of the STA1 transcript in the presence of glucose. The expression of NRG1 itself is inhibited in the absence of glucose. DNA-bound LexA-Nrg1 represses transcription of a target gene 10.7-fold in a glucose-dependent manner, and this repression is abolished in both ssn6 and tup1 mutants. Two-hybrid and glutathione S-transferase pull-down experiments show an interaction of Nrg1 with Ssn6 both in vivo and in vitro. These findings indicate that Nrg1 acts as a DNA-binding repressor and mediates glucose repression of the STA1 gene expression by recruiting the Ssn6-Tup1 complex. (+info)
Interaction of inflammatory cells and oral microorganisms. II. Modulation of rabbit polymorphonuclear leukocyte hydrolase release by polysaccharides in response to Streptococcus mutans and Streptococcus sanguis.
The release of lysosomal hydrolases from polymorphonuclear leukocytes (PMNs) has been postulated in the pathogenesis of tissue injury in periodontal disease. In the present study, lysosomal enzyme release was monitored from rabbit peritoneal exudate PMNs exposed to Streptocccus mutans or Streptococcus sanguis. S. mutans grown in brain heart infusion (BHI) broth failed to promote significant PMN enzyme release. S. sanguis grown in BHI broth, although more effective than S. mutants, was a weak stimulus for promotion of PMN hydrolase release. Preincubation of washed, viable S. mutans in sucrose or in different-molecular-weight dextrans resulted in the ability of the organisms to provoke PMN release reactions. This effect could bot be demonstrated with boiled or trypsinized S. mutans or with viable S. sanguis. However, when grown in BHI broth supplemented with sucrose, but not with glucose, both S. mutans and S. sanguis triggered discharge of PMN enzymes. The mechanism(s) whereby dextran or sucrose modulates PMN-bacterial interaction may in some manner be related to promotion of microbial adhesiveness or aggregation by dextran and by bacterial synthesis of glucans from sucrose. (+info)
Downregulation of metallothionein-IIA expression occurs at immortalization.
Metallothioneins (MTs) may modulate a variety of cellular processes by regulating the activity of zinc-binding proteins. These proteins have been implicated in cell growth regulation, and their expression is abnormal in some tumors. In particular, MT-IIA is expressed 27-fold less in human colorectal tumors and tumor cell lines compared with normal tissue (Zhang et al., 1997). Here we demonstrate that MT-IIA downregulation occurs when human cells become immortal, a key event in tumorigenesis. After immortalization MT-IIA expression remains inducible but the basal activity of the MT-IIA promoter is decreased. MT-IIA downregulation at immortalization is one of the most common immortalization-related changes identified to date, suggesting that MT-IIA has a role in this process. (+info)
Estrogen-dependent and independent activation of the P1 promoter of the p53 gene in transiently transfected breast cancer cells.
Loss of p53 function by mutational inactivation is the most common marker of the cancerous phenotype. Previous studies from our laboratory have demonstrated 17 beta estradiol (E2) induction of p53 protein expression in breast cancer cells. Although direct effects of E2 on the expression of p53 gene are not known, the steroid is a potent regulator of c-Myc transcription. In the present studies, we have examined the ability of E2 and antiestrogens to regulate the P1 promoter of the p53 gene which contains a c-Myc responsive element. Estrogen receptor (ER)-positive T47D and MCF-7 cells were transiently transfected with the P1CAT reporter plasmid and levels of CAT activity in response to serum, E2 and antiestrogens were monitored. Factors in serum were noted to be the dominant inducers of chloramphenicol acetyltransferase (CAT) expression in MCF-7 cells. The levels of CAT were drastically reduced when cells were maintained in serum free medium (SFM). However, a subtle ER-mediated induction of CAT expression was detectable when MCF-7 cells, cultured in SFM, were treated with E2. In serum-stimulated T47D cells, the CAT expression was minimal. The full ER antagonist, ICI 182 780 (ICI) had no effect. Treatment with E2 or 4-hydroxy tamoxifen (OHT) resulted in P1CAT induction; OHT was more effective than E2. Consistent with c-Myc regulation of the P1 promoter, E2 stimulated endogenous c-Myc in both cell lines. Two forms of c-Myc were expressed independent of E2 stimuli. The expression of a third more rapidly migrating form was E2-dependent and ER-mediated since it was blocked by the full ER antagonist, ICI, but not by the ER agonist/antagonist OHT. These data demonstrate both ER-mediated and ER-independent regulation of c-Myc and the P1 promoter of the p53 gene, and show differential effects of the two classes of antiestrogens in their ability to induce the P1 promoter of the p53 gene in breast cancer cells. (+info)