Plasmid expression and maintenance during long-term starvation-survival of bacteria in well water.

Strains of enteric bacteria and pseudomonads containing plasmid R388::Tnl721 (Tpr, Tcr) or pRO101 (Hgr, Tcr) were starved for over 250 days in sterile well water to evaluate effects of starvation-survival on plasmid expression and maintenance. Viable populations dropped to between approximately 0.1 and 1% of the initial populations. Escherichia coli(pRO101) and Pseudomonas cepacia(pRO101) lost both viability and plasmid expression at a lower rate than strains containing R388::Tnl721. Three patterns of host-plasmid interaction were detected: (i) no apparent loss of plasmid expression, (ii) loss of plasmid expression on initial recovery with subsequent expression upon resuscitation, and (iii) loss of capability to produce functional plasmid resistance.     

Effect of Growth Rate and Starvation-Survival on the Viability and Stability of a Psychrophilic Marine Bacterium

Cell populations of the marine bacterium ANT-300, from either batch or continuous culture with dilution rates ranging from D = 0.015 h⁻¹ to D = 0.200 h⁻¹, were monitored for viability, direct counts, and optical density for 98 days under starvation conditions. Three stages of starvation survival were observed for each of the cell populations. Although direct counts remained at 2 × 10⁷ to 3 × 10⁷ cells ml⁻¹ throughout the starvation period, large fluctuations occurred in cell viability during stage 1 (0 to 14 days) of starvation survival. Stage 2 (14 to 70 days) involved an overall decrease in viability for each of the cell populations; the rate of viability loss was dependent upon the growth rate. Cell viability stabilized at approximately 0.3% of the direct count in stage 3 (70 to 98 days). Long-term starvation corresponded to the prolongation of stage 3 starvation survival. Cell volumes for each of the cell populations decreased with the length of the starvation period. However, the cell volume of starved cells was also dependent more on growth rate than on the length of the time starved. We hypothesize that the cell population with the slowest growth rate is most closely representative of cells found in the oligotrophic marine environment.     

Effect of 24,25-dihydroxyvitamin D3 on 1,25-dihydroxyvitamin D3 metabolism in calcium-deficient rats.

The effect of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] on 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] metabolism was examined in rats fed on a low-calcium diet. These rats exhibit hypocalcaemia, high urinary cyclic AMP excretion, a markedly elevated serum 1,25(OH)2D concentration and low serum concentrations of both 24,25(OH)2D and 25(OH)D. When the rats are treated orally with 1, 5 or 10 micrograms of 24,25(OH)2D3/100 g every day, there is a dramatic decrease in serum 1,25(OH)2D concentration in a dose-dependent manner concomitant with an increase in serum 24,25(OH)2D concentration. Serum calcium concentration and urinary cyclic AMP excretion are not significantly affected by the 24,25(OH)2D3 treatment, which suggests that parathyroid function is not affected by the 24,25(OH)2D3 treatment. The 25(OH)D3 1 alpha-hydroxylase activity measured in kidney homogenates is markedly elevated in rats on a low-calcium diet but is not affected by any doses of 24,25(OH)2D3. In contrast, recovery of intravenously injected [3H]1,25(OH)2D3 in the serum is decreased in 24,25(OH)2D3-treated rats. Furthermore, when [3H]1,25(OH)2D3 is incubated in vitro with kidney or intestinal homogenates of 24,25(OH)2D3-treated rats there is a decrease in the recovery of radioactivity in the total lipid extract as well as in the 1,25(OH)2D3 fraction along with an increase in the recovery of radioactivity in the water-soluble phase. These results are consistent with the possibility that 24,25(OH)2D3 has an effect on 1,25(OH)2D3 metabolism, namely that of enhancing the degradation of 1,25(OH)2D3. However, because a considerable proportion of the injected 24,25(OH)2D3 is expected to be converted into 1,24,25(OH)3D3 by renal 1 alpha-hydroxylase in 24,25(OH)2D3-treated rats, at least a part of the decrease in serum 1,25(OH)2D concentration may be due to a competitive inhibition by 24,25(OH)2D3 of the synthesis of 1,25(OH)2D3 from 25(OH)D3. Thus the physiological importance of the role of 24,25(OH)2D3 in regulating the serum 1,25(OH)2D concentration as well as the mechanism and metabolic pathway of degradation of 1,25(OH)2D3 remain to be clarified.     

Localization of cytochrome P-450 in the colonic mucosa of 3-methylcholanthrene-pretreated and untreated rats An immunohistochemical study

Summary Immunohistochemical localization of cytochrome P-450 in the colonic mucosa of 3-methylcholanthrene-pretreated and untreated rats was studied by indirect fluorescent antibody staining technique. A polyclonal antibody for cytochrome P-450_MC purified from hepatic microsomes of 3-methylcholanthrene-pretreated rats was used for this experiment. A strong immunofluorescence was found to be localized in the cytoplasm of the surface epithelium of the mucosa in the colon of 3-methylcholanthrene-pretreated rats. A faint immunofluorescence was also observed in the epithelium of untreated rats. 7-Ethoxycoumarin O-deethylase activity of colonic microsomes was significantly enhanced by 3-methylcholanthrene-pretreatment in parallel with an increase in the intensity of immunostaining for cytochrome P-450_MC in Western blotting analysis. This is the first report on the localization of cytochrome P-450 in the colonic mucosa.     

Preventive effect of MCI-186 on 15-HPETE induced vascular endothelial cell injury in vitro

Using cultured bovine aortic endothelial cells, the effects of MCI-186, a radical scavenger, were studied on arachidonic acid metabolism and on the cell injury caused by 15-HPETE. MCI-186 at 3 X 10(-5) M enhanced prostacyclin production in the intact endothelial cells without affecting phospholipase A2. When endothelial cell homogenates were used as an enzyme source, it was found that MCI-186 stimulated the conversion of arachidonic acid to prostacyclin like phenol, perhaps by trapping OH radicals produced in the process of the conversion of PGG2 to PGH2. On the other hand, MCI-186 was found to inhibit lipoxygenase metabolism of arachidonic acid in cell free homogenates of rat basophilic leukemia cells. The lipoxygenase inhibition caused by 3 X 10(-5) M MCI-186 was almost equivalent to that caused by 3 X 10(-6) M BW 755C. MCI-186 remarkably protected against endothelial cell damage caused by 15-HPETE. 3 X 10(-5) M of 15-HPETE caused endothelial cell death in about 60% of the population: however, pretreatment of the cells with 10(-5) M of MCI-186 or concomitant addition of 10(-5) M of MCI-186 with 15-HPETE to the cultures prevented the cell death completely. These results suggest that MCI-186 may become an unique anti-ischemic drug.     

Further studies on the relationship between tyrosine supply and catecholamine production in cultured adrenal chromaffin cells.

To elucidate a possible role of tyrosine supply as a factor modulating catecholamine biosynthesis in the adrenergic cell, the transport of [14C]tyrosine into cultured bovine adrenal chromaffin cells was first examined, and the relationship between [14C]tyrosine transport and [14C]catecholamine formation was then investigated. Under the conditions which were routinely employed to determine the rate of catecholamine biosynthesis, tyrosine was taken up into the cells in a manner independent of extracellular Na+ and Ca2+, and this uptake was also insensitive to ouabain and various metabolic inhibitors. The stimulation of these cells with high K+ and other secretagogues caused no significant alteration in the uptake. While, tyrosine transport was markedly inhibited by tyrosine analogues and other L-aromatic amino acids, and this inhibition was accompanied by the reduction of [14C]catecholamine formation. In contrast, tyrosine transport was markedly enhanced by flavone, and this enhancement was also accompanied by the augmentation of catecholamine production under the same experimental conditions. These results seem to indicate that the transport of tyrosine into the cells may be closely related to catecholamine formation within the cells, thus providing an evidence for a possible role of tyrosine supply as one of the factors affecting catecholamine production in the adrenal chromaffin cell.     

Lymphokine production by human melanoma tumor-infiltrating lymphocytes

Summary Lymphokine production by human melanoma tumor-infiltrating lymphocytes (TIL) was studied. Uncultured TIL produced interferon (IFN), but not interleukin-2 (IL-2) or IL-4, in response to anti-CD3 mAb or IL-2. In bulk cultures, IL-2-activated TIL displaying autologous tumor-specific cytotoxicity (CTL-TIL) produced IFN in culture with medium alone, whereas IL-2-activated noncytotoxic TIL did not. Addition of anti-CD3 mAb or autologous tumor cells up-regulated IFN production in IL-2-activated TIL from 10 of 12 or 6 of 12 cases respectively. Those from 4 of 12 cases (2 CTL-TIL and 2 noncytotoxic TIL) produced IL-2 in culture with medium alone. At the clonal level, 5 (4 CD4⁺ and 1 CD8⁺) of 7 autologous tumor-specific CTL clones derived from TIL and 3 (2 CD4⁺ and 1 CD8⁺) of 7 noncytotoxic TIL clones produced IFN in culture with medium alone, which was up-regulated by adding anti-CD3 mAb. Two IFN-producing CTL clones tested produced IL-2 in 4 ×-concentrated supernatants from a 3.5-h culture with medium alone. Furthermore, 2 IFN-producing CTL clones tested expressed mRNA for both IFN and IL-2. IL-2 production and its mRNA expression were up- or down-regulated, respectively, by adding anti-CD3 mAb or autologous tumor cells. IL-4 production was not observed in culture either with medium alone or with IL-2 in any of the cells described above. Anti-CD3 mAb was required for IL-4 production in 3 of 12 IL-2-activated TIL, 2 of 6 CTL clones, and none of 5 noncytotoxic TIL clones. In summary, IFN production was characteristic of melanoma TIL. Some autologous tumor-specific CTL in TIL are suggested to be productive of IL-2 and IFN under unstimulated conditions, both being required for self-activation in an autocrine loop.This work was supported in part by grant CA-47891 from the National Cancer Institute     

Correlation of activity with phenotypes of Escherichia coli partial function mutants of rnh, the gene encoding RNase H

Summary The rnh gene of Escherichia coli encodes RNase H. rnh mutants display at least two phenotypes: (1) they require functional RecBCD enzyme for growth; thus rnh-339::cat recB270 (Ts) and rnh-339::cat recC271 (Ts) strains are temperature sensitive for growth; (2) rnh mutants permit replication that is independent of the chromosomal origin, presumably by failing to remove RNA-DNA hybrids from which extra-original replication can be primed. We report here that manifestation of these two phenotypes occurs at different levels of RNase H function; we have examined partially functional rnh mutants for their in vitro RNase H activity, their ability to rescue viability in recB or recC cells and their ability to permit growth of mutants incapable of using oriC [dnaA (Ts)].