Changes in Escherichia coli cells starved in seawater or grown in seawater-wastewater mixtures.

Some metabolic modifications of Escherichia coli cells during starvation in seawater were studied in laboratory microcosms. The apparent die-off of this bacterium under such conditions, as observed by comparing the enumeration of CFU in conventional freshwater media and direct epifluorescence counts, was partially prevented when cells were previously grown in salted organic medium or on seawater-wastewater agar. beta-Galactosidase activity of starved cells disappeared gradually with time, even though some other enzymatic activities, such as that of alkaline phosphatase, increased. Moreover, some modifications of sensitivity to antibiotics, heavy metals, and bacteriophages in seawater- and wastewater-grown cells suggested that the cells undergo structural changes under natural marine conditions. These results provide additional experimental data indicating the possible active adaptation of E. coli cells to seawater.     

Changes in Escherichia coli cells starved in seawater or grown in seawater-wastewater mixtures

Some metabolic modifications of Escherichia coli cells during starvation in seawater were studied in laboratory microcosms. The apparent die-off of this bacterium under such conditions, as observed by comparing the enumeration of CFU in conventional freshwater media and direct epifluorescence counts, was partially prevented when cells were previously grown in salted organic medium or on seawater-wastewater agar. beta-Galactosidase activity of starved cells disappeared gradually with time, even though some other enzymatic activities, such as that of alkaline phosphatase, increased. Moreover, some modifications of sensitivity to antibiotics, heavy metals, and bacteriophages in seawater- and wastewater-grown cells suggested that the cells undergo structural changes under natural marine conditions. These results provide additional experimental data indicating the possible active adaptation of E. coli cells to seawater.     

Regulation of Dipeptidyl Aminopeptidase I and Angiotensin Converting Enzyme Activities in Cultured Murine Brain Cells by Cortisol and Thyroid Hormone

Cultures of dissociated brain cells from 15-day-old fetal mice were grown in the presence and absence of 20 or 50 nM triiodothyronine (T3), 30 or 300 nM cortisol, and 30 nM cortisol plus 50 nM T3 added to chemically defined media or in media supplemented with 15% serum from control and hypothyroid calves. The specific activities of five lysosomal enzymes--N-acetyl galactosaminidase, beta-glucuronidase, beta-galactosidase, cathepsin B, and dipeptidyl aminopeptidase I (DAP-I)--were higher in cells grown in calf serum than in cells grown in defined media. Of these enzymes, only DAP-I was elevated in activity when the cells were grown in hypothyroid calf serum instead of control calf serum. Elevation of DAP-I activity was reversed by addition of 20 nM T3 to hypothyroid calf serum. The enzymatic properties of DAP-I were similar whether the cells were grown in control or hypothyroid calf serum and were similar to those reported for human fibroblasts and the purified enzyme. When the cells were grown in defined media, cortisol decreased the activities of all lysosomal enzymes, with 300 nM cortisol being more effective than 30 nM cortisol. Addition of 50 nM T3 to 30 nM cortisol decreased DAP-I activity more than 30 nM cortisol alone, but 50 nM T3 alone in defined media did not alter DAP-I levels. The reduction of DAP-I activity in these cells by T3 required cortisol, unidentified components in serum, or both.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteolytic enzymes and arachidonic acid metabolites produced by MRC-5 cells on various microcarrier substrates

Summary Human diploid fibroblasts were cultured on microcarriers made from DEAE-dextran, denatured collagen, DEAE-dextran linked to denatured collagen, and glass. Cells grown on these four substrates were examined for the production of proteolytic enzymes and arachidonic acid metabolites. Culture fluids from cells grown on the DEAE-dextran microcarriers contained the highest amounts of proteolytic enzyme activity. Both plasminogen-independent and plasminogen-dependent fibrinolytic activities were present and the plasminogen-dependent activity seemed to result from the presence of both urokinase and tissue plasminogen activator. Culture fluid from the cells grown on the glass microcarriers contained the least amount of protease activity, and nearly all of the plasminogen-activator activity seemed to be of the urokinase type. Protease activity in the culture fluids of cells grown on the other two substrates were intermediate. With regard to arachidonic acid metabolites, cells grown on the DEAE-dextran microcarriers produced the highest amounts of cyclooxygenase products but very low levels of lipoxygenase metabolites. Cells grown on the other three substrates produced comparable amounts of various cyclooxygenase products (lower than that produced by cells on the DEAE-dextrans substrate). Cells grown on the glass microcarriers also produced detectable amounts of two lipoxygenase metabolites—leukotriene B₄ and leukotriene C₄. Inasmuch as both proteolytic enzymes and arachidonic acid metabolites regulate basic cell properties, the differential amount of these metabolites observed in the culture fluids on the various substrates may contribute to the biological differences that exist on these substrates. This study was supported in part by grants R44 CA 36656 and IK08HL01332-01 from the Public Health Service, U. S. Department of Health and Human Services and by grant BC-512 from the American Cancer Society. JDH is a research fellow of the American Lung Association.     

Sublethal stress in Escherichia coli: a function of salinity.

Sublethal stress in Escherichia coli was detected in various test media after exposure (in vitro) to seawater of various salinites. Stress was measured with an electrochemical detection technique and a beta-galactosidase assay. Test media included EC medium, medium A-1, and tryptic soy broth modified to contain lactose for beta-galactosidase assay experiments. Stress was defined as the difference between a predicted electrochemical response time calculated for unstarved cells from a standard curve and the observed electrochemical response time for cells starved in seawater. The higher the salinity, the greater the stress for all test media examined. Stress was most pronounced in EC and was attributed primarily to initial die-off of starved cells exposed to the test medium at the elevated temperature of 44.5 degrees C. Lag time and growth rates in test media were not significantly affected by salinity. beta-Galactosidase specific activity, assayed in starved cells after transfer to an induction medium at 44.5 degrees C for 150 min, was inversely related to the salinity of the starved cell suspension. The consequences of these observations with respect to coliform enumeration methods are discussed.     

Sublethal stress in Escherichia coli: a function of salinity.

Sublethal stress in Escherichia coli was detected in various test media after exposure (in vitro) to seawater of various salinites. Stress was measured with an electrochemical detection technique and a beta-galactosidase assay. Test media included EC medium, medium A-1, and tryptic soy broth modified to contain lactose for beta-galactosidase assay experiments. Stress was defined as the difference between a predicted electrochemical response time calculated for unstarved cells from a standard curve and the observed electrochemical response time for cells starved in seawater. The higher the salinity, the greater the stress for all test media examined. Stress was most pronounced in EC and was attributed primarily to initial die-off of starved cells exposed to the test medium at the elevated temperature of 44.5 degrees C. Lag time and growth rates in test media were not significantly affected by salinity. beta-Galactosidase specific activity, assayed in starved cells after transfer to an induction medium at 44.5 degrees C for 150 min, was inversely related to the salinity of the starved cell suspension. The consequences of these observations with respect to coliform enumeration methods are discussed.     

THE INFLUENCE OF THE MODE OF NUTRITION ON THE DIGESTIVE SYSTEM OF OCHROMONAS MALHAMENSIS

The intracellular distribution and level of acid hydrolases in Ochromonas malhamensis were studied in cells grown osmotrophically in a defined medium, in a carbon-free starvation medium, and during phagotrophy in each of these media. By cytochemical techniques, little enzymic reaction product was observed in the vacuoles of osmotrophic cells grown in the defined medium. Starved cells, however, contained autophagic vacuoles and cannibalized other Ochromonas cells. Dense enzymic reaction product was observed in the digestive vacuoles and in the Golgi cisternae of these starved cells. Moreover, starved cells and cells grown in a nutritionally complete medium ingested Escherichia coli which appeared in digestive vacuoles containing enzymic reaction product. Biochemical assays for lysosomal acid phosphatase (E.C. 3.1.3.2 orthophosphoric monoester phosphohydrolase) and acid ribonuclease (E.C. 2.7.7.16 ribonucleate nucleotido-2'-transferase) were done on Ochromonas cultures in the same experimental treatments and under identical assay conditions as the cytochemical study. During starvation, the acid hydrolase specific activities were consistently twice those found in cells grown in an osmotrophic complete medium. Ochromonas fed E. coli showed no increase in acid hydrolase specific activity as compared to controls not fed E. coli. The latency of lysosomal acid hydrolases in cells fixed with glutaraldehyde was reduced, suggesting that this fixative increases lysosomal membrane permeability and may release enzymes or their reaction products into the cytoplasmic matrix during cytochemical analysis. This could explain the cytoplasmic staining artifact sometimes observed with glutaraldehyde-fixed cells when studied by the Gomori technique. This study confirms that Ochromonas malhamensis, a phytoflagellate, does produce digestive vacuoles and can ingest bacteria, thereby fulfilling its role as a heterotroph in an aquatic food chain. When Ochromonas is grown in a nutritionally complete osmotrophic medium, phagocytosis causes appearance of acid hydrolases in the digestive vacuoles, whereas the total activity of the enzymes remains unchanged. An organic carbon-free medium strongly stimulates acid hydrolaes activity and causes these enzymes to appear in the digestive vacuoles whether phagocytosis occurs or not.     

Nitrogen Starvation and the Regulation of Glutamine Synthetase in Agmenellum quadruplicatum

The level of glutamine synthetase activity in Agmenellum quadruplicatum strain PR-6 was dependent on the nitrogen source used for growth and on the nutritional status of the cells. During exponential growth, glutamine synthetase activity was low in cells grown on ammonia, urea, or nitrate. During the transition from nitrogen replete to nitrogen starved growth, glutamine synthetase activity began to rise. With ammonia as a nitrogen source, glutamine synthetase activity as determined in whole cells increased from 1 nanomole per minute per milliliter during exponential growth to 22 nanomoles per minute per milliliter during severe nitrogen starvation. In cells grown on nitrate the increase was from 5 to 39 nanomoles per minute per milliliter, and in cells grown on urea the increase was from 4 to 31 nanomoles per minute per milliliter.     

α-glucosidase in Mycoplasma mycoides subspecies capri

Abstract Mycoplasma mycoides subsp. capri utilisede maltose in medium lacking serum and hence serum saccharolytic enzymes. The presence of α-glucosidase activity was demonstrated by p-nitrophenyl-α- d -glucoside hydrolysis in toluene-treated cells. Specific activities were approx. 4-fold higher in cells grown in the presence of maltose than in cells grown with other sugars or with glucose plus maltose. Extracellular activity was < 2% of cellular activity in growing cultures. α-Glucosidase activity was also demonstrated in cells grown in medium containing serum. It is suggested that the presence of α-glucosidase might be of value in mycoplasma chatacterisation; in a previous study, α-glucosidase activity was not detected in Mycoplasma mycoides subsp. mycoides .     

Distribution of two urinary ribonuclease-like enzymes in human organs and body fluids

In order to determine the distribution of two human urinary RNase (RNase Us and RNase UL)-like enzymes in human tissues and body fluids, enzyme immunoassay systems were established using rabbit anti-RNase sera. The sensitivity of the assay systems was of similar order to that of radioimmunoassay systems previously reported. In the enzyme immunoassay, the cross reactivities of anti-RNase UL serum towards RNase Us, bovine kidney RNase K2, bovine RNase A, and bovine seminal RNase Vs were less than 1%. The cross reactivity of anti-RNase Us-serum towards RNase UL was less than 0.5% and cross reactivities were minimal for RNase A, RNase K2, and RNase Vs. The RNase levels in human organs and body fluids were measured by enzyme immunoassay. In milk, semen and saliva, only RNase UL-like enzyme was found. Both RNase Us- and RNase UL-like enzymes were found in kidney, stomach, and pancreas and the RNase Us/RNase UL ratios were 0.49, 1.35, and 0.34, respectively. In lung, liver, spleen, and leukocytes, most of the RNase activity was accounted for by RNase Us-like enzyme. The activity of RNase Us-like enzyme was especially high in lung, spleen, and leukocytes. The crude extracts of several tissues and body fluids were separated by phosphocellulose column chromatography and the contents of the two urinary RNase-like enzymes were determined by enzyme immunoassay. In stomach, kidney, pancreas, and serum, both enzymes were present in multiple forms. In spleen and lung, both the major RNase (RNase Us) and minor RNase (RNase UL) existed in two forms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of growth rate and starvation on fluorescent in situ hybridization of Rhodopseudomonas palustris

In situ hybridization with a fluorescently labeled 16S rRNA-targeted probe was examined using Rhodopseudomonas palustris as a model organism, which had been grown at different rates and under different conditions of growth and starvation. The specific growth rate did not affect the percentage of hybridized cells in aerobically grown R. palustris cultures. However, significant changes in the percentage of hybridized cells occurred during extended periods of starvation. These changes were observed both in batch cultures grown and starved aerobically in the dark, and in cultures grown phototrophically and starved anaerobically in the dark. Aerobic growth in batch culture and subsequent starvation resulted in a complete lack of detectable hybridization after 20 days of starvation. In contrast, even after 30 days of starvation, 50% of all cells were still detectable in cultures grown aerobically at growth rates <0.06 h(-1) and then starved aerobically in the dark. The same was true for phototrophically grown cells that were starved anaerobically in the light. During starvation there was a clear, though non-linear, positive correlation between the percentage of hybridized cells and the RNA content. In contrast, no direct correlation was observed between the number of hybridized cells in a culture and the viability of this culture. Thus, in habitats with growing, non-growing, and starving bacteria, data on quantitative detection of populations based on 16S rRNA-targeted probing should be used with extreme caution as the detectability of the individual cells is strongly influenced by their physiological history and current physiological state.     

Influence of phosphate ions and alkaline phosphatase activity of cells on survival ofEscherichia coli in seawater

When grown in a minimal medium and suspended for 2 hours in distilled water, seawater, phosphate buffer or a polyphosphate solution,E. coli MC4100 cells with high alkaline phosphatase activity survived in seawater for longer periods than cells with low or no activity. However, mutant cells totally deprived of alkaline phosphatase activity held in phosphate-containing media before transfer to seawater showed survival almost as high as the wild type strain, indicating that alkaline phosphatase activity is not the only factor influencing survival. Alkaline phosphatase activity also increased the protection of cells provided by glycine betaine. Survival was enhanced when cells were preincubated in the presence of phosphate or polyphosphate. Thus, the transfer of cells in wastewater could influence their subsequent survival in seawater.     

Sulphur acquisition by Neisseria meningitidis

Group B Neisseria meningitidis (SD1C) was grown on defined medium supplemented with each of a variety of sulphur compounds as the sole source of sulphur. The organism grew on sulphate, sulphite, bisulphite, thiosulphate, dithionite, hydrosulphide, thiocyanate, L-cysteine, L-cystine, reduced glutathione, methionine, mercaptosuccinate, and lanthionine, but not on dithionate unless previously sulphur starved. Good growth was seen on concentrations of sulphate or thiosulphate as low as 10 microM. When pregrown on and subsequently starved for sulphate, the meningococcus showed enhanced transport capacity for this ion. Optimal conditions for assessing sulphur transport by active sulphur-limited cells were determined. The maximal sulphate uptake velocity was 9.3 nmol sulphate X mg protein-1 X min-1, and the apparent Km was 1.4 microM, far below human nasopharyngeal or serum sulphate levels.     

Distribution and ontogeny of digestive enzymes in larval yellowtail and winter flounder

The distribution of digestive enzymes was studied using histoenzymological methods in yellowtail and winter flounder larvae reared on three different diets: live food, weaned at day 15, or starved. Alkaline phosphatase, dipeptidyl peptidase IV, aminopeptidase M and esterase were present at 3 days post-hatch and became differentially distributed coinciding with morphological development. For larvae fed a live diet, activity of these enzymes was present in the intestine of both species and rectum of yellowtail flounder. Alkaline phosphatase was also present in the post-oesophageal swelling (stomach anlage) of winter flounder. Activity of all enzymes was absent in starved winter flounder larvae and a decrease in aminopeptidase M and esterase activity occurred in starved yellowtail flounder larvae. Acid phosphatase was not identified in either species. The eVect of weaning on enzymatic activity was not evaluated fully as the larvae did not survive long enough after the introduction of the artificial diet to complete experimentation.     

Secretion and formation of perimicrovillar membrane in the digestive system of the Sunn pest, Eurygaster integriceps (Hemiptera: Scutelleridae) in response to feeding

In this study, development of perimicrovillar membrane (PMM) from midgut cells of starved and fed Eurygaster integriceps (Hemiptera: Scutelleridae) was studied. Three different approaches, including scanning electron microscopy (SEM), transmission electron microscopy (TEM), marker enzymes of the PMMs (α-glucosidase), perimicrovillar space (aminopeptidase), and microvillar membranes (β-glucosidase) were used. Activities of these enzymes were remarkably low in the starved insects. Moreover, microscopic observations revealed that PMM is not present in the starved insect. Activities of enzymatic markers increased at 5 h postfeeding, and TEM and SEM observations showed the formation of PMM as well as migration of double-membrane vesicles from center of the columnar cell to the cell apex. The highest PMM was observed at 20 h postfeeding which at this time marker enzyme activity, such as α-glucosidase activity, was high, too. Thus, at 20 h postfeeding, PMM system was evident and epithelial cells were completely covered by PMM system. After 20 h postfeeding, presence of the fine holes in PMM started to be seen and at 40 h post-feeding, observation showed degradation of PMM system. Thus, it could be concluded that PMM in E. integriceps is secreted by epithelial cell membrane when needed and its secretion and formation is regulated by feeding. This system was not present in the starved insects as its development takes place at 5 h postfeeding.     

The effects of newborn calf serum and foetal bovine serum on the Na+ + K+-activated adenosine triphosphatase activity in 3T3 and SV3T3 cells grown to confluency

The effects of cell density and growth in 10% foetal bovine serum and 10% newborn calf serum on the activity of the enzyme (Na+ + K+)-ATPase were studied in 3T3 and SV3T3 cells. The enzyme activity decreases in 3T3 cells grown in foetal bovine serum as the cells approach confluency while in those grown in newborn calf serum the enzyme activity increases. The (Na+ + K+)-ATPase activity does not change with increase in cell density in SV3T3 cells grown in foetal bovine serum while the enzyme activity in those grown in newborn calf serum increases with increase in cells density up to about 1.35 x 10(5) cells/sq. cm. and then decreases with further increase in cell number. At confluency it was found that the enzyme activity is higher in the SV3T3 as compared to the 3T3 cells when the cells were grown in 10% foetal bovine serum, whereas in those grown in 10% newborn calf serum the enzyme activity is higher in the 3T3 as compared to the SV3T3 cells.     

Retardation of fetal brain cell growth during maternal starvation: Circulating factors versus altered cellular response

Maternal starvation inhibits fetal brain development during late gestation in the rat. To determine whether intrinsic or extrinsic factors might be the principal contributor to altered growth, brain cells from 20 day fetuses were cultured in a 96 well plate with MEM and 10% adult rat serum. Tissue growth was monitored by spectrophotometric measurement of the mitochondrial reduction of a chromagen 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT). After 1, 4 or 6 days incubation, MTT activity in non confluent cultures was shown to be directly related to tissue mass. When fetal brain cell cultures were incubated with 1% and 10% concentrations of adult rat serum, an 11-fold increase in MTT activity paralleled a 15-fold increase in tritiated thymidine incorporation. The impact of maternal starvation on fetal brain cell growth was examined by measuring MTT activity in fetal brain cells from fed and starved mothers. When cultures were incubated for 6 days with graded concentrations of fed adult serum (1.25–10%), the MTT response was slightly but consistently lower in cells from starved when compared with cells from fed mothers. By contrast, a marked difference in MTT activity which was paralleled by a lower DNA content became apparent when fetal rat brain cells were incubated with starved adult serum. Fetal serum and adult male serum were found to support growth equally well, while incubation of fetal brain cells with maternal sera resulted in lower MIT values than with the corresponding fetal sera. When cells were incubated with fetal sera pooled from starved mothers, MTT activity was decreased by 42 to 45%. A relative decrease in MTT activity was also apparent when cells were exposed to sera from starved mothers. Graded concentrations of starved fetal serum (2.5–10%) produced an increase in MTT activity that was consistently lower than similar concentrations of fed fetal serum, a finding suggesting a decrease in growth factors. Mixing fasted with fed serum did not correct the diminished growth, and indicated that an inhibitor might also be functioning to restrict growth. These findings therefore suggest that the principal determinants of diminished fetal brain growth during maternal starvation are not only intrinsic to the cells but are importantly related to the altered extrinsic factors in the fetal circulation.     

Histochemical findings in the rat gastric mucosa during starvation

Summary The influence of starving on the activity of enzymes of the rat gastric mucosa was investigated by selected histochemical methods. Beside the conventional methods of enzymatic histochemistry the technique of semipermeable membranes was used in the proof of lysosomal enzymes. Dehydrogenases were proved in aqueous and also in gel media with PMS.During the starvation in the parietal cells a marked increase took place in the activity of acid phosphatase, E-600 resistant esterase, less in -glucuronidase. High activity of the lysosomal enzymes in macrophages did not change during starvation. Nor did any changes took place in the activity of alkaline phosphatase in the endothelium of the capillaries. The chief cells in the control and starving animals, in contrast to the human gastric mucosa, did not contain any non-specific esterase. Concerning dehydrogenase, parietal cells with a different activity of these enzymes were observed both in starved and control animals.In the rat gastric mucosa starving induced changes in the activity of the enzymes which mark important organelles of the cells. Thus it is possible to consider the observed histochemical changes as a functional manifestation of morphological damage of cellular structures which are affected during starvation.     

Compartmental analysis of sulphate transport in Lemna minor L., taking plant growth and sulphate metabolization into consideration

Rat hepatoma cells accumulate considerably less 2-aminoisobutyrate after cultivating in the absence of serum the change in rate of aminoisobutyrate uptake takes place within 1 h of serum starvation. Starvation of amino acids by contrast raises aminoisobutyrate uptake in the presence or absence of serum, but the cells are much less responsive to amino acid supply than to availability of serum. Phosphate (10 mM) reduced aminoisobutyrate uptake by cells grown in serum to that exhibited by serum-starved cells. Aminoisobutyrate uptake by cells grown in serum was reduced by glycine, proline, alanine, serine, glutamine, methylaminoisobutyrate and 2-aminonorbornane-2-carboxylate, the effects of methylaminoisobutyrate and 2-aminonorbornane-2-carboxylate being additive. However, similar inhibition phenomena were not seen for cells deprived of serum where aminoisobutyrate uptake tended to a relatively constant level insensitive to inhibitory influences, yet substantially greater than that arising by simple diffusion. The comparative insensitivity of our hepatoma line when starved of serum to competition and repression phenomena is in contrast to findings of others. Our results also suggest a lack of clear delineation of specificities for the A and L transport systems as usually defined.