Increases in enzyme levels during the formation of phenolic acids in carrot cell cultures

The levels of glutamic acid dehydrogenase (GDH), phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (CAH) and O-methyltransferase (OMT) were measured during the formation of phenolic acids in carrot cells in suspension culture. Caffeic, ferulic and p-hydroxybenzoic acids were always present as the culture proceded. Total content of these acids increased at the early logarithmic and linear phases. GDH showed high activity at the early logarithmic and stationary phases. PAL activity was much enhanced at the linear and stationary phases. CAH activity was found in actively growing cells, especially at the early and late logarithmic phases OMT behaved similarly to PAL. The increases in GDH and CAH might be responsible for the rapid synthesis of phenolic acid at the early logarithmic phase. The increase in phenolic acid at the linear phase would certainly be due to enhancements of both PAL and OMT. On the other hand, the accumulation of vanillic acid was observed in cells which were transferred and cultured on an agar medium, but not in cells in suspension culture. This accumulation is related to increases in OMT levels and also to changes in the degree of β-oxidation.     

Development of phenotypic resistance to direct lethal miconazole action by Candida albicans entering stationary phase

In the late logarithmic or very early stationary phase of the growth cycle, yeast cells of Candida albicans undergo a shift from susceptibility to resistance to the direct lethal action of miconazole. Regulation of this phenotypic shift was examined. Experiments based on viable count determinations and the construction of time-kill curves showed that reestablishment of resistance is independent of both pH and the attainment of some critical viable cell density. However, it was found that development of resistance requires the continued availability of an appropriate energy source toward the end of exponential growth.     

Proteolysis in Euglena gracilis

Endoproteolytic activities (EC 3.4.22. and 23.) of cell-free extracts of Euglena gracilis, measured by autolysis and azocaseinolysis, vary considerably during the culture growth cycle. They are high in the lag phase, drop sharply up to the mid-logarithmic phase, and then rise again reaching the initial high levels in the stationary phase. This pattern has been observed for both the soluble and the particulate proteolytic activities of four cell types differing with regard to the developmental state of the chloroplast: dark-grown, light-induced, and light-grown wild-type cells, as well as light-grown apoplastic W₃BUL mutant cells, all on a glucose-based medium. Therefore, the activity of the main intracellular proteinases is neither directly nor indirectly light-regulated, but seems to be controlled by the availability of nutrients. Endogenous inhibitors of proteinases could not be detected. Cysteine proteinase activity has been found in the soluble and the particulate fractions, but aspartic proteinase activity in the latter ones only. Different cysteine proteinases may be present in the two fractions, during the different growth phases, and in the four cell types studied.Abbreviations CBB Coomassie Brilliant Blue G-250 - DFP diisopropyl fluorophosphate - EDTA disodium ethylendiaminetetraacetic acid - E-64 l-transepoxysuccinyl-leucyl-amido(4-guanidino)butane - Iog phase logarithmic growth phase - MET 2-mercaptoethanol - PMSF phenylmethylsulfonyl fluoride - Z benzyloxycarbonyl Paper I of this series is Krauspe and Scheer (1986). A preliminary publication appeared (Krauspe et al. 1982)     

The Mechanism of Onset of the Stationary Phase in Euglena gracilis Grown with 10 mM Succinate: Intracellular pH Values*

SYNOPSIS. When Euglena gracilis were grown with 10mM succinate at pH 3.5 the extracellular pH averaged 3.62 and the cultures had produced 6 × 10⁵ cells/ml when the stationary phase began. Oxygen consumption values reached a maximum of 30 μliters/10⁶ cells/hr. Total protein and dry weights per cell remained constant during the logarithmic phase and began to decline when the late logarithmic phase was reached. Added succinate caused the cultures in stationary phase to commence logarithmic growth once more. Onset of the stationary phase in cultures grown at pH 3.5 was due to depletion of succinate. When cultures were grown at pH 6.9 the extracellular pH averaged 7.62 and the cultures produced 3 × 10⁵ cells/ml when the stationary phase began. Oxygen consumption values reached a maximum of 20 μliters/10⁶ cells/hr during the logarithmic phase. The decline in total protein and dry weights per cell began at the beginning of the logarithmic phase and continued into the stationary phase of growth. Cultures grown at pH 3.5 should produce a larger number of cells/ml than cultures grown at pH 6.9 if the cells are responding to the unionized moiety of succinate and not the ionized moiety. At pH 3.5 83% of the succinate is unionized, whereas at pH 6.9 0.20% of the succinate is unionized. The onset of the stationary phase in cultures grown at pH 3.5 and pH 6.9 is due to lack of an adequate amount of extracellular unionized succinate. Intracellular pH values were determined in cultures grown at pH 6.9 using the weak acid DMO (5.5-dimethyl-2,4-oxazolidinedione). As the extracellular pH increased from 6.90 to 7.62, the intracellular pH increased from 5.89 to 6.89. As the extracellular pH increased from 7.62 to 8.44, the intracellular pH increased from 6.89 to 7.50.     

Morphological,growth, and chromosomal changes in bovine pancreatic duct epithelial cells exposed toN-methyl-N′-nitro-N-nitrosoguanidine

Summary Epithelial cells cultured from bovine pancreatic ducts were given a single treatment ofN-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Multinucleated cells and giant cells were observed more frequently in carcinogen-treated cultures than in controls. The MNNG-treated cultures also contained a sizeable population of small, dense cells that were not observed in control cultures. At the concentration of 1.0 μg/ml, MNNG caused an initial depression in the growth rate of the cells followed by growth stimulation for several weeks. The MNNG produced chromosomal damage in the cells as indicated by the observation that a substantial proportion of carcinogen-treated cells were heteroploid and contained a high frequency of metacentric and submetacentric chromosomes and a dicentric marker chromosome. The MNNG treated and control cultures did not acquire the ability to grow in soft agar or to produce tumors after transplantation into athymic, nude mice. This work was supported in part by Public Health Service Contract and Interagency Agreement Y01-CP60204 from the Division of Cancer Cause and Prevention, National Cancer Institute.     

The Fate of RNA Degradation Products in Starved Cultures of Tetrahymena pyriformis W.*

The RNA content of a population of Tetrahymena pyriformis W was followed during the growth phases of the culture. The cellular RNA levels were found to reach a maximum in early log phase and to decrease throughout the remainder of the log and deceleration phases. There was a 25% decrease in RNA amount when cells in late stationary phase were compared to those in deceleration. This loss of RNA was mimicked when cells from the deceleration phase were suspended in a non-nutrient buffered medium. Procedures were established to determine RNA content and the intra- and extracellular distribution of RNA degradation products, namely purine and pyrimidine bases and orthophosphate. Balance sheets are presented to show that the decrease in RNA levels was accompanied by an equivalent increase in purine and pyrimidine bases and phosphorus derivatives. The validity of the procedures employed was demonstrated. The influence of magnesium, cholesterol and glucose on the cells suspended in a non-nutrient buffer was examined. Each was found to affect the ultimate distribution of RNA products in a characteristic fashion suggesting that each compound acts by a different mode of action.     

Diacetyl and Acetoin Production by Lactobacillus casei

Agitation of broth cultures of Lactobacillus casei retarded cellular dry weight accumulation but enhanced production of both diacetyl and acetoin. Addition of pyruvate overcame this retardation, but addition of sulfhydryl-protecting reagents did not. Both pyruvate and citrate enhanced accumulated dry weight of L. casei incubated without agitation, but only pyruvate increased diacetyl accumulation. Both actively dividing cells and cells suspended in buffer converted pyruvate to diacetyl and acetoin. Maximum production of diacetyl and acetoin occurred during the late logarithmic or early stationary phases. Cells isolated from pyruvate- or citrate-containing cultures showed the greatest ability to convert pyruvate to diacetyl and acetoin. The optimum pH for diacetyl and acetoin formation by whole cells was in the range of 4.5 to 5.5. The presence of citrate or acetate enhanced diacetyl and acetoin formation by L. casei cells in buffer suspension.     

The Effect of Carbohydrate and Nitrogen Concentration on Phenol Synthesis in Paul's Scarlet Rose Cells Grown in Tissue Culture

The effects of exogenous concentrations of glucose and nitrate on total ethanol extractable phenols and leucoanthocyanins were studied in Paul's Scarlet Rose cells grown in either liquid suspension or solid culture. Aliquots of liquid suspension cultured cells were harvested during logarithmic, early stationary, and late stationary periods of growth for determination of fresh weight, dry weight, total ethanol extractable phenols and leucoanthocyanins. Cells produced phenols during all phases of growth, but at stationary phase, the production was greatest. Increasing concentrations of exogenous glucose in the culture medium resulted in an increased synthesis of total phenols in logarithmic cells, and an increased synthesis of total phenols and leucoanthocyanin in stationary cells. Addition of increased concentrations of exogenous nitrate to the stationary cells grown in suspension culture markedly reduced synthesis of leucoanthocyanins although total phenol synthesis was not significantly affected. Similar observations were made in cells cultured on solid medium in respect to exogenous glucose concentration, however these cells differed from the suspension cultured cells by having increased amounts of total phenol synthesis and decreased synthesis of leucoanthocyanins in the presence of increasing concentrations of exogenous nitrate in the culture medium.     

Secretion of an antibacterial factor during resuscitation of dormant cells inMicrococcus luteus cultures held in an extended stationary phase

A high proportion ofMicrococcus luteus cells in cultures starved for 3–6 months in spent medium following growth to stationary phase in batch culture lost the ability to grow and form colonies on agar plates, but could be resuscitated from dormancy by incubation in liquid medium containing supernatant taken from the late log phase of viable cultures of the same organism (Kaprelyants et al. 1994). In the present work, we found that during the first 50–70 h of such resuscitation the dormant cells actually divide for 10–17 generations in lactate minimal medium containing yeast extract whilst remaining nonculturable on agar plates. Further incubation results in a decrease in the total cell number in liquid medium. The addition of viable (culturable)Micrococcus luteus cells in concentrations of up to 10⁴ ml^–1 to test tubes containing either resuscitating cells or supernatant from these cultures revealed the excretion of a factor or factors which inhibited the proliferation of otherwise viable cells. The maximum production of this factor took place after some 96 h of incubation of starved cells in resuscitation medium. Supernatant from late logarithmic phase batch cultures ofM. luteus abolished the antibacterial effect of starved cultures incubated in resuscitation medium. It is concluded that the stimulating effect of viable cells, and of supernatant taken from batch cultures, on the resuscitation of dormant cells might be connected in part with overcoming the activity of an antibacterial factor causing self-poisoning of dormant cells during their resuscitation.     

Two types of antimutagenic effects of gallic and tannic acids towards N-nitroso-compounds-induced mutagenicity in the amesSalmonella Assay

The frequency ofhis ⁺ revertants induced by N-methyl-N-nitrosourea (MNU) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) in the strain TA100 ofSalmonella typhimurium was decreased by gallic and tannic acid. In weak buffer solutions, the inhibition effects of gallic acid towards MNU and MNNG mutagenicity was caused primarily by a decrease of pH in the incubation mixtures. At adjusted pH (pH 5.0 and 6.5), the antimutagenic effects are largely the result of an interaction between MNU or MNNG with phenolic acids outside the cells.     

Effect of presensitization of Nocardia asteroides with specific antibody on the viability of the organism

Nocardia asteroides from various growth phases was treated in vitro with normal rabbit sera, immune rabbit sera containing nocardial polyclonal antibodies and a monoclonal antibody. At intervals, samples were grown in broth or on blood agar plates to determine their viability. Log and stationary phase cells were injected intra-peritoneally into female BALB/c mice and their survival rates in the liver and spleen were determined. Presensitization with antibodies reduced the viability of the log phase cells by 48% and that of the late stationary phase by 4%. The antibody-treated log phase organisms were less viable on the blood agar medium and in the spleen and liver than the control organisms. This indicates that pretreatment with antibody has a lethal effect on N. asteroides and affects its survival in vivo.     

A method for the immobilization of living Tetrahymena

The surfaces of plastic (polystyrene) Petri dishes from several suppliers were discovered to have the useful property of immobilizing cells of the ciliate Tetrahymena thermophilia upon contact in nutrient-free buffer (10 mM Tris, pH 7.4). The procedure works with cells in both logarithmic and stationary growth phase, so long as they are first transferred to nutrient-free buffer, and then added to dishes already containing buffer to a depth of 2–10 mm. Dish surfaces specially treated for tissue cultures are unsuitable for this purpose. Cells can be released from the dish surfaces by the simple addition of growth medium (1% proteose peptone). Immobilized cells are fully competent to complete conjugation or cell division. The technique offers promise for facilitating experiments requiring microinjection, microsurgery, or simply detailed observation of living protozoan cells.     

Secondary cell wall formation in Cryptococcus neoformans as a rescue mechanism against acid-induced autolysis

Growth of the opportunistic yeast pathogen Cryptococcus neoformans in a synthetic medium containing yeast nitrogen base and 1.0–3.0% glucose is accompanied by spontaneous acidification of the medium, with its pH decreasing from the initial 5.5 to around 2.5 in the stationary phase. During the transition from the late exponential to the stationary phase of growth, many cells died as a consequence of autolytic erosion of their cell walls. Simultaneously, there was an increase in an ecto-glucanase active towards β-1,3-glucan and having a pH optimum between pH 3.0 and 3.5. As a response to cell wall degradation, some cells developed an unusual survival strategy by forming 'secondary' cell walls underneath the original ones. Electron microscopy revealed that the secondary cell walls were thicker than the primary ones, exposing bundles of polysaccharide microfibrils only partially masked by an amorphous cell wall matrix on their surfaces. The cells bearing secondary cell walls had a three to five times higher content of the alkali-insoluble cell wall polysaccharides glucan and chitin, and their chitin/glucan ratio was about twofold higher than in cells from the logarithmic phase of growth. The cell lysis and the formation of the secondary cell walls could be suppressed by buffering the growth medium between pH 4.5 and 6.5.     

Antimutagenic activity of Lactobacillus plantarum KLAB21 isolated from kimchi Korean fermented vegetables

Antimutagenic activity of Lactobacillus plantarum KLAB21, isolated from Korean kimchi, was investigated against MNNG (N-methyl-N-nitro-N-nitrosoguanidine), NQO (4-nitroquinoline-1-oxide), NPD (4-nitro-O-phenylenediamine) and aflatoxin B1 using Salmonella typhimurium strains TA100 and TA98. Although all the cell fractions including the culture supernatant, dry cells and cell-free extract exhibited antimutagenic activity against MNNG and NQO, the culture supernatant possessed the highest activity. The antimutagenic ratio of the culture supernatant was 98.4% against MNNG on strain TA100 and 57.3% against NQO on strain TA98. Its antimutagenic activity was reconfirmed by a Bacillus subtilis spore-rec assay. Levels of the antimutagenic ratios of other lactic acid bacteria originating from fermented milk ranged between 26.8 to 53% against MNNG and 28.5 to 43.4% against NQO. The antimutagenic activities of the strain KLAB21 against NPD were 72.6% on TA100 and 62.8% on TA98, and those against aflatoxin B1 were 82.5% on TA100 and 78.2% on TA98.     

Effect of external high potassium and pH on the uptake of choline in glial and neuronal cells in culture

TheV _max of the uptake of choline was increased in nerve cell cultures by lowering (from 7.4 to 6.5) or increasing (from 7.4 to 8.1) the pH. In neurons no effect was observed on the value of theK _m's of the uptake of either the apparent high or low affinity components. In glial cells only a low affinity component was measured at pH 6.5 and diffusion was observed at pH 8.1. An excess of K⁺ ions in the incubation medium reproduced the increase inV _max observed with changes in pH suggesting a possible dependence of the uptake of choline upon the H⁺ and OH^– gradients. Taking into account the characteristics already known of the transport of choline into nerve cells, such a dependence adds new insight in the mechanisms underlying the transport and indicates another possible regulation of choline entry, eventually directed towards the synthesis of acetylcholine.     

Ecological Application of Antibiotics as Respiratory Inhibitors of Bacterial Populations

Two terregenous and four marine bacterial isolates were treated with six antibiotics and antibiotic combinations. Comparisons made between responses of cells in early and late logarithmic and stationary growth phases indicated variable sensitivity to the agents. Bacteria in stationary and late log-phase cultures exhibited the greatest resistance, whereas the early log-phase cells exhibited greatest antibiotic susceptibility. We conclude that the tested antibiotics cannot be used for ecological purposes to delineate bacterial respiration in mixed microbial communities.     

The relationship between growth phase and cyanogenesis inPseudomonas aeruginosa

In batch cultures ofPseudomonas aeruginosa, hydrogen cyanide is produced primarily during the transition between logarithmic and stationary phases. This transient response is due to the synthesis of the enzyme system of cyanogenesis during mid to late logorithmic and the inactivation of this system in early stationary phase. Although glycine, the metabolic precursor of cyanide, stimulates cyanogenesis, it is not necessary to incorporate this amino acid in the growth medium to produce elevated enzyme levels. Under conditions of iron limitation (1×10⁻⁶ M), phosphate limitation (0.1 mM), and excess phosphate (250 mM), the culture produces low levels of the cyanogenic enzyme system. Increasing the carbon and energy source,l-glutamate, prolongs cyanogenesis and postpones the inactivation of the cyanogenic enzyme system.