Stationary Phase Of Cell Growth Research Articles

Nitrate use by tobacco cells in response to N-stress and ammonium nutrition

Characterization of NO 3 (-) use by suspension cultured tobacco cells during a culture cycle is needed to take advantage of cell cultures for further study of the biochemical regulation of NO 3 (-) uptake induction and decay processes. Tobacco (Nicotiana tabacum L., cv. Ky14) cells were cultured with media containing different N sources. Cells cultured with a mixture of NO 3 (-) and NH 4 (+) (40 mM NO 3 (-) plus 20 mM NH 4 (+) , in Murashige and Skoog media) initially grew slightly faster but attained the same maximum cell culture density as those cultured with 40 mM NO 3 (-) only. Cells subcultured with N-free media grew at a similar rate for the first 3 d as those cells grown with N, then ceased further growth. The cessation of growth of cells subcultured with N-free media coincided with depletion of cell NO 3 (-) . The NO 3 (-) influx of cells subcultured with N-free media increased eleven-fold and those grown with N increased four- to five-fold before declining. Maximal NO 3 (-) influx rates occurred at the onset of the stationary growth phase for N-stressed cells, while cells grown with N reached maximums prior to the stationary phase of cell growth. Cells grown with a mixture of NO 3 (-) and NH 4 (+) had lower NO 3 (-) reductase (NR) activity and higher cell NO 3 (-) levels than those of cells grown with NO 3 (-) only. The NR activity of cells subcultured with N-free media peaked within 1 d after subculture before declining to a constitutive level when cell NO 3 (-) was depleted. The level of cell NO 3 (-) plays a critical role in the expression of the NO 3 (-) uptake and reduction processes. The transitions in the expression of NO 3 (-) uptake and reduction activities of tobacco cell suspension cultures should prove valuable for further study of the biochemical and molecular basis for the regulation of these processes.

In situ berberine separation with immobilized adsorbent in cell suspension cultures ofThalictrum rugosum

The use of suspended and alginate-entrapped XAD-7, polycarboxyl ester resin, for thein situ separation of berberine, isoquinoline alkaloid, produced from plant cell culture ofThalictrum rugosum was investigated. XAD-7 could adsorb the berberine and the amount of berberine adsorbed on XAD-7 depended on pH. The neutral form of berberine was adsorbed onto XAD-7 and the adsorption isotherm for berberine showed a Langmuir-type appearance.In situ berberine removal enhanced the production of secondary metabolites in cell suspension culture ofThalictrum rugosum. Addition of XAD-7 at the exponential phase of cell growth was the most effective for enhancement of berberine production. In chitosan-treated cell culture to permeabilize intracellular berberine, berberine secretion was significantly accelerated by addition of alginate-entrapped XAD-7 at the stationary phase of cell growth and thus more than 70% of the produced berberine could be adsorbed to alginate-entrapped XAD-7.

Selection of anti-SCLC antibodies for diagnosis of bone marrow metastasis : Myklebust AT, Beiske K, Pharo A, De L, Davies C, Aamdal S, Fodstad O. Institute for Cancer Research, 0310 Oslo 3. Br J Cancer 1991;63 Suppl 14:49–53

The binding profile of 17 monoclonal antibodies (MAbs) to small lung cancer cell lines and normal bone marrow and peripheral blood cells was studied by immunocytochemistry and flow cytometry. At antibody concentrations that stained PJD tumour cells, only four MAbs (MOC1, MOC31, NrLu10, 81A6) were devoid of cross-reactivity with normal cells, whereas significant binding to subtypes of bone marrow and blood cells was seen for 13 antibodies. For the eight most promising MAbs the binding to ten SCLC cell lines was moderate to strong in 47 MAb/cell line combinations, and low or insignificantly in 33 combinations. Three cell lines lacked antigen for all MAbs studied. Flow cytometry was significantly less sensitive than immunocytochemistry in assessing MAb binding to both normal and tumour cells. The antigen expression was for several MAbs higher in exponentially growing tumour cells than in cells in stationary growth phase. Seven of the MAbs, which originally showed low to moderate cross-reactivity with normal cells, were titrated down to the lowest concentrations at which they stained H-146 tumour cells with high levels of antigen expression. At these concentrations five (MLuC1, Oat-1, SM-1, NCC-Lu-243, LAM2) of the seven Mabs showed acceptably low binding to bone marrow cells. At optimal concentrations altogether four to nine of the 17 antibodies studied may be used to detect tumour cell involvement in bone marrow of SCLC patients.

Mössbauer spectroscopic studies of iron in Pseudomonas aeruginosa

The present Mössbauer spectroscopic studies of isolated bacterioferritin and whole cells of Pseudomonas aeruginosa have shown that the iron core of bacterioferritin is not altered on isolation. These studies have also shown that the bacterioferritin core is typically 85% oxidized within the cell and may contain a significant proportion of its iron as small clusters during the early stage of the stationary phase of cell growth.

Induction of recombinant gene expression in Escherichia coli using an alkaline pH shift

A commonly used approach to control recombinant protein production in Escherichia coli utilizes the λ p L promoter-operator and the λ repressor. Inactivation of the λ repressor function allows transcription to proceed. However, induction of the RecA-mediated cleavage of λ repressor by the addition of nalidixic acid or inactivation of a temperature-sensitive λ repressor by growth at the nonpermissive temperature can have detrimental effects on protein production. This paper describes the use of an alkaline shift in the pH of the growth medium that allows expression of genes from the p L promoter in a RecA-independent manner. This procedure results in high-level production of recombinant protein. The pH shift is performed in the stationary phase of cell growth, using culture volumes ranging from 1–1000 ml. This method can result in the production of over 15-fold more active protein than when using a temperature shift.

Growth stage-dependent expression of 6-hydroxy-d-nicotine oxidase of the nicotine regulon of Arthrobacter oxidans

In the gram-positive soil bacterium Arthrobacter oxidansd,l-nicotine induces specific flavoenzymes involved in the catabolism of the alkaloid. The delayed appearance of 6-HdNO activity as compared to that of the other enzymes of the nicotine regulon was investigated. Immunological methods and specific labeling of the 6-HdNO polypeptide with [14C]riboflavin showed that enzyme activity, occurrence of 6-HdNO antigen and covalent flavinylation of the 6-HdNO polypeptide all coincide with the stationary phase of cell growth. Northern blots, hybridized with an intragenic 6-HdNO probe, revealed a 6-HdNO-specific mRNA species of unique size only in stationary phase cells. Our results suggest that 6-HdNO is expressed from an independent transcription unit in a growth stage-dependent manner.

Multivalent regulation of the nusA operon of Escherichia coli

The rate of synthesis and intracellular content of the NusA protein, a transcription termination factor, were determined for wild-type and nusA and/or nusB mutants of Escherichia coli. Both the rate and content of NusA in wild-type strains were similar to that of the RNA polymerase sigma subunit, a transcription initiation factor, on a molar basis, and about 30%-40% the levels of RNA polymerase beta beta' subunits. At the stationary phase of cell growth, the values increased in parallel for both transcription factors up to approximately the level of the beta beta' subunits. In nus mutants, the rate of synthesis and the content of the sigma subunit were significantly increased. These observations together suggest that the two transcription factors are coordinately regulated.

Light-illumination effects on the cellular concentration of photolyase molecules in yeast.

The effects of light-illumination in the absence or presence of cycloheximide (CHX) on the number of photoreactivating enzyme molecules per haploid cell of Saccharomyces cerevisiae (NPRE) were investigated. NPRE increased, when the cells were held in buffer in light in the absence of CHX for 24 or 48 h before UV-irradiation (buffer-holding). The increase of NPRE was clearly observed in cells, whose NPRE before bufferholding was small, that is, in stationary growth phase cells grown at 37 degrees C and in logarithmic growing cells incubated at 30 degrees C. In the case of buffer-holding in light in the presence of CHX or in the dark in the absence or presence of CHX, the increase of NPRE was small or not observed.

X-Ray Responses of Human Colon Tumor Cells Grown in Artificial Capillary Culture<xref ref-type="fn" rid="FN2">2</xref>

Clone A human colon adenocarcinoma cells were grown in three-dimensional artificial capillary culture (ACC) to determine responses of capillaries treated 3 weeks after tumor cell inoculation with a specific, easily quantifiable cytotoxic agent, ionizing radiation. The high-density growth of tumor cells in ACC can be considered to be an in vitro analogue of a solid tumor. Changes in extracapillary space (ECS) fluid concentrations of lactate dehydrogenase (LDH) and aspartate aminotransferase (GOT) and the utilization of glucose in circulating medium were monitored after a supralethal radiation dose (90 Gy) of X-rays. Immediately after irradiation, increased levels of LDH and GOT were found that reached maximum levels about four to five times those found in nonirradiated control capillaries at 10-13 days post irradiation and then declined. Patterns of enzyme production appeared to correlate with the numbers of nonviable tumor cells collected from the ECS of the artificial capillaries. In contrast, glucose utilization showed little correlation with either enzyme concentration or dead cell production. It was determined that, while capillaries grown and treated in this manner appear to respond in a dose-dependent manner to ionizing radiation (as indicated by changes in LDH and GOT levels), these particular end points are relatively insensitive and are not suitable for studies in which therapeutic levels of X-radiation might be given. In other studies, tumor cells were removed from unirradiated capillaries by trypsinization and used to obtain complete survival curves after graded doses of X-radiation. The dose-response curves obtained indicate that clone A colon tumor cells grown in ACC show a marked decrease in their ability to accumulate sublethal radiation injury as compared to responses of these cells growing exponentially in asynchronous monolayer cultures, to synchronized mid-G1 tumor cells, or to tumor cells in stationary growth phase. These data suggest that ACC is a potentially useful model to study the effects of cytotoxic agents on human tumor cells.

The release of cytosolic proteins from cells treated with concanavalin A during scraping from plastic culture dishes

When rat hepatoma cells (HTC and R117-21B), treated with concanavalin A (conA) at 37 °C, were scraped from plastic culture dishes with a silicone-rubber policeman, the cell membranes were broken and the cytoplasm was released. This phenomenon was also observed in cells treated with conA at 4 °C, even though it took a longer time to show the same effect. The effect of 10 μg/ml of conA on the release of the cellular proteins reached a plateau when the treatment was carried out at 37 °C. Ninety percent of this effect was abolished by 10 mM of α-methyl- d-mannoside. The effect was completely nullified by 100 mM. At 4 °C, however, even 100 mM of this sugar could not abolish this effect. The apparent decrease in the cellular proteins with conA after scraping was observed not only in the logarithmic phase, but also in the stationary phase of cell growth. The breakdown of plasma membranes with conA eventually caused decrease in tyrosine aminotransferase activity, even though the lectin induced the enzyme activity in cultured cells.

Cytochrome P-450 factors determining synthesis in strain D7 Saccharomyces cerevisiae An alternative system to microsomal assay

Certain aspects of cytochrome P-450 induction were studied in a diploid strain (D7) of the yeast Saccharomyces cerevisiae in order to obtain cells containing a high level of metabolizing enzymes. The highest level of cytochrome P-450 was reached during the logarithmic growth phase in a 20%-glucose liquid medium. Yeast cells harvested in these conditions were used in the mutagenesis test with dimethyl nitrosamine (DMNA) as a positive control and with styrene (Sty). Both substances gave positive results, whereas Sty never showed any mutagenic activity in the conventional test with stationary growth phase cells and external metabolic activation. The test with cells from the logarithmic growth phase is proposed as a possible alternative to the liver-microsome assay, and its reliability is discussed.

Cyclic Adenosine 3′,5′‐Monophosphate Binding Protein in Tetrahymena: Properties and Subcellular Distribution1

ABSTRACT. Cyclic AMP binding activity was determined in the ciliate Tetrahymena pyriformis NT‐1 strain. The fractions having the binding activity were eluted in a single peak coincident with a protein kinase activity. Although metal ions were not essential for activity, the binding was slightly activated by Mg2+ or Ca2+. The binding activity was sensitive to temperature, ionic strength, and pH of the reaction mixture and was decreased by treatment of the cytosol protein with trypsin or by heating at 100°C. The binding was specific for cyclic AMP, with an estimated apparent Kd of 40 nM. When the cyclic AMP binding activity in subcellular fractions was measured, an increase in the activity of ciliary, mitochondrial, and microsomal fractions was observed during the transition from the exponential to the stationary phase of cell growth, whereas no significant change occurred in the binding activity of the whole cell homogenate. These results suggest that the redistribution of cyclic AMP binding proteins may be implicated in the regulation of cyclic AMP concentration in the cell.

Heterotrophic tobacco cell cultures during greening

Structural changes in cells and plastids are described that occur during the greening of an initially dark‐grown cell suspension of tobacco (Nicotiana tabacum L. cv. Xanthi). The pattern of cell growth during greening, expressed in dry weight or cell number, showed a classical sigmoid curve with a lag phase (T0–T2), an exponential phase (T3–T9) and a stationary phase (T10–T21). Achlorophyllous vacuolated cells (T0), obtained after 3 culture cycles in the dark, contained amyloplasts devoid of lamellae. Exposure to light brought about an enrichment in cytoplasm and an amyloplast to proplastid transformation (starch loss) accompanied by chlorophyll synthesis. By T3, many cells appeared meristematic and contained dividing proplastids with rudimentary single lamellae typical of those in intact meristematic leaf cells. As cell division occurred (T3 to T9), plastids replicated and their internal membrane system developed progressively into defined grana‐intergrana thylakoids. By the stationary phase of cell growth (T14), the lamellar system had reached a highly structured grana‐intergrana network typical of higher plant chloroplasts. We have emphasized the analogies between the sequence of events (proplastid to chloroplast transition) during the greening of tobacco cells and that in developing intact leaves; in this respect the cell cultures provide a useful material for studies dealing with the biogenesis of structural or physiological events.

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.

Effects of Low-Level α Radiation on Intracellular Energy Metabolism

The effect on the adenosine triphosphate (ATP) content of cell samples irradiated with ..cap alpha.. particles of a mean LET of 135 to 141 keV/..mu..m is described. Cells in logarithmic growth phase showed an ATP content about 50% below the unirradiated control value 5 to 15 sec after receiving a mean cellular dose of 25 rad (0.25 Gy). This was followed by a slow recovery up to control values after about 30 sec. Stationary cells responded with an increase in ATP content about threefold over that of the controls 20 sec after irradiation. Within the following 20 sec these elevated ATP values decreased to control levels. Additional measurements of /sup 14/C uptake also showed pronounced differentiated changes in carbon uptake after irradiation for cells in logarithmic and stationary growth phase. The possible importance of these effects on bioenergetic processes as a compensatory reaction of the cellular system to an increased demand for energy is discussed.

The Kinetics and Metabolism of the Cells of Hibernating Animals during Hibernation

Publisher Summary The period of quiescence in the cell cycle of hibernating animals is an example of the evolutionary adaptation at the cellular level for survival during unfavorable environmental conditions in these organisms. A peculiar type of metabolism in the quiescent cells helps them to survive successfully during this unfavorable period. This chapter presents the mechanisms responsible for the transition from the normal cell cycle. In the tissues of animals, the cells in the resting phase are distributed among the proliferating cells. The absence of morphological criteria makes it impossible to study their structure and function in vivo. One of the possible approaches is the search for definite physiological states. In vivo study of the resting period of hibernation has a number of advantages as compared to the model system of the resting cell now widely used—the stationary phase of cell growth in tissue culture—because in the former instance, the humoral factors that control mitotic homeostasis are retained.

Sensitivity of cells in exponential and stationary growth phase to combined treatment with radiation and quinacrine.

The effect of Quinacrine on the recovery of cells from sublethal radiation injury was analysed by testing cellular survival after exposure to low and high radiation doses, and after irradiation with split doses. Quinacrine was found to inhibit recovery in cell cultures in exponential growth phase but not, or only slightly, in cells in stationary growth. This finding is related to the inhibitory effect of Quinacrine on the repair of radiation induced DNA strand breaks.

Release of Tumor-Associated Antigens by Murine Melanoma Cells

Synthesis and release of radiolabeled macromolecules and tumor-associated antigens (MAA) by murine B16 melanoma was studied by pulse labeling cells in culture with 3H-leucine. Approximately 36% of newly synthesized macromolecules and 44% of newly synthesized MAA were released in 48 hr. MAA release was slightly, but consistently, more rapid than the average release of other macromolecules. Release of MAA did not result solely from cell death since it was greater than that of 51Cr-labeled molecules and cell viability was over 98%. The rate of release of newly synthesized MAA was not significantly influenced by cell replication. However, synthesis of MAA was much greater during the logarithmic than the stationary phase of cell growth, suggesting a concomitant increase in the amount of MAA available for release. These findings indicate that antigens and other macromolecules can be rapidly released by viable tumor cells.

Adrenergic mechanism in Tetrahymena. 3. Cyclic adenosine 3', 5'-monophosphate and cell proliferation.

The presence of cyclic adenosine 3', 5'-monophosphate (cAMP) in the ciliated protozoan, Tetrahymena pyrifurmis W, was demonstrated by thin-layer chromatography (TLC). The content of cAMP in the cell increased abruptly in the early exponential phase, and then diminished with further culture. When cells in the exponential or stationary growth phase were cultured in a fresh medium, growth was inhibited by addition of dibutyryl cAMP (Bt2-cAMP) or methylxanthines. In synchronized cultures of Tetrahymena, Bt2-cAMP inhibited protein and RNA syntheses in the G1 phase and DNA synthesis in the S phase. These results indicate that cAMP regulates the growth of the protozoan in the G1 phase.

An ultrastructural study of ingestion and digestion in Tetrahymena pyriformis.

SYNOPSIS. When the structures involved in digestive events in T. pyriformis are examined at the electron microscope level, some information is added to that long known from light microscopy. The food trapping mechanism consists of the three membranelles, undulating membrane, oral ribs, and a “valve” apparently closing the opening to the cytopharynx. Both of the latter structures are supported by microtubules. Fibers extend internally from the cytopharynx and are closely associated with the food vacuole as it forms.Clear vacuoles resembling pinocytic vacuoles appear to arise from differentiated areas of the pellicle and plasma membrane. These vacuoles may fuse with primary lysosomes. Hydrolases are thus contributed to the pinocytic vacuoles which may then fuse with food vacuoles. When first formed food vacuoles contain no hydrolases but may acquire them directly, from primary lysosomes or from pinocytic vacuoles. Digestion proceeds to completion in the food vacuole, at which time soluble food products are released to the cytoplasm. Undigested materials are lost through the cytopyge. In stationary growth phase cells autophagic vacuoles form containing mitochondria and other cellular particulates. Such vacuoles probably contain hydrolases when formed and they may receive others by fusion with primary lysosomes.