Effect of indoleacetic acid on the growth ofRhizobium in culture

After inoculation ofRhizobium lupini strain A98 andR. leguminosarum strain PRE into a medium containing IAA, growth was initially suppressed. However, when IAA was added in the course of the logarithmic phase, growth was not inhibited. Apparently, IAA affects primarily the lag phase cells.Neither adaptation ofRhizobium to IAA was observed, nor spontaneous breakdown or biological degradation of IAA.The lag phase prolongation depended on the ratio: amount of IA A/number of cells.The authors wish to thank Professor Dr. A. Quispel for his interest and valuable discussions.     

Effect of Ethyl Picolinate on Germination, Growth and Sporulation of Bacillus spp.

Ethyl picolinate inhibited outgrowth and sporulation of Bacillus cereus T and laboratory isolates of B. megaterium AH2 and AV1 and B. brevis AG4. The outgrowth-inhibition was relieved by supplementing the medium before inoculation with any of the following: aspartate, asparagine, leucine, lysine, phenylalanine, tyrosine and vitamin-free casamino acids. The uptake of radioactive precursors in the synthesis of proteins, ribonucleic acid and deoxyribonucleic acid was prevented when ethyl picolinate was added before inoculation. Cultures incubated for a short period before the addition of ethyl picolinate assimilated radioactive precursors, outgrew and divided after a lag. None of the cultures containing ethyl picolinate sporulated. Growth was not inhibited when a portion of vegetatively growing culture was transferred to medium containing ethyl picolinate.     

The oxidative modification of low-density lipoproteins by macrophages.

1. Mouse resident peritoneal macrophages in culture modified human 125I-labelled low-density lipoprotein (LDL) to a form that other macrophages took up about 10 times as fast as unmodified LDL. The modified LDL was toxic to macrophages in the absence of serum. 2. There was a lag phase of about 4-6 h before the LDL was modified so that macrophages took it up faster. A similar time lag was observed when LDL was oxidized by 5 microM-CuSO4 in the absence of cells. 3. LDL modification was maximal when about 1.5 x 10(6) peritoneal cells were plated per 22.6 mm-diam. well. 4. Re-isolated macrophage-modified LDL was also taken up much faster by macrophages, indicating that the increased uptake was due to a change in the LDL particle itself. 5. Micromolar concentrations of iron were required for the modification of LDL by macrophages to take place. The nature of the other components in the culture medium was also important. Macrophages would modify LDL in Ham's F-10 medium but not in Dulbecco's modified Eagle's medium, even when iron was added to it. 6. The macrophage-modified LDL appeared to be taken up almost entirely via the acetyl-LDL receptor. 7. LDL modification by macrophages was inhibited partially by EDTA and desferrioxamine and completely by the general free radical scavengers butylated hydroxytoluene, vitamin E and nordihydroguaiaretic acid. It was also inhibited completely by low concentrations of foetal calf serum and by the anti-atherosclerotic drug probucol. It was not inhibited by the cyclo-oxygenase inhibitors acetylsalicylic acid and indomethacin. 8. Macrophages are a major cellular component of atherosclerotic lesions and the local oxidation of LDL by these cells may contribute to their conversion into cholesterol-laden foam cells in the arterial wall.     

Glucose upshift of carbon-starved marine Vibrio sp. strain S14 causes amino acid starvation and induction of the stringent response.

The physiological status of carbon-starved cells of the marine Vibrio sp. strain S14 has been investigated by the analysis of their immediate response to carbon and energy sources. During the first minute after glucose addition to 48-h-starved cells, the pools of ATP and GTP increased rapidly, and the [ATP]/[ADP] ratio reached the level typical for growing cells within 4 min. The total rates of RNA and protein synthesis increased initially but were inhibited 4 to 5 min after glucose addition by the induction of the stringent response. A mutation in the relA gene abolished stringent control during the recovery and significantly prolonged the lag phase, before the starved cells regrew, after the addition of a single source of carbon. However, both the wild-type and the relA cells regrew without a significant lag phase when given glucose supplemented with amino acids. On the basis of these results, it is suggested that carbon-starved cells are deficient in amino acid biosynthesis and that ppGpp and the stringent response are involved in overcoming this deficiency, presumably by depressing the synthesis of amino acid biosynthetic enzymes. Furthermore, the data suggest that the starved cells primarily are starved for energy, and evidence is presented that the step-up in the rate of protein synthesis after refeeding is partially dependent on de novo RNA synthesis.     

Effect of tunicamycin on germ tube and yeast bud formation in Candida albicans

Tunicamycin is an antimicrobial agent which inhibits the first reaction of the dolichol pathway leading to N-glycosylation of proteins. The effect of tunicamycin on the growth of the dimorphic fungus Candida albicans differed depending on the growth phase of the organism. Addition of tunicamycin to stationary phase yeast cells inhibited the resumption of growth of those cells in either morphology, as cultures failed to initiate either yeast bud or germ tube formation. When tunicamycin was added to growing cells, growth was inhibited but not immediately. When it was added to germ tube cultures, nuclear division and septum formation continued for some time before ceasing. Addition of the drug to exponential phase yeast cultures resulted in an approximately 45% increase in cell number before cell division ceased and yeast accumulated in both budded and unbudded stages of the cell cycle. Accumulation of trichloroacetic acid precipitable radiolabelled protein and nucleic acid continued unchanged for some time following addition of tunicamycin; however, after a while a reduced rate of accumulation was noted.     

Mechanism of acid tolerance in a rhizobium strain isolated from Pueraria lobata (Willd.) Ohwi

The Rhizobium sp. strain PR389 was isolated from the root nodules of Pueraria lobata (Willd.) Ohwi, which grows in acidic (pH 4.6) yellow soil of the Jinyun Mountains of Beibei, Chongqing, China. While rhizobia generally have a pH range of 6.5-7.5 for optimum growth, strain PR389 grew in a liquid yeast extract - mannitol agar medium at pH 4.6, as well as in a pH 4.1 soil suspension, suggesting acid tolerance in this specific strain of rhizobium . However, at pH 4.6, the lag phase before vigorous growth was 40 h compared with 4 h under neutral conditions (pH 7.0). For PR389, the generation time after the lag phase remained the same at different pH levels despite the different durations of the lag phase. Except in the pH 4.4 treatment, the pH of the culturing media increased from 4.6, 4.8, 5.0, and 5.5 to neutral and slightly alkaline after 70 h of culture. Chloramphenicol was added to determine if protein production was involved in the increasing pH process. Chloramphenicol significantly inhibited PR389 growth under acid stress but had little effect under neutral conditions. Proton flux measured during a short acid shock (pH 3.8) revealed that this strain has an intrinsic ability to prevent H(+) from entering cells when compared with acid-sensitive rhizobia. We propose that the mechanism for acid tolerance in PR389 involves both intracellular and extracellular processes. When the extracellular pH is lower than pH 4.4, the cell membrane blocks hydrogen from entering the cell. When the pH exceeds 4.4, the rhizobium strain has the ability to raise the extracellular pH, thereby, potentially decreasing the toxicity of aluminum in acid soil.     

Contact inhibition, polyribosomes, and cell surface membranes in cultured mammalian cells

An “overlay” method for rapidly and synchronously inducing contact inhibition in normal cultured cells has been developed. Using this method, disaggregation of cytoplasmic polyribosomes has been observed to occur within a matter of hours after overlay, followed by a decrease in cellular ribosomal RNA. Polysome disaggregation was influenced by the extent of cell-cell interaction and was inhibited by pretreatment of overlay cells with cycloheximide. Treatment of underlay cells with cytosine arabinoside also induced polysome disaggregation, but only after an appreciable lag as compared to that observed in overlaid cultures. Disaggregation could be induced by this method in cultured cells derived from normal tissue but not in cells derived from cancerous tissue. Polysome synthesis in growing “normal” cells (as measured by incorporation of tracer uridine into RNA) was markedly decreased when a cell surface membrane preparation was added to cultures.     

Effect of levulinic acid on pigment biosynthesis in Agmenellum quadruplicatum.

When levulinic acid was added to a growing culture of the cyanobacterium (blue-green alga) Agmenellum quadruplicatum PR-6, delta-aminoelevulinic acid accumulated in the medium and chlorophyll a synthesis and cell growth were inhibited, but there was a small amount of c-phycocyanin synthesis. The amount of delta-aminolevulinic acid produced in the treated culture did not fully account for the amount of pigment synthesized in the untreated control. Levulinic acid and either sodium nitrate or ammonium chloride were added to nitrogen-starved cultures of PR-6, and delta-aminolevulinic acid production and chlorophyll a and c-phycocyanin content were monitored. When ammonium chloride was added as a nitrogen source after nitrogen starvation, the cells recovered more rapidly than when sodium nitrate was added as a nitrogen source. In cultures recovering from nitrogen starvation, synthesis of c-phycocyanin occurred before synthesis of chlorophyll a.     

The stimulation of the initiation of DNA synthesis by fibroblast growth factor in swiss 3T3 cells: Interactions with hormones during the pre-replicative phase

Fibroblast Growth Factor (FGF) stimulates quiescent Swiss 3T3 cells to initiate DNA synthesis and divide. Cells begin to enter the S-phase after a lag of 13–15 hr, and the rate of initiation of DNA synthesis in the population can be quantified by a first order rate constant, k. A subsaturating concentration of FGF may establish the lag phase, while the value of k is dependent on the FGF concentration present during the second half of the lag phase. Insulin and hydrocortisone enhance the effect of FGF by increasing k without changing the lag phase, and they can act when added at any time after FGF. Prostaglandin E₁ (PGE₁) causes a decrease in k and a lengthening of the lag phase, and acts only when added during the first 8 hr. None of these agents stimulate DNA synthesis in the absence of FGF. These results show that the stimulation of growth by FGF follows the same basic pattern as was previously shown with Prostaglandin F_2α (PGF_2α). However, since hydrocortisone inhibits stimulation by PGF_2α when added during the first 4 hr of the lag phase, there are clearly differences in some events stimulated by the two growth factors.     

Growth Stimulating Substance for Microorganisms Produced by Escherichia coli Causing the Reduction of the Lag Phase in Microbial Growth and Identity of the Substance with Pyrroloquinoline Quinone

The finding that most strains of microbes produce a growth stimulating substance for microorganisms was demonstrated and confirmed with the culture broth of Escherichia coli grown on a glucose-mineral medium. Addition of culture broth of E. coli to the culture media of the others markedly reduced the lag phase in microbial growth but not growth rate in the subsequent exponential phase nor the total cell yield in the stationary phase. The growth stimulation causing reduction of the lag phase was dependent on the amount of culture broth added. Occurrence of cell growth was essential for the excretion of the growth stimulating substance by E. coli. Under identical inoculum size, even with a heavy inoculum, a further reduction of the lag phase was observed by the addition of culture broth of E. coli. The substance was only effective at the initial growth phase but inert when the substance was added to a growing culture at the exponential phase. Finally, the substance was identified as pyrroloquinoline quinone, a newly established coenzyme, through chromatographic, spectroscopic and enzymatic criteria.     

ABA-induced proline accumulation in barley leaf segments: Dependence on protein synthesis

The kinetics of proline accumulation in barley ( Hordeum vulgare L. cv. Georgie) leaf segments showed a lag phase of ca 3 h when the increase was induced by abscisic acid (ABA), but not when the accumulation of the imino acid was promoted by isobutyric acid (IBA). Cycloheximide (CHI) supplied together with ABA, either from the beginning of the treatment or some time before the end of the lag phase, completely abolished ABA-induced proline accumulation, whereas no block was observed when the inhibitor was supplied after the lag phase. Cordycepin (COR) exibited a similar effect. The IBA-induced increase in proline was not influenced by CHI for at least 5 h.
When segments were pretreated with ABA for a period longer than the lag phase in the absence of salts in the external medium, there was no significant increase in proline. If KCI was added to the incubation medium after such a pretreatment, however, proline increased even after removal of the hormone from the external medium. This increase in proline occurred without any lag phase, and was only partially inbibited by CHI and rapidly and totally blocked by fusicoccin (FC). These results suggest that some protein, characterized by a fast turnover and possibly conferring the sensitivity to KCI, is synthesized during the early hours (lag phase) of the ABA treatment.
The synthesis of this protein(s) does not seem to be involved in the increase in proline induced by IBA and is thus a peculiar aspect of that mediated by ABA.     

Mouse lymphoma L1210 cells can be arrested in the G1 phase by adjusting cellular cysteine and glutathione

Mouse lymphoma L1210 cells (NCI line) that have low ability to take up cystine became deficient in cellular cysteine and glutathione in normal culture media. The cells entered the resting state during culture when they were seeded at high cell densities. They remained viable and were mostly present in the G1 or G0 phase. In the growth-arrested state, the cellular glutathione content was one order of magnitude lower than in the exponentially growing phase in the presence of 2-mercaptoethanol. In the arrested state, DNA synthesis was almost inhibited, and RNA and protein synthesis decreased markedly. Transfer of the cells to medium containing 2-mercaptoethanol, which improves the utilization of cystine by these cells, produced the rapid recovery of RNA and protein synthesis. DNA synthesis slowly increased, reaching a maximum after a lag period.     

Regulation of amino acid transport in growing cells of Streptomyces hydrogenans

(1) The active uptake of different amino acids by growing cells of Streptomyces hydrogenans was shown to be correlated with the physiological age of the cells. During the lag phase of growth the transport capacity increased and attained its highest level when the growth rate was maximum. During further growth the transport capacity declined progressively. The lowest transport activity was observed when the culture shifted into the stationary growth phase. (2) Such modulation of transport capacity was independent on the presence or absence of amino acids in the growth medium of the cells. (3) The size and the composition of the pool of free intracellular amino acids was also undergoing substantial variations during the growth cycle of the culture. In the lag phase, the levels of all amino acids decreased markedly and attained their lowest values at the end of this phase. During further growth the pool size was slowly replenished. (4) Removal of the pool resulted in a considerable gain of transport capacity. Therefore, it was concluded that active amino acid transport in growing Streptomyces hydrogenans is under feedback control by intracellular amino acids. (5) Quantitatively, the modulation of the pool size could not fully account for the variation of the transport capacity. Since a pool-independent stimulation of transport was found to be correlated with the increase of the growth rate of the cells, the possibility is discussed that the stimulation of transport is either due to increased levels of distinct RNA species, which might provide positive feedback signals for transport, or by increased rates of de novo synthesis of transport limiting proteins.List of Abbreviations AIB 2-aminoisobutyric acid - CM complete medium - MM mineral medium     

Effects of free long-chain fatty acids on thermophilic anaerobic digestion

Summary Low concentrations of the long-chain fatty acids oleate and stearate inhibited all steps of the anaerobic thermophilic biogas process during digestion of cattle manure. The lag phase increased when the concentrations of oleate and stearate were 0.2 g/l and 0.5 g/l, respectively, and no growth was found at concentrations of 0.5 g/l for oleate and 1.0 g/l for stearate. The toxic effect of these acids was permanent as growth did not occur when inhibited cultures were diluted to a non-inhibitory concentration. No adaptation to the fatty acids toxicity was observed by pre-exposing the cultures to non-inhibitory concentrations and the inhibitory response was the same as for cultures not pre-exposed to the fatty acids. Oleate was less inhibitory when added as a neutral oil in the form of the glycerol ester. This indicates that it is the free fatty acid that influences the bacterial activity. Correspondence to: B. K. Ahring     

Late G1 amino acid restriction point in human dermal fibroblasts

Human dermal fibroblasts arrested in G0 by maintenance in medium supplemented with 0.1% serum were not restimulated to divide when fresh medium containing 10% dialyzed serum but lacking group B amino acids (cystine, isoleucine, lysine, phenylalanine and tyrosine) was added. Unlike rodent cells, the addition of fresh serum-supplemented medium lacking only isoleucine did not cause a growth arrest. The amino acid sensitive growth arrest in human fibroblasts was dependent both on presynchronization in G0 as well as a prestarvation for amino acids prior to stimulation with high serum. When cells were restimulated in the absence of amino acids, they arrested predominantly in G1, although a small percentage of cells entered early S phase. When medium containing a complete complement of amino acids was then added, cells initiated DNA synthesis following a minimum lag of 2-3 hr. Growth arrested cells initiated DNA synthesis even when complete unsupplemented medium was added, although the addition of high concentrations of insulin or 10% serum increased the rate of entry.     

Effect of glucose starvation on the nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase of yeast.

Yeast cells growing on mineral medium plus ammonia and glucose contained high levels of nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase activity, as measured in crude extracts. After suspension of cells in fresh medium lacking glucose, there was a loss of the glutamate dehydrogenase activity. Loss of activity was inhibited by 2,4-dinitrophenol, sodium azide, iodoacetic acid, and cycloheximide. The enzyme activity was restored when glucose was added back to the medium, and this recovery was fully prevented in the presence of cycloheximide.     

Effect of Phosphate and Other Anions on Trimethylarsine Formation by Candida humicola

Phosphate inhibited the formation of trimethylarsine from arsenite, arsenate, and monomethylarsonate, but not from dimethylarsinate, by growing cultures of Candida humicola. Phosphite suppressed trimethylarsine production by growing cultures from monomethylarsonate but not from arsenate and dimethylarsinate, and hypophosphite caused a temporary inhibition of both proliferation and the conversion of these three arsenic sources to trimethylarsine. Resting cells of C. humicola derived from cultures grown in arsenic-free media generated the volatile arsenical only after a lag phase. High antimonate concentrations reduced the rate of conversion of arsenate to trimethylarsine by resting cells, but nitrate was without effect.     

Juvenile hormones inhibit murine cell cycle progression and expression of type c viruses

Summary Juvenile hormones (JH), congeners of retinoic acid, were examined for their capacity to inhibit cell cycle progression and chemically induced expression of endogenous xenotropic retrovirus in Kirsten sarcoma virus-transformed BALB (K-BALB) mouse cells. JHI, II, and III were found to inhibit induction of virus by 5-iododeoxyuridine (IUdR) and histidinol (Hdl) in a concentration-dependent fashion. Some inhibition of macromolecular synthesis was observed upon culture of the cells with JH; the most affected was RNA synthesis, which was reduced 27 to 40% within 4 h by the juvenoids. Epoxide hydrase (EH) activity, as determined by high-pressure liquid chromatography (HPLC), was present in amounts sufficient for the cells to convert the hormones metabolically to an ultimate form. A contact-inhibited K-BALB variant was synchronized by mitotic arrest and the cell cyclespecific effect of JHIII on virus induction during S phase was studied. JHIII added during G₁ phase, and followed by induction, inhibited virus expression 95 and 76% by IUdR and Hdl, respectively. Induction was inhibited only 35% when JHIII was added during S phase concomitantly with the inducers and no inhibition was observed when JHIII was added during G₂ phase followed by the inducers. JHIII added to synchronous cells in G₁ phase inhibited progression of cells into S phase and the onset of DNa synthesis. The results indicate that mouse fibroblasts have a juvenile hormone-sensitive restriction point in G₁ phase that might relate to the effects these hormones have on cell replication and differentiation. This work was supported under Contract NO-1-CO-75380 with the National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205.     

Hormonal regulation of initiation of DNA synthesis and of differentiated function in Y-1 adrenal cortical cells.

ACTH, 8-Br-cAMP, and serum deprivation arrested Y-1 functional mouse adrenal tumor cells in the G1 phase of the cell cycle. Though ACTH and 8-Br-cAMP treated cells were larger with increased macromolecular synthetic rates compared to cells arrested in G1 by serum removal, a similar 8- to 10-hours lag to initiation of DNA synthesis was observed after either ACTH or 8-Br-cAMP removal or after serum addition. After the 8- to 10-hour lag period, cells entered S phase exponentially. ACTH or 8-Br-cAMP opposed serum induced DNA synthesis initiation only when added prior to S. Once commitment to DNA synthesis occurred, ACTH or 8-Br-cAMP addition did not inhibit DNA synthesis although 8-Br-cAMP induced a secondary block in G2. Though ACTH and 8-Br-cAMP inhibited serum induced initiation of DNA synthesis and did not affect serum induced cellular hypertrophy, both substances increased the steroidogenic capacity of the cell. ACTH and 8-Br-cAMP thus appear to specifically oppose the stimulatory effects of serum on initiation of DNA synthesis while inducing the differentiated function of the cell.     

Effect of actinomycin D on the formation of enzymes in Jerusalem artichoke tuber slices

Summary The formation of both peroxidase and phenol oxidase was induced by culturing slices of Jerusalem artichoke tubers. Actinomycin D inhibited the formation of enzymes only when added before the termination of the lag phase (10 h after slicing). The data suggest that 4 to 6 h after slicing, tissues produce a mRNA which does not become fully operative until a translation mechanism has been activated.