Growth Characteristics of Kilham Rat Virus and Its Effect on Cellular Macromolecular Synthesis

Kilham rat virus (KRV) is adsorbed into the rat nephroma cell within 1 hr after infection. There follows a latent period of about 12 hr during which less than 1% of the input infectious virus can be accounted for. New infectious virions can be detected at about 12 hr and the maximal yield of virus is attained by 23 hr after infection. The increase in final virus yield is about 200-fold over that found in the latent period. During this 23-hr period of virus growth, the rate of protein synthesis remains 75 to 100% of that in the uninfected cell. Ribonucleic acid (RNA) synthesis during this period is maintained at 100 to 150% of that found in the control cells. The addition of the inhibitor of deoxyribonucleic acid (DNA) synthesis, 5-fluoro-deoxyuridine (FUDR), up to 8 hr after infection completely suppresses virus production. After 8 hr, viral DNA production has started and FUDR inhibition progressively decreases until by 23 hr the addition of the inhibitor no longer causes a reduced virus yield. Viral DNA synthesis once initiated is required for the remainder of the 23-hr virus cycle. Viral DNA synthesis probably begins about 4 hr before the production of infectious virions. In the KRV-infected cells, DNA synthesis decreased sharply for 6 to 7 hr after infection in comparison to the uninfected cell. At 7 to 8 hr after infection, DNA synthesis in the infected cell increased and was maintained at a higher level than in the control cells for the rest of the virus growth period.     

Metabolic Regulation in Glucose-Limited Chemostat Cultures of Escherichia coli

Glucose-limited chemostat cultures of Escherichia coli, growing at dilution rates above 0.3/hr, continue to grow at the restricted rate after removal of glucose restriction. In a glycogenless strain, the specific rates of increase of mass, protein, and ribonucleic acid (RNA) were equal before and after supplementation with 0.05% glucose and did not increase detectably until after 30 to 60 min. The unrestricted specific growth rate was reached after two to three doublings of cell mass. Supplementation with glucose plus 20 amino acids, but not with glucose plus vitamins or ribosides, produced an immediate increase in the specific rates of mass and RNA synthesis followed by an increase in the specific rate of protein synthesis. In a wild-type strain, synthesis of protein and RNA continued at the restricted rate after glucose supplementation, but the specific rate of increase of mass immediately increased due to rapid synthesis of glycogen. At dilution rates less than 0.3/hr, the specific rates of increase of mass, protein, and RNA increased immediately after supplementation with glucose, but did not immediately attain the unrestricted growth. The results at dilution rates greater than 0.3/hr are interpreted to mean that the regulation of a number of enzymatic reactions is entirely through control of enzyme synthesis, without modulation of enzyme function. The levels of such enzymes are controlled so that operation with zero-order kinetics precisely meets the demands for balanced growth. It was shown that glutamic dehydrogenase and glutamic-oxalacetic transaminase are regulated in this manner.     

Heat transient related changes in stress-protein synthesis

A slow temperature transient from 37 to 42 degrees C over 3 hr instead of the usual rapid 4- to 7-min transient increases thermal resistance twofold in MTC tumor cells and yet reduces the rates of synthesis of the 70- and 22-kDa heat-stress proteins (hsp) immediately prior to and during expression of thermal resistance--2 to 8 hr after reaching 42 degrees C [S. P. Tomasovic, P. A. Steck, and D. Heitzman, Radiat. Res. 95, 399-413 (1983)]. However, examination of hsp synthesis at earlier times reaching 42 degrees C (0.5 to 2 hr) has revealed differential expression of the individual hsp that is dependent on the rate of heating. Within 30 min of reaching 42 degrees C, cells exposed to slow transients had higher rates of synthesis of the 112- and 90- but not the 70-kDa hsp. However, cells exposed to rapid transients had a higher rate of synthesis of the 70-kDa hsp by 1 hr after reaching 42 degrees C. The rate of synthesis of the 22-kDa hsp was similar in cells heated by either method. Rates of synthesis of the 112-, 90-, and 22-kDa hsp in cells exposed to rapid transients did not equal or surpass the rates for cells exposed to slow transients until between 2 and 3 hr of heating, just before expression of thermal resistance. Rate of heating also had differential effects on total protein synthesized and transport. The total protein synthesized was observed to be 40% higher in slow-transient-treated cells over the first 2 hr. Transport of an amino acid analog, aminoisobutyric acid, was significantly inhibited in rapid-transient cells immediately after reaching 42 degrees C and had not recovered 1 or 5 hr later. Similar to total protein synthesis transport in slow-transient-treated cells was unaffected. There was no significant difference between slow- and rapid-transient-treated cells in hsp degradation, cell-cycle distribution, or amino acid pool sizes in the first 4 to 6 hr after reaching 42 degrees C. These results suggest that although the ultimate thermal dose was about 10-fold higher under slow-transient conditions, the cells receiving this treatment made regulatory or metabolic adjustments, including altered hsp synthesis patterns, that reduced initial heat damage. Either the protection of total protein synthesis or that combined with higher initial rates of synthesis of some hsp could explain the previously reported increased initial D0, increased thermotolerance, and reductions in latter hsp synthesis rates seen following slow temperature transients.     

Effect of Rehydration on Recovery, Repair, and Growth of Injured Freeze-Dried Salmonella anatum

From 70 to 90% of the Salmonella anatum cells that survived freeze-drying in nonfat milk solids were injured. After rehydration, these injured survivors failed to grow on a selective plating medium containing deoxycholate but could form colonies on a nonselective medium. In a suitable environment after rehydration, injury disappeared in most of these cells. The rate of this repair at 25 C was very rapid initially and, in a medium containing milk solids, was completed within 1 hr after rehydration. The repaired cells initiated growth about 1 hr later than normal cells and grew at a slower rate. In a medium containing milk solids, initial recovery, extent of repair of injury, initiation of growth, and rate of growth were not influenced by supplementation with extra nutrients in other rehydration media. Rehydration controlled by modifying the concentrations of lactose, sucrose, or milk solids in the rehydration medium influenced the recovery of cells and the time that growth was initiated. Glycerol failed to increase recovery. Higher numbers of cells were recovered by rehydrating at 15 to 25 C, but an earlier initiation of growth and more rapid growth were observed at 35 C.     

Intracellular protein breakdown in non-growing cells of Escherichia coli

1. When Escherichia coli leu(-) was incubated at 35 degrees in a medium based on minimal medium, but with the omission of phosphate ions, or glucose, or NH(4) (+) ions and leucine, intracellular protein was degraded at a rate of about 5%/hr. in each case. If Mg(2+) ions were omitted, however, the rate of degradation was 2.9%/hr. 2. Under certain conditions of incubation, protein degradation was inhibited. The inhibitor was neither NH(4) (+) ions nor amino acids, and its properties were not those of a protein, but it might be an unstable species of RNA. 3. Although a large part of the cell protein was degraded at about 5%/hr. during starvation of NH(4) (+) ions and leucine, some proteins were lost at more rapid rates, whereas others were lost at lower rates or not at all. 4. In particular, beta-galactosidase activity was lost at about 8%/hr. during starvation of NH(4) (+) ions and leucine, whereas d-serine-deaminase and alkaline-phosphatase activities were stable. During starvation of Mg(2+) ions, all three enzyme activities were stable.     

Macromolecular Synthesis in Saccharomyces cerevisiae in Different Growth Media

Synthesis of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein was determined in Saccharomyces cerevisiae during amino acid and pyrimidine starvation and during shift-up and shift-down conditions. During amino acid starvation, cell mass, cell number, and RNA continued to increase for varying periods. During amino acid and pyrimidine starvation, cell mass and RNA showed little increase, whereas total DNA increased 11 to 17%. After a shift from broth medium to a minimal defined medium, increase in RNA and protein remained at the preshift rate before assuming a lower rate. DNA increase remained at an intermediate rate during shift-down, and then dropped to a low rate. During shift-up from minimal to broth medium, increase in cell number, protein, and DNA showed varying lag periods before increasing to the new rate characteristic of broth medium; each of these quantities exhibited a step sometime in the first 2 hr after transfer to rich medium, suggesting a partial synchronous division. Immediately after shift-up, RNA synthesis assumed a high rate, and then dropped to a rate characteristic of growth in the rich medium after about 1 hr.     

Bioreactor operated production of lipase: castor oil hydrolysis using partially-purified lipase.

A highly stable lipase from Pseudomonas aeruginosa KKA-5 was produced by batch cultivation technique employing shake flask and 5 L-bioreactor. The bioreactor was run at different airflow rates. Low airflow rates (1 and 3 L/min), did not lead to effective growth and lipase production. Growth increased by about one order and lipase production increased by about 6 times, at an airflow rate of 5 L/min. Lipase production occurred during decelerated cell growth. A highly stable lipase was produced which retained its activity in the running bioreactor, even after a period of one month. This stable lipase was partially-purified using ammonium sulphate precipitation technique. Castor oil was hydrolyzed using 300U crude and partially-purified lipase, each. Approximately 21-fold, partially-purified lipase could hydrolyze 81% castor oil within a period of 96 hr, where as only 63% hydrolysis was obtained, in 216 hour, when crude lipase was used.     

Approximation of the cell cycle in synchronized populations of Streptococcus faecium.

Slowly growing populations (TD = 70 to 80 min) of Streptococcus faecium (S. faecalis ATCC 9790) were synchronized by selection after sucrose gradient fractionation. The cell cycle was approximated by correlating the patterns of DNA accumulation and cell division. More specifically, the beginning of cell cycle was equated with the beginning of a rapid linear increase in DNA accumulation. The DNA content of the culture approximately doubled during the period of accumulation, which lasted about 51 min. The period of rapid DNA accumulation, was followed by a period of reduced accumulation that lasted about 24 min. During synchronized growth, cell numbers increased rapidly in coordination with the period of rapid DNA accumulation and exhibited a plateau during the period of reduced DNA accumulation. In contrast, RNA and protein appeared to accumulate exponentially throughout the cell cycle at the same rate as culture mass.     

Further characterization of the spontaneous growth response in Zea coleoptile segments

Excised segments of corn (Zea mays L., Bear Hybrid WF 9?38)coleoptiles show a strong "spontaneous" increase in growth rateabout 4.0 hr after excision. The response can be delayed about2 hr using a brief (10 min) exposure to IAA during the latentperiod. An established spontaneous growth response can be suppressedby a 30 to 60 min exposure to auxin and does not reappear untilabout 2.5 hr after withdrawal of the hormone. During the 3 hrperiod following withdrawal of exogenous auxin there is a two-foldincrease in magnitude and a three-fold decrease in latent periodof a growth response to a sub-optimal level of auxin. The dataare consistent with the hypothesis that the spontaneous growthresponse is caused by a time-dependent change in sensitivityof isolated tissue to auxin and/or a change in the endogenouslevel of auxin. Apical sections of Zea coleoptiles with the tip intact do notgrow at the rapid rate one might expect of tissue with an endogenousauxin supply. Instead they grow very poorly and exhibit botha weak spontaneous growth response and a poor response to exogenouslysupplied auxin. Indirect evidence suggests that this is dueto the production of a growth inhibitor by the tip. (Received August 3, 1976; )     

Compartments of protein metabolism in the developing brain.

We investigated whether the higher rate of amino acid incorporation into immature than into mature brain protein is due to (a) rapid growth, (b) a small rapidly metabolized protein pool, or (c) a higher turnover rate of most of the protein. We measured net growth and the incorporation of [14C]tyrosine or [14C]valine into brain proteins in young rats and mice. The specific activity of the free amino acid pool was kept constant in the tyrosine experiments. Incorporation of tyrosine into protein was continued for up to 30 h by which time the specific activity of protein-bound amino acid reached 1/3 of that of the free (precursor) amino acid. The growth (accretion) of brain proteins was approx. 0.635% per h in mice and rats in the 1-4 day period after birth. In previous studies we found that the turnover rate of the bulk (about 96%) of adult brain proteins is below 0.3% per h. Because of the presence of a small (about 4%) active pool the average turnover rate is 0.6% per h. The present experiments show a degradation rate of 0.7-1.1% per h in the brain proteins of the young. This high metabolic rate is not due to a small rapidly degraded fraction of protein. The very rapid protein fraction previously seen in adult rats is either very small (below 1%) or absent in the young. Thus most of the proteins in the immature brain during the rapid growth phase are formed and broken down at a rate that is approximately three times higher than that of the bulk of proteins in the adult brain. The small active protein pool in the adult on the other hand has a metabolic rate higher than that of the immature brain proteins.     

CHANGES IN THE PROLIFERATION RATE OF MOUSE EPIDERMIS AFTER IRRADIATION: CONTINUOUS LABELLING STUDIES

The proliferative response of mouse skin to damage caused by X-irradiation has been tested by giving repeated injections of tritiated thymidine and scoring the percentage of labelled cells in high resolution autoradiographs. Four, nine and fourteen daily fractions of 300 rads of X-rays were used and labelling commenced 4 days after the last fraction. The epidermis of the upper surface and the sole of the foot were scored separately and were compared with the skin of unirradiated feet. In unirradiated skin the proliferation rate of the basal layer cells is more rapid on the sole than on the upper surface. The cell cycle times deduced from continuous labelling curves were 81 hr and 111 hr respectively and the growth fractions were 97% and 85%. After irradiation with small daily doses the homeostatic response to cell killing was slow. More rapid proliferation occurred after nine fractions in the sole, but was not apparent in the skin of the upper surface until fourteen fractions had been given. After fourteen fractions the cell cycle time was about 24 hr on both surfaces and the growth fraction was about 90%. The initial labelling index after a single thymidine injection was a poor measure of proliferation rate. The delay in the time of onset of faster proliferation is similar, both qualitatively and quantitatively, to that measured previously from the additional dose increments needed if large doses were given at different times after the multifraction treatments (Denekamp, 1973).     

LPP-1 Infection of the Blue-Green Alga Plectonema boryanum: I. Electron Microscopy

One-step growth and intracellular growth experiments were performed at high multiplicities of the virus LPP-1 during the infection of the blue-green alga Plectonema boryanum. The eclipse period lasts until 4 hr after infection, the latent period terminates at 6 hr, and the rise period continues until 14 to 16 hr after infection. The burst size was independent of multiplicity of infection over the ranges from 1 to 50. The burst size was 3,000 to 5,000 plaque-forming units (PFU) per infectious center or about 200 PFU per cell. Samples for electron microscopy were taken at characteristic times during the lytic cycle. The first sign of viral infection was the invagination of the photosynthetic lamellae at 3 hr after infection. Mature virions were visible at 4 hr. By 6 to 7 hr, many mature intracellular viral particles could be seen, with lysis beginning at 7 hr. By 10 hr after infection, all infected cells contained mature virions. No evidence for mass migration of performed viral precursors was obtained. The invagination of the lamellae could be prevented by the early addition of chloramphenicol, which implies that this process requires protein synthesis.     

Changes in the proliferation rate of mouse epidermis after irradiat-on continuous labelling studies.

The proliferative response of mouse skin to damage caused by X-irradiation has been tested by giving repeated injections of tritiated thymidine and scoring the percentage of labelled cells in high resolution autoradiography. Four, nine and fourteen daily fractions of 300 rads of X-rays were used and labelling commenced 4 days after the last fraction. The epidermis of the upper surface and the sole of the foot were scored separately and were compared with the skin of unirradiated feet. In unirradiated skin the proliferation rate of the basal layer cells is more rapid on the sole than on the upper surface. The cell cycle times deduced from continuous labelling curves were 81 hr and 111 hr respectively and the growth fractions were 97% and85%. After irradiation with small daily doses the homeostatic response to cell killing was slow. More rapid proliferation occurred after nine fractions in the sole, but was not apparent in the skin of the upper surface until fourteen fractions had been given. After fourteen fractions the cell cycle time was about 24 hr on both surfaces and the growth fraction was about 90%. The initial labelling index after a single thymidine injection was a poor measure of proliferation rate. The delay in the time of onset of faster proliferation is similar, both qualitatively and quantitatively, to that measured previously from the additional dose increments needed if large doses were given at different times after the multifraction treatments (Denekamp, 1973).     

Synthesis of macromolecules by Escherichia coli near the minimal temperature for growth

When a culture of Escherichia coli ML30 growing exponentially at 37 C in a glucose minimal medium was shifted abruptly to 10 C, growth decreased for about 4.5 hr. There was no net synthesis of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein. The cells, however, respired at a rate characteristic of cells growing in the steady state at 10 C and were able to accumulate alpha-methyl-d-glucoside. When growth recommenced at 10 C, protein synthesis started at 4 hr, RNA synthesis, with a burst at 6 hr, and DNA synthesis, with a burst at 7 hr. One synchronous division occurred at about 11 hr after shifting to 10 C. There was no alteration in the steady-state RNA to protein ratio. The results are discussed in relation to other reported effects of shifts in environmental conditions. The lag at 10 C was dependent on prior conditions of growth at 37 C. Growth at 37 C under conditions giving catabolite repression were necessary for the lag to be established on shifting to 10 C.     

Inhibitory proteins in the Newcastle disease virus-induced suppression of cell protein synthesis

Bolognesi, D. P. (Rensselaer Polytechnic Institute, Troy, N.Y.), and D. E. Wilson. Inhibitory proteins in the Newcastle disease virus-induced suppression of cell protein synthesis. J. Bacteriol. 91:1896-1901. 1966.-Infection by Newcastle disease virus brings about a rapid and marked inhibition of cell protein synthesis (CPS) in chick embryo fibroblast monolayers. The block to CPS is initiated about 5 hr after infection, and by 9 hr about 85% of the host protein synthesis is shut off. Azauridine (3 mg/ml), a ribonucleic acid (RNA) synthesis inhibitor, prevents the virus-induced inhibition of CPS when added at the time of infection; but it does not prevent the inhibition when added at 3 hr after infection. When puromycin (60 mug/ml), a protein synthesis inhibitor, was added at 3.5 hr after infection, viral RNA was synthesized in normal amounts, but the virus-induced inhibition of CPS was prevented. Actinomycin D added at the time of infection does not, however, prevent the virus-induced inhibition of CPS. The results of these experiments indicate that proteins synthesized during Newcastle disease virus replication are responsible for the inhibition of host-cell protein synthesis. The synthesis of these inhibitory proteins depends on the prior synthesis of viral RNA.     

Effect of Mengovirus Replication on Choline Metabolism and Membrane Formation in Novikoff Hepatoma Cells

Infection of Novikoff rat hepatoma cells (subline NlSL-67) with mengovirus resulted in a two- to threefold increase in the rate of choline incorporation into membrane phosphatidylcholine at about 3 hr after infection, without affecting the rate of transport of choline into the cell or its phosphorylation. The time course of virus-stimulated phosphatidylcholine synthesis was compared with the time courses of other virus-induced processes during a single cycle of replication. The formation of viral ribonucleic acid (RNA) polymerase and of viral RNA commenced about 1 hr earlier than the virus-stimulated choline incorporation. Further, isopycnic centrifugation of cytoplasmic extracts indicated that the excess of phosphatidylcholine synthesized by infected cells is not located in the membrane structures associated with the viral RNA replication complex, but with structures of a lower density (1.08 to 1.14 g/cc). These membrane structures probably represent the smooth vesicles which accumulate in the cytoplasm of infected cells during the period of increased phosphatidylcholine synthesis between 3 and 5 hr after infection. They are formed with both newly synthesized phosphatidylcholine and phosphatidylcholine present prior to infection. However, concomitant protein synthesis is not required for the stimulated synthesis of membranes; the effect was not inhibited by treating the cells with inhibitors of protein synthesis at 3 hr after infection, although virus production was inhibited about 90% and virus-induced cell degeneration was markedly reduced and delayed. Production of mature virus began normally at about the same time as the stimulation of phosphatidylcholine synthesis. Treatment of infected cells with puromycin at 2 hr, on the other hand, completely inhibited the stimulation of phosphatidylcholine synthesis.     

Steady-State Measurement of the Turnover of Amino Acid in the Cellular Proteins of Growing Escherichia coli: Existence of Two Kinetically Distinct Reactions

Turnover of cellular protein has been estimated in Escherichia coli during continuous exponential growth and in the absence of extensive experimental manipulation. Estimation is based upon the cumulative release into carrier pools of free leucine-1-(14)C over a number of time intervals after its pulsed incorporation into protein. Breakdown rates obtained with other labeled amino acids are similar to those obtained with leucine. Two kinetically separate processes have been shown. First, a very rapid turnover of 5% of the amino acid label occurs within 45 sec after its incorporation, most likely indicating maturative cleavages within the proteins after their assembly. A slower heterogeneous rate of true protein turnover follows, falling by 39% in the remaining proteins for each doubling of turnover time. At 36 C, the total breakdown rate of cellular protein is 2.5 and 3.0% per hr over a threefold range of growth rate in glucose and acetate medium, respectively. This relatively constant breakdown rate is maintained during slower growth by more extensive protein replacement, one fifth of the protein synthesized at any time in the acetate medium being replaced after 4.6 doubling times. Intracellular proteolysis thus appears to be a normal and integral reaction of the growing cell. The total rate equals minimal estimates obtained by others for arrested or decelerated growth but is kinetically more heterogeneous. Quantitatively proteolysis is not directly affected by growth arrestment per se as caused by alpha-methylhistidine, chloramphenicol, or uncouplers of oxidative phosphorylation, but qualitatively it can gradually become more homogeneous kinetically as a secondary event of starvation. Under more extreme conditions as with extensive washing, prolonged phosphorylative uncoupling, or acidification of the growth medium, the proteolytic rate can increase severalfold.