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PubMed1999

Glucose starvation and hypoxia induce nuclear accumulation of proteasome in cancer cells.

Solid tumors commonly contain regions with glucose-starved and hypoxic conditions. Tumor cells under the adverse conditions can survive through the stress response, such as cell cycle arrest. In this study, we found that the stress conditions stimulated nuclear accumulation of proteasomes, large multicatalytic protease complexes, in human colon cancer HT-29 cells. The nuclear proteasome levels both in amount and in activity were increased approximately 4 and 2 times by glucose starvation and hypoxia, respectively. No changes were detected in the total expression levels of proteasome. The nuclear proteasome accumulation was also observed in ovarian cancer A2780 cells under glucose starvation, suggesting that this response was regardless of the origin of cancer cells. Our results indicate that the nuclear proteasome distribution is enhanced by glucose starvation and hypoxia, and suggest that the proteolysis by proteasome in the nucleus may play roles in the stress response of solid tumor cells.

Cell Cycle↗

PubMed2000

Glucose starvation reduces IGF-I mRNA in tumor cells: evidence for an effect on mRNA stability.

The purpose of this study was to characterize the mechanisms by which glucose regulates IGF-I gene expression in rat C6 glioma cells and in rat GH3 pituitary adenoma cells. Glucose starvation for periods of 12 to 48 h decreased IGF-I mRNA levels. In contrast, there was no stimulation of IGF-I mRNA by medium glucose between 1 and 25 mM over a 24-h period. Studies with hexoses and glycolytic metabolites suggested that glucose metabolism was required to maintain IGF-I mRNA. Glucose starvation lowered IGF-I mRNA half-life in both C6 and GH3 cells. Protein synthesis inhibition lowered IGF-I mRNA by about 20% in glucose-fed C6 and GH3 cells, while potently increasing IGF-I mRNA in glucose-starved C6 cells and not altering IGF-I mRNA in glucose-starved GH3 cells. Our results suggest that in these tumor cells, IGF-I mRNA stability is reduced by glucose starvation, secondary to a deficiency in intracellular glucose metabolism. Ongoing protein synthesis is not required for this mRNA de-stabilizing effect in GH3 cells. Rather, in glucose-starved C6 cells, decreased IGF-I mRNA stability may result from the action of a labile protein.

Animals↗

PubMed2002

Different effects of glucose starvation on expression and stability of VEGF mRNA isoforms in murine ovarian cancer cells.

Vascular endothelial growth factor (VEGF) has been implicated as a potent regulator of angiogenesis in tumors, and its protein exists as at least five isoforms with distinct biologic activities and clinical significance. Tumors under metabolic stress conditions dramatically increase VEGF expression due to both increased transcription and decreased mRNA degradation. However, it is not known how stress conditions regulate expression of each VEGF isoform. Here, we report a novel Taqman real-time RT-PCR strategy for quantification of all murine VEGF isoforms and find that (1) glucose starvation dramatically up-regulates the mRNA level of all VEGF isoforms, with the three abundant isoforms, VEGF120, VEGF164, and VEGF188, increasing at a similar rate, while the rare isoform VEGF144 is more markedly up-regulated; (2) glucose starvation induces a significant increase of the relative abundance of VEGF144 mRNA, but not the more prevalent isoforms VEGF120, VEGF164, and VEGF188, compared to total VEGF; and (3) the stability of each isoform mRNA differs under the control conditions as well as glucose starvation. The latter significantly stabilizes mRNA of all VEGF isoforms at a different rate, with VEGF144 most significantly stabilized. Our results indicate that under metabolic stress conditions VEGF144 is the most dramatically up-regulated VEGF isoform, probably through mechanism(s) different from the three abundant VEGF isoforms.

Alternative Splicing↗

PubMed1994

High concentrations of ppGpp decrease the RNA chain growth rate. Implications for protein synthesis and translational fidelity during amino acid starvation in Escherichia coli.

We show that the RNA chain growth rate on lacZ is reduced by an elevated ppGpp level even in the absence of starvation. Under these conditions the polypeptide chain elongation rate is affected little, if at all. These results lead us to re-examine the role of ppGpp in the reduction of protein synthesis and translational fidelity during amino acid starvation. We find that ppGpp has little or no direct effect on translation rate or fidelity. Rather, the effects of ppGpp on translation are indirectly caused by the fact that ppGpp inhibits mRNA synthesis, making mRNA limiting for translation during amino acid starvation. The reduced level of mRNA thereby reduces the severity of the aminoacyl-tRNA limitation and, in turn, decreases mistranslation. Mistranslation in the starved relA strain therefore results from an increased severity of aminoacyl-tRNA limitation due to the failure of this strain to reduce mRNA levels by increasing the level of ppGpp. Finally, the initial rise of the ppGpp level in the starved stringent strain, followed by a characteristic reduction to a steady poststarved level, can now be explained by the initially high, and then decreasing number of "hungry" codons adjusted through the mRNA pool.

Amino Acids↗

PubMed1978

Genetic damage during thymidylate starvation in Saccharomyces cerevisiae.

Thymidylate starvation in a yeast mutant auxotrophic for dTMP caused cell death and the induction of mutations in the mitochondrial genome. After 24 h of starvation almost all surviving cells were respiratory deficient petites. In addition, shorter episodes of dTMP starvation induced chloramphenicol and erythromycin resistant mutants, indicating the occurrence of mitochondrial point mutations. Suboptimal concentrations of exogenous thymidylate were also found to induce petites and a decline in cell viability and the magnitude of these effects was acutely dependent upon the dTMP concentration. Cesium chloride gradient analysis of DNA from cells undergoing thymineless incubation revealed a progressive loss of mitochondrial DNA, and a decrease in the molecular weight of nuclear DNA.

Chloramphenicol↗

PubMed1976

Effect of glucose starvation on the expression of transferred tsx genes in Escherichia coli K12 zygotes.

Escherichia coli K12 Hfr H Tsxs Strs and F- Pro- Tsxr His- Arg- Strr bacteria were conjugated in the absence of arginine with or without glucose. The efficiency of conjugation, measured by the frequency of Pro+ and His+ recombinants was not affected. Arginine starvation alone did not affect the tsxs gene expression which occurred in all the zygotes which had received the gene. In contrast, argine and glucose starvation allows tsxs expression only in those zygotes in which the donor gene had been integrated in the genome. As the glucose starvation brings on a destabilization of the messenger RNA synthesized by the F- cells in absence of arginine, the results can be interpreted as follows: the transferred tsxs genes are transitorily expressed in all the zygotes at the unintegrated state. After this transient period, only thsoe genes integrated in the chromosomes of the zygotes continue to be expressed.

Arginine↗

PubMed1996

Respiratory gas exchange, nitrogenous waste excretion, and fuel usage during starvation in juvenile rainbow trout, Oncorhynchus mykiss.

Oxygen consumption, CO2 excretion, and nitrogenous waste excretion (75% ammonia-N and 25% urea-N) were measured daily in 4-g rainbow trout over a 15-day starvation period. Oxygen consumption and CO2 excretion declined while N excretion increased transiently in the mid-part of the starvation period but was unchanged from control levels at the end. Component losses (as percentage of total fuel used) of protein, lipid, and carbohydrate were 66.5, 31.1, and 2.4% respectively, as measured from changes in body weight and body composition, the latter relative to a control group at day 0. Instantaneous fuel use, as calculated from the respiratory quotients and nitrogen quotients, indicated that relative protein use rose during starvation, but contributed at most 24% of the aerobic fuel (as carbon). Lipid metabolism fell from about 68 to 37%, and was largely replaced by carbohydrate metabolism which rose from 20 to 37%. We conclude that the two approaches measure different processes, and that the instantaneous method is preferred for physiological studies. The compositional method is influenced by greater error, and measures the fuels depleted, not necessarily burned, because of possible interconversion and excretion of fuels.

Analysis of Variance↗

PubMed1986

The production and release of an extracellular polysaccharide during starvation of a marine Pseudomonas sp. and the effect thereof on adhesion.

A marine Pseudomonas sp. S9 produced and released an extracellular polysaccharide during complete energy and nutrient starvation in static conditions. The presence of the polysaccharide on the cell surface, demonstrable by immune transmission electron microscopy, correlated with changes in the degree of adhesion to hydrophobic surfaces. Polysaccharide coated cells showed a lower degree of adhesion than did cells devoid of the polymer. After 10 h of starvation, no ruthenium red stained antibody stabilized polysaccharides could be observed on the cell surface. The polysaccharide was not produced during growth since lysates of mid-log phase cells did not precipitate the antiserum. The relative proportions of sugars in the polysaccharide were 28% glucose, 35% N-acetyl-glucosamine and 37% N-acetylgalactosamine. The released polysaccharide did not significantly alter the physical parameters of surface tension and viscosity of the starvation regime. Cells starved in agitated conditions did not produce any extracellular polysaccharides and exhibited a different adhesion pattern to hydrophobic surfaces.

Bacterial Adhesion↗

PubMed1995

Assessment of survival during starvation of Escherichia coli and Klebsiella pneumoniae in artificial urine: analysis of the kinetics of colony formation.

A kinetic model of colony formation was proposed by Hattori, based on a count of the colonies that appear on a plate in successive short intervals of time. In this model, three parameters (lambda, tr and N infinity) are defined, which reflect the ability of a bacterium to yield colonies and allow us to described the dynamics of bacterial populations in soil and of E. coli at different growth phases. In this paper we report a reparametrization of the kinetic model of colony formation, with the aim of facilitating more accurate calculation of lambda and tr. Moreover, we observed that during the starvation of E. coli and K. pneumoniae in urine, lambda can be used to assess survival, since this parameter clearly decreases during starvation. Retardation time values (tr) were similar in E. coli and K. pneumoniae throughout the starvation experimental period.

Culture Media↗

PubMed2000

Enhanced expression of a calcium-dependent protein kinase from the moss Funaria hygrometrica under nutritional starvation.

Among the downstream targets of calcium in plants, calcium-dependent protein kinases (CDPKs) form an interesting class of kinases which are activated by calcium binding. They have been implicated in a diverse array of responses to hormonal and environmental stimuli. In order to dissect the role of CDPKs in the moss Funaria hygrometrica, a polymerase chain reaction (PCR)-based approach was adopted to clone the gene. Using degenerate PCR primers against conserved regions of CDPKs, a 900 bp amplicon was obtained from the genomic DNA of Funaria. Southern hybridization under low stringency conditions indicated the presence of several CDPK related sequences in the Funaria genome. This observation is consistent with reports of multigene families of CDPKs in other plants. The 900 bp fragment was subsequently used to isolate a 2.2 kb partial genomic clone of the CDPK gene from Funaria. The genomic clone encodes an open reading frame (ORF) of 518 amino acids. Interestingly, unlike other CDPK genes from plants, the entire 1.5 kb ORF is not interrupted by introns. The deduced amino acid sequence of the Funaria gene shows extensive homology with CDPKs from higher plants, 73% identity with the Fragaria CDPK and 71% identity with CDPK isoform 7 of Arabidopsis. Phylogenetic analysis revealed that the Funaria CDPK is closer to the CDPKs from higher plants like strawberry and Arabidopsis as compared to those from lower plants such as the liverwort Marchantia, the green alga Chlamydomonas or another moss Tortula. Northern analysis shows enhanced expression of the CDPK transcript within 24-48 h of starvation for nitrogen, phosphorus or sulphur. So far the only other kinase which is known to be induced by nutrient starvation in plants is the wpk 4 which is a snf-1 related kinase (SnRKs). To our knowledge this is the first report that implicates a CDPK in the starvation response.

Amino Acid Sequence↗

PubMed2000

Effect of starvation and chloramphenicol on acceleration of bacterial dihexyl sulfosuccinate biotransformation.

Starvation for carbon and energy sources accelerated the biotransformation of the anion-active surfactant dihexyl sulfosuccinate (DHS) by Comamonas terrigena cells. Chloramphenicol (Cm) added at different time intervals to non-starved cells inhibited the DHS transformation. The largest difference between cells treated and non-treated by Cm was observed for a 16-h-starvation period. Protein synthesis de novo during starvation enhanced the DHS biotransformation efficiency. A partial transformation of DHS in the presence of Cm indicated the constitutive character of enzymes involved in primary DHS biodegradation.

Bacterial Proteins↗

PubMed2005

Protein profiling of 3T3-L1 adipocyte differentiation and (tumor necrosis factor alpha-mediated) starvation.

The increased incidence of obesity and related disorders in Western societies requires a thorough understanding of the adipogenic process. Data at the protein level of this process are scarce. Therefore we performed a proteome analysis of differentiating and starving 3T3-L1 cells using two-dimensional gel electrophoresis combined with mass spectrometry. Effects of different starvation conditions were examined by subjecting 3T3-L1 adipocytes to caloric restriction, either in the absence or the presence of the lipolysis inducer tumor necrosis factor-alpha. Ninety-three differentially expressed proteins were found during differentiation and starvation of 3T3-L1 cells, 50 of which were identified. GenMAPP/MAPP-finder software revealed a non-reciprocal regulation of the glycolytic pathway during 3T3-L1 differentiation followed by starvation. Furthermore, proteins involved in growth regulation, cytoskeletal rearrangements and protein modification, 16 of which have not been described before in 3T3-L1 cells, were identified. In conclusion, our data provide valuable information for further understanding of the adipogenic process.

3T3-L1 Cells↗

PubMed1997

Disruption of the phosphate-starvation response of oilseed rape suspension cells by the fungicide phosphonate.

The influence of the anti-fungal agent phosphonate (Phi) on the response of oilseed rape (Brassica napus L. cv. Jet Neuf) cell suspensions to inorganic phosphate (Pi) starvation was examined. Subculture of the cells for 7 d in the absence of Pi increased acid phosphate (APase; EC 3.1.3.2) and pyrophosphate (PPi)-dependent phosphofructokinase (PFP; EC 2.7.1.90) activities by 4.5- and 2.8-fold, respectively, and led to a 19-fold increase in Vmax and a 14-fold decrease in Km (Pi) and Pi uptake. Addition of 2 mM Pi to the nutrient media caused dramatic reductions in the growth and Pi content of the Pi-starved, but not Pi-sufficient cells, and largely abolished the Pi-starvation-dependent induction of PFP, APase, and the high-affinity plasmalemma Pi translocator. Immunoblotting indicated the cells contain three APase isoforms that are synthesized de novo following Pi stress, and that Pi treatment represses this process. Phosphonate treatment of Pi-starved cells significantly altered the relative extent of in-vivo 32P-labelling of polypeptides having M(rs) of 66, 55, 45 and 40 kDa. However, Phi had no effect on the total adenylate pool of Pi-starved cells which was about 32% lower than that of Pi-sufficient cells by day 7. Soluble protein levels, and activities of pyruvate kinase (EC 2.7.1.40) and ATP-dependent phosphofructokinase (EC 2.7.1.11) were unaffected by Pi starvation and/or Phi treatment. The effects of Phi on the growth, and APase and PFP activities of Pi-starved B. napus seedlings were similar to those observed in the suspension cells. The results re consistent with the hypothesis that a primary site of Phi action in higher plants is at the level of the signal transduction chain by which plants perceive and respond to Pi stress at the molecular level.

Acid Phosphatase↗

PubMed2006

Effects of N-starvation and C-source on Bradyrhizobium japonicum exopolysaccharide production and composition, and bacterial infectivity to soybean roots.

The exopolysaccharide (EPS) is an extracellular molecule that in Bradyrhizobium japonicum affects bacterial efficiency to nodulate soybean. Culture conditions such as N availability, type of C-source, or culture age can modify the amount and composition of EPS. To better understand the relationship among these conditions for EPS production, we analyzed their influence on EPS in B. japonicum USDA 110 and its derived mutant DeltaP22. This mutant has a deletion including the 3' region of exoP, exoT, and the 5' region of exoB, and produces a shorter EPS devoid of galactose. The studies were carried out in minimal media with the N-source at starving or sufficient levels, and mannitol or malate as the only C-source. Under N-starvation there was a net EPS accumulation, the levels being similar in the wild type and the mutant with malate as the C-source. By contrast, the amount of EPS diminished in N-sufficient conditions, being poyhydroxybutyrate accumulated with culture age. Hexoses composition was the same in both N-situations, either with mannitol or malate as the only C-source, in contrast to previous observations made with different strains. This result suggests that the change in EPS composition in response to the environment is not general in B. japonicum. The wild type EPS composition was 1 glucose:0.5 galactose:0.5 galacturonic acid:0.17 mannose. In DeltaP22 the EPS had no galactose but had galacturonic acid, thus indicating that it was not produced from oxidation of UDP-galactose. Infectivity was lower in DeltaP22 than in USDA 110. When the mutant infectivity was compared between N-starved or N-sufficient cultures, the N-starved were not less infective, despite the fact that the amounts of altered EPS produced by this mutant under N-starvation were higher than in N-sufficiency. Since this altered EPS does not bind soybean lectin, the interaction of EPS with this protein was not involved in increasing DeltaP22 infectivity under N-starvation.

Bacterial Adhesion↗

PubMed1999

Nitrogen starvation in synechococcus PCC 7942: involvement of glutamine synthetase and NtcA in phycobiliprotein degradation and survival

The nondiazotrophic cyanobacterium Synechococcus sp. strain PCC 7942 responds to nitrogen deprivation by differentiating into nonpigmented resting cells able to survive prolonged periods of starvation. The degradation of photosynthetic pigments, termed chlorosis, proceeds in an ordered manner in which the light-harvesting phycobiliproteins are degraded prior to chlorophyll. Here, we show that the function of the global transcription activator of nitrogen-regulated genes, NtcA, is required for the sequential pigment degradation and cell survival. The P(II) protein, known to signal the nitrogen status of the cells, is most probably not involved in the perception of the nitrogen-starvation-specific signal since in a mutant lacking P(II), chlorosis proceeded in the same manner as in the wild type. Inhibition of glutamine synthetase with l-methionine sulfoximine led to a rapid decrease of apc mRNA and to an increase of nblA mRNA levels, which is characteristic for nitrogen deprivation, suggesting that nitrogen starvation is sensed by a metabolic signal connected to glutamine synthetase activity. However, l-methionine sulfoximine treatment did not induce phycobiliprotein degradation, but led to an immediate cessation of this proteolytic process after its induction by nitrogen deprivation. This suggests that the proteolytic activity elicited by the expression of nblA has to be supported by glutamine synthetase activity.

Journal Article↗

PubMed1999

Theoretical Analysis of the Starvation Response under Substrate Pulses.

A simulation model was constructed in which two model bacteria competed with each other in a closed system to which periodic substrate additions were made. One bacterium responded to starvation by entering into a dormant state characterized by a decreased death rate; the other bacterium did not make this transition. The periodicity of substrate addition was varied, and the magnitude of selection for one bacterial type was calculated. For model parameters typical of those measured in Escherichia coli, positive selection for the bacterium capable of the starvation response only occurred if the time interval of substrate addition exceeded 54 times the minimum doubling time of the bacterium. The magnitude of the selection coefficient was most sensitive to two model parameters: the time constant for conversion of dormant cells back to actively growing ones, and the magnitude of the death rate of cells which did not undergo the starvation response.

Journal Article↗

PubMed1999

Starvation in yeast increases non-adaptive mutation.

The frequency of reversion in a histidine-requiring mutant of Saccharomyces cerevisiae increases about ten-fold in stationary cells during histidine starvation. Histidine starvation enhances a similar frequency of reversion in a tryptophan-requiring mutant. Starvation, therefore, enhances mutation frequencies in a non-adaptive manner. The base analogue 6-N-hydroxylaminopurine (HAP) added prior to plating on medium with limited histidine strongly increases reversion of the histidine mutant. HAP-induced reversion increases further in stationary starving cells with the same kinetics as that which increases spontaneous reversion. Adding HAP to the stationary starving cells does not produce any effect.

Adenine↗

PubMed2001

Fission yeast tor1 functions in response to various stresses including nitrogen starvation, high osmolarity, and high temperature.

A target of rapamycin (TOR) protein is a protein kinase that exerts cellular signal transduction to regulate cell growth in response to extracellular nutrient conditions. In the Schizosaccharomyces pombe genome database, there are two genes encoding TOR-related proteins, but their functions have not been analyzed. Here we report that one of the genes, referred to as tor1+, is required for sexual development induced by nitrogen starvation. Ste11 is a key transcription factor for the initiation of sexual development. The expression of ste11+ is normally regulated in tor1- cells; and overexpression of ste11+ hardly rescues the defect in fertility in tor1-. Upon nitrogen starvation, tor1+ cells promote two rounds of the cell cycle to become arrested at the G1 phase before initiation of sexual development. The tor1- cells do not promote such a cell cycle, suggesting that Tor1 is necessary for the response to nitrogen starvation. The tor1- cells show no growth or very slow growth under various stress conditions, including external high pH, high concentrations of salts or sorbitol, and high temperature. These results suggest that Tor1 is necessary for any response to a wide range of stresses. The vegetative growth of tor1- cells is inhibited by rapamycin, although tor1+ cells are resistant to the drug. The tor1- cells are hypersensitive to fluphenazine and cyclosporin A, which specifically inhibit calmodulin and calcineurin, respectively.

Amino Acid Sequence↗