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Combination of culture on collagen gels and glucose starvation for cloning human colon cancer cells. Obtention of clones exhibiting different patterns of enterocytic differentiation.

Glucose starvation has been widely used to select differentiated subpopulations from the heterogenous human colon cancer cell line HT29. We observed that the important cell loss elicited by culturing these cells in glucose-free medium could be limited when type I collagen gel was used as substratum instead of conventional plastic support. We took advantage of this property to develop a new protocol, which combined glucose starvation and culture on collagen gels, for cloning HT29 cells. Using this procedure we have isolated four clones that were characterized on the basis of morphological (optical and transmission electron microscopy), electrophysiological (determination of transepithelial electrical parameters) and biochemical (detection of villin, sucrase-isomaltase and carcinoembryonic antigen) criteria. These four clones expressed different patterns of enterocytic differentiation regarding to these criteria. These results confirmed the heterogeneity of the HT29 cell line. One of these clones, HT29-A7, which displayed numerous intercellular cysts that disappeared at confluency, appears as a complementary model in the study of epithelial biogenesis.

Cell Differentiation↗

Evolution of digestion of carbohydrates in the separate parts of the digestive tract of the edible snail Helix lucorum (Gastropoda: Pulmonata: Stylommatophora) during a complete 24-hour cycle and the first days of starvation.

In the present study we examined carbohydrase activities during a complete 24-h cycle and during the first days of starvation in both adult and juvenile snails. The results indicated the predominant role of the digestive gland in the secretions of the enzymes responsible for degradation of most of the carbohydrates tested. Salivary glands secreted some digestive enzymes but in amounts lower than secreted by digestive gland. Enzymatic activities fluctuated during the first hours of digestion and also after the digestive tract was empty. The relatively high enzymatic activities recorded 24 h after the intake of food and during starvation could be due to the circadian rhythm of this species and/or to the participation of an existing microflora in the digestive tract of Helix lucorum. The double origin (exogenous and endogenous) of some digestive enzymes such as cellulases is discussed.

Aging↗

Noncoordinate control of the synthesis of different species of RNA in Escherichia coli K12 during uridine starvation.

During uridine starvation in Escherichia coli K12, the rate of RNA accumulation comes down to about 7% of the nonstarved rate. This is achieved, in part, by an eight-fold increase in the assembly time of stable RNA molecules. However, the assembly time of mRNA molecules is not enhanced as much, being longer by a factor of 3 in starved cells compared to nonstarved ones. It, therefore, appears that the rate of synthesis of these two RNA species is noncoordinately controlled during uridine starvation. This control does not seem to be mediated by guanosine tetraphosphate.

Escherichia coli↗

Activation of 5-HT1C-receptors suppresses excessive wheel running induced by semi-starvation in the rat.

Male Wistar rats were housed in cages linked to running wheels and fed on a schedule designed to reduce their body weight by 20-30%. During this period of semi-starvation the rats increased their daily running wheel activity (RWA) by up to 30 km/day. RWA could be kept at this level provided that body weight was kept constant. Different serotonin receptor (5-HT) agonists and antagonists were tested for their effects on RWA and it was found that RWA could be suppressed only by agonists with high affinity for the 5-HT1C receptor (TFMPP, mCPP, DOI and quipazine). Serotonin receptor agonists, which do not pass the blood-brain barrier, and 5-HT itself had no effect on RWA. The inhibitory effect of the agonists on RWA was prevented by pretreatment with antagonists that also had high affinity for 5-HT1C receptors (mianserin, metergoline and mesulergine). From these results we conclude that semi-starvation-induced hyperactivity can be blocked by 5-HT1C agonists. Furthermore we suggest that the animal model presented in this study might be a useful tool for in vivo studies on selective 5-HT1C receptor activation.

Animals↗

Development of papillae on colonies of two isopolyauxotrophic strains of Saccharomyces cerevisiae allelic in RAD6 during adenine starvation.

Papilla formation on colonies of two isopolyauxotrophic strains (ade2 his3 leu2 trp1 ura3) allelic in RAD6 was compared in order to find proper conditions for selecting mutants of Saccharomyces cerevisiae with altered starvation-induced mutability. The most promising for this purpose appeared to be culturing low numbers of colonies on suboptimal plates with a growth-limiting amount of adenine at 28 degrees C for 20 d. Inactivation of the RAD6 gene which suppresses the level of starvation-associated mutagenesis markedly enhanced papilla formation under these conditions. Formation of almost all papillae on 20-d-old colonies of BJC3 was caused by mutation. Most of the papillae (75%) were white Ade+ revertants. Three groups of these papillae were distinguished (Ade+, Ade+Rad6+ and Ade+Trp+). Both, Ade+Rad6+ and Ade+Trp+ double reversions were very probably caused by a suppressor mutation. The less frequent red papillae had the same auxotrophic markers and UV sensitivity as BJC3 but their outgrowth in liquid media was greater. It appears that creation of these papillae is caused by mutation affecting the cell response to growth limitation by low concentrations of adenine.

Adaptation, Biological↗

DNA synthesis - dependent cell division of Escherichia coli 15 TAU after arginine and uracil starvation.

Extensive cell division after synchronization of Escherichia coli 15 TAU by arginine and uracil starvation occurs only when DNA synthesis is permitted to proceed by at least a short pulse of thymine applied between 30 and 60 min after transfer of synchronized culture to thymine-free medium with arginine and uracil. The time schedule of synchronized cell division in dependence on the schedule of intervals of DNA synthesis and inhibition of DNA synthesis was determined. The termination of replication cycles which were not completed to the very end during arginine and uracil starvation seems to be the decisive event for subsequent cell division after synchronization.

Arginine↗

High-level expression of ice nuclei in Erwinia herbicola is induced by phosphate starvation and low temperature.

In laboratory cultures of ice nucleation-active (Ice+) Erwinia herbicola isolates, it has been difficult to achieve high-level expression of ice nuclei, especially nuclei active at temperatures warmer than -5 degrees C (i.e., type 1 ice nuclei). Here we demonstrate that starvation for phosphate and exposure to low temperature triggers expression of ice nuclei in E. herbicola cultures. Starvation for nitrogen, sulfur, or iron was less effective. Under optimal conditions with two different strains, essentially all cells produced ice nuclei active at -10 degrees C or warmer, with an average of 22% containing type 1 ice nuclei within 1 h of a low-temperature shift. These conditions did not greatly enhance the shedding of ice nucleation-active membrane vesicles that are known to be produced by Ice+ E. herbicola isolates. These results support the theory that the Ice+ phenotype may allow nutrient-limited epiphytes to trigger freezing damage, releasing nutrients from host plants.

Erwinia↗

Rhizobacterial glutathione levels as affected by starvation and cadmium exposure.

The rhizosphere is a continuously fluctuating environment in which severe stresses are put on its inhabitants, and glutathione, a reducing tripeptide, and related compounds probably have important roles in cellular protection. In the present study the metabolism of glutathione was examined in rhizobacteria subjected to stress. The plant-growth-promoting rhizobacterium Pseudomonas fluorescens 5.014 and its mutant 5-2/4 were exposed to starvation, either by resuspension or exhaustion, and to cadmium. Glutathione levels, cell protein, and viable count were determined and compared in different conditions. Both starvation and cadmium exposure decreased the amount of glutathione in the cell. No changes of the glutathione concentration in the medium were observed with or without the presence of rhizobacteria, indicating that there was no transport over the cell membrane. The glutathione levels within the rhizobacteria may give valuable information on how different stresses affect the bacteria. In this study, the involvement of glutathione in the increased stress resistance earlier observed in nutrient-starved P. fluorescens was not supported. The concentration of bacterial glutathione is suggested as a possible marker for rhizosphere competence, which, however, needs to be further evaluated with several strains of rhizobacteria.

Cadmium↗

A starvation-specific serine protease gene, isp6+, is involved in both autophagy and sexual development in Schizosaccharomyces pombe.

Schizosaccharomyces pombe isp6(+) gene encodes a vacuolar serine protease, which is specifically induced during nitrogen starvation. An isp6-disruption mutant, isp6Delta, grew normally under normal conditions but was defective in large-scale protein degradation during nitrogen starvation, a hallmark of autophagy. Vacuoles are the organelles for such drastic protein degradation but those of isp6Delta were apparently aberrant. isp6Delta was infertile under nitrogen source-free conditions with poor expression of ste11(+), a gene critical for sexual development. A protein kinase A-disruption mutant, pka1Delta, is prone to sexual development because expression of ste11(+) is derepressed. However, isp6Deltapka1Delta still showed defects in ste11(+) expression and sexual development under nitrogen source-free conditions. isp6Delta and isp6Deltapka1Delta were able to initiate sexual development to produce spores when only a small amount of a nitrogen source was present. Pat1 protein kinase negatively controls meiosis, and a temperature-sensitive mutant of pat1, pat1-114, initiates meiosis irrespective of ploidy at the restrictive temperature. However, isp6Deltapat1-114 did not start meiosis under nitrogen source-free conditions even at the restrictive temperature. These observations suggest that isp6(+) contributes to sexual development by providing a nitrogen source through autophagy.

Autophagy↗

The Ste16 WD-repeat protein regulates cell-cycle progression under starvation through the Rum1 protein in Schizosaccharomyces pombe.

The haploid cells of the fission yeast, Schizosaccharomyces pombe, are arrested in the G1-phase by nitrogen starvation and are committed to sexual reproduction (mating and sporulation). We isolated the sterile mutants which were defective in G1 arrest following nitrogen starvation. Genetic analysis of these mutants defined a single locus designated as ste16. The nucleotide sequence revealed that ste16+ encodes an 82-kDa protein containing eight WD40-repeats in its carboxy terminal half. The ste16 disruptant was viable, but arrested the cell cycle in the G2-phase after the nutritional down-shift. When transferred to fresh growth medium, the G2-arrested ste16Delta haploids resumed the mitotic cycle from the S-phase, resulting in diploidization. This diploidization phenomenon was completely suppressed by the null mutation of rum1 encoding the inhibitor of Cdc2 kinase. As the Rum1 protein level was remarkably elevated in the ste16Delta, the Ste16 protein negatively controls the Rum1 level. The loss of function of ste16 disturbs the cell-cycle progression and impairs the mechanism for the maintenance of ploidy.

Cell Cycle↗

Induction of jlbA mRNA synthesis for a putative bZIP protein of Aspergillus nidulans by amino acid starvation.

The jlbA (jun-like hZIP) gene of Aspergillus nidulans was isolated. The deduced amino acid motif of the C-terminal region of jlhA encodes a putative DNA-binding site composed of a basic amino acid domain and an adjacent leucine zipper motif. This region shares highest similarities to the C-terminal DNA-binding domain and the basic zipper (bZIP)-motifs of transcription factors like CPCA from A. niger, Gcn4p from Saccharomyces cerevisiae, human JUNB and c-JUN. The putative jlbA protein contains a PEST-rich region (an instability region rich in the amino acids proline, glutamic acid, serine and threonine) described to be implicated in protein stability. The jlbA mRNA formation is elevated up to 40-fold upon amino acid starvation induced by the addition of the false feedback inhibitor 3-amino-1,2,4-triazole. This induction is partially dependent and partially independent on the presence of the transcription factor CPCA. Therefore jlbA is a novel gene of A. nidulans which is transcriptionally activated by amino acid starvation conditions.

Amino Acid Sequence↗

Aerenchyma formation in roots of maize during sulphate starvation.

Young maize ( Zea mays L., Poaceae) plants were grown in a complete, well-oxygenated nutrient solution and then deprived of their external source of sulphate. This treatment induced the formation of aerenchyma in roots. In addition to the effect of sulphate starvation on root anatomy, the presence and location of superoxide anions and hydrogen peroxide, and changes in calcium and pH were examined. By day 6 of sulphate deprivation, aerenchyma started to form in the roots of plants and the first aerenchymatous spaces were apparent in the middle of the cortex. S-starvation also induced thickening of the cell walls of the endodermis. Active oxygen species appeared in groups of intact mid-cortex cells. Formation of superoxide anion and hydrogen peroxide was found in degenerating cells of the mid-cortex. Very few nuclei in the cortex of S-starved roots fluoresced, being shrunken and near to the cell wall. By day 12 of S-deprivation, a fully developed aerenchyma was apparent and there were only a few 'chains' of cells bridging hypodermis to endodermis and stele of roots. Cell walls of endodermis of S-starved roots increased 68% in thickness. Intensive fluorescence in the cell walls of the endodermal, hypodermal and to a lesser extent of epidermal cells was observed due to the formation of active oxygen species, while there was no fluorescence in the cortical cells. There was a higher Ca concentration in the cells walls of the endodermis and epidermis, compared to the rest of the S-starved root tissues. A higher pH was observed, mainly in the cell walls of the hypodermis and to a lesser extent in the cell walls of the endodermis. Superoxide anion and hydrogen peroxide was found in degenerating cells of the root cortex. There was no fluorescence of nuclei in the cortex of S-starved roots.

Acridine Orange↗

Phosphate starvation responses are mediated by sugar signaling in Arabidopsis.

Phosphate (Pi) is one of the least available plant nutrients in soils. It is associated with dynamic changes in carbon fluxes and several crucial processes that regulate plant growth and development. Pi levels regulate the expression of large number of genes including those involved in photosynthesis and carbon metabolism. Herein we show that sugar is required for Pi starvation responses including changes in root architecture and expression of phosphate starvation induced (PSI) genes in Arabidopsis. Active photosynthesis or the supplementation of sugar in the medium was essential for the expression of PSI genes under Pi limiting conditions. Expression of these genes was not only induced by sucrose but also detected, albeit at reduced levels, with other metabolizable sugars. Non-metabolizable sugar analogs did not induce the expression of PSI genes. Although sugar input appears to be down-stream of initial Pi sensing, it is absolutely required for the completion of the PSI signaling pathway. Altered expression of PSI genes in the hexokinase signaling mutant gin2 indicates that hexokinase-dependent signaling is involved in this process. The study provides evidence for requirement of sugars in PSI signaling and evokes a role for hexokinase in some components of Pi response mechanism.

Arabidopsis↗

Modification of p53 protein profile by gamma irradiation followed by methyl donor starvation.

The possible beneficial radio-protective effects of one-carbon transfer agents namely folate, choline and methionine have been the subject of extensive investigation. Ionizing radiation is known to extensively damage the DNA. One-carbon transfer agents have been proposed to have important role in context of DNA repair via their role in purine and thymidylate synthesis and in DNA methylation. Sufficient dietary availability of one-carbon transfer agents therefore, might have ability to modify radiation effects. In present study modifications in level of tumor suppressor protein p53 by gamma irradiation followed by methyl donor starvation was observed. Experiments showed an increase in nuclear and cytoplasmic p53 protein concentration in liver, spleen and thymus. The overall rise in the level of p53 protein in liver was found to be less than that in spleen and thymus. Moreover significant heterogeneity in the basal level of expression of the p53 protein in liver, spleen and thymus was observed as the level of p53 protein in spleen and thymus was found to be 7-8 fold more than that in liver. Results indicated that radiation stress followed by methyl donor starvation could significantly induce p53 protein in spleen and thymus where there was a dramatic accumulation of p53 following irradiation, while in other tissues, particularly the liver, no such dramatic response was seen. Folate contribution of intestinal bacteria was found to influence p53 protein levels. Our observations indicated a prominent role played by the methyl donors in protecting the cell against harmful effects of ionizing radiation.

Animals↗

Modulation of DNA methyltransferase profile by methyl donor starvation followed by gamma irradiation.

DNA methylation is an important epigenetic mechanism of transcriptional control, which plays an essential role in maintaining cellular function. Role of one-carbon transfer agents/methyl donors namely folate, choline and methionine in DNA methylation has been the subject of extensive investigation. The methylation pattern of DNA is established during embryogenesis by DNA methyltransferase 3 (dnmt3) and is subsequently maintained by maintenance methylation activity of the enzyme DNA methyltransferase 1 (dnmt1). Ionizing radiation is known to extensively damage the DNA. Sufficient dietary availability of methyl donors is known to contribute towards one-carbon transfer mediated repair of damaged DNA where folate is involved in nucleotide base synthesis. In the present study, modification in activities of dnmt1 and dnmt3 by methyl donor starvation followed by gamma-irradiation was observed. Assays were based on the catalytic transfer of (3)H-methyl groups from S-adenosyl-L: -methionine to a DNA substrate. Experiments showed a dose and methyl donors starvation dependent attenuation in dnmt1 activity. Attenuation of dnmt1 activity was most significant for diet deprived of all the three-methyl donors. No significant change in nuclear or cytoplasmic dnmt3 activity was observed when either or all the three possible source of dietary methyl group supply were removed. Ionizing radiation and methyl donor deficiency were observed to act synergistically towards inhibiting dnmt1 activity. Present results suggested possibility of interaction among folate, methionine and choline deficiency to potentiate symptoms of ionizing radiation stress. These enzymatic modifications might contribute to altered DNA methylation after chronic feeding of methyl donor free diets followed by gamma irradiation. These results suggested that dietary availability of methyl donors and gamma-radiation stress might significantly alter the dnmt1 profile.

Animals↗

Protein synthesis in rat maternal and fetal tissues with starvation.

The incorporation a of 3H-leucine was studied in male rats and female rats 14 and 19 days pregnant that were either fed or starved for 48 hours. 3H-leucine incorporation into the liver was similar among all the fed animals. There was a 40% to 50% increase in the 3H-leucine incorporation of the placenta and fetus per milligram of ribonucleic acid from the fourteenth to the nineteenth day of pregnancy. With starvation there was a significant increase in 3H-leucine incorporation in both the fetus and the placenta of the 19-day-pregnant animal associated with a significant decrease in liver incorporation. Yolk sac placenta demonstrated a greater increase than the allantoic placenta. Both the male and 14-day-pregnant animals had minimal changes with starvation. The problems inherent in studying protein synthesis, especially during pregnancy, are presented.

Animals↗

Synthesis of inducible enzyme in Escherichia coli recovering from prolonged energy starvation.

A marked breakdown of ribosomes and rRNA occurs in Escherichia coli cells during prolonged deprivation of a carbon source (energy starvation). In E. coli recovering from energy starvation: (a) synthesis of RNA started immediately, total protein synthesis showed a delay of 5 to 10 minutes; (b) beta-galactosidase, tryptophanase and serine deaminase could not be induced in the first 50--70 min; (c) a lag of 60 min in the synthesis of beta-galactosidase was observed in a lac constitutive mutant of E. coli; synthesis of the constitutive enzyme malate dehydrogenase did not shown any delay. RNA synthesized in the early stages of recovery contained a higher percentage of low molecular weight molecules than RNA synthesized after 70 min of recovery or during exponential growth. Messenger RNA specific for beta-galactosidase was not synthesized for the first 50--60 min of recovery even when the specific inducer was added to the cultures.

Carbon↗

General and specific effects of amino acid starvation on the formation of undermodified Escherichia coli phenylalanine tRNA.

The heterogeneity of undermodified phenylalanine tRNA produced in relaxed control E. coli during amino acid starvation was investigated. Examination of the RPC-5 elution profiles of tRNAPhe prepared from non-starved cells and cells starved of a variety of amino acids, including some known to be involved in the formation of modified bases revealed that: (1) only one species of fully modified tRNAPhe appears to occur in cells grown in enriched medium; (2) at least two chromatographically unique isoacceptor species are observed in addition to the normal tRNAPhe in starved cells; (3) the unique, undermodified species of tRNAPhe from leucine-starved cells, known to be deficient in dihydrouridine, pseudouridine, 2-thiomethyl-N6-(delta2-isopentenyl) adenosine and 3-(3-amino-3-carboxypropyl) uridine, co-elute with the unique species produced in cells starved of histidine or arginine or treated with puromycin or chloramphenicol; (4) additional unique species of tRNAPhe can be detected in methyl- and sulfur-deficient tRNA from methionine- and cysteine-starved cells; (5) analysis of phenoxyacetylated tRNA revealed that the chromatographically unique and normal species from starved cells contain subspecies deficient in 3-(3-amino-3-carboxypropyl) uridine; and (6) using phenoxyacetylation as a means of effecting the resolution of undermodified subspecies, a total of at least ten chromatographically unique subspecies of rRNAPhe were detected in an organism that appears to posses only one gene for tRNAPhe. Taken together, the results support the view that there are both general and specific effects of amino acid starvation on the post-transcriptional modification of tRNA.

Amino Acids↗