Sizes of G1 and G2 populations in starved Tetrahymena

Feulgen cytophotometry and autoradiography were used to study DNA content and DNA synthesis in starved and starved-refed Tetrahymena pyriformis GL-C. It was found that (1) the cell population shows a limited increase in cell number during starvation and this increase is restricted to the first 7 h of starvation; (2) at the end of starvation, there is a portion of the cell population whose DNA content is similar to that for standard G2 cells; (3) a significant portion of the dividing cells at the first division following refeeding in the presence of [³H]TdR are unlabeled; (4) these unlabeled cells are among the first to divide and, upon division, generally enter into a cell cycle either lacking a G1 phase or with a shortened G1 phase.     

The influence of starvation and different diets on the hindgut of isopoda (Mesidotea entomon,Oniscus asellus,Porcellio scaber)

Summary Under conditions of food deprivation, the hindgut epithelium of the experimental animals (Mesidotea entomon, Oniscus asellus, Porcellio scaber) undergoes ultrastructural changes. After the application of different diets it was demonstrated that this part of the alimentary canal contains nutrients, though it is lined by a cuticle. Experimental evidence for the formation of glycogen from glucose offered as the only diet comes from autoradiographic experiments. Amino acids, too, were detected in the hindgut cells soon after refeeding. Lipids, on the other hand, which are first absorbed by the large cells of the midgut glands, were not found in the hindgut epithelium. The existence of lipid inclusions in the hindgut epithelium some weeks after refeeding, however, supports the hypothesis that lipids reach the epithelial cells of the hindgutvia midgut glands and hemolymph.     

Nuclear DNA in normal and refed Amoeba proteus

Amoeba proteus synthesizes DNA in G2 phase of the cell cycle upon feeding after starvation. The characteristics of the DNA synthesized in G2 have been studied by microscope photometry of individual Feulgen-stained nuclei and by buoyant density centrifugation of nuclear DNA in CsCl. Amoeba nuclei were found to contain 42.8 pg of DNA. This DNA bands in CsCl at a density of 1.693 g/cm³ with a satellite at 1.714 g/cm³ which makes up 24% of nuclear DNA. DNA from whole cells has an additional non-nuclear satellite at 1.726 g/cm³. When cells are starved and re-fed with food labeled with [³H]thymidine, the DNA synthesized is predominantly the 1.714 satellite. The amount of DNA synthesized in G2 is small since there is no measurable difference in Feulgen dye binding to nuclei of starved vs starved and re-fed cells. The data suggest that refeeding induces a resumption of late S phase DNA synthesis, or the preferential synthesis of specific DNA sequences such as rRNA genes.     

Divider size and the cell cycle after prolonged starvation ofTetrahymena corlissi

Cell growth and division of the ciliateTetrahymena corlissi were examined upon refeeding after prolonged starvation of up to 12 days. Division did not automatically occur when a certain critical cell size was reached. Rather, it varied both with the nutritional history of the cell and the nutrient conditions in which the cell was growing. Upon refeeding, cells starved for 12 days divided at a smaller size and later than cells starved for 6 days. Cells refed at high density took longer to begin division than cells refed at low density. The results are discussed with respect to the relative starvation and critical constituent models of the cell cycle and in terms of the polymorphic life cycle ofTetrahymena species.     

Timing of tRNA and 5S rRNA gene replication in Tetrahymena pyriformis

The timing for replication of the genes coding for tRNA and 5S rRNA has been studied in Tetrahymena pyriformis. The cells were synchronized by two different procedures, known to synchronize not only cell divisions but also the macronuclear DNA replication, namely (1) the heat-shock procedure described by Zeuthen [12] and (2) the starvation/refeeding procedure described by Cameron & Jeter [13]. The DNA replication was followed by addition of 5-bromodeoxyuridine (BUdR) prior to a synchronous DNA replication. DNA was isolated at various times during replication, analysed by CsCl gradient centrifugation and hybridization with tRNA and 5S rRNA. The results show that the replication of the genes for tRNA and 5S rRNA follows the replication of the bulk macronuclear DNA.     

Mating type differentiation in tetrahymena thermophila: Characterization of the delayed refeeding effect and its implications concerning intranuclear coordination

Mating type determination in Tetrahymena thermophila involves developmentally programmed, heritable alterations of the macronucleus, localized to the mtd locus. This determination can be predictably controlled by the environmental conditions during macronuclear development, eg, temperature and time of refeeding. In this article we have further characterized the effects of delayed refeeding on mating type determination, as revealed by the frequency of mating types among the progeny of a cross. Our results show that 1) the magnitude of this starvation effect decreases with temperature of conjugation and becomes undetectable at 18°C; 2) starvation during the interval 14 to 22 hr (after conjugation is induced at 30°C) is a necessary and sufficient condition for the induction of starvation effects; 3) relative mating type frequencies vary monotonically with nutrient concentration present during this critical period; and 4) sister macronuclei, developing under starvation conditions in the same cytoplasm, differentiate majority mating types characteristic of early or late refeeding; sister macronuclei show no apparent correlation with each other. On the basis of our observations on early and late refed cells, we propose that the composition of the newly developed macronucleus is the outcome of two key events: 1) mating type determination at the mtd locus and 2) differential molecular cloning of generally one or two autonomously replicating fragments (ARFs) of the macronuclear DNA bearing the mtd locus.     

Uncoordinate synthesis of histone H1 in cells arrested in the G1 phase

It has been known for several years that DNA replication and histone synthesis occur concomitantly in cultured mammalian cells. Normally all five classes of histones are synthesized coordinately. However, mouse myeloma cells, synchronized by starvation for isoleucine, synthesize increased amounts of histone H1 relative to the four nucleosomal core histones. This unscheduled synthesis of histone H1 is reduced within 1 h after refeeding isoleucine, and is not a normal component of G1. The synthesis of H1 increases coordinately again with other histones during the S phase. The DNA synthesis inhibitors, cytosine arabinoside and hydroxyurea, block all histone synthesis in S-phase cells. The levels of histone H1 mRNA, relative to the other histone mRNAs, is increased in isoeleucine-starved cells and decreases rapidly after refeeding isoleucine. The increased incorporation of histone H1 is at least partially due to the low isoleucine content of histone H1. Starvation of cells for lysine resulted in a decrease in H1 synthesis relative to core histones. Again the ratio was altered on refeeding the amino acid. 3T3 cells starved for serum also incorporated only H1 histones into chromatin. The ratio of different H1 proteins also changed. The synthesis of the H1⁰ protein was predominant in G₀ cells, and reduced in S-phase cells. These data indicate the metabolism of H1 is independent of the other histones when cell growth is arrested.     

The effect of nitrogen refeeding on the carbohydrate content into nitrogen-starved cells of Platymonas striata Butcher

Studies on the mean cellular carbohydrate contents of Platymonas striata Butcher under conditions of nitrogen-starvation, and after refeeding these starved cultures with either nitrate or ammonium ions (growing under continuous illumination or with an alternating light/dark regime) have shown that nitrogen-starved cells accumulated abnormal amounts of cellular carbohydrate and that nitrogen refeeding produced a marked drop in the cellular carbohydrate. Cells grown in a light/dark regime accumulated less carbohydrates than those grown in continuous light. The mean cellular carbohydrate levels 16 h after nitrogen refeeding were still much in excess of those of cells grown with normal nutrition. It was therefore suggested that the differences in nitrogen uptakes in this period — when comparing either the uptake of cells grown in continuous light with that of cells grown in a light/dark regime; or when comparing the uptakes of cells presented with either nitrate or ammonium ions and grown in a light/dark regime —cannot be directly due to shortages of carbohydrate for the provision of carbon skeletons for nitrogen assimilation.     

The increases in the rates of synthesis of ribosomal proteins and ribosomal RNA during refeeding of starved Tetrahymena cells are not dependent on DNA replication

We have analysed the effects of an inhibition of DNA replication by hydroxyurea on the synthesis of ribosomal proteins (r-proteins) and ribosomal RNA (rRNA) in Tetrahymena cells resuming growth after long-term starvation. The coordinate regulation of the synthesis of individual r-proteins and their increased rate of synthesis during refeeding are not impaired by inhibition of DNA replication. Moreover, the presence of hydroxyurea does not prevent an increase in the rate of synthesis of rRNA around 70-80 min after refeeding. Previously, this increase was claimed to be gene dose-dependent. Up to 180 min after refeeding, the synthesis of r-proteins appears to be closely coupled with that of rRNA and proceed in stoichiometric balance, irrespective of whether hydroxyurea is present or not. After 180 min of refeeding in the presence of hydroxyurea, this stoichiometric balance breaks down, and the rate of synthesis of r-proteins clearly exceeds that of the rRNA synthesis.     

Re-examination of some species of the genus Geotrichum Link: Fr

The nutritional physiology and the growth rate of thirty-four strains representing species of Geotrichum without known teleomorph states were examined. From twenty-seven strains the mol% G+C were calculated from the DNA melting curves. The first derivatives of the melting curves of seven strains, including the type strain of Geotrichum clavatum, demonstrated the presence of two peaks, 12% away from each other; the remaining strains showed only a single broad peak. DNA homology values among strains of the former group were high, indicating their conspecificity. The strains of the latter group could be subdivided into six DNA homology groups, four of which could be identified with recognized species and two may represent novel taxa. A combined key of Geotrichum and its teleomorph states Galactomyces and Dipodascus is presented.     

Effect of cell population density on G2 arrest in Tetrahymena

The ciliated protozoan, Tetrahymena pyriformis strain GL-C, has been used to study the effect of cell population density during starvation on the synchrony obtained after refeeding and on the number of cells arrested in G2 phase of the cell cycle. At high cell densities two peaks of division indices were observed after refeeding while only one was observed at low cell densities. Cell division began earlier in cultures starved at high cell densities. Most importantly, the proportion of cells in G2 was considerably higher in populations starved at high cell densities. When tritiated thymidine was present during the refeeding period, radioautographs of cell samples at different times showed that the first cells to exhibit division furrows contained unlabeled nuclei. The first peak in the division index after refeeding was observed only at higher cell densities and is attributed to the cells arrested in G2. These results suggest that Tetrahymena is an excellent organism to study the concept of resting stages in the cell cycle and their control.     

Nutritional supplement chromium picolinate generates chromosomal aberrations and impedes progeny development in Drosophila melanogaster

Chromium picolinate, [Cr(pic)₃], is a popular nutritional supplement found in a variety of consumer products. Despite its popularity, safety concerns over its use have arisen. The supplement has been shown to generate clastogenic damage, mitochondrial damage, oxidative damage, and mutagenic effects in cultured cells and oxidative DNA damage and lipid peroxidation in rats. Recently [Cr(pic)₃] has been demonstrated to generate heritable genetic change and delays in progeny development in Drosophila melanogaster. Based on the damage to chromosomes of cultured cells and of animal models, similar chromosome damage appeared to be a likely source of the mutagenic effects of the supplement in Drosophila. The current three-part study examines the effects of several chromium-containing supplements and their components on hatching and eclosion rates and success of development of first generation progeny of adult Drosophila fed food containing these compounds. It further examines the effects of the compounds on longevity of virgin male and female adults. Finally, the chromosomes in the salivary glands of Drosophila late in the third instar larval stage, which were the progeny of Drosophila whose diets were supplemented with nutritional levels of [Cr(pic)₃], are shown to contain on average over one chromosomal aberration per two identifiable chromosomal arms. No aberrations were observed in chromosomes of progeny of untreated flies. The results suggest that human consumption of the supplement should be a matter of concern and continued investigation to provide insight into the requirements of chromium-containing supplements to give rise to genotoxic effects.     

Autogamy in Paramecium cell cycle stage-specific commitment to meiosis

Autogamy is a process of meiosis and fertilization which takes place in unpaired Paramecium cells, and which is triggered by starvation. This study examines the consequences of nutritional down-shift at various points within the cell cycle on the occurrence of autogamy. It shows that cells become committed to autogamy in a two-step process. An initial point of commitment to autogamy occurs about 100 min prior to the median time of cell division (cell cycle duration, 330 min). Cells which have become committed to autogamy initiate meiosis following the next fission, others complete another vegetative cell cycle before undergoing meiosis. Treatments that perturb the cell cycle and displace the point of commitment to division also displace the point of initial commitment to autogamy to the same extent.The initial commitment to autogamy can be reversed by refeeding. The second, final, point of commitment to autogamy occurs about 30 min after the fission, immediately prior to initiation of meiosis, and coincides with the beginning of meiosis. If cells are refed at this point, or at later stages, autogamy continues.Autogamy is not well synchronized either in naturally starved cultures or in those subjected to abrupt nutritional down-shift. This is a consequence of the cell cycle stage dependence of entry into autogamy. Autogamy occurs synchronously in samples of dividers selected from asynchronous cultures 2 or more hours after nutritional down-shift. The timing of the events of conjugation and autogamy coincide when the pre-autogamous fission is aligned temporally with the initial contact of mating cells.     

THE OCCURRENCE AND PHYLOGENETIC SIGNIFICANCE OF PUTATIVE PLACENTAL TRANSFER CELLS IN THE GREEN ALGA COLEOCHAETE

Following fertilization, zygotes of the green alga Coleochaete orbicularis, which are retained on the haploid thallus, first enlarge, then become covered with a layer of vegetative cells. Light microscopy and high-voltage electron microscopy revealed the presence of localized wall ingrowths in vegetative cells adjacent to zygotes. These covering cells resemble the gametophytic placental transfer cells of embryophytes in their morphology, location, and time of development. If Coleochaete cells with wall protuberances function as do placental transfer cells of embryophytes, their presence is evidence that photosynthates may be transported between haploid thallus cells and zygotes. Thus, a nutritional relationship between different phases of the life cycle, similar to that which occurs in embryophytes, may also have evolved in green algae. This first report of putative placental transfer cells in a green alga supports Bower's (1908) ideas concerning the origin of land plant sporophytes and alternation of generations. The presence or absence of cells with wall ingrowths in several species of Coleochaete was correlated with estimates of zygote-plant area ratios.     

Resumption of DNA Synthesis and Cell Division in Wheat Roots as Related to Lateral Root Initiation

The arrest of DNA synthesis and termination of cell division in basal meristematic cells as well as the resumption of these processes as related to the initiation of lateral root primordia (LRP) were studied in tissues of Triticum aestivumroots incubated with ³H-thymidine. All cells of the stelar parenchyma and cortex as well as most endodermal and pericycle cells left the mitotic cycle and ceased proliferative activity at the basal end of the meristem and at the beginning of the elongation zone. Some endodermal and pericycle cells started DNA synthesis in the basal part of the meristem and completed it later on during their elongation, but they did not divide. In the cells of these tissues, DNA synthesis resumed above the elongation zone, the cells being located much closer to the root tip than the first newly dividing cells. Thus, the initiation of LRP started much closer to the root tip than it was previously believed judging from the distance of the first dividing pericycle cells from the root tip. DNA synthesizing and dividing cells first appeared in the stelar parenchyma, then, in the pericycle, and later, in the endodermis and cortex. It seems likely that a release from the inhibition of DNA synthesis allows the cells that completed mitotic cycle in the basal part of meristem in the G₁phase to cease the proliferative arrest above the elongation zone and to continue their cycling. The location of the first DNA synthesizing and dividing cells in the stelar parenchyma and pericycle did not strictly correspond to the LRP initiation sites and proximity to the xylem or phloem poles. This indicates that LRP initiation results from the resumption of DNA synthesis in all pericycle and stelar parenchyma cells that retained the ability to synthesize DNA and occurs only in the pericycle sector situated between the two tracheal protoxylem strands, all cells of which terminated their mitotic cycles in the G₁phase.     

An examination of the efficiency of glucose and glutamine as energy sources for cultured chick pigment epithelial cells

Embryonic chick pigment epithelial cells in culture require glucose as their major energy source for long-term growth, pigment formation, and colony organization. Cell number increases with glucose concentration at least up to 5.0 mM. Cells can be grown with glutamine as the major energy source but produce comparable cell numbers for only the first 3 days in culture, after which they cease growing. However, they are able to metabolize glutamine at a two to sixfoid higher rate than cells grown in the presence of glucose as measured by CO₂ release and by incorporation into protein. In cells grown in the presence of both glucose and glutamine, basal ATP levels were 31.1 nmoles/mg protein; P-creatine averaged 15.2 nmoles/mg protein and showed marked variability between experimental groups. During starvation, P-creatine levels fell while ATP levels remained relatively constant. Glucose was required for the recovery of P-creatine to prestarvation levels when measured 5 min after refeeding. Because of these marked changes in P-creatine concentration as a function of nutritional status, the ATP/P-creatine ratio becomes a useful measure of the energy state of the cell.     

RELATIONSHIP OF NUTRITIONAL FACTORS TO IN VITRO TUMOR CELL GROWTH AND CYTOTOXICITY PRODUCED BY CYTOSINE ARABINOSIDE

The in vitro relationship between nutritional factors, proliferative status of tumor cells, and the cytotoxic action of cytosine arabinoside (ara-C) was investigated. The reduction in the concentration of only one essential amino acid, L-isoleucine, in the growth medium of A(T₁)Cl-3 hamster fibrosarcoma cells decreased DNA synthesis in this cell population and slowed the rate of progression of G₁ phase cells into S phase of the cell cycle. The complete omission of isoleucine from the growth medium blocked the progression of G₁ phase cells into S phase and prevented the cytotoxic action of ara-C. The addition of isoleucine to the isoleucine-deprived cells permitted these cells to enter the S phase and restored their sensitivity to the cytotoxic action of ara-C. When G₁ phase cells were placed in a medium containing reduced levels of all the amino acids and vitamins there was a prolongation of the G₁ phase. Since medium with low levels of amino acids produced a delay in the entry of G₁ phase cells into the S phase, the time interval in which these cells were most sensitive to the cytotoxic action of ara-C was different for G₁ phase cells placed in medium with adequate levels of all the amino acids. These in vitro data indicate that nutritional factors can markedly effect the proliferation of tumor cells and the cytotoxic action of ara-C.     

DNA synthesis in Escherichia coli during a nutritional shift-up

Summary DNA synthesis in a thymine-requiring Escherichia coli K12 strain was studied by exploiting deoxyguanosine, so simulating the behaviour of Thy⁺ strains. DNA synthesis is inhibited during the first 25 min after a nutritional shift-up. The new DNA/mass is lower than that predicted by current models for initiation control.Dedicated to the memory of Shmuel Zabrovitz, whose high spirits and good humor enabled him to complete the work while struggling with his lethal diseaseDeceased     

Assessing the capacity for compensatory growth in growth‐hormone transgenic coho salmon Oncorhynchus kisutch

The effect of feed cycling (consisting of periods of starvation followed by periods of refeeding to satiation) on compensatory growth was evaluated in growth hormone transgenic and non‐transgenic wild‐type coho salmon Oncorhynchus kisutch. The specific growth rate (G_SR) of feed‐restricted non‐transgenic O. kisutch was not significantly different from the G_SR of fully‐fed non‐transgenic O. kisutch during two refeeding periods, whereas the G_SR of feed‐restricted transgenic O. kisutch was significantly higher in relation to the G_SR of fully‐fed transgenic O. kisutch during the second refeeding period, but not during the first, indicating that growth compensation mechanisms are different between non‐transgenic and growth‐hormone (GH)‐transgenic O. kisutch and may depend on life history (i.e. previous starvation). Despite the non‐significant growth rate compensation in non‐transgenic O. kisutch, these fish showed a level of body mass catch‐up growth not displayed by transgenic O. kisutch.     

Stem cell dynamics in response to nutrient availability

When nutrient availability becomes limited, animals must actively adjust their metabolism to allocate limited resources and maintain tissue homeostasis. However, it is poorly understood how tissues maintained by adult stem cells respond to chronic changes in metabolism. To begin to address this question, we fed flies a diet lacking protein (protein starvation) and assayed both germline and intestinal stem cells. Our results revealed a decrease in stem cell proliferation and a reduction in stem cell number; however, a small pool of active stem cells remained. Upon refeeding, stem cell number increased dramatically, indicating that the remaining stem cells are competent to respond quickly to changes in nutritional status. Stem cell maintenance is critically dependent upon intrinsic and extrinsic factors that act to regulate stem cell behavior. Activation of the insulin/IGF signaling pathway in stem cells and adjacent support cells in the germline was sufficient to suppress stem cell loss during starvation. Therefore, our data indicate that stem cells can directly sense changes in the systemic environment to coordinate their behavior with the nutritional status of the animal, providing a paradigm for maintaining tissue homeostasis under metabolic stress.