Biochemical changes accompanying the long-term starvation of Micrococcus luteus cells in spent growth medium

Changes in the biochemical properties of Micrococcus luteus cells were studied during the transition to a dormant state after incubation in an extended stationary phase. The overall DNA content after 150 days of starvation was similar to its initial level, while the RNA content decreased by 50%. Total lipids and protein, phospholipids and membrane proteins declined rapidly within the first 1–10 days of starvation. After 180 days of starvation, cells contained 43% of the protein and 35% of the lipid initially present. Starvation for 120 days resulted in the loss of phosphatidylglycerol and, to some extent, of phosphatidylinositol, giving a membrane whose phospholipids consisted mainly of cardiolipin. The membrane fluidity declined during starvation, as judged by diphenyl hexatriene fluorescence anisotropy measurements. Oxidase activities declined to zero within the first 20–30 days of starvation, while the dehydrogenases and cytochromes were more stable. The activities of some cytoplasmic enzymes were lost very rapidly, while NADPH-linked isocitrate dehydrogenase had 30% of its initial activity after 120 days of starvation. For all parameters tested there were significant fluctuations during the first 10–20 days of starvation, which may reflect cryptic growth in the culture.Abbreviations MPN Most probable number - DPH Diphenyl hexatriene     

Effect of low-nutrient seawater on morphology,chemical composition,and virulence of Salmonella typhimurium

The response of Salmonella typhimurium to low nutrient levels was determined by measuring the concentrations of lipids, carbohydrates, DNA, RNA, and proteins over a 32-day starvation period. Ultrastructural integrity was observed by transmission electron microscopy. Lipid and carbohydrate content of bacterial cells rapidly declined within the first 16 days, while DNA and proteins exhibited a more gradual decline over the 32 days of starvation. In contrast, RNA content did not decrease appreciably upon nutrient starvation. Structural damage occurred especially after 16 days of starvation. After 32 days of nutrient deprivation, we recorded degenerative cellular forms, a coccoidal cell shape, a decrease in cellular volume, and the loss of the three-layered outer membrane. The morphological and structural alterations correlated with virulence in infected animals. We observed a decrease in virulence of S. typhimurium after 9, 16, and 32 days of starvation, reaching a maximal decrease after 32 days of nutrient deprivation. The decrease in virulence correlated to surface hydrophobicity alterations, adherence to eukaryotic cells, and phagocytosis.Abbreviations BHI brain heart infusion - PBS phosphate-buffered saline - TE Tris-EDTA buffer - F _t phagocytic index - K _t Killing index     

On the mechanism of conjugation in Escherichia coli K 12

Summary The phenomenon of conjugation consists of many stages. The most important are: the formation of contacts between mating cells, the transfer of DNA from the donor to the recipient, and the integration of the transfered DNA fragments into the chromosome of the recipient. Only after completion of all these stages are recombinants formed. With the aid of specific inhibitiors (nalidixic acid, FUDR), thymine starvation, and use of special thermosensitive mutants it is possible to study the role of DNA synthesis during every stage of conjugation. It was demonstrated that the genetic transfer is due to semiconservative DNA-replication in the donor cell. The fragments of DNA transfered are synthesized in the period of mating by a special replication system (F-replicon). In case of T _DNA ^S mutants unable to grow at 41°, the ability to synthesize DNA during conjugation is preserved.The inhibition of the DNA synthesis in the donor cell by poisons leads to complete inhibition of genetic transfer. The third stage — formation of recombinants requires DNA synthesis in the recipient cell and is inhibited by poisoning, thymine starvation or T _DNA ^S mutations in the recipient. In cases where recombination is not involved (i.e. sexduction) the inhibition of DNA synthesis in the recipient has no significant effect.     

CELL ARREST IN Gl AND G2 OF THE MITOTIC CYCLE OF VICIA FABA ROOT MERISTEMS

Experiments were performed with cultured excised primary root tips of Vicia faba ‘Longpod’ to determine: (1) the proportion of meristematic cells arrested in Gl and in G2 during carbohydrate starvation, and to determine if the proportion is fixed or can be varied experimentally; (2) the effect of increased starvation on the ability of arrested cells in Gl and G2 to initiate DNA synthesis and mitosis, respectively, when exogenous sucrose was supplied; and (3) whether puromycin, cycloheximide, or actinomycin D prevented the initiation of DNA synthesis and the onset of mitosis. Microspectrophotometry of nuclear DNA and autoradiographic measurements of incorporated ³H-thymidine showed that 72 hr of starvation immediately after excision produced tissue with more than 70 % of the cells arrested in G2 and less than 30 % in Gl. If cultured for three days and then starved for 72 hr, the tissue had nearly equal numbers of cells arrested in Gl and G2. As the duration of starvation increased, the time required to initiate DNA synthesis and to divide when carbohydrate was replenished also increased. Inhibition of protein synthesis by puromycin and cycloheximide prevented the initiation of DNA synthesis and mitosis, but actinomycin D, an inhibitor of RNA synthesis, did not prevent division of cells from G2 nor DNA synthesis by cells from Gl. The experiments demonstrated that the mitotic cycle of Vicia has two major controls, one in Gl and another in G2, and that other factors determine how many cells are affected by either of these cycle controls.     

Starvation/stationary-phase survival of <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> SQ1: a physiological and genetic analysis

The adaptation of Rhodocccus erythropolis SQ1 to energy and carbon starvation was investigated in terms of both the capacity to survive starvation and the contribution of a nutrient-induced stationary phase to cross-protection to other types of environmental stress. It was found that R. erythropolis SQ1 survives for at least 43 days in LB and distilled water, and 65 days in chemically defined medium (CDM) containing high (1%) or low (0.1%) glucose. Furthermore, early stationary-phase R. erythropolis SQ1 grown in CDM 0.1% exhibited enhanced resistance to heat and oxidative stress compared with exponential-phase cells. A second objective of this study was to identify genetic elements involved in starvation/stationary-phase survival. A mutant bank of R. erythropolis SQ1 generated by random transposon insertion mutagenesis was screened; four mutants lost culturability when grown in CDM 1%. No drop in culturability was observed when these mutants were grown in CDM 0.1%. The DNA flanking transposon insertion could be recovered from three mutants. Transposon insertions were found in uvrB (UvrB, part of the DNA excision repair mechanism), between a putative guaB gene and another guaB-like gene, and between a gene encoding a putative phosphoglycerate mutase and putative thioredoxin/cytochrome c biogenesis genes. This represents a first study of the starvation/stationary-phase survival response of Rhodococcus, an organism of immense significance in environmental bioremediation and a number of industrial processes.     

不同方法处理供核细胞对细胞周期和重构胚发育的影响

利用流式细胞仪和细胞染色体核型分析技术,比较奶牛的转基因体细胞和正常细胞经血清饥饿、抑制培养周期同步化处理后的G0/G1期细胞比例;并将同步化处理的核供体细胞进行核移植,然后统计囊胚发育率.结果表明,血清饥饿和抑制培养均能获得较高比例的G0/G1期细胞,两组间差异不显著(P>0.05),但均显著高于未处理对照组(P<0.05);血清饥饿组的囊胚率显著高于抑制培养组和非处理对照组(P<0.05);但细胞同步化处理6 d后细胞染色体核型异常率增加.因此,要获得正常核型的G0/G1核移植供体细胞和较高的囊胚率,同步化处理时间以不超过4 d为宜.     

家兔供体细胞的发育周期与重构胚发育的关系

采用血清饥饿法处理体外培养的兔子胎儿成纤维细胞,并将其作为供体细胞移入去核卵母细胞内构建重构胚胎。检查供体细胞的细胞周期对重构胚的融合率、分裂率和着床率的影响。实验结果表明：培养基中血清含量在0．5％的情况下,G0／G1期的细胞比例由正常培养条件下(培乔液中含有10％FCS)的73．2％明显地增加到86％以上。饥饿1～3天的细胞作为供体细胞构建重构胚时,可明显提高重构胚的融合率,但是不同的饥饿时间其融合率并无显著的差异。饥饿处理可明显增加重构胚的分裂率,以饥饿处理3天为最佳。     

Studies on genes and biochemical events induced by carbon-source starvation in plant cells

Carrot (Daucus carota L.) suspension cells exhibit a number of physiological responses when carbon sources in the medium are depleted (i.e., carbon-source starvation). We previously reported that activities of several phospholipid catabolic enzymes, such as phospholipase D (PLD) and lipolytic acyl hydrolase (LAH), are induced to provide cells with alternative carbon sources. In this study we report sequence of PLD cDNA. When starvation was prolonged over approximately five days, cells started to die. To analyze the initiation of cell death, we examined the degradation of DNA and activity of DNA endonuclease. Preliminary results showed that DNA degradation occurred at the onset of cell death. Our findings suggest that carrot cells exhibit two different phases-acclimation response and cell death-during starvation. In working toward a long-term objective of understanding the whole scope of biochemical events during starvation, we have also catalogued the genes induced by starvation.     

短期饥饿和再投喂对岩原鲤仔鱼生存生长以及RNA/DNA和RNA/蛋白质比率的影响(英文)

研究通过对岩原鲤仔鱼在饥饿和再投喂条件下其生存、生长率、RNA/DNA和RNA/蛋白质比率的测定,评估了仔鱼对饥饿的耐受能力和恢复能力。在(19.5±0.5)℃水温下,将岀膜后第16天的岩原鲤仔鱼随机分成6个组:1个持续投饲对照组,实验组分别禁食1、2、3、4、5d后再投喂,实验共进行10d。每天分别从各组取9尾鱼测定体重、体长、RNA、DNA、蛋白质含量。实验结果显示,饥饿处理组仔鱼存活率和以上各项生长指标均随饥饿时间的增加而下降,在恢复投喂后均表现不同程度的补偿生长,其中饥饿1、2、3d的仔鱼在恢复投喂后显示出完全补偿生长,几乎弥补了饥饿所产生的影响,平均终体重与对照组比较无显著差异。饥饿4、5d的仔鱼显示部分补偿生长,恢复投喂只少量减轻了饥饿的影响,平均终体重与对照组相比存在显著差异。饥饿1、2、3d的仔鱼和4、5d的仔鱼在恢复投喂后分别需要1—2d和4d时间才能达到与对照组无显著差异水平。仔鱼生长率变动范围从0.59%到8.00%WW/day,仔鱼RNA/DNA比率、RNA/蛋白质比率与生长率的回归方程为:GR=3.63RNA/DNA 1.74(R2=0.80)和GR=120.14RNA/Protein 2.33(R2=0.31),两种比率均与生长率呈显著线性相关,RNA/DNA比率对生长变化的拟合度更好。结果表明,仔鱼阶段食物缺乏很可能是影响岩原鲤仔鱼存活、生长的主要因素。RNA/DNA更适合作为评定岩原鲤仔鱼营养条件和生长的指标。     

Inhibition of apoptosis in serum starved porcine embryonic fibroblasts

In nuclear transplantation, serum starvation is a general method to synchronize donor cells at the quiescent stage (G(0)) of the cell cycle. However, serum starvation during culture of mammalian cells may induce cell death, especially through apoptosis, thus contributing to the low efficiency of nuclear transplantation. This study was performed to characterize apoptosis during serum starvation and to determine the effects of apoptosis inhibitors such as a protease inhibitor [alpha(2)-macroglobulin (MAC)] and antioxidants [N-acetylcysteine (NAC), glutathione (GSH)] on serum starved porcine embryonic fibroblasts (PEF). PEF, collected from day 25-30 porcine fetuses, were cultured for 5 days in media containing 0.5% FBS to induce quiescence. Serum starved PEF showed typical morphology of apoptotic cells and stained for DNA fragmentation by TUNEL assay (26.7%). All apoptosis inhibitors tested in this study significantly (P < 0.05) reduced apoptosis of serum starved PEF, with antioxidants having better results (MAC: 7.4% vs. NAC: 1.0%, and GSH: 0.8%). Equally and importantly, the treatment with apoptosis inhibitors did not change the proportion of G(0)/G(1) stage cells. Therefore, the addition of MAC and antioxidants during serum starvation of PEF reduces apoptosis of quiescent fibroblasts and may contribute to increasing the efficiency of nuclear transplantation by improving the quality of donor nuclei.     

A Study of Factors Affecting the Efficiency of Electrofusion of Enucleated Eggs and Cells-Donors of Nuclei

We studied the capacity of rabbit oocytes for electrofusion with morula blastomeres and fetal fibroblasts. The morula blastomeres fused with aging ooplasts more readily than the fetal fibroblasts: 92.9 versus 63.0%, p < 0.001. The fetal fibroblasts fused with young enucleated oocytes more efficiently than with the aging ones: 98.4 versus 63.0%, p < 0.001. Serum starvation of the fetal fibroblasts in DMEM medium for 7–14 days reduced their capacity for fusion with young ooplasts, as compared to that after starvation for 0–4 days: 67.2 versus 98.9%, p < 0.01). The increased time of starvation in an impoverished medium reduced the capacity of fetal fibroblasts with aging ooplasts as compared to the fibroblasts cultivated in the full medium and in the impoverished medium for one or two days: 64.5 versus 37.4%, p < 0.01. Hence, the efficiency of the fusion of the oocytes with nuclear donor cells depends on the age of the recipient oocyte, the origin of nuclear donor cells, and the conditions of cultivation.     

Temporal and metabolic overlap between lipid accumulation and programmed cell death due to nitrogen starvation in the unicellular chlorophyte Chlamydomonas reinhardtii

Lipid accumulation due to nitrogen depletion has been studied extensively in Chlamydomonas reinhardtii and the metabolic changes that lead to triacylglycerol biosynthesis have been of particular interest to researchers in the biodiesel industry. The induction of programmed cell death (PCD) in response to nitrogen starvation has also been documented in related chlorophytes. Here, we examined the temporal and metabolic overlap of lipid accumulation and PCD in response to nitrogen starvation in the important model organism C. reinhardtii. Nitrogen starvation induced physiological stress, measured by the progressive decline in chlorophyll a fluorescence, reduced photosynthetic efficiency and decreased growth. In keeping with previous reports, cells accumulated lipids reaching a peak after 2–3 days. At the same time, DNA nicking and caspase‐like protease activity was observed in a proportion of cells, and ultrastructural observations confirmed that death was via PCD. Our results demonstrate that DNA nicking and caspase‐like activity are observed during PCD in C. reinhardtii in response to nitrogen starvation, and that death occurs at the same time as lipid biosynthesis. Microalgal lipid production due to nitrogen depletion in C. reinhardtii is limited by the decrease in culture growth and knowing that the loss of culture density is, at least in part, due to PCD is important for the biotechnology industry.     

Effect of Photoperiod and Nitrogen Supply on Basal Shoots Development in Rhododendron Catawbiense

In order to control young plant form by modifying culture conditions, plants of Rhododendron catawbiense from in vitro culture were grown in a greenhouse under different photoperiodic treatments (long or short days) combined or not with a several-week nitrogen starvation. After 12 weeks of culture under long days (16 h) with nitrogen supply, plants showed a rhythmic acrotonous development. When long days were combined with a six-week nitrogen starvation, the apical growth pause was extended leading to an increase of the number of acrotonous lateral ramifications. Short-day (8 h) treatment affected distal burst potential and moreover when a concomitant nitrogen starvation was applied. This lack of distal development allowed basal buds swelling, leading to basitonous plants. When plants were returned back to long days after 2, 4 or 6 weeks under short days, distal buds resumption competed with basal shoots development. Durable basitonous plants were obtained by a 12-week short days treatment combined with a 6-week nitrogen starvation.     

Quantifying food limitation of arthropod predators in the field

A method for quantifying food limitation of arthropod predators in the field is presented and applied to species of ground beetles (Carabidae) and sheet-web spiders (Linyphiidae) from a cereal field. Food limitation is expressed quantitatively as accumulated hunger (=starvation) by transforming 24-h food consumption at 20°C of animals newly collected in the field into days of starvation at 20°C. This is done by means of a reference curve relating 24-h food intake at 20°C to starvation periods (days) at 20°C. Such a reference curve was obtained for the carabid beetle Agonum dorsale in the laboratory. For other species the reference curve was modified with species-specific data. The procedure makes it possible to compare the feeding conditions of different species populations that are part of the same community. Hunger levels in the field for females of A.␣dorsale were equivalent to c. 15 days of starvation in early spring, c. 5 days in June, increasing to c. 10 days in late summer. Two ground beetles occurring during summer also showed increasing hunger levels from June to July, probably the result of a dry summer. Two spider species experienced a hunger level in the range of 4–8 days of starvation. Received: 15 August 1997 / Accepted: 22 January 1998     

Effects of different states of sheep fetal fibroblasts as donor cells on the early development in vitro of reconstructed sheep embryos

To investigate the effects of different states of donor cells on the development of reconstructed sheep embryos, we designed five treatments of donor cells, including cell passage, cell size, serum starvation, colchicine treatment and gene transfection. Results are as follows: (Ⅰ) Compared with 16-18 passage cells, the morula/blastocyst rate of 5-7 passage cells as donor nuclei was significantly higher (17.3% vs. 4.9%, P<0.05), suggesting the advantage of short-time cultured cells in supporting the development of reconstructed embryos. (Ⅱ) The morula/blastocyst rate of reconstructed embryos derived from medium cells (15-25μm) as donor nuclei was higher than that from large cells (25-33μm) and small cells (8-15μm)( 20.0% vs. 8.0%, 9.7%), indicating that reconstructed embryos from medium cells had a greater potentiality to develop into morula/blastocysts than those from small or large ones. (Ⅲ) The morula/blastocyst rate of reconstructed embryos from donor cells of SS (serum starvation) was lower than that from donor cells of NSS (non-serum starvation), but no significant difference was detected between SS and NSS(11.8% vs. 18.6%, P>0.05). (Ⅳ) Fetal fibroblasts treated with 0.05μmol/L colchicine exhibited a higher morula/blastocyst rate of reconstructed embryos than those treated with 0.10 μmol/L colchicine and untreated ones (27.5% vs. 12.1%, 17.1%), however, no significant difference among the three treatments was detected (P>0.05). (Ⅴ) The morula/blastocyst rate of reconstructed embryos from fetal fibroblasts transfected with GFP gene only was 3.1%, significantly lower than that from non-transgenic cells (3.1% vs. 20.4%, P<0.05). In conclusion, our results demonstrated that fetal fibroblasts of fewer passages, medium size could ensure a higher morula/blastocyst rate of reconstructed embryos. Serum starvation of donor cells might be unnecessary to the development of reconstructed embryos. Donor cells treated with 0.05μmol/L colchicine could facilitate the development of reconstructed embryos. Additionally, as cells transfected with GFP gene were used as donor nuclei, adverse effect on the development of reconstructed embryos was observed. Therefore, the developmental efficiency of reconstructed embryos could be improved if proper treatments to donor cells were used.     

Regulation of expression of the cucumber isocitrate lyase gene in cotyledons upon seed germination and by sucrose

A 6.5 kb cucumber genomic DNA fragment containing the icl gene was introduced into Nicotiana plumbaginifolia and shown to direct isocitrate lyase (ICL) mRNA synthesis in transgenic seedlings upon germination, in a temporally regulated manner. Two putative icl promoter fragments, of 2900 and 572 bp, were subsequently linked to the GUS reporter gene and introduced into N. plumbaginifolia. Both constructs directed GUS expression after transgenic seed germination, and although the 572 bp fragment gave only 1% of the activity of the 2900 bp fragment, it directed expression in the same cotyledon-specific and temporally regulated pattern. Seedlings were transferred to darkness after 18 days growth in the light, to induce a starvation response. The 2900 bp construct was activated by starvation and repressed by exogenous sucrose, whereas the 572 bp construct was not starvation-responsive. To localize the region of the 2900 bp promoter fragment which is responsible for regulation by sucrose, further deletions were make, linked to GUS, and assayed in a cucumber protoplast transient assay system. Constructs with promoters of 2900, 2142 and 1663 bp were activated by starvation and repressed by sucrose, but promoters of 1142 and 572 bp showed no such response. We conclude that the icl gene promoter contains at least two distinct cis-acting elements, one required for the response to sucrose and the other which participates in expression upon seed germination.     

Fate in soil of a recombinant plasmid carrying aDrosophila gene

A recombinant plasmid (C357; 3.5 Mdal) containing heterologous DNA (pBR322 [2.6 Mdal] with cDNA for an egg yolk protein fromDrosophila grimshawi) inEscherichia coli strain HB101 survived in and was recovered on selective media from sterile and nonsterile soil during 27 days at frequencies similar to those of theE. coli(pBR322) system. In sterile saline, the numbers of all cells decreased during 34 days, but the numbers of the plasmidless host declined less. There was no selective loss of the heterologous DNA in either soil or saline, as determined by colony hybridization with a³²P-labeled DNA probe for the cDNA, but the HB101(C357) appeared to be less able than HB101(pBR322) to cope with conditions of starvation. These results suggested that nonessential eucaryotic DNA inserted into plasmid DNA has little effect on the survival in soil or saline of the bacterial host and the maintenance of the vector.     

The effect of starvation on RNA: DNA ratios and growth of larval striped bass, Morone saxatalis

Hatchery reared larval striped bass, Morone saxatilis , 8-days-post-hatching were subjected to various feeding/starvation regimes over a period of 14 days.
Batches of larvae from each treatment were sampled over the 14-day period and subdivided for determination of notochord length and RNA:DNA ratio. The best growth was found in fully fed F1000 larvae (exposed to 1000 Artermia nauplii l⁻¹), which reached 8.2 mm after 11 days and 9.6 mm after 14 days. Starved animals after 11 days had notochord lengths of 4.9 mm. Growth curves from feeding-delayed larvae indicated that animals fed after up to 5 days starvation were capable of complete recovery. F100 larvae (exposed to 100 Anemia nauplii 1^−l) had a slower growth rate than F1000 larvae, reaching a notochord length of 7.3 mm after 14 days. RNA:DNA ratios over time closely followed notochord growth curves, with clear differences between starved, F100 and F1000 larvae being established after only 2 days. Equilibrium RNA:DNA ratios of 3.0 and 2.25 were established in F1000 and F100 larvae, respectively, 6.8 days after the beginning of the experiment. The average lag time between a change from the starved to the fed condition and a change in RNA:DNA ratio as determined by the divergence of the nucleic acid curve from the starved condition was 0.66 days.
In treatments where starvation followed various periods of feeding, larvae regressed in notochord length such that the final length at 14 days reflected the degree of feeding. RNA:DNA ratios in these animals again closely followed growth curves with a lag time of 0.81 days.
It was concluded that RNA:DNA ratios provided very accurate indices of growth in striped bass larvae which were highly sensitive to feeding status.     

Resource allocation and time budgeting in adults of the cockroach,Periplaneta americana: The interaction of behaviour and metabolic reserves

Activity (to food, water and shelter) and resource allocation (mass budget, and size of various body components) were examined in populations of adult Periplaneta americana subjected to periods of starvation or sugar feeding. Following 13 days of starvation, roaches ate 5 times their normal daily ration and feeding required about 20 days to return to pre-starvation levels. When sucrose was substituted for the usual dog-food diet for 13 days, there was also a large increase in feeding initially. When the dog food was re-established, however, there was very little feeding for about one week. Although reproduction was markedly curtailed by starvation, females fed sucrose continued reproducing for at least 59 days. The results suggested that the roaches were mainly energy limited. Although the consumption of roaches was strongly affected by reserve depletion, eating was a small component of the time budget, and overall activity was relatively unchanged by starvation or sugar-feeding. There was a slight decrease in activity during starvation, but the circadian pattern remained unchanged. Females carrying oöthecae were highly active and were apparently attracted to food (even though they ate little). Thus the activity of the population was dominated by circadian rhythmicity and the reproductive cycle.     

DNA methylation in bovine adult and fetal fibroblast cells

The aim of this project was to develop a simple screening tool to measure the DNA methylation of fibroblast cells, and to determine if differences in DNA methylation could be detected in adult and fetal fibroblast cells after serum starvation (SS). Four adult and four fetal tissue explants were collected to produce presumptive fibroblast cell cultures for this experiment. All cell lines underwent three repetitions of serum starvation for 0 (control), 2, 5, or 7 days. The DNA was extracted from the cells and analyzed for DNA methylation content using methylation sensitive restriction enzyme digestion, gel electrophoresis and image analysis. There was no difference (p = 0.11) between the DNA methylation of the adult and fetal nonclonal cell lines. A cubic trend (p = 0.09) of increased DNA methylation at 2 days of serum starvation followed by periods of decreasing DNA methylation at 5 and 7 days were observed for the adult nonclonal cell lines. A significant interaction (p = 0.03) was observed between fetal cell line and day. This simple, rapid DNA methylation assay may be beneficial when evaluating cells' DNA methylation content.