Low concentrations of dimethyl sulphoxide accelerate conjugation and incorporation of glycine and glucosamine intetrahymena

Summary Dimethyl sulphoxide at relatively low comentrations, 0.01 to 1 mM, enhanced the conjugation and cell-to-cell adhesion of complementary strains of matingTetrahymena thermophila. The time required to form stable conjugates was reduced by dimethyl sulphoxide. This chemical stimulated the uptake of glycine and glucosamine from the suspending media. Incorporation of 2-¹⁴C-glycine and 6-³H-D-glucosamine into protein and glycoprotein was enhanced in whole cells, surface membrane and cilia. Incorporation of glucosamine into the microsomal fraction was increased in the dimethyl sulphoxidetreated cells while there was little change in glycine incorporation. There were no detectable changes in glycine and glucosamine incorporation into the nuclear fractions isolated from conjugatingTetrahymena exposed to dimethyl sulphoxide.     

Doublet cells inTetrahymena as indicators of culture media composition

Stomatogenesis in ciliates is a complex and carefully orchestrated event. The exo⁻ mutant SB255 ofTetrahymena thermophila has defects in mucocyst formation and docking and can also have one or two mouths. Three common culture media (proteose peptone, Medium 357, and yeast extract) were analyzed for total C, N, and inorganic elements and then tested for their effect on the number of mouths present in SB255. Cultures of SB255 grown in Medium 357 consisted of a mixed population of cells with either two mouths (doublet) or one mouth. Cultures from the same original stock grown in Medium 357 (SBm) and in 1% proteose peptone (SBpp) had different percentages of doublet cells in 1-, 2-, 3-, and 4-d-old cultures. When transferred to and grown in 1% yeast medium, both SBpp and SBm cultures had increased percentages of doublets over a 4-d culture period. When grown in 0.1, 0.5, or 1% yeast medium for 2 d, both SBpp and SBm cultures had more doublets in 1% than in either 0.1 or 0.5% yeast medium. Cultures of SBm grown in Medium 357. or 1% yeast medium for 2 d had a 10-fold increase in doublet cells compared to the inoculum. After 2 d in 1% proteose peptone, SBm cultures had percentages of doublet cells almost equal to that of the inoculum. Immunofluorescence and scanning electron microscopy (SEM) were used to examine cellular morphology of the doublet cells. These findings suggest that enriched media promote the growth of doublet cells. Furthermore, these doublets could prove to be a useful model system for the study of biological roles of trace elements.     

ATP synthesis and storage

Since 1929, when it was discovered that ATP is a substrate for muscle contraction, the knowledge about this purine nucleotide has been greatly expanded. Many aspects of cell metabolism revolve around ATP production and consumption. It is important to understand the concepts of glucose and oxygen consumption in aerobic and anaerobic life and to link bioenergetics with the vast amount of reactions occurring within cells. ATP is universally seen as the energy exchange factor that connects anabolism and catabolism but also fuels processes such as motile contraction, phosphorylations, and active transport. It is also a signalling molecule in the purinergic signalling mechanisms. In this review, we will discuss all the main mechanisms of ATP production linked to ADP phosphorylation as well the regulation of these mechanisms during stress conditions and in connection with calcium signalling events. Recent advances regarding ATP storage and its special significance for purinergic signalling will also be reviewed.     

Effects of copper on phagocytosis inTetrahymena

Summary Addition of copper, corresponding to 100 ppm, to the normal 2% proteose peptone medium is tolerated byTetrahymena. This concentration of copper stimulates phagocytosis to a maximum value which is reached gradually during the first 1 hour exposure, and which is maintained during continuous exposures. Cell proliferation is resumed after a lag period, although at a decreased rate. Cells exposed to copper contain small refractile granules, previously proposed to represent an ion-regulating system; the number of granules remains constant in proliferating cells. Higher concentrations of copper also resulted in an elevated rate of phagocytosis but at the same time cell mortality was observed; this lack of transition between inhibited phagocytosis and cell mortality may be ascribed to the physiological role of copper. The high amount of organic matter in the growth medium protects against the toxic effects of copper, thus in the absence of organic matterTetrahymena tolerated only a 100-fold lower concentration of copper than that tolerated in the growth medium. However, cells which had initiated granule formation (for example for regulation of calcium) prior to starvation and exposure to copper, were more resistant to copper than cells which had not yet activated this mechanism, perhaps because of the low capacity of starved cells for protein synthesis.     

Cytokinin effect on protein synthesis in vivo in higher plants

The influence of naphthalene-1-acetic acid and kinetin on protein synthesis in vivo was investigated by measuring the incorporation of radioactive amino acids into polypeptides of synthesizing polysomes. The second subculture of sterile pith tissue of Nicotiana tabacum L. cv. Wisconsin 38 grown on a medium containing only a minimum of growth substances was further starved in a small volume of medium lacking auxin and cytokinin for 12 h. An incubation period of 4 h with [¹⁴C]amino acids followed. The last 30 min of those incubations were carried out in the presence of actinomycin D and the last 20 min were performed under different conditions: a) without any growth substances, b) with naphthalene-l-acetic acid, c) with kinetin. By measuring the differences of the specific radioactivities of the polysomes the following results were revealed: 1) Kinetin increases the protein synthesis by an average of 35%-2) Auxin has no effect on protein synthesis.Abbreviations Act. D actinomycin D - NAA naphthalene-1-acetic acidPart of a Diplomarbeit, University of Bonn 1976     

ATP synthesis associated with the conversion of hexachlorocyclohexane related compounds

Clostridium rectum strain S-17 converts -1,2,3,4,5,6-hexachlorocyclohexane (HCH) related compounds to chlorobenzenes. The metabolites from -1,2,3,4,5,6-hexachlorocyclohexene and -1,3,4,5,6-pentachlorocyclohexene are identified as 1,2,4-trichlorobenzene and 1,4-dichlorobenzene, respectively. ATP synthesis, converting these chlorinated compounds, is observed in the cell suspension of C. rectum as indicated by luciferase-luciferin reaction and phosphorylation of ³²P-labeled phosphate. These observation lead to the conclusion that HCH and related compounds serve as artificial electron acceptors of the Stickland reaction, and therefore, the reductive dechlorination is associated with ATP synthesis.Abbreviations HCH -1,2,3,4,5,6-hexachlorocyclohexane - HCCH -1,2,3,4,5,6-hexachlorocyclohexene - PCCH -1,3,4,5,6-pentachlorocyclohexene - TCCH -3,4,5,6-tetrachlorocyclohexene - 1,2,4-TCB 1,2,4-trichlorobenzene - 1,4-DCB 1,4-dichlorobenzene - MCB monochlorobenzene - DTT 1,4-dithiothreitol - IAA monoiodoacetic acid     

ATP浓度和缺氧暴露对大鼠脑线粒体RNA和蛋白质体外合成的影响

本文探讨介质中ATP浓度和急,慢性缺氧暴露对大鼠脑线粒体内RNA和蛋白质合成的影响。用差速离心法分离正常和低压舱模拟4000m高原急性连续缺氧暴露3d和慢性连续缺氧暴露40d大鼠脑线粒体,用体外无细胞（cell-free in vitro)^3H-UTP和^3H-Leucine掺入法分别测定线粒体RNA和蛋白质合成活性,结果显示,大鼠急性缺氧暴露后大脑皮质线粒体RNA体外合成活性降低40%,蛋白质合成活性降低60%;慢性缺氧暴露后线粒体RNA和蛋白质合成活性分别为对照的72%和76%;ATP对正常大鼠脑线粒体RNA以及蛋白质的体外合成活性的影响均呈双相性,大于或小于1mmol/L均可产生不同程度的抑制效应,结果提示,缺氧可在转录和翻译两个水平上影响脑线粒体mtDNA的表达,而慢性缺氧暴露时,线粒体半自主性功能的改善可能是机体对缺氧适应的细胞机制之一;ATP对脑线粒体内转录和释放活性的调节是一种经济有效的反馈调节方式。     

Hydrolysis and synthesis of ATP by membrane-bound ATPase from a motile Streptococcus

ATPase was detected in the membranes of a motile Streptococcus. Maximal enzymic activity was observed at pH 8 and ATP/Mg²⁺ ratio of 2. Mn²⁺ and Ca²⁺ could replace Mg²⁺ to some extent. Besides ATP, GTP and ITP were substrates. The enzyme was inhibited by N,N-dicyclohexylcarbodiimide but not by sodium azide, uncouplers or bathophenanthroline.An electrochemical gradient of protons, which was artificially imposed across the membranes of Streptococcus cells by manipulation of either the K⁺ diffusion potential or the transmembrane pH gradient, led to ATP synthesis. ATP synthesis was abolished by proton conductors, an inhibitor of the ATPase or an increase in the extracellular K⁺ concentration. A comparison between the phosphate potential and the electrochemical proton gradient showed that the data found are in agreement with a stoichiometry of 2 protons translocated per molecule ATP synthesized.Abbreviations electrochemical gradient of protons - DMO 5,5-dimethyl-2,4-oxazolidinedione - CCCP carbonylcyanide m-chlorophenylhydrazone - FCCP carbonylcyanide p-trifluoromethoxyphenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DNP 2,4-dimitrophenol     

RNA and protein synthesis in sperm cells isolated from Zea mays L. pollen

Summary Sperm cells are thought to be quiescent in pollen and activated upon pollen germination. To test this hypothesis, protein, RNA and DNA synthesis were assessed in Zea mays sperm cells at different times after isolation from pollen. Protein synthesis changed with time; while some proteins were found to be constitutive in both 0 and 24 h cells, others were synthesized and some disappeared. Overall, the number of proteins detected at 24 h doubled compared with freshly isolated cells. Incorporation of [³H]leucine in 24 h cells was about 50 times that in freshly isolated cells, and that of [5, 6-³H]uridine, about 7 times. Very low incorporation of [6-³H]thymidine into the cells was detected; there was no difference between freshly isolated and 24 h cells. It is possible that the differences in synthetic activity between freshly isolated and 24-h-old cells might correspond to sperm cell activation during pollen tube growth. If so, these metabolic changes may play an important role in fertilization.Supported by funds from a Strategic Grant (D.D.C.) and an Operating Grant (D.J.G.) from the Natural Sciences and Engineering Research Council of Canada     

Regulation of respiration and ATP synthesis in higher organisms: Hypothesis

The present view on the regulation of respiration and ATP synthesis in higher organisms implies only Michaelis-Menten type kinetics and respiratory control as regulatory principles. Recent experimental observations, suggesting further regulatory mechanisms at respiratory chain complexes, are reviewed. A new hypothesis is presented implying regulation of respiration and ATP synthesis in higher organisms mainly via allosteric modification of respiratory chain complexes, in particular of cytochromec oxidase. The allosteric effectors, e.g., metabolites, cofactors, ions, hormones, and the membrane potential are suggested to change the activity and the coupling degree of cytochromec oxidase by binding to specific sites at nuclear coded subunits. Recent results on the structure and activity of cytochromec oxidase, supporting the hypothesis, are reviewed.Dedicated to Professor Dr. Carl Martius on the occasion of his 80th birthday.     

Necessity of protein synthesis for metamorphosis in the marine hydroidHydractinia echinata

Summary In the marine colonial hydroidHydractinia echinata metamorphosis from the larval to the adult (polyp) stage is induced by various agents, including CsCI and dioctanoylglycerol (diC8). Induction is prevented when the inhibitors of protein synthesis cycloheximide or ementine were applied simultaneously with the metamorphosis-inducing agents. With diC8 treatment, the inhibitors caused most animals to transform into mosaics consisting of larval and polyp body parts instead of normal shaped polyps. In contrast, treatment with cycloheximide or ementine just before or after incubation with the metamorphosis-inducing agents did not prevent larvae from metamorphosis. No substantial quantitative changes in protein synthesis occur during induction of metamorphosis, however, the protein pattern is changed upon induction. The most prominent new polypeptides (25 and 73 kD) were observed when CsCI was used to trigger metamorphosis. In addition, both in CsCl- and in diC8-treated larvae, the synthesis of a new 23 kD protein occurred, whilst synthesis of others ceased (41 and 44 kD).     

Amino acid uptake and protein synthesis during early meiosis in Saccharomyces cerevisiae

Uptake and incorporation into proteins of an externally supplied amino acid were followed during early meiosis in yeast. Under conditions optimal for development, an insufficient permeability of the cell leads to an incorporation pattern which reflects the changes in the activity of the amino acid transporting system rather than those in protein synthesis. A more correct picture of protein synthesis during early meiosis is obtained by the use of a mutant with an enhanced level of amino acid uptake.Abbreviation SPM Sporulation medium     

Intracellular location of ATP citrate lyase in leaves of Pisum sativum L.

The aim of this work was to discover if there is enough ATP citrate lyase (EC 4.1.3.8) in the cytosol of the leaves of Pisum sativum L. to catalyse the synthesis of the acetyl CoA needed for terpenoid synthesis. Estimates of the maximum catalytic activity of the enzyme in leaves of 7-d-old peas gave values of 113 nmol min^-1 g^-1 fresh weight. The rate of carotenoid accumulation in these leaves corresponded to a requirement for acetyl CoA of 0.7 nmol min^-1 g^-1 fresh weight. The distribution of marker enzymes during fractionation of homogenates of leaves from 7 to 10-d-old peas showed that differential centrifugation led to the isolation in reasonable yields of chloroplasts, mitochondria, peroxisomes and the endomembrane system. None of the above components of the leaf contained appreciable detectable activity of ATP citrate lyase, the distribution of which closely paralleled that of the cytosolic marker. It was concluded that in young leaves of pea most of the ATP citrate lyase is in the cytosol.     

Cytoplasmic membrane changes in conjugating Tetrahymena

Summary The formation of stacks of smooth-surfaced cisternae was studied in samples of Tetrahymena pyriformis that were prepared for electron microscopy after starvation for 2 days in buffer, and in samples of organisms of different mating type that were starved for 2 days and then mixed to induce conjugation. The number of stacks of cisternae was greater in starved ciliates than in those from stock cultures, and the size and number of stacks increased further after mixing animals of different mating type. When cells were either starved or mixed in buffer to which the protein synthesis inhibitor cycloheximide had been added, the formation of the membranous stacks was almost completely abolished. Addition of the RNA synthesis inhibitor actinomycin D, however, did not result in a significant decrease in the size and number of the stacks of saccules. Conjugation did not occur in the presence of either cycloheximide or actinomycin. The results suggest that protein synthesis is required for the formation of the stacks, but RNA synthesis is no longer necessary for their formation at this stage. The previous identification of the stacks as a Golgi apparatus and the possible functions of these membranes are considered.Supported by grants from NSF (GB-32285) and the American Cancer Society (E-500).The authors acknowledge the technical assistance of Mrs. Sue Thompson.     

Differentiated protein synthesis with polysomes from Petunia ovaries before fertilization

Polysomes from cross-, self- and unpollinated ovaries have been injected in Xenopus laevis eggcells. The synthesised proteins have been identified by means of gel-electrophoresis. It appeared that few hours after pollination the protein pattern of the pollinated ovary has changed in comparison with that of unpollinated ones. However, in this early stage, there is only little difference found in the protein pattern of selfed and crossed ovaries. From 12 h after pollination and onwards, the difference between pollinated and unpollinated ovaries becomes very obvious, as does also the difference between crossed and selfed ovaries. At 24 h after pollination many different proteins exist in crossed and selfed ovaries. From 32 h to 48 h after pollination the crossed ovaries differentiate further on, whereas the ovaries after self-pollination show more and more proteins which were also found in the unpollinated ovaries of that age. Possible explanations and hypothesis for these phenomena are discussed as well as the probable occurrence of signal(s) arriving from the stigma or the style at the ovary.     

Seed-specific expression of a lysine rich protein sb401 gene significantly increases both lysine and total protein content in maize seeds

The sb401 gene from potato (Solanum berthaultii) encoding a pollen-specific protein with high lysine content was successfully integrated into the genome of maize plants and its expression was correlated with increased levels of lysine and total protein content in maize seeds. A plasmid vector containing the sb401 gene under the control of a maize seed-specific expression storage protein promoter (P19z) was constructed and introduced into maize calli using microprojectile bombardment. The integration of the sb401 gene into the maize genome was confirmed by Southern blot analysis and its expression was confirmed by Western blot analysis. Quantification of lysine and protein content in R1 maize seeds showed that, compared to the non-transgenic maize control, the lysine content increased by 16.1% to 54.8%, and total protein content increased by 11.6% to 39.0%. There was no visible morphological change in vegetative parts and seeds of the transgenic maize plants. Lysine and protein analysis of the transgenic maize grains showed that the levels of lysine and total protein remained high for six continuous generations, indicating that the elevated lysine and total protein levels were heritable. These results indicate that the sb401 gene could be successfully employed in breeding programmes aimed at improving the nutritional value of maize.     

Changes in protein synthesis during the initial stage of life of tobacco protoplasts

The biosynthesis of Fraction I protein in isolated protoplasts is compared with that in the plant. Radioactive precursors were incorporated into isolated protoplasts (in vitro labeling) and into leaves, from which the protoplasts were isolated later (in situ labeling). The biosynthesis of Fraction I protein stopped almost completely as soon as the protoplasts were incubated in the culture medium.     

Patterns of protein synthesis in salivary glands ofDrosophila melanogaster during larval and prepupal development

Summary Patterns of protein synthesis in the salivary glands ofDrosophila melanogaster have been studied throughout late larval and prepupal development by pulse labelling the tissues with³⁵S-methionine. Specific changes to the pattern of proteins synthesized during development are found and the significance of these changes is discussed in view of the known changes in gene (puffing) activity which occur at the same times. We review the problem of salivary gland function in prepupalDrosophila.     

Spatio-temporal organization of intracellular ATP content and oscillation patterns in response to blue light byPhysarum polycephalum

Summary Phototactic responses in a giant amoeboid cell ofPhysarum plasmodium were studied both by analyzing intracellular ATP content and by applying image processing for recording oscillatory changes in thickness with use of a microcomputer. The ATP content fluctuated and deviated from the initial level upon exposure to light with varying wavelength from 400 to 600 nm. The maximum decrease in the integrated value dt with T=9 minutes occurred at the wavelength 450 nm. The ATP in a migrating plasmodium distributed twice as high in the front as in the rear. This polar pattern became unstable, and a new wavy pattern appeared by stimulating a local frontal part with blue light. In a concentrically extending plasmodium, peripheral and inside regions oscillated in opposite phase to each other. When part of this organism was exposed to light, stimulated and unslimulated regions began to oscillate in opposite phase, and phase waves propagated inward the stimulated region. And the protoplasm there became thinner, the strongest avoidance reaction occurring to 450 nm light as in ATP response. Phototactic behavior inPhysarum is discussed in terms of bifurcation in spatio-temporal organization appearing in a self-organizing system.