Changes in adenosine 5'-triphosphate, adenylate energy charge and adenosine 3'5'-cyclic monophosphate during the freezing of buffalo semen

In freshly ejaculated buffalo semen (N = 4) there were 24.61 +/- 5.28 nmol ATP and 40.39 +/- 5.94 nmol total adenylate/10(8) spermatozoa, and 97.75 +/- 7.06 pmol cAMP/10(9) spermatozoa. The semen was frozen in 4 steps (I, dilution; II, cooling; III, glycerolization and equilibration; IV, freezing and thawing). Motility, ATP, total adenylate and cAMP were significantly lower after Step IV than after Step I. Motility and ATP concentration were significantly correlated in egg-yolk--Tris (r = 0.530, P less than 0.05), skim milk--egg yolk (r = 0.754, P less than 0.01), egg yolk--citrate--glucose (r = 0.784, P less than 0.01) and citric acid--whey (r = 0.551, P less than 0.05). Cyclic AMP and motility in egg yolk--Tris were also correlated (r = 0.714, P less than 0.01). The adenylate energy charge was stable in all 4 freezing steps.     

ATP and adenylate energy charge during phosphate-mediated control of antibiotic synthesis.

The addition of inorganic phosphate or guanosine-5′-monophosphate to a phosphate-limited mycelial system of $\text{Streptomyces griseus}$ inhibited candicidin production. Accompanying the inhibition was a rapid increase in intracellular ATP concentration. Adenylate energy charge increased only slightly indicating that ATP is a more likely intracellular effector than energy charge in mediating phosphate control of antibiotic biosynthesis.     

Reincorporation of adenosine 5'-diphosphate/adenosine 5'-triphosphate carrier into phospholipid membranes. Phospholipid-protein interaction as studied by phosphorus-31 nuclear magnetic resonance and electron microscopy

Combined phosphorus-31 nuclear magnetic resonance (31P NMR) and electron microscopic studies were performed on the ADP/ATP carrier protein from beef heart mitochondria. The protein was incorporated into phospholipids by addition of Triton-protein micelles to a lipid suspension or to the dry lipid. All of the phospholipid (egg phosphatidylcholine or mixtures of egg phosphatidylcholine and egg phosphatidylethanolamine) that contributed to the observed 31P NMR signal under these conditions appeared to be in a bilayer configuration. Freeze-fracturing and negative-staining electron microscopy showed unilamellar vesicles and multilayers. An isotropic signal could be attributed to vesicle rotation, judging from its sensitivity to increasing viscosity. The presence of small vesicles was also noticeable in the 31P NMR spectra of planar oriented membranes. In the presence of phosphatidylethanolamine, aggregation of protein particles was observed. Gel chromatography of the protein-Triton-phospholipid mixture revealed that, before Triton removal, large amounts of protein are associated with multibilayers. Separation of loaded and unloaded membranes by centrifugation in D2O showed that, upon stepwise addition, protein incorporates preferentially into unloaded liposomes. From these findings a mechanism of protein reincorporation was deduced.     

Membrane changes during cartilage maturation. Increase in 5'- nucleotidase and decrease in adenosine inhibition of adenylate cyclase

To examine the potential participation of the plasma membrane in differentiation, we studied the enzymatic activities of 5'-nucleotidase and adenylate cyclase as a function of chondrocyte maturation. 16-day- old chick embryo tibiae epiphyses were dissected into proliferative, growing and hypertrophying zones. Partially purified membrane fractions prepared by differential centrifugation from the respective tissue segments were assayed for enzymatic activity. Cell suspensions from the same segments were examined cytochemically for the presence of 5'- nucleotidase. The findings show that the 5'-nucleotidase activity of the chick embryo epiphyseal cartilage has the following characteristics: (a) it has a Km of about 25 muM for 5'AMP, and is inhibited by a mixture of 2' and 3'AMP (apparent Ki about 10(-4) M) and by AOPCP; (b) it is predominantly localized at the cell surface but is also detected in the cytoplasm and in association with nuclear heterochromatin; and (c) it increases 10-fold (on a DNA basis) during the maturation of the epiphyseal cartilage cells. The adenylate cyclase activity has these characteristics: (a) it does not change during chondrocyte maturation (on a DNA basis); (b) its susceptibility to adenosine inhibition decreases at least 10-fold. The implication of these findings relative to a possible role of adenosine in cellular communication is discussed.     

Concomitant changes in adenylate cyclase and cytolytic activities of lymphoid cells during graft versus host reaction.

Spleen cells from adult F1 hybrid mice undergoing the graft versus host reaction due to the inoculation of lymphoid cells of parental origin showed increased adenylate cyclase activity and cytolytic activity. A time-course study revealed that both the adenylate cyclase and cytolytic activities started increasing at Day 4 and reached maximum at Day 8 of initiation of the graft versus host reaction. Furthermore, the magnitude of both adenylate cyclase activation and cytolytic activity were dependent upon the number of parental cells injected into the F1 hybrid recipients. Elevated adenylate cyclase activity was also observed in spleen cells from irradiated animals undergoing the graft versus host reaction in which the nonspecific, but not the specific cytolytic activity was markedly depressed.     

In vivo adaptive control of beta-receptors and adenylate cyclase during short-term heat exposure in rat parotid glands.

1. Adaptation of beta-adrenergic receptors (beta-AR) and adenylate cyclase (AC) in rat parotid glands during short-term heat exposure (33 degrees C) were studied. 2. Heat exposure reduced AC activity in response to isoproterenol (IPR). 3. The number of beta-AR on the cell surface significantly increased after 24 hr but returned to control level after 48 hr. 4. IPR-induced [3H]GDP release was significantly reduced throughout exposure. 5. The data suggest that the major factor which results in the desensitization of AC during short-term heat exposure is a blunted coupling between beta-AR and GTP binding protein(s).     

In vivo and in vitro influence of selenium on DNA/RNA synthesis in spleen and lymphocytes in culture--possible mediation of changes in GSH/GSSG ratio

Effect of superanutritional levels of selenium (Se) as sodium selenite (0.5 and 1.5 ppm) given orally to Balb/c mice for one and two weeks was observed on the rate of DNA/RNA synthesis, levels of reduced as well as oxidized glutathione (GSH and GSSG) and glutathione peroxidase (GSH-Px)/glutathione-S-transferase (GSH-S-transferase) activities in spleen. Similar effect of three different concentrations of Se (10(-7), 10(-5) and 10(-3) M) in culture media was also observed on the rate of DNA/RNA synthesis in proliferating lymphocytes taken from mice spleen. The results of the present study indicated that with increasing concentration and duration of Se treatment in vivo and in vitro, a marked inhibition of the rate of DNA/RNA synthesis was observed. Levels of total glutathione and GSSG in spleen were elevated significantly only after two weeks in 1.5 ppm treatments. Glutathione peroxidase activities in spleen decreased (p < 0.05) in 1.5 ppm group at one week and in 0.5 ppm group at two week treatment. At higher Se treatment, the activity recovered towards control. However, GSH-S-transferase in spleen remained unchanged at all treatment intervals. The results indicated that changes in glutathione system by increasing Se concentration might account for inhibition of rate of DNA/RNA synthesis.     

The opposing effects of calmodulin, adenosine 5'-triphosphate, and pertussis toxin on phorbol ester induced inhibition of atrial natriuretic factor stimulated guanylate cyclase in SK-NEP-1 cells.

In the present study, we investigated the effects of calmodulin, adenosine 5'-triphosphate (ATP) and pertussis toxin (PT) on phorbol ester (PMA) (a protein kinase C activator) induced inhibition of ANF-stimulated cyclic GMP formation in cells from the human renal cell line, SK-NEP-1. PMA inhibited ANF-stimulated guanylate cyclase activity in particulate membranes by about 65%. Calmodulin reversed this inhibition in a dose dependent manner. ATP potentiated Mg++ but not Mn++ supported guanylate cyclase activity. In PMA treated membranes, ATP potentiating effects were abolished. PMA also inhibited ANF-stimulated cGMP accumulation, but pretreatment with PT prevented this PMA inhibition. PT did not affect basal or ANF-stimulated cGMP accumulation. In conclusion, these results demonstrated that PMA (activated protein kinase C) inhibited ANF stimulation of particulate guanylate cyclase in opposition to the activating effects of calmodulin or ATP in SK-NEP-1 cells. The protein kinase C inhibitory effects appeared to be mediated via a PT-sensitive G protein.     

Evidence for new factors in the coordinate regulation of energy metabolism in Escherichia coli. Effects of hypoxia, chloramphenicol succinate, and 2,4-dinitrophenol on glucose utilization, glycogen synthesis, adenylate energy charge, and hexose phosphates during the first two periods of nitrogen starvation.

We studied the effects of decreased aeration, chloramphenicol succinate, and 2,4-dinitrophenol on the cellular rates of glycogen synthesis and glucose utilization and on the cellular concentrations of adenine nucleotides, glucose 6-phosphate, fructose 1,6-diphosphate, and phosphoenolpyruvate during the first two periods of nitrogen starvation of Escherichia coli W4597(K). A quantitative relationship between the changes in the rates and the accompanying changes in the hexose phosphates is demonstrated. However, the relationship for glycogen synthesis is different in different sets of metabolic conditions. We suggest that this difference reflects a change in the steady state level of a previously unknown effector of ADP-glucose synthetase (glucose 1-phosphate adenylyltransferase, EC 2.7.7.27) the rate-limiting enzyme of bacterial glycogen synthesis. We show that the properties of the hypothetical in vivo effector are consistent with the inhibitory effects of ppGpp (guanosine 3'-diphosphate 5'-diphosphate) and pppGpp (guanosine 3'-diphosphate 5'-triphosphate) on this enzyme in vitro. In addition, tetracycline, an inhibitor of the synthesis of these nucleotides, apparently prevents the change in the quantitative relationship. The relationship between glucose utilization and the hexose phosphates is altered at the transition to Period II of nitrogen starvation. We propose that this change reflects the alteration of the cellular steady state level of an unknown effector of the glucose phosphotransferase system. In contrast to the ATP-hexose phosphate system of shared regulatory effects, the specific effects of the unknown effectors allow the rates of glucose utilization and glycogen synthesis to be altered independently of each other and independently of changes in the rate of glycolysis. This independence allows a greater latitude of response for the individual pathways in more severe metabolic stress or in accommodating the metabolic changes necessary for long term survival.     

Regulation of deoxyribonucleotide biosynthesis during in vivo bacteriophage T4 DNA replication. Intrinsic control of synthesis of thymine and 5-hydroxymethylcytosine deoxyribonucleotides at precise ratio found in DNA.

The kinetics of the de novo formation of pyrimidine deoxyribonucleotides is the same after infection by wild type bacteriophage T4, which generate very low steady state levels of deoxytibonucleotides, and by T4 DNA synthesis-negative mutatants (Dna-), which accumulate high levels, suggesting that the control is not by a feedback mechanism. In this study, the ratio of the de novo synthesis of dTMP to HmdCMP derivatives was measured by determining the total thymine and 5-hydroxylxytosine (HmCyt) deoxyribonucleotides synthesized by the reductive pathways from [6-3H]uracil including those in DNA and any degradation products excreted into the medium. The ratio of the de novo synthesis of Thy/HmCyt derivatives remained constant at 2.1 +/- 0.1 for at least 45 min after infection by wild type phage, i.e. precisely at the Thy/HmCyt ratio in T4 DNA. On infection by phage mutated in the Dna-genes 32, 41, 44, or 45, the ratio still remained close to 2 to 1 for at least 25 min. Only after the pyrimidine deoxyribonucleotide concentrations reached levels about 100-fold greater than the initial values did the ratio begin to increase. However, a mutant of the structural gene for T4 DNA polymerase showed some increase in ratio by 15 min. Mutants of gene 1 (HmdCMP kinase) were distinct in that the Thy/HmCyt ratio dropped to about 1.0 by 25 min, and then remained quite constant. Uniquely, in these mutants a significant quantity of 5-hydroxymethyluracil or a derivative was found, about 40% being in the medium. The product was shown to be derived by deamination of a 5-HmCyt derivative. All Dna- mutants tested excreted 35 to 50% of their thymine derivatives, mostly as thymine, into the medium. Neither thymine nor 5-hydroxymethyluracil derivates were excreted after wild type phage infection. We propose that pyrimidine deoxyribonucleotide synthesis is regulated at a Thy:HmCyt ratio of 2:1 as an intrinsic property of a complex of enzymes synthesizing and channeling deoxyribonucleotides for T4 DNA replication and not exclusively by effector-sensitive mechanisms.     

Effects of NaCl on growth and nitrogen fixation and assimilation of inoculated and KNO3 fertilized Vicia faba L. and Pisum sativum L. plants

Faba bean (Vicia faba L. var. minor cv. Alborea) and pea (Pisum sativum L. cv. Lincoln) plants, inoculated with Rhizobium leguminosarum biovar. viciae strain GRA19, were treated with salt (100 mM NaCl) and/or nitrate (8 mM KNO₃) to test whether plants grown with inorganic-nitrogen are more tolerant to salinity than plants entirely reliant upon fixed nitrogen. According to the growth inhibition recorded, pea plants dependent on dinitrogen fixation proved more tolerant to salt stress than those N-fertilized, in contrast to results obtained for faba bean plants. This study therefore confirms that plants dependent on nitrogen fixation are not always more sensitive to salinity than are N-fertilized plants. Nitrate addition did not reduce the specific nitrogenase activity in pea, but did in faba bean. However, nodulation was inhibited in both legumes. The specific nitrogenase activity was more affected by salt treatment in N-fertilized plants for both legumes. The activity of the enzymes mediating ammonium assimilation in nodules (GS, NADH-GOGAT) was inhibited by salt stress both in N-fixing and in N-fertilized pea and faba bean plants.     

In vitro effects of growth factors and hormones on three Perkinsus species and increased proliferation of P. marinus during cloning

Clonal cultures are essential for the genotypic and phenotypic characterization of Perkinsus species but their cloning, especially of P. marinus, can be tedious. The use of a growth factor and hormone supplement to facilitate cloning was, therefore, investigated. Many of the 16 supplements tested significantly increased P. marinus and P. olseni proliferation but only two significantly increased P. chesapeaki proliferation. The concentration of the most effective supplement for all three Perkinsus species (i.e., endothelial cell growth supplement, ECGS) and medium dilution were then optimized for P. marinus cultured at low densities. Finally, the advantage of using conditioned culture medium, a feeder layer, and ECGS alone and in different combinations to improve cloning of P. marinus were compared. Using conditioned culture medium, a feeder layer and ECGS in combination, each cell (N = 7) seeded singly yielded clonal cultures with 253 ± 167 cells after 21 days. In contrast, only 4 out of 7 cells seeded singly in culture medium yielded clonal cultures with 5 ± 4 cells after 21 days.     

Mineral nutrition, of cultured chlorophyllous cells of tobacco VII. Effects of the NH4/NO3 ratio and of Cl in the media on the growth and ionic balance of cells

NG, a strain of cultured tobacco cells of Nicotiana glutinosahad high growth rates and carboxylate contents (C—A) of100 to 130 meq/100 g of dry cells on media containing 42 meqNO₃/liter as the sole N source. (C—A) is the amount ofinorganic cations minus inorganic anions in meq per 100 g ofdry cells. NG, cultured on media containing NH₄ 10+NO₃ 42 in meq/liter,had lower growth rates and lower (C—A) values as comparedwith NG on media containing NO₃ as the sole N source. NG, cultured on media containing NH₄ 30+NO₃ 42 in meq/liter,had high growth rates and (A—C) values of 22 to 53 meq/100gof dry cells. In this case, the (A—C) content may correspondto organic cations, basic organic N compounds such as free asprotein-bound basic amino acids. The easily absorbed Cl mayhave been required maintain good growth conditions such as ionicbalance and a favorable pH in the cells. Thus cultured cells of Nicotiana glutinosa may have physiologicaladaptability against variations in a relatively wide range of|C—A| contents [|C—A| being the absolute valuesof (C—A)]. (Received May 15, 1980; )     

The importance of the ratio UV-B/photosynthetic active radiation (PAR) during leaf development as determining factor of plant sensitivity to increased UV-B irradiance: effects on growth, gas exchange and pigmentation of bean plants (Phaseolus vulgaris cv. Label)

To evaluate the effect of different naturally occurring irradiation conditions on the sensitivity of bean (Phaseolus vulgaris cv. Label) to increased UV-B levels, plants were grown under six different light treatments. In the control series (at ambient levels of UV-B), UV-B and visible light were decreased in parallel, resulting in three different total irradiation treatments with the same UV-B/PAR ratio. A second series with a 15% increase in UV-B irradiation at each PAR level was used to investigate the effect of UV-B under the varying total irradiance levels. The different total irradiance levels resulted in large differences in total dry weight, specific leaf weight, photosynthesis-light response and pigment concentrations. Nevertheless, the 15% increase in UV-B resulted in equal reductions in total dry weight (from 24.5 to 34.3%) and effective photosynthesis for all light levels. The accumulation of protective pigments in the primary bean leaves was strongly correlated to the total irradiance level (200% increase from the lowest to the highest light level), but was not influenced by increasing UV-B levels. As the UV-B/PAR ratio outside increases with decreasing total irradiance (when induced by cloud cover) this implies that low radiation levels are potentially dangerous to some plants, even though the UV-B levels may seem negligible.