Metabolism of Extracellular Adenine Nucleotides by Cultured Rat Brain Astrocytes

Intact astrocytes cultured from newborn rat cerebral cortex rapidly converted extracellular ATP to ADP. The ATPase responsible was apparently not saturated, even at 750 microM ATP. In contrast, the conversion of ADP to AMP was slow, and the reaction was limiting for the subsequent dephosphorylation process. Adenosine formation was the only fate for AMP. The reaction was catalyzed by 5'-nucleotidase with an apparent Km of 55 microM for AMP and appeared to be inhibited by high concentrations of ATP and ADP. Astrocytes were able to take up adenosine with an apparent Km value of 45 microM. Uptake was inhibited by dipyridamole but not by anti-5'-nucleotidase IgG. The results support the proposal that astrocytes play a role in modulating synaptic events involving ATP and adenosine.     

Neonatal Hypothyroidism Affects the Adenine Nucleotides Metabolism in Astrocyte Cultures from Rat Brain

Neonatal hypothyroidism is associated with multiple and severe brain alterations. We recently demonstrated a significant increase in hydrolysis of AMP to adenosine in brain of hypothyroid rats at different ages. However, the origin of this effect was unclear. Considering the effects of adenine nucleotides to brain functions and the harmful effects of neonatal hypothyroidism to normal development of the central nervous system, in this study we investigated the metabolism of adenine nucleotides in hippocampal, cortical and cerebellar astrocyte cultures from rats submitted to neonatal hypothyroidism. ATP and AMP hydrolysis were enhanced by 52 and 210%, respectively, in cerebellar astrocytes from hypothyroid rats. In hippocampus of hypothyroid rats, the 47% increase in AMP hydrolysis was significantly reverted when the astrocytes were treated with T3. Therefore, the imbalance in the ATP and adenosine levels in astrocytes, during brain development, may contribute to some of the effects described in neonatal hypothyroidism.Elizandra Braganhol and Alessandra Nejar Bruno are first authors.     

Regulation of Energy Transduction and Electron Transfer in Cytochrome c Oxidase by Adenine Nucleotides

Cytochrome c oxidase from bovine heart contains seven high-affinity binding sites for ATP or ADP and three additional only for ADP. One binding site for ATP or ADP, located at the matrix-oriented domain of the heart-type subunit VIaH, increases the H⁺/e^– stoichiometry of the enzyme from heart or skeletal muscle from 0.5 to 1.0 when bound ATP is exchanged by ADP. Two further binding sites for ATP or ADP, located at the cytosolic and the matrix domain of subunit IV, increases the K _M for Cytochrome c and inhibit the respiratory activity at high ATP/ADP ratios, respectively. We propose that thermogenesis in mammals is related to subunit VIaL of cytochrome c oxidase with a H⁺/e^– stoichiometry of 0.5 compared to 1.0 in the enzyme from bacteria or ectotherm animals. This hypothesis is supported by the lack of subunit VIa isoforms in cytochrome c oxidase from fish.     

Nucleotide Metabolism in Salt-Stressed Zea mays L. Root Tips: I. Adenine and Uridine Nucleotides

Corn plants (Zea mays L. cv Pioneer 3906) were grown in a glass house on control and saline nutrient solutions, in winter and summer. There were two saline treatments, both with osmotic potential = −0.4 megapascal but with different Ca²⁺/Na⁺ ratios: 0.03 and 0.73. Root tips and shoot meristems (culm tissue) of 26 day-old plants were analyzed for nucleotides to ascertain if there were correlations between nucleotide pool size and the reduced growth on saline cultures. Several other cell components also were determined. Plants grown in winter were only half as large as those grown in summer mainly because of the lower light intensity and lower temperature. But the relative yield reduction on salt treatment compared to the control was similar in winter and summer. The two different salt treatments caused similar yield reductions. Neither salt treatment affected nucleotide pools in culm tissue, with the possible exception of UDPG in winter. In the case of root tips, salt treatment had little or no effect on nucleotide pool sizes in winter when many already seemed near a critical minimum, but in summer it reduced several pools including ATP, total adenine nucleotide, UTP, total uridine nucleotide, and UDP-glucose. The reductions were greatest on the salt treatment with low Ca²⁺/Na⁺. There was no simple correlation between the effects of salt stress on growth and on nucleotide pool size. The nucleotide pools of culm tissue indicated that in some respects this tissue was effectively insulated from the salt stress. Roots that were in direct contact with the saline solution indicated significant reductions in nucleotide pools only in the summer whereas growth was reduced both summer and winter. It is possible that the nucleotide concentrations of root cells in winter were already near a critical minimum so that nucleotide synthesis and growth were tightly linked. Significant reductions in nucleotide pools that would be expected to affect growth were more evident in summer when pools were larger and growth was more rapid. But even where ATP and total adenine nucleotides were reduced, the ratio of ATP:ADP and the adenylate energy charge remained unchanged indicating an active adenylate kinase that had access to most of the adenine nucleotide pools, and possible catabolism of excess AMP.     

Effect of Vacuum Ultraviolet Irradiation on Abiogenous Synthesis of Adenine Nucleotides in the Presence of Lunar Ground

Vacuum ultraviolet (VUV) radiation, a powerful energy source in free Space, have been established to promote synthesis of natural nucleotides. It is shown that lunar ground protects from destruction (up to 1.5–2.0% ) the nucleotides formed after VUV-irradiation of dry films (adenosine and inorganic phosphate). Identification and quantitative determination of the products of synthesis and destruction is performed by the method of high performance liquid chromatography. The following products of synthesis are found: 5'-AMP > 2'3'-cAMP > 2'-AMP > 3'5'-cAMP > 3'-AMP. The obtained results are discussed from the viewpoint of hypothesis about the Space (extraterrestrial) origin of biologically important compounds that were initial for evolution on the Earth.     

Nucleotide Pools and Adenylate Energy Charge in Balanced and Unbalanced Growth of Chromatium

Adenine nucleotide pools and their energy charge were measured during balanced and unbalanced growth of photoheterotrophic Chromatium cultures. The methods used involved rapid sampling, accurate to within 1 s, from isotopically labeled cultures followed by chromatographic separation of individual nucleotides. During balanced growth, both energy charge and adenosine triphosphate (ATP) concentrations, whether expressed as a function of cell protein or intracellular water, were slightly higher in limiting light intensities than in cultures growing at their maximal rate in bright light. The ATP found corresponded to 4.67 +/- 0.08 nmol/mg of protein or 1.34 +/- 0.57 mM for low-light cells and to 4.41 +/- 0.58 mmol/mg of protein or 0.85 +/- 0.12 mM for high-light cells. Corresponding energy charges were 0.85 +/- 0.02 and 0.81 +/- 0.02. Illumination shifts caused differential synthesis of photosynthetic pigments lasting 2 to 3 h without corresponding perturbation of adenine nucleotide levels. Cultures in intermittent illumination were severely affected by some cycle durations; they had abnormal morphology and very high bacteriochlorophyll-to-protein ratios. In such cultures, energy charge and nucleotide concentrations were within normal limits and relaxed to the dark steady state during the dark periods. Arsenate at AsO(4) (3-) to PO(4) (3-) ratios of 10:1 in the medium retarded growth, but no abnormality of charge or quantity of phosphate-containing nucleotides was found. These experiments therefore suggest that, within experimental error, neither the size nor the charge of the adenylate pools governs growth rate in Chromatium. Moreover, these parameters do not appear to be concerned in regulating the synthesis of photosynthetic apparatus in this organism.     

Serosurvey of Rickettsia typhi and Rickettsia felis in HIV‐infected patients

Consistent with the effects of HIV on cell‐mediated immunity, an increased susceptibility to intracellular microorganisms has been observed. Rickettsiae are obligate intracellular microorganisms. The aim of this study was to examine Rickettsia typhi and Rickettsia felis infections in HIV+ population. Sera of 341 HIV+ patients were evaluated by indirect immunofluorescent assay. Age, sex, residential locality, risk behavior, stage according to criteria of the Center for Disease Control and Prevention, CD4+/CD8+ T cells, Hepatitis B antigen, and Hepatitis C serology were surveyed. Seroprevalences of R. typhi and R. felis infection were 7.6% and 4.4%, respectively. No associations were found between seropositivities and the assessed variables. Findings were similar to those obtained in healthy subjects from the same region.     

Cross-Reactivity of Rickettsia japonica and Rickettsia typhi Demonstrated by Immunofluorescence and Western Immunoblotting

Cross-reactivity between Rickettsia japonica and R. typhi was observed by immunofluorescence tests using sera from patients with Oriental spotted fever (OSF), from whom the causative agent was isolated and identified as R. japonica. Western immunoblotting with these sera revealed that only the 120-kilodalton surface polypeptide, i.e., rickettsial outer membrane protein (rOmp) B, has a common antigenicity with the 105-kilodalton surface polypeptide of R. typhi. In some cases, antibodies specifically reactive with R. typhi were detected in acute-phase sera followed by a significant rise in titers, possibly because of an anamnestic response to a previous infection with an R. typhi-like agent; the sera retained reactivity to R. typhi even after absorption by a homologous strain. A lipopolysaccharide (LPS)-like antigen of R. typhi was found to be reactive with some sera of OSF patients. The ladder bands on Western immunoblot of rickettsial organisms were confirmed to be polysaccharide in nature, which was demonstrated by comparing them with the pattern of silver-stained gel of proteinase K-treated rickettsial specimens after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Light and Gravity Effects on Adenine Nucleotide Content and Energy Charge in Maize Roots

Two millimeter apical segments of maize (cv. LG 11) primaryroots were analysed in relation to the effects of light andgravity on adenine nucleotide content. Adenosine triphosphate(ATP) content is very sensitive to these stimuli. ATP levelsare lower in roots exposed to light than in those kept in thedark. The energy charge (E.C.) decreases markedly after exposureto light and gravity. For the vertical roots E.C. is stable.Present data confirm the fact that light and gravity may acton cell metabolism, modifying the energy requirements. Thiswill be discussed in relation to some hormone actions. (Received June 23, 1981; Accepted September 25, 1981)     

Effect of Adenine Nucleotides on the Respiration of Carrot Root Slices

Sodium pyruvate and dinitrophenol stimulated O(2) uptake of freshly cut phloem parenchyma from carrot roots by 63 and 120% at optimal concentrations, indicating that production of pyruvate by glycolysis regulates over-all respiratory rate. Adding 0.5 to 6.7 mm Na(3)ADP and Na(3)ATP to slices rapidly stimulates respiration rate by 20 to 85%. The effect is greater at the lower end of this concentration range and is not due to change in pH or active cation uptake. It is suggested that treating tissue with both nucleotides stimulates pyruvate kinase, the rate-limiting step in respiration of freshly cut slices, by increasing the concentration of endogenous ADP. Adenosine diphosphate continued to stimulate O(2) uptake until the peak of induced respiration, but ATP inhibited respiration during development and decline of this peak. Absence of respiratory stimulation by NaH(2)PO(4) and of respiratory inhibition by added nucleosides confirms that inorganic phosphate is not a limiting factor of respiration in freshly cut slices. The stimulation of respiration rate of these slices by dinitrophenol is consistent with results from experiments in which ADP and ATP were applied to the tissue.     

Energy Metabolism of Rumex Leaf Tissue in the Presence of Senescence-regulating Hormones and Sucrose

Hormones which inhibit senescence of Rumex leaf tissue in the dark include gibberellin A₃, and the cytokinins 6-benzylamino purine and zeatin. These hormones inhibit respiratory metabolism in this tissue, but do not change the pattern or total amount of oxygen consumption during senescence. Abscisic acid, a senescence accelerator, correspondingly stimulates oxygen consumption. This correlation of senescence rate and respiration rate holds with regard to the hormone concentrations effective and the continued activity of the hormones when added after the lag phase of chlorophyll breakdown. Transfer experiments show that the respiratory inhibition due to gibberellin A₃ and the promotion due to abscisic acid become established within 3 hours of hormone addition. When gibberellin A₃ and zeatin were rapidly added to narrow strips of tissue, no inhibitions of oxygen uptake were observed in the first 12 minutes. Senescence-inhibiting concentrations of sucrose strongly stimulate respiratory meabolism, raise the respiratory quotient, and cause inhibition of chlorophyll and protein breakdown which is distinct from the effect of gibberellins or cytokinins.     

Expression and Purification of the Crystalline Surface Layer Protein of Rickettsia typhi

The crystalline surface layer (S-layer) protein (SLP) of Rickettsia typhi is known as the protective antigen against murine typhus. We previously reported a cloning and sequence analysis of the SLP gene of R. typhi (slpT) and showed that the open reading frame of this gene encodes both the SLP and a 32-kDa protein. To express only the SLP from this gene, the putative signal sequence and the 32-kDa protein portion were removed from the slpT. This protein was expressed in Escherichia coli as a fusion protein, consisting of the SLP and maltose binding protein. The recombinant protein reacted strongly with polyclonal antiserum of a patient with murine typhus.     

Changes in Adenine Nucleotides of Intact Chromatium D Produced by Illumination

The total adenine nucleotide content of suspensions of Chromatium D averaged 14 nmoles/mg of dry weight. Of this, one-third to one-half was adenosine triphosphate (ATP), even in suspensions incubated in darkness. Illumination with high intensities caused a rise in ATP and a drop mainly in adenosine diphosphate, the new steady state being reached in 5 to 15 sec at room temperature. The dark steady state was re-established 15 to 30 sec after returning the suspensions to darkness. The rates of these changes were little affected by the presence of electron donors or CO(2), though their magnitude was reduced when substrates were added to starved suspensions. At limiting light intensities, complex kinetics characterized the transition from both dark to light and light to dark, and, at lower light intensities, more ATP was produced in suspensions supplemented with electron donors than in starved cells. The results show that photophosphorylation accompanying cyclic electron flow occurred in intact cells, and suggest that noncyclic phosphorylation can also occur.     

Cloning and sequencing of the gene encoding the candidate HtrA of Rickettsia typhi

We screened a phage library of Rickettsia typhi with a polyclonal antiserum to clone genes which encode immunogenic proteins of R. typhi. Among several clones obtained, one clone codes for a 466-amino-acid protein similar to the heat-shock protein, HtrA. The deduced rickettsial HtrA contains a putative signal peptide sequence at the N-terminus, a serine protease-like domain, and two PDZ domains. The recombinant protein of rickettsial HtrA reacted with sera from patients with murine typhus and tsutsugamushi disease. We suggest that this gene and its recombinant protein would be valuable for the immunologic diagnosis of rickettsial diseases.     

Deoxyribonucleic Acid Adenine and Cytosine Methylation in Salmonella typhimurium and Salmonella typhi

The methylations of adenine in the sequence -GATC- and of the second cytosine in the sequence - [Formula: see text] - were studied in Salmonella typhimurium and in Salmonella typhi. The study was carried out by using endonucleases which restrict the plasmid pBR322 by cleavage at the sequences -GATC- (DpnI and MboI) and - [Formula: see text] - (EcoRII). The restriction patterns obtained for this plasmid isolated from transformed S. typhimurium and S. typhi were compared with those of pBR322 isolated from Escherichia coli K-12. In E. coli K-12, adenines at the sequence -GATC- and the second cytosines at - [Formula: see text] - are met hylated by enzymes coded for by the genes dam and dem, respectively. From comparison of the restriction patterns obtained, it is concluded that S. typhimurium and S. typhi contain genes responsible for deoxyribonucleic acid methylation equivalent to E. coli K-12 genes dam and dcm.     

Oxypurinol-Enhanced Postischemic Recovery of the Rat Brain Involves Preservation of Adenine Nucleotides

Abstract: The present study investigated the effect of the administration of oxypurinol (40 mg/kg), an inhibitor of xanthine oxidase, on adenosine and adenine nucleotide levels in the rat brain during ischemia and reperfusion. The brains of the animals were microwaved before, at the end of a 20-min period of cerebral ischemia, and after 5, 10, 45, and 90 min of reperfusion. Cerebral ischemia was elicited by four-vessel occlusion with arterial hypotension to 45–50 mm Hg. Adenosine and adenine nucleotide levels in the oxypurinol-pretreated (administered intravenously 20 min before ischemia) rats were compared with those in nontreated animals exposed to the same periods of ischemia and reperfusion. Oxypurinol administration resulted in significantly elevated ATP levels at the end of ischemia and 5 min after ischemia, but not at 10 min after ischemia. ADP levels were also elevated, in comparison with those in the control rats, at the end of the ischemic period. Conversely, AMP levels were significantly reduced at the end of ischemia and during the initial (5 min) period of reperfusion. Adenosine levels were lower in oxypurinol-treated rats, during ischemia, and in the initial reperfusion phase. Oxypurinol administration resulted in a significant increase in the energy charge both during ischemia and after 5 min of reperfusion. Physiological indices, namely, time to recovery of mean arterial blood pressure and time to onset of respiration, were also shortened in the oxypurinol-treated animals. These beneficial effects of oxypurinol may have been a result of its purine-sparing (salvage) effects and of its ability to inhibit free radical formation by the enzyme xanthine oxidase. Preservation of high-energy phosphates during ischemia likely contributes to the cerebroprotective potency of oxypurinol.     

Effect of Nucleotides and Other Inhibitors on the Inactivation of Glutamate Decarboxylase

Abstract: The effects of 17 nucleotides and nucleotide analogs and 11 other compounds on the glutamate-promoted inactivation of brain glutamate decarboxylase were examined. Among the nucleotides, the major determinant of potency was the polyphosphate chain, Glutamate-promoted inactivation was strongly enhanced by low concentrations (<100 μM) of adenosine tetraphosphate and all eight nucleoside triphosphates tested. Nucleoside diphosphates enhanced inactivation, but were much less effective than the nucleoside triphosphates; nucleoside monophosphates were not effective. Modification of the polyphosphate chain of the nucleoside triphosphates also affected potency; adenylylimidodiphosphate and α,β-methylene ATP were about as effective as nucleoside diphosphates, but α,β-methylene ATP was nearly as effective as ATP. The nucleoside base had only a small effect on potency; purine nucleotides were more potent than pyrimidine nucleotides, and one nucleotide with a tricyclic base, 1, N⁶-etheno ATP, was as effective as the purine nucleoside triphosphates. The 2'-hydroxyl group of ribose was unimportant, since deoxy ATP was as effective as ATP. Three nonnucleotide polyanions were strong promoters of inactivation; inositol hexasulfate and 5-phosphorylribose 1-pyrophosphate were at least as effective as ATP; inositol hexaphosphate (phytate) was as effective as the nucleoside diphosphates. These results suggest that a major determinant of potency was a strong negative charge on the molecule. Negative charge was not sufficient, however, since fructose 1,6-bisphosphate did not promote inactivation. Inactivation by all of these compounds was slow, requiring more than 20 min for full effect. Two competitive inhibitors, chloride and glutarate, acted immediately and also reduced rather than enhanced glutamate-promoted inactivation.     

牡丹切花能荷水平及能量代谢与瓶插品质的关系

为了揭示牡丹切花能量调控规律及其与切花瓶插品质的关系,以发育至二级状态的‘洛阳红’切花为试材,研究高能荷水平(大田自然生长,FNG)和低能荷水平(自然生长采后5～6 ℃贮藏15 d,PCS)状态下切花瓶插品质与能荷以及能量代谢的敏感基因SnRK1表达的关系。结果表明：(1)与FNG(高能荷)相比,PCS(低能荷)明显加快瓶插过程中牡丹切花开放和衰老进程,显著缩短瓶插寿命和减小最大花径,并提早呼吸跃变峰值,提高呼吸速率,加快蔗糖降解,导致花瓣琥珀酸脱氢酶(SDH)和线粒体复合体Ⅳ(CCO)活性下降加快,造成ATP和EC水平快速下降,引起PsSnRK1的表达水平上调时间提前,表达水平提高。(2)PsSnRK1 基因能够对牡丹花瓣能量匮乏快速响应,能荷水平下降可诱导 PsSnRK1基因表达上调。研究发现,冷藏后花瓣能荷水平下降是导致牡丹切花瓶插过程中花朵快速衰老的重要因素之一,花瓣能量状况在调控牡丹切花瓶插品质过程中起着重要作用,并为通过调节牡丹切花能量水平提高切花瓶插品质提供一种新策略。