Fusion of Self-Assembling Amphipathic Oligopeptides with Cyclodextrin Glycosyltransferase Improves 2-O-d-Glucopyranosyl-l-Ascorbic Acid Synthesis with Soluble Starch as the Glycosyl Donor

In this study, we fused six self-assembling amphipathic peptides (SAPs) with cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans to catalyze 2-O-d-glucopyranosyl-l-ascorbic acid (AA-2G) production with cheap substrates, including maltose, maltodextrin, and soluble starch as glycosyl donors. The results showed that two fusion enzymes, SAP5-CGTase and SAP6-CGTase, increased AA-2G yields to 2.33- and 3.36-fold that of wild-type CGTase when soluble starch was used as a substrate. The cyclization activities of these enzymes decreased, while disproportionation activities increased. Enzymatic characterization of the two fusion enzymes was performed, and kinetics analysis of AA-2G synthesis confirmed the enhanced soluble starch specificity of SAP5-CGTase and SAP6-CGTase compared to that in the wild-type CGTase. As revealed by structure modeling of the fusion and wild-type CGTases, enhanced substrate-binding capacity may result from the increased number of hydrogen bonds present after fusion. This study demonstrates an effective protein fusion approach to improving the substrate specificity of CGTase for AA-2G synthesis. Fusion enzymes, especially SAP6-CGTase, are promising starting points for further development through protein engineering.     

MD-2 Residues Tyrosine 42, Arginine 69, Aspartic Acid 122, and Leucine 125 Provide Species Specificity for Lipid IVA

Lipopolysaccharide (LPS) activates the innate immune response through the Toll-like receptor 4 (TLR4)·MD-2 complex. A synthetic lipid A precursor, lipid IV_A, induces an innate immune response in mice but not in humans. Both TLR4 and MD-2 are required for the agonist activity of lipid IV_A in mice, with TLR4 interacting through specific surface charges at the dimerization interface. In this study, we used site-directed mutagenesis to identify the MD-2 residues that determine lipid IV_A species specificity. A single mutation of murine MD-2 at the hydrophobic pocket entrance, E122K, substantially reduced the response to lipid IV_A. Combining the murine MD-2 E122K with the murine TLR4 K367E/S386K/R434Q mutations completely abolished the response to lipid IV_A, effectively converting the murine cellular response to a human-like response. In human cells, however, simultaneous mutations of K122E, K125L, Y41F, and R69G on human MD-2 were required to promote a response to lipid IV_A. Combining the human MD-2 quadruple mutations with the human TLR4 E369K/Q436R mutations completely converted the human MD-2/human TLR4 receptor to a murine-like receptor. Because MD-2 residues 122 and 125 reside at the dimerization interface near the pocket entrance, surface charge differences here directly affect receptor dimerization. In comparison, residues 42 and 69 reside at the MD-2/TLR4 interaction surface opposite the dimerization interface. Surface charge differences there likely affect the binding angle and/or rigidity between MD-2 and TLR4, exerting an indirect influence on receptor dimerization and activation. Thus, surface charge differences at the two MD-2/TLR4 interfaces determine the species-specific activation of lipid IV_A.     

Site-saturation engineering of lysine 47 in cyclodextrin glycosyltransferase from Paenibacillus macerans to enhance substrate specificity towards maltodextrin for enzymatic synthesis of 2-O-d-glucopyranosyl-l-ascorbic acid (AA-2G)

In this work, the site-saturation engineering of lysine 47 in cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans was conducted to improve the specificity of CGTase towards maltodextrin, which can be used as a cheap and easily soluble glycosyl donor for the enzymatic synthesis of 2-O-d-glucopyranosyl-l-ascorbic acid (AA-2G) by CGTase. When using maltodextrin as glycosyl donor, four mutants K47F (lysine→ phenylalanine), K47L (lysine→ leucine), K47V (lysine→ valine) and K47W (lysine→ tryptophan) showed higher AA-2G yield as compared with that produced by the wild-type CGTase. The transformation conditions (temperature, pH and the mass ratio of l-ascorbic acid to maltodextrin) were optimized and the highest titer of AA-2G produced by the mutant K47L could reach 1.97 g/l, which was 64.2 % higher than that (1.20 g/l) produced by the wild-type CGTase. The reaction kinetics analysis confirmed the enhanced maltodextrin specificity, and it was also found that compared with the wild-type CGTase, the four mutants had relatively lower cyclization activities and higher disproportionation activities, which was favorable for AA-2G synthesis. The mechanism responsible for the enhanced substrate specificity was further explored by structure modeling and it was indicated that the enhancement of maltodextrin specificity may be due to the short residue chain and the removal of hydrogen bonding interactions between the side chain of residue 47 and the sugar at ?3 subsite. Here the obtained mutant CGTases, especially the K47L, has a great potential in the production of AA-2G with maltodextrin as a cheap and easily soluble substrate.     

High-Affinity Transport of γ-Aminobutyric Acid, Glycine, Taurine, L-Aspartic Acid, and L-Glutamic Acid in Synaptosomal (P2) Tissue: A Kinetic and Substrate Specificity Analysis

In a cortical P2 fraction, [14C]gamma-aminobutyric acid ([14C]GABA), [14C]glycine, [14C]taurine, and [14C]glutamic and [14C]aspartic acids are transported by four separate high-affinity transport systems with L-glutamic acid and L-aspartic acid transported by a common system. GABA transport in cortical synaptosomal tissue occurs by one high-affinity system, with no second, low-affinity, transport system detectable. Only one high-affinity system is observed for the transport of aspartic/glutamic acids; as with GABA transport, no low-affinity transport is detectable. In the uptake of taurine and glycine (cerebral cortex and pons-medulla-spinal cord) both high- and low-affinity transport processes could be detected. The high-affinity GABA and high-affinity taurine transport classes exhibit some overlap, with the GABA transport system being more specific and having a much higher Vmax value. High-affinity GABA transport exhibits no overlap with either the high-affinity glycine or the high-affinity aspartic/glutamic acid transport class, and in fact they demonstrate somewhat negative correlations in inhibition profiles. The inhibition profiles of high-affinity cortical glycine transport and those of high-affinity cortical taurine and aspartic/glutamic acid transport also show no significant positive relationship. The inhibition profiles of high-affinity glycine transport in the cerebral cortex and in the pons-medulla-spinal cord show a significant positive correlation with each other; however, high-affinity glycine uptake in the pons-medulla-spinal cord is more specific than that in the cerebral cortex. The inhibition profile of high-affinity taurine transport exhibits a nonsignificant negative correlation with that of the aspartic/glutamic acid transport class.     

Alternative Routes for the Synthesis of 5-Aminolevulinic Acid in Maize Leaves : I. Formation from 2-Ketoglutarate via 4,5-Dioxovaleric Acid

Cell-free extracts from greening maize (Zea mays L.) leaves catalyze the conversion of [¹⁴C]2-ketoglutarate (KG) to [¹⁴C]5-aminolevulinic acid (ALA) in a reaction which requires NADH and an amino donor and shows maximal activity around pH 6.5. The enzymic system is located in the cytosol. This cell fraction contains a low level of `KG dehydrogenase' activity and a transaminase which catalyzes the conversion of 4,5-dioxovaleric acid (DOVA) to ALA. The transaminase can use glutamate, aspartate, or alanine as amino donor. It is effectively inhibited by aminooxyacetate and ethylenediamine tetraacetate and shows maximal activity at pH 6.7. The activity of DOVA transaminase is only slightly affected by preillumination of leaves and can also be detected in green leaves and in roots.

DOVA was isolated from leaves and roots and determined as its benzoquinoxaline derivative. Significant amounts were found only in tissues in which ALA had accumulated or after it was exogenously supplied. DOVA was labeled in vivo by both [¹⁴C]ALA and [¹⁴C]KG. Small amounts were also formed from ALA in a cell-free system.

It is suggested that DOVA may be an intermediate in the diversion of ALA to respiratory metabolism and that it is not involved in the biosynthesis of this porphyrin precursor.

     

Photorespiratory Amino Donors, Sucrose Synthesis and the Induction of CO2 Fixation in Barley Deficient in Glutamine Synthetase and/or Glutamate Synthase

Murray, A. J. S., Black well, R. D., Lea, P. J. and Joy, K.W. 1988. Photorespiratory amino donors, sucrose synthesis andthe induction of CO₂ fixation in barley deficient in glutaminesynthetase and/or glutamate synthase.—J. exp. Bot. 39:845–858. A number of mutants of barley have been produced which lackboth chloroplastic glutamine synthetase and ferredoxin-dependentglutamate synthase activities. The plants accumulated ammoniato the same extent as mutants deficient in only glutamine synthetasebut shared the gas-exchange characteristics of the glutamatesynthase deficient parent. These mutants have been used to demonstratedirectly the ability of alanine to ameliorate the dramatic dropin fixation rate normally exhibited by glutamate synthase deficientmutants on transfer to photorespiratory conditions. Immediatelyafter transfer to air, the mutants deficient in glutamate synthaseactivity demonstrated a reduced ability to incorporate ¹⁴C derivedfrom ¹⁴CO₂ into sucrose. This effect was, however, dependenton the previous induction of CO₂ fixation. Use of ¹⁴CO₂ revealedthat the induction phase of CO₂ fixation was altered in allthree mutants. Neither of the parents nor the double mutantaccumulated sucrose in air under conditions which promote sucroseaccumulation by the wild type. The implications of these resultsfor photosynthesis and the control of sucrose synthesis arediscussed. Key words: Photorespiratory barley mutant, amino donors, sucrose, GS, glutamate synthase.     

Effects of Light and Inhibitors on Glutamate Metabolism in Leaf Discs of Vicia faba L: Sources of ATP for Glutamine Synthesis and Photoregulation of Tricarboxylic Acid Cycle Metabolism

Metabolism of [¹⁴C]glutamate was studied in leaf discs of Vicia faba L. in light and in darkness. In white light glutamine was the main labeled product. In the dark label was principally in compounds closely associated with tricarboxylic acid cycle metabolism, predominantly aspartate. Entry of label from glutamate into tricarboxylic acid metabolism appeared to be at least partially by decarboxylation of glutamate to γ-amino butyric acid, followed by conversion to succinate. 3-(3,4-dichlorophenyl)-1, 1-Dimethylurea inhibited light-enhanced synthesis of glutamine and caused reversion toward the dark pattern of metabolism. Methionine sulfoximine severely inhibited glutamine synthesis and caused accumulation of labeled malate.     

Effects of Arachidonic Acid on Dopamine Synthesis, Spontaneous Release, and Uptake in Striatal Synaptosomes from the Rat

Abstract: Arachidonic acid (AA) markedly stimulated, in a dose-dependent manner, the spontaneous release of [³H]dopamine ([³H]DA) continuously synthesized from [³H]tyrosine in purified synaptosomes from the rat striatum. As estimated by simultaneous measurement of the rate of [³H]H₂O formation (an index of [³H]tyrosine conversion into [³H]DOPA), the AA response was associated with a progressive and dose-dependent reduction of [³H]DA synthesis. In contrast to AA, arachidic acid, oleic acid, and the methyl ester of AA (all at 10⁻⁴ M ) did not modify [³H]DA release. The AA (3 × 10⁻⁵ M )-evoked release of [³H]DA was not affected by inhibiting AA metabolism, with either 5,8,11,14-eicosatetraynoic acid or metyrapone, suggesting that AA acts directly and not through one of its metabolites. AA also inhibited in a dose-dependent manner [³H]DA uptake into synaptosomes, with a complete blockade observed at 10⁻⁴ M . However, AA (10⁻⁴ M ) still stimulated [³H]DA spontaneous release in the presence of either nomifensine or other DA uptake inhibitors, indicating that AA both inhibits DA reuptake and facilitates its release process. Finally, the AA (10⁻⁴ M )-evoked release of [³H]DA was not affected by protein kinase A inhibitors (H-89 or Rp -8-Br-cAMPS) but was markedly reduced in the presence of protein kinase C inhibitors (Ro 31-7549 or chelerythrine).     

Cys31, Cys47, and Cys195 in BinA Are Essential for Toxicity of a Binary Toxin from Bacillus sphaericus

The mosquito larvicidal binary toxin produced by Bacillus sphaericus is composed of 2 proteins called BinA and BinB. While BinB acts as specificity determinant, BinA is expected to bind to BinB, translocates into cytosol, and exerts its activity via an unknown mechanism. To study the role of cysteine in BinA, 3 cysteine residues were substituted by alanine and serine. Substitution at Cys195 significantly reduced the toxin activity, whereas substitution at Cys31 and Cys47 abolished its toxicity. Intrinsic fluorescent analysis suggested that all mutant proteins should have similar tertiary structure to that of the wild type. Analysis of the mutant protein on sodium dodecyl sulfate–polyacrylamide gel electrophoresis with and without a reducing agent indicated that all 3 cysteine residues were not involved in disulfide bond formation within the BinA molecule. This is the first report to demonstrate that cysteine residues at 3 positions in BinA are required for full toxicity of the binary toxin. They may play a critical role during oligomerization or interaction between BinA and BinB to form the active complex.     

Elicitor-Induced Changes in Ca2+ Influx,K+ Efflux,and 4-Hydroxybenzoic Acid Synthesis in Protoplasts of Daucus carota L

Suspension-cultured carrot cells (Daucus carota) and their protoplasts respond to a fungal elicitor prepared from the culture medium of Pythium aphanidermatum by accumulating 4-hydroxybenzoic acid (4-HBA). Protoplasts release the compound into the culture medium. Using 45CaCl2 as a tracer, we were able to demonstrate that the secretion of 4-HBA is preceded by a rapid increase in the Ca2+ influx and a concomitant K+ efflux. If the increased Ca2+ influx was prevented by ethyleneglycol-bis([beta]-aminoethylether)-N,N[prime]-tetraacetic acid, 4-HBA synthesis was inhibited by 70%. These results are discussed with regard to signal transduction from the plasma membrane to the nucleus of carrot protoplasts.     

Aspartate Aminotransferase for Synthesis of Transmitter Glutamate in the Medulla Oblongata: Effect of Aminooxyacetic Acid and 2-Oxoglutarate

The effects of aminooxyacetic acid (AOAA), a transaminase inhibitor, and 2-oxoglutarate, a precursor to glutamate by the activity of aspartate aminotransferase (AAT), on slices of rat medulla oblongata, cerebellum, cerebral cortex, and hippocampus were studied. The slices were superfused and electrically stimulated. There was a Ca2+-dependent stimulus-evoked release of endogenous glutamate, gamma-aminobutyric acid (GABA), and beta-alanine in all regions examined. AOAA (10(-4) and 10(-3) M) decreased the release of glutamate in the medulla oblongata and cerebellum but not in the hippocampus. L-Canaline, a specific inhibitor of ornithine aminotransferase, did not affect the glutamate release in the medulla. 2-Oxoglutarate (10(-3) M) increased the release of glutamate in the medulla oblongata and cerebellum but not in the cerebral cortex and hippocampus. Treatment with AOAA (10(-4) M) almost abolished the activities of AAT in all regions studied. AOAA (10(-4) and 10(-3) M) increased the stimulus-evoked release of GABA in the cerebellum, cerebral cortex, and hippocampus, whereas the stimulus-evoked release of beta-alanine was decreased by this agent in all regions studied. These results suggest the participation of AAT in the synthesis of the transmitter glutamate in the medulla oblongata and cerebellum of the rat.     

Synthesis of the Neurotoxin Quinolinic Acid in Apoptotic Tissue from Suberites domuncula: Cell Biological, Molecular Biological, and Chemical Analyses

Sessile marine animals, such as sponges, are prone to infection by prokaryotic as well as by eukaryotic attacking organisms. Using the sponge Suberites domuncula we document for the first time that in its apoptotic tissue a toxic compound is produced that very likely controls the elimination of the dying tissue. Apoptosis was induced by exposing the sponges to 2,2'-dipyridyl or by maintaining them under nonaeration conditions. After that treatment at least one eukaryotic epibiont (Bittium sp.) could be found grazing on apoptotic tissue. Cell proliferation assays demonstrated that aqueous extracts from unaffected sponge tissue displayed no cytotoxicity. However, addition of an extract from apoptotic tissue to neuronal cells from rat brain exerted strong toxicity. The underlying compound was identified as quinolinic acid; quantitative determination showed that quinolinic acid is present only in apoptotic tissue (4.8 mg/g dry wet weight). The complementary DNA encoding the key enzyme of the quinolinic acid pathway, 3-hydroxyanthranilate 3,4-dioxygenase, was cloned and characterized. The expression of this gene is up-regulated in apoptotic tissue. These data suggest that a complex molecular network controls apoptotic elimination of sponge tissue, which results in the synthesis of the bioactive compound quinolinic acid that controls the elimination of the tissue, perhaps via differential effects on grazing epibionts.     

Thermodynamic Potential for the Abiotic Synthesis of Adenine, Cytosine, Guanine, Thymine, Uracil, Ribose, and Deoxyribose in Hydrothermal Systems

The thermodynamic potential for the abiotic synthesis of the five common nucleobases (adenine, cytosine, guanine, thymine, and uracil) and two monosaccharides (ribose and deoxyribose) from formaldehyde and hydrogen cyanide has been quantified under temperature, pressure, and bulk composition conditions that are representative of hydrothermal systems. The activities of the precursor molecules (formaldehyde and hydrogen cyanide) required to evaluate the thermodynamics of biomolecule synthesis were computed using the concentrations of aqueous N₂, CO, CO₂ and H₂ reported in the modern Rainbow hydrothermal system. The concentrations of precursor molecules that can be synthesized are strongly dependent on temperature with larger concentrations prevailing at lower temperatures. Similarly, the thermodynamic drive to synthesize nucleobases, ribose and deoxyribose varies considerably as a function of temperature: all of the biomolecules considered in this study are thermodynamically favored to be synthesized throughout the temperature range from 0°C to between 150°C and 250°C, depending on the biomolecule. Furthermore, activity diagrams have been generated to illustrate that activities in the range of 10⁻²– 10⁻⁶ for nucleobases, ribose and deoxyribose can be in equilibrium with a range of precursor molecule activities at 150°C and 500 bars. The results presented here support the notion that hydrothermal systems could have played a fundamental role in the origin of life, and can be used to plan and constrain experimental investigation of the abiotic synthesis of nucleic-acid related biomolecules.     

Effects of Ammonia on Respiration, Adenylate Levels, Amino Acid Synthesis and CO2 Fixation in Cells of Chlorella vulgaris 11h in Darkness

The effects of NH₄Cl on respiration, adenylate and free aminoacid levels as well as dark CO₂ fixation were investigated usingnitrogen-starved Chlorella vulgaris 11h cells with or withoutaddition of methionine sulfoximine (MSX), an inhibitor of glutaminesynthetase. Upon addition of NH₄Cl (1 mM) to the cells not treatedwith MSX, respiration was stimulated and the level of ATP droppedrapidly, while the levels of ADP and AMP increased. NH₄Cl alsostimulated amino acid synthesis, especially of glutamine, andmarkedly enhanced dark CO₂ fixation. Addition of NH₄Cl to MSX-treatedcells stimulated respiration and lowered the level of ATP, butdid not enhance glutamine synthesis and only slightly stimulateddark CO₂ fixation. ⁴On leave from Institute of Medical Science, Advance R &D Co. Minami-Hashimoto, Sagamihara, Kanagawa-ken 220, Japan (Received January 28, 1984; Accepted April 19, 1984)     

兽疫链球菌透明质酸合成相关基因hasB的克隆以及性质研究

透明质酸是链球菌荚膜的主要组成部分,有着重要的生理功能。UDP-葡萄糖脱氢酶(HasB)是透明质酸合成中的一个关键酶,而C类链球菌的UDP-葡萄糖脱氢酶编码基因(hasB)尚未被克隆。通过hasB基因的上下游序列设计引物从兽疫链球茵的基因组中克隆出一段序列,测序结果显示其包含一个由1206个碱基组成的开放阅读框,所编码的蛋白序列同化脓链球菌和乳链球菌的UDP-葡萄糖脱氢酶蛋白序列分别有63．1％和70．6％的相似性。将这段基因置于T7启动子下,并在大肠杆菌中进行表达,能够得到一个约47kDa的蛋白,酶活测定显示其具有UDP-葡萄糖脱氢酶活性。这些结果表明所克隆的基因是兽疫链球菌的UDP-葡萄糖脱氢酶编码基因。     

Total Synthesis of Acetate from CO2: Methyltetrahydrofolate, an Intermediate, and a Procedure for Separation of the Folates

Whole-cell preparations of Clostridium thermoaceticum were exposed to a short pulse of (14)CO(2) under conditions in which double-labeled acetate was synthesized. Radioactive methyltetrahydrofolate monoglutamate, diglutamate, and triglutamates were isolated from extracts of the cells. The radioactivity was found to be exclusively in the five methyl position. The specific activities of the methyltetrahydrofolate derivatives were very high and were in accord with the proposal that methyltetrahydrofolates are the precursors of the methyl of acetate. A new method of separation of folates employing QAE-Sephadex chromatography and a linear gradient with triethylammonium bicarbonate is presented which completely resolves the common folate monoglutamates and, upon freeze-drying, yields salt-free preparations.     

Immunohistochemical analysis of bFGF, TGF-β1 and catalase in rectus abdominis muscle from cattle foetuses at 180 and 260 days post-conception

The potential for muscle growth depends on myoblast proliferation, which occurs essentially during the first two thirds of the foetal period in cattle. Thereafter, myofibres acquire their contractile and metabolic properties. Proliferation is regulated by molecular growth factors and by the tissue oxidative activity. The aim of this study was the quantification by immunochemistry of basic fibroblast growth factor (bFGF) and transforming growth factor beta 1 (TGF-β1) and also of enzyme catalase (CAT) activity in rectus abdominis muscle. Samples were collected from cattle foetuses of different growth potential at 180 and 260 days post-conception (dpc). One major conclusion from this work is that protein contents of the muscle tissue bFGF and, to a lower extent, CAT activity decreased with increasing age during the foetal life. No differences were found between the different genotypes of cattle. However, the CAT to bFGF ratio tended to be lower in fast-growing cattle and increased with foetal age. TGF-β1 did not change with age and was localised mostly at the vascular bed. CAT was detected in smooth and rough reticulum in striated muscles at 180 dpc, and additionally in mitochondria at 260 dpc. In conclusion, the balance between intracellular growth factors (bFGF and TGF-β1) and the activity of antioxidant enzyme CAT may participate in the regulation of the transition from myoblast proliferation to differentiation. Thus, increased ratio of CAT to bFGF might be a good index indicating initiation of muscle maturation in cattle foetus prior to birth.     

Inhibition of Protein Synthesis in Cell-Free Systems from Interferon-Treated, Infected Cells: Further Characterization and Effect of Formylmethionyl-tRNA(F)

The translation of encephalomyocarditis virus (EMC) RNA is markedly inhibited in cell-free systems from interferon-treated, vaccinia virus-infected L-cells (10, 11). The polypeptide products synthesized in response to EMC RNA in cell-free systems from these and untreated infected cells have been analyzed by electrophoresis on polyacrylamide gels. Qualitatively, the same EMC-specific polypeptides were synthesized throughout. In experiments using preincubated microsomes from normal Krebs cells to assay cell sap from L-cells which had been exposed to interferon prior to infection, only the amount of the EMC-specific polypeptide products was reduced. This result suggests that there is an inhibition very early in translation in interferon-treated, infected cells. Initiation seems a priori the more attractive site for this inhibition, but an effect shortly after initiation cannot be excluded. With unfractionated cell-free systems from interferon-treated infected L-cells, however, there appeared to be an additional minor inhibitory effect on polypeptide chain elongation, in that the EMC-specific polypeptides synthesized showed not only a reduction in amount but also a bias towards lower molecular weight. The formylated methionyl initiator tRNA (Fmet-tRNA_F) was used as a further probe into the apparent effect on intiation. With this reagent we have confirmed that there is one major initiation site for the translation of EMC RNA in these cell-free systems. In addition, the results have shown that EMC-specific polypeptide chains initiated with Fmet escape the major interferon-mediated inhibition at or shortly after initiation.     

Influence of Na+, K+, and Ca2+ on Glutamine Synthesis and Distribution in Rat Brain Cortex Slices: A Possible Linkage of Glutamine Synthetase with Cerebral Transport Processes and Energetics in the Astrocytes

The ouabain-induced suppression of glutamine synthesis and retention in incubating rat brain cortex slices was found to be mimicked by changes in the cationic content of the incubation medium, which cause an increase in the intracellular [Na+] and a decrease in the intracellular [K+]. The suppression of glutamine synthesis (and fixation of ammonia) was also found to take place when Ca2+ was omitted from the incubation medium. This occurred whether endogenous or exogenous glutamate was the substrate for glutamine synthesis. The suppressions cannot be due solely to an effect on glutamate uptake, because the uptake is not markedly affected by these conditions. The results show that Na+, K+, and Ca2+ influence the synthesis and distribution of glutamine in the brain. They suggest that Ca2+ and the Na+, K+ pump may serve a role in regulating the activity of ATP-dependent glutamine synthetase, a key enzyme of the glutamate-glutamine cycle, located in the astrocytes. This may be mediated via a direct effect on the enzyme or through an effect on the production of ATP.     

The Cell Cycle in Crithidia fasciculata. Temporal Relationships between Synthesis of Deoxyribonucleic Acid in the Nucleus and in the Kinetoplast1, 2

SYNOPSIS. Autoradiographic technics with tritium-labeled thymidine have been used to determine G1, S, G2 and D for the kinetoplast and the nucleus of Crithidia fasciculata at 15, 25 and 32 C. The kinetoplast completes division before the nucleus at all 3 temperatures. The S phases of both organelles occur in approximate synchrony and are approximately equal in length but the nucleus begins and completes S before the kinetoplast at the 2 lower temperatures. This relationship is reversed at 32 C. Most of the effect of temperature on generation time is due to its effect on the length of S. The results are compared with similar studies on C. luciliae, Trypanosoma mega, other protozoa and tissue cells in culture. The role of the approximate synchrony of nuclear and kinetoplastic cycles in maintenance of the kinetoplastic condition is discussed and the hypothesis is proposed that this synchrony results from the sharing by nucleus and kinetoplast of the same mechanism for the production of the deoxyribonucleotides used in replication of their respective DNAs.