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A single, modified nucleoside in Escherichia coli tRNA, 6-(3-methyl-2-butenylamino)-2-methyl-thio-9-beta-D-ribofuranosylpurine, has been desulfurized with Raney nickel to afford its probable biosynthetic precursor. The limitations of the reaction at the nucleoside and tRNA levels and its lack of inhibition of the amino acid acceptor activity of tRNA are described.
The highly sensitive quantitation of virus-specific CD8(+) T cells using major histocompatibility complex-peptide tetramer assays has revealed higher levels of cytotoxic T lymphocytes (CTLs) in acute and chronic virus infections than were recognized previously. However, studies in lymphocytic choriomeningitis virus infection have shown that tetramer assays may include measurement of a substantial number of tetramer-binding cells that are functionally inert. Such phenotypically silent CTLs, which lack cytolytic function and do not produce interferon (IFN)-gamma, have been hypothesized to explain the persistence of virus in the face of a quantitatively large immune response, particularly when CD4 help is impaired. In this study, we examined the role of functionally inert CTLs in chronic HIV infection. Subjects studied included children and adults (n = 42) whose viral loads ranged from <50 to >100,000 RNA copies/ml plasma. Tetramer assays were compared with three functional assays: enzyme-linked immunospot (Elispot), intracellular cytokine staining, and precursor frequency (limiting dilution assay [LDA]) cytotoxicity assays. Strong positive associations were observed between cell numbers derived by the Elispot and the tetramer assay (r = 0.90). An even stronger association between tetramer-derived numbers and intracellular cytokine staining for IFN-gamma was present (r = 0.97). The majority (median 76%) of tetramer-binding cells were consistently detectable via intracellular IFN-gamma cytokine staining. Furthermore, modifications to the LDA, using a low input cell number into each well, enabled LDAs to reach equivalence with the other methods of CTL enumeration. These data together show that functionally inert CTLs do not play a significant role in chronic pediatric or adult HIV infection.
Many peptide hormones in a variety of species are produced from larger precursors by limited proteolysis at basic amino acid-containing sites. The marine mollusc Aplysia has homologs of mammalian peptide-processing enzymes, including furin, prohormone convertase 1 (PC1), PC2, and carboxypeptidase E (CPE). A novel neuronal Aplysia enzyme was recently identified that was most closely related to carboxypeptidase D (CPD; Fan and Nagle, DNA Cell Biol. 15, 937-945, 1996), a second carboxypeptidase thought to be present in the secretory pathway and to contribute to peptide hormone processing. We have identified and cloned multiple overlapping bag-cell neuron cDNAs that encode two proteins that are members of the CPD family. Sequence analyses demonstrate that the longer CPD protein (1446 residues) contains an N-terminal signal peptide and four carboxypeptidase-like domains; the third and fourth domains are not predicted to form active enzymes, as several critical residues are absent. The shorter CPD protein is predicted to contain two active carboxypeptidase-like domains. Northern blot analysis identified a major Aplysia CPD mRNA (5.3 kb) and several smaller minor transcripts in central nervous system tissue. The CPD was purified from Aplysia ovotestis using a method previously developed for mammalian CPD. The purified Aplysia CPD binds antisera raised against regions of the protein encoded by the Aplysia cDNA clone, as well as an antiserum raised against duck CPD. The enzymatic properties of purified Aplysia CPD are generally similar to those of mammalian CPD. Aplysia CPD is a candidate prohormone-processing enzyme that may play a role in the processing of Aplysia prohormones in the secretory pathway.
Since mitochondrial oxidative phosphorylation does not produce sufficient adenosine triphosphate for rapid restoration of contractile function in myocardium reoxygenated after ischaemia alternative non-oxidative routes by which purine nucleosides or bases may be used to increase the cytoplasmic adenine nucleotide pool were studied. Comparative rates of uptake and salvage of adenosine, adenine, and hypoxanthine were determined using myocytes isolated from adult rat heart. For each precursor reactions limiting the overall rate at which substrate in the extracellular fluid is incorporated into the intracellular adenosine triphosphate pool were identified. Adenosine was salvaged at twice the rate seen with adenine in the presence of ribose, whereas the rate of salvage of hypoxanthine, in the presence of ribose, was only 5% of that for adenosine. Adenine may be an advantageous substrate since high concentrations of adenine are not inhibitory to salvage and do not influence cardiac haemodynamics. Salvage of hypoxanthine appeared to be limited by the rate of adenylosuccinate synthetase, which was present at less than 1% of the adenylosuccinase rate in rat, rabbit, and beef heart. In addition, since salvage of bases is dependent on the availability of phosphoribosylpyrophosphate rates of synthesis of phosphoribosylpyrophosphate were measured in whole myocytes from ribose and in cytoplasmic extracts from ribose and from ribose-5-phosphate. Metabolic sites were identified at which interventions designed to accelerate salvage rates might best be directed.
Feeding medium-chain triglycerides (MCT) is reported to result in less weight gain as fat than feeding long-chain triglycerides (LCT). In this experiment, fatty acid metabolism was studied in hepatocytes isolated from rats fed high fat diets containing MCT (70.9% of total energy), LCT (72.3% total energy) or a low fat (LF) (10.8% total energy). In the MCT group, hepatocyte utilization of [1-14C]oleate or [1-14C]octanoate, unlabeled ketone body (KB) production and lipogenesis (LG) were greater than in the other groups. Esterified products (EST) from both fatty acids were greater in the MCT groups, and 5-tetradecycloxy-2-furoic acid, an effective inhibitor of LG, did not abolish this. Lactate/pyruvate increased EST from octanoate and total LG but had little effect on octanoate oxidation. These data suggest that the capacity of medium-chain fatty acid esterification is adaptable to MCT content of the diet and may help regulate their metabolic fate. Despite high rates of KB production in the MCT group, the NADH-NAD ratio was not elevated. Rapid lipogenesis may contribute to the utilization of the excess energy equivalents formed. High LG and EST in the presence of a glycerol precursor may limit the usefulness of MCT in dietetic products for weight control. The animal fed an MCT diet is unusual in that the antithetical pathways of fatty acid catabolism and anabolism are being carried out simultaneously and at rapid rates. The lesser food efficiency of MCT may be related to the energy losses necessarily associated with cycling between these processes.(ABSTRACT TRUNCATED AT 250 WORDS)
Endogenous synthesis of taurine by cats is limited. Putative precursors of taurine, cysteinesulfinic acid and cysteic acid, were fed to cats to determine whether they were utilized. Groups of five cats were depleted of taurine by a resin (Colestipol(R)) diet, then given 6 dietary treatments containing (g/kg diet): 0.0, 0.4, or 0.8 taurine; or 0.98 or 1.96 cysteinesulfinic acid, or 0.4 taurine + 1.0 cysteic acid for 12 wk. Plasma and whole blood taurine concentrations and body weights were measured weekly. Concentration of taurine in semitendinosus muscle was measured initially, after 2 wk of taurine depletion (after resin-diet), and monthly thereafter. The resin diet decreased concentrations of taurine in plasma, whole blood, and muscle to 0.20, 0.49, and 0.37 of initial values, respectively. Cysteinesulfinic acid diets resulted in no significant (P > 0.05) increase in the concentration of taurine in plasma, whole blood, or muscle, and no increased excretion of cysteinesulfinate or taurine in urine or feces. Cats fed the diets containing 1.0 g cysteic acid + 0.4 g taurine, or 0.8 g taurine/kg diet had similar concentrations of taurine in plasma, whole blood, and muscle. Aminotransferase activity for cysteinesulfinic acid in the liver and intestinal mucosa of cats and rats was higher than that for aspartic or cysteic acids. Transamination of dietary cysteinesulfinic acid to beta-sulfinylpyruvate (which spontaneously decomposes), rather than decarboxylation is postulated as the basis for no detectable conversion to taurine. In contrast, cysteic acid is reversibly transaminated to beta-sulfopyruvate which is stable and thereby is a precursor for taurine in cats.
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High levels of deoxyadenosine and deoxyguanosine in patients with inherited deficiency of either adenosine deaminase or purine-nucleoside phosphorylase, respectively, are considered to be responsible for the associated immunological disorder. The mechanism involves phosphorylation to the corresponding deoxyribonucleoside triphosphates which subsequently inhibit the CDP-reducing activity of ribonucleotide reductase. Addition of deoxycytidine protects cells from the cytotoxic effects of deoxyadenosine and deoxyguanosine by competition for phosphorylation and by replenishing dCTP, the apparent limiting DNA precursor. Addition of cytidine, but not uridine, led to a reversal of deoxyguanosine and thymidine growth inhibition, comparable to that obtained with deoxycytidine. Analysis of the intracellular nucleotide pools showed that increased levels of cytidine ribonucleotides were sufficient to overcome the inhibitory effects of dGTP and dTTP on CDP reduction, thereby circumventing a depletion of the dCTP pool. A partial reversal of deoxyadenosine toxicity was also obtained with addition of cytidine. In this case little change in the dCTP level was observed, but a decreased dGTP pool appeared to be correlated with growth inhibition. High cytidine ribonucleotide levels partially prevented this effect. The present results may encourage the use of cytidine in combination with deoxycytidine as a pharmacological regime in treatment of immunodeficiency disease associated with increased deoxyribonucleotide levels.
Myelinogenesis in developing rats was studied following chronic dosing with triethyl tin (TET), at a level of 1.0 mg TET/kg body wt/day. Experiments included starved controls with body weights depressed by 17 to 40% to equal those of the TET-treated groups. Rats at ages of 16, 21, and 30 days showed decreases relative to well-nourished controls in body weight, forebrain weight, myelin yield, cerebroside level, and specific activity of brain 2',3'-cyclic nucleotide-3'-phosphohydrolase when dosed with TET. At 30 days, myelin and cerebroside yields were reduced by approximately 55%, while CNP activity was reduced by less than 20%. No differences in the forebrain myelin protein composition between control, starved, and TET animals were noted. The rate of myelin protein synthesis relative to brain total protein (assayed by incorporation of intracranially injected [3H]glycine into brain homogenate and myelin proteins) was decreased in the TET rats in proportion to the decreased yield of myelin, but no particular myelin protein was preferentially affected. Matching starved controls exhibited similar body weight decreases, less pronounced forebrain weight decreases, and little or no decrease in myelin concentration. There was a relative increase in the myelin protein synthesis rate in the starved rats, indicating preferential utilization of limited protein precursors for myelin protein synthesis. Spinal cord myelin was also decreased in the TET rats, but less severely than in the forebrain. At all ages optic, but not sciatic, nerves showed decreases in myelin concentration with TET treatment. We conclude that TET inhibits forebrain growth and CNS myelination more severely than can be accounted for by a general metabolic insult.
The wine yeast Saccharomyces cerevisiae is central in the production of aroma compounds during fermentation. Some of the most important yeast-derived aroma compounds produced are esters. The esters ethyl acetate and isoamyl acetate are formed from alcohols and acetyl-CoA in a reaction catalysed by alcohol acetyltransferases. The pool of acetyl-CoA available in yeast cells could play a key role in the development of ester aromas. Carnitine acetyltransferases catalyse the reversible reaction between carnitine and acetyl-CoA to form acetylcarnitine and free CoA. This reaction is important in transferring activated acetyl groups to the mitochondria and in regulating the acetyl-CoA/CoA pools within the cell. We investigated the effect of overexpressing CAT2, which encodes the major mitochondrial and peroxisomal carnitine acetyltransferase, on the formation of esters and other flavour compounds during fermentation. We also overexpressed a modified CAT2 that results in a protein that localizes to the cytosol. In general, the overexpression of both forms of CAT2 resulted in a reduction in ester concentrations, especially in ethyl acetate and isoamyl acetate. We hypothesize that overproduction of Cat2p favours the formation of acetylcarnitine and CoA and therefore limits the precursor for ester production. Carnitine acetyltransferase expression could potentially to be used successfully in order to modulate wine flavour.
Antigen-specific T-cell responses may be described by combining three categories: (i) the specificity and effector functions of a T-cell population, (ii) the quantity of T-cell responses (i.e., the number of responding T cells within the CD4/CD8 population), and (iii) the "quality" of T cells (defined by the T-cell receptor [TCR] structure). Several methods to measure T-cell responses are now available including evaluation of T-cell precursors using limiting dilution, the enzyme-linked immunospot assay, ex vivo TCR variable (v)-segment analysis determined by flow cytometry, and TCR-CDR3 length analysis (spectratyping), as well as identification of peptide-specific T cells using major histocompatibility complex (MHC) class I tetramers containing appropriate peptides. Until now, only a limited set of MHC-peptide complexes have been available as tetramer complexes. We demonstrate that CD8(+) or CD4(+) T cells in patients with cancer can be molecularly defined using a combination of spectratyping (TCR structure and "molecular composition") plus the implementation of an antibody panel directed against 21 individual VB TCR chains ("quantity" of T-cell families). This approach is instrumental in defining and comparing the magnitudes of CD4(+) or CD8(+) T-cell responses over time in individual patients, in comparing the TCR VA and VB repertoire in different anatomic compartments, and in comparing the TCR VA-VB diversity with that in normal healthy controls. This method provides the means of objectively defining and comparing the TCR repertoire in patients undergoing vaccination protocols and underlines the necessity to calibrate the TCR-CDR3 analysis with a qualitative assessment of individual TCR VB families.
Although live attenuated vaccine strains of simian immunodeficiency virus (SIV) have proven highly effective in protecting macaques against challenge with pathogenic SIV strains, little is known about the mechanisms of protective immunity induced by these vaccines. We examined cytotoxic T-lymphocyte (CTL) responses against SIV in animals infected with SIVmac239delta nef (deficient in nef) or SIVmac239delta 3 (deficient in nef, vpr, and upstream sequences in U3). To enhance detection of SIV-specific CTL activity, we stimulated peripheral blood mononuclear cells with autologous B-lymphoblastoid cell lines which had been infected with recombinant vaccinia viruses expressing SIV proteins and subsequently inactivated with psoralen and UV light. Animals chronically infected with SIV239delta nef or SIV239delta 3 mounted vigorous CTL responses against the SIV Gag and Env proteins. This CTL activity was major histocompatibility class restricted and mediated by CD8+ T lymphocytes. CTL responses persisted at relatively high levels for more than 6 years after infection. Limiting dilution precursor frequency assays demonstrated that the frequency of SIV-specific CTLs was as high as 234 CTL precursors per 100,000 cells. Animals acutely infected with SIV239delta nef developed CTL activity by day 14 after infection, coincident with decreases in viral load. Animals acutely infected with SIV239delta 3 developed CTL responses within 4 weeks of infection. Thus, vaccination of juvenile or adult animals with SIV239delta nef or SIV239delta 3 results in the induction of a vigorous CTL response which arises early in the course of infection and persists for years after a single inoculation of virus.
Two intrastrain variants of herpes simplex virus type 1 (HSV-1) were isolated from a newborn with fatal disseminated infection. A small-plaque-producing variant (SP7) was the predominant virus (>99%) in the brain, and a large-plaque-producing variant (LP5) was the predominant virus (>99%) in the lung and gastrointestinal tract. EcoRI and BamHI restriction fragment patterns indicated that SP7 and LP5 are related strains. The large-plaque variants produced plaques similar in size to those produced by HSV-1 KOS. Unlike LP5 or KOS, SP7 was highly cell associated and processing of glycoprotein C and glycoprotein D was limited to precursor forms in infected Vero cells. The large-plaque phenotype from KOS could be transferred into SP7 by cotransfection of plasmids containing the EK or JK EcoRI fragment or a 3-kb plasmid with the UL34.5 gene of HSV-1 KOS together with SP7 DNA. PCR analysis using primers from within the ICP34.5 gene indicated differences for SP7, LP5, and KOS. Sequencing data indicated two sets of deletions in the UL34.5 gene that distinguish SP7 from LP5. Both SP7 and LP5 variants were neurovirulent (lethal following intracranial inoculation of young BALB/c mice); however, the LP5 variant was much less able to cause lethal neuroinvasive disease (footpad inoculation) whereas KOS caused no disease. Passage of SP7 selected for viruses (SLP-5 and SLP-10) which were attenuated for lethal neuroinvasive disease, were not cell-associated, and differed in the UL34.5 gene. UL34.5 from SLP-5 or SLP-10 resembled that of KOS. These findings support a role for UL34.5 in promoting virus egress and for neuroinvasive disease.
To observe changes in the rate of RNA synthesis during Dictyostelium discoideum spore germination, we have pulse labeled germinating spores with [3H]uracil at stage-specific intervals. The labeled RNAs have been fractionated on 1.7 and 10% polyacrylamide gels. Additionally we have monitored the total changes in ribonucleic acid content during germination. In general, the rate of synthesis of various RNA species appears to increase noncoordinately, while the total RNA content remains unchanged during germination. This may indicate extensive RNA turnover during germination. Little or no RNA synthesis is observed during the first pulse period (postactivation lag). During the 1-2-h pulse period, 25, 17, 5.8, and 5 S RNA were all synthesized. The synthesis of these species increased with each pulse period, with the greatest amount of synthesis occurring during the final 3-4-h pulse. The synthesis of 4 S RNA initiated during the 2-3-h pulse and increased during the final 3-4-h pulse. The percentage of poly(A) + RNA increased with each time period from 0.1% of the total labeled RNA at the 0-1-h pulse period to 10% of the total RNA synthesized at the 2-3-h pulse time. During the final pulse period (3-4 h), this level dropped to 6%. Analysis of acid-soluble extracts from dormant spores revealed relatively high levels of the four ribonucleoside 5'-triphosphates. These levels remained unchanged during the 1st h of germination. Thus RNA synthesis during the postactivation lag stage of germination does not appear to be limited by precursor availability and may involve other regulatory mechanisms.
Macaca nemestrina has been described as an animal model for acute HIV-1 infection. This animal, unlike most infected humans, appears to contain HIV-1 replication. Therefore analysis of HIV-1-specific proliferative and cytotoxic T lymphocyte (CTL) responses following HIV-1 challenge of M. nemestrina may provide information into the role of such responses in both the control of acute HIV infection and protective immunity. Although CD4+ T cell responses to HIV-1 are generally difficult to detect in HIV-1-infected humans, early and persistent CD4+ T cell proliferative responses to HIV-1 antigens were detected in all HIV-1-inoculated M. nemestrina. HIV-1-specific CD8+ CTL responses were evaluated in PBMC by stimulation with autologous cells expressing HIV-1 genes, limiting dilution precursor frequency analysis, and T cell cloning. CTL reactive with gag, env, and nef were present 4-8 wk after infection, and persisted to 140 wk after infection. The presence of both CD4+ and CD8+ T cell responses before and after clearance of HIV-1 viremia is consistent with a role for these responses in the successful control of HIV-1 viral replication observed in M. nemestrina. Further studies of T cell immunity in these animals that resist disease should provide insights into the immunobiology of HIV-1 infection.
[35S]Cysteine-labeled putative precursors for vasopressin-associated neurophysin (NP-VP) and oxytocin-associated neurophysin (NP-OT) were isolated from the supraoptic nuclei (SONs) of normal rats. Homozygous Brattleboro rats were deficient in one of these precursors, the NP-VP precursor. Direct support for the hypothesis that OT and its NP- and VP and its NP are synthesized from two separate macromolecular common precursors was obtained by limited proteolysis of the precursors with trypsin and identification of the fragments as NPs, VP, or OT by a number of criteria (14). In this paper, these common precursors designated propressophysin (precursor for NP-VP and arginine VP) and prooxyphysin (precursor for NP-OT and OT) were further characterized. Propressophysin was specifically bound by Concanavalin-A-Sepharose and was eluted by 0.2 M alpha-methyl mannoside, showing that it is glycosylated. In contrast, prooxyphysin does not appear to be glycosylated. Using [3H]fucose as label injected near the SONs, two proteins (mol wt, approximately 10,000) were identified, which appear to be derived from propressophysin, that are rapidly transported to the posterior pituitary of normal rats. These two proteins were absent in homozygous Brattleboro animals. Both propressophysin and prooxyphysin were bound by a NP-Sepharose affinity support. The binding was different from that of arginine VP or OT to NP, since it was independent of pH and was hydrophobic in nature. In addition to prooxyphysin, the SONs of homozygous Brattleboro rats also contained other [35S]cysteine-labeled protein (mol wt, 20,000) composed of a NP-like protein and NP-binding peptides. The tryptic peptides derived from these proteins were very different in their chromatographic (high performance liquid chromatography) properties than the tryptic peptides derived from propressophysin and prooxyphysin. This protein ('X') was not bound by a Concanavalin-A-Sepharose affinity column, although it had chromatographic properties similar to propressophysin on Sephadex G-75.
A study was conducted to investigate the effects of dietary phenylalanine and lysine deficiencies on thyroid morphology in chicks fed crystalline amino acid diets. Both mean follicle diameter and epithelial cell height were significantly reduced in birds fed either of the deficient diets as compared with controls. These results agree with previous studies on serum T4 and T3 concentrations where the effects of an amino acid deficiency were general and not limited to precursors of thyroxine.