Synthesis of functional human immunodeficiency virus tat protein in baculovirus as determined by a cell-cell fusion assay.

The human immunodeficiency virus tat protein is a strong trans-activator of the expression of mRNAs originating from the viral long terminal repeat. We have expressed the first 72 amino acids (coding exon 1) of this protein in eucaryotic Spodoptera frugiperda SF9 cells by using a baculovirus vector, Autographa californica nuclear polyhedrosis virus. We show that the baculovirus vector stably produced the 72-amino-acid form of the tat protein but was unable to stably synthesize a larger 101-amino-acid full-length version of the same polypeptide. The 72-amino-acid tat protein, when introduced into mammalian fibroblasts by using a cell-cell fusion technique, functionally trans-activated the expression of the human immunodeficiency virus long terminal repeat.     

Relative sensitivity of 32P-postlabelling of DNA and the autoradiographic UDS assay in the liver of rats exposed to 2-acetylaminofluorene (2AAF)

Groups of male Alderley Park rats were dosed concomitantly with 2-acetylaminofluorene (2AAF) by gavage at doses between 0.01 mg/kg and 40 mg/kg, and livers sampled 2-72 h later. The liver of one group of animals was perfused to yield hepatocytes which were assayed in vitro for unscheduled DNA synthesis (UDS) via incorporation of tritiated thymidine and autoradiography. DNA was extracted from the livers of the other group and DNA adduct levels determined using the 32P-postlabelling technique. The major C-8 2-aminofluorene/guanosine adduct and 3 minor adducts were quantitated, enabling the relative sensitivity of the 2 techniques to be compared. A dose- and time-related UDS response was observed, which, at the most sensitive time-point (12 h) enabled DNA repair to be discerned at a dose level of 0.1-1 mg/kg of 2AAF, a response classified as formally positive at 5 mg/kg 2AAF. Only the C-8 adduct, as determined by 32P-postlabelling, was discernible at 0.01 mg/kg of 2AAF, although other adducts were visible on autoradiograms at higher dose levels. It is concluded that as part of a well-defined dose response, UDS can be discerned with confidence for doses of 2AAF between approximately 0.1 and 5 mg/kg, and DNA adducts for doses of 2AAF between approximately 0.01 and 1 mg/kg. Discernible UDS for 2AAF in the rat liver is apparent at approximately 13 DNA (total) adducts/10(8) nucleotides, or approximately 8 DNA (C-8) adducts/10(8) nucleotides. The presumed C-8 2-acetylaminofluorene/guanosine adduct, prepared by reaction of 2-acetoxy-2-acetylaminofluorene (2AAAF) with DNA, was a significant but unreliable marker of 2AAF/DNA adducts in the rat liver in vivo. DNA repair did not appear to remove DNA adducts selectively, and adducts remained in DNA when discernible DNA repair had ceased.     

α-Ketoglutarate and Malate Uptake and Metabolism by Synaptosomes: Further Evidence for an Astrocyte-to-Neuron Metabolic Shuttle

This study was undertaken to provide further evidence relevant to the hypothesis that astrocytes supply one or more citric acid cycle intermediates to synaptic terminals, thereby serving an anaplerotic function necessitated by the synthesis and release of amino acid neurotransmitters. In our experiments, two populations of synaptosomes obtained from the brain of rats were separated from myelin and mitochondria by using Percoll to generate continuous density gradients. Both synaptosomal populations readily accumulated 14C-labelled alpha-ketoglutarate and L-malate by high-affinity transport systems. Hofstee plots of uptake velocity as a function of substrate concentration were highly nonlinear, indicating that uptake was mediated by two or more carriers, or was subject to negative cooperativity. At least one carrier was selective for alpha-ketoglutarate and another for malate, whereas a third carrier appeared to be present which transported both substrates. At low concentrations (approximately 1 microM), alpha-ketoglutarate transport was almost totally Na+-dependent, whereas malate uptake exhibited little Na+-dependency. The transport of alpha-ketoglutarate was associated with a net influx, and therefore was not due to a homoexchange process. alpha-Ketoglutarate and malate were metabolized rapidly to glutamate and aspartate, respectively, by both synaptosomal preparations; however, in all cases, label accumulated in gamma-aminobutyric acid rather slowly. The incorporation of label into glutamine from alpha-ketoglutarate was much greater in the high-density synaptosomes that in low-density synaptosomes, an indication that the former contained a higher proportion of astrogliasomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cerebral Metabolic Compartmentation as Revealed by Nuclear Magnetic Resonance Analysis of D-[1-13C]Glucose Metabolism

Abstract: Nuclear magnetic resonance (NMR) was used to study the metabolic pathways involved in the conversion of glucose to glutamate, γ-aminobutyrate (GABA), glutamine, and aspartate. d -[1^-13C]Glucose was administered to rats intraperitoneally, and 6, 15, 30, or 45 min later the rats were killed and extracts from the forebrain were prepared for ¹³C-NMR analysis and amino acid analysis. The absolute amount of ¹³C present within each carbon-atom pool was determined for C-2, C-3, and C-4 of glutamate, glutamine, and GABA, for C-2 and C-3 of aspartate, and for C-3 of lactate. The natural abundance ¹³C present in extracts from control rats was also determined for each of these compounds and for N-acetylaspartate and taurine. The pattern of labeling within glutamate and GABA indicates that these amino acids were synthesized primarily within compartments in which glucose was metabolized to pyruvate, followed by decarboxylation to acetyl-CoA for entry into the tricarboxylic acid cycle. In contrast, the labeling pattern for glutamine and aspartate indicates that appreciable amounts of these amino acids were synthesized within a compartment in which glucose was metabolized to pyruvate, followed by carboxylation to oxaloacetate. These results are consistent with the concept that pyruvate carboxylase and glutamine synthetase are glia-specific enzymes, and that this partially accounts for the unusual metabolic compartmentation in CNS tissues. The results of our study also support the concept that there are several pools of glutamate, with different metabolic turnover rates. Our results also are consistent with the concept that glutamine and/or a tricarboxylic acid cycle intermediate is supplied by astrocytes to neurons for replenishing the neurotransmitter pool of GABA. However, a similar role for astrocytes in replenishing the transmitter pool of glutamate was not substantiated, possibly due to difficulties in quantitating satellite peaks arising from ¹³C-¹³C coupling.     

Anticonvulsant sugar sulfamates. Potent cyclic sulfate and cyclic sulfite analogues of topiramate

Exploration structure-activity relationships surrounding the clinically effective antiepileptic drug topiramate (1) led to a series of potent anticonvulsants with a 4,5-cyclic sulfate or 4,5-cycli sulfite functionality. Key derivative 2 (RWJ-37947) is ca. 8 times more potent than topiramate in mice; it also features a long duration of action and a very favorable neurotoxicity index.     

SEQUENCES OF THE RRN18 GENES OF CHLAMYDOMONAS HUMICOLA AND C. DYSOSMOS ARE IDENTICAL,IN AGREEMENT WITH THEIR COMBINATION IN THE SPECIES C. APPLANATA (CHLOROPHYTA)1

The nuclear Rrn18 gene coding for small-subunit ribosomal RNA was amplified from Chlamydomonas humicola and C. dysosmos. The sequences were identical, in agreement with the combination of these two species under the name C. applanata on morphological and physiological grounds by Ettl and Schlösser (1992).     

Quantitative high-pressure liquid chromatographic analysis of methylated purines in DNA of rats treated with chemical carcinogens

A rapid, sensitive method for the quantitative measurement of certain major and modified purines in DNA of carcinogen-treated animals is presented. DNA hydrolysates are analyzed by high-pressure liquid chromatography combined with fluorescence detection and electronic integration of peaks. Limits of detection are approximately 7 ng for 7-methylguanine and 150 pg for O⁶-methylguanine. Between 100 and 250 μg target organ DNA from animals treated with several carcinogens was shown to contain readily detectable amounts of these methylated bases. The method provides results comparable to those obtained with conventional methods using radioactively labeled carcinogens.     

Characterization of an immunoglobulin binding protein homolog in the maize floury-2 endosperm mutant.

The maize b-70 protein is an endoplasmic reticulum protein overproduced in the floury-2 (fl2) endosperm mutant. The increase in b-70 levels in fl2 plants occurs during seed maturation and is endosperm specific. We have used amino acid sequence homology to identify b-70 as a homolog of mammalian immunoglobulin binding protein (BiP). Purified b-70 fractions contain two 75-kilodalton polypeptides with pl values of 5.3 and 5.4. Both 75-kilodalton polypeptides share several properties with BiP, including the ability to bind ATP and localization within the lumen of the endoplasmic reticulum. In addition, both b-70 polypeptides can be induced in maize cell cultures with tunicamycin treatment. Like BiP, the pl 5.3 form of b-70 is post-translationally modified by phosphorylation and ADP-ribosylation. However, modification of the pl 5.4 species was not detected in vitro or in vivo. Although the b-70 gene is unlinked to fl2, b-70 overproduction is positively correlated with the fl2 gene and is regulated at the mRNA level. In contrast, the fl2 allele negatively affects the accumulation of the major endosperm storage proteins. The physical similarity of b-70 to BiP and its association with abnormal protein accumulation in fl2 endoplasmic reticulum may reflect a biological function to mediate protein folding and assembly in maize endosperm.     

Modulation of transmission at a glutamate synapse.

The amino acid L-aspartate markedly potentiates the responses elicited by L-glutamate at excitatory neuromuscular synapses in lobster walking limbs. Results are consistent with the idea that aspartate increases the affinity between glutamate and its binding sites in the postsynaptic receptor. Although complications due to release from other amino acid sources are a serious qualification, studies of neurally induced release of glutamate and aspartate suggest that both amino acids are released from excitatory nerve terminals. Experiments comparing the potentiating action of a variety of amino acids with their ability to inhibit glutamate uptake are not supportive of the notion that inhibition of agonist removal is the primary mode of action in the potentiation process. However, this idea, as well as the suggestion that aspartate may induce release of glutamate from extrajunctional entrapment sites, are not ruled out. Indeed, it is likely that the modulatory process embodies a multiplicity of reactions with given ones dominating from preparation to preparation.