Metabolic differences between primary cultures of astrocytes and neurons from cerebellum and cerebral cortex. Effects of fluorocitrate

Astrocytes and neurons cultured from mouse cerebellum and cerebral cortex were analyzed with respect to content and synthesis of amino acids as well as export of metabolites to the culture medium and the response to fluorocitrate, an, inhibitor of aconitase. The intracellular levels of amino acids were similar in the two astrocytic populations. The release of citrate, lactate and glutamine, however, was markedly higher from cerebellar than from cortical astrocytes. Neurons contained higher levels of glutamate, aspartate and GABA than astrocytic cultures. Cortical neurons were especially high in GABA and aspartate, and the level of aspartate increased specifically when the extracellular level of glutamine was elevated. Fluorocitrate inhibited the TCA cycle in the astrocytes, but was less effective in cerebellar neurons. Whereas neurons responded to fluorocitrate with an increase in the formation of lactate, reflecting, glycolysis, astrocytes decreased the formation of lactate in the presence of fluorocitrate, indicating that astrocytes to a high degree synthesize pyruvate and hence lactate from TCA cycle intermediates.     

The human genome contains multiple sequences of varying homology to mouse mammary tumour virus DNA

Sequences related to the mouse mammary tumour virus (MuMTV) DNA were isolated from a genomic library of human DNA by screening under conditions of relaxed stringency. It is estimated that there are in the order of 50 MuMTV-like sequences per haploid genome and that the homology between the different human sequences and MuMTV varies by 15%.     

Integration of foreign genome fragments into cells transformed or cotransformed with fragmented adenoviral DNA

The integration of DNA of highly oncogenic simian adenovirus type 7 (SA7) and non-oncogenic human adenovirus type 6 (Ad6) into the genome of newborn rat kidney cells transformed by fragmented DNA preparations was studied using reassociation kinetics and spot hybridization. Transforming DNA was fragmented with the specific endonuclease SalI (SA7) and BglII (Ad6). In contrast to the cell transformation by intact viral DNA, transformation by fragmented DNA resulted in integration into the cellular genome of not only the lefthand fragment with the oncogene but also of other regions of the viral genome. Additionally integrated fragments were stable and preserved during numerous passages of cells lines, although they were no expressed, at least in the case of the Ad6-transformed cell line. The integration of the fragments of SA7 DNA was accompanied by loss of 25-50% of the mass of each fragment. Adding the linear form of the pBR322 plasmid to the preparation of transforming Ad6 DNA also contributed to its cointegration into the genome of the transformed cell. This technique of cell cotransformation with any foreign DNAs together with the viral oncogens may be used as an equivalent of an integration vector for eukaryotic cells.     

Recombination between satellite phage P4 and its helper P2. I. In vivo and in vitro construction of P4: :P2 hybrid satellite phage

P4::P2 hybrid satellite phages which carry a portion (including the P2 head gene Q and the cohesive end) of the left end of the P2 chromosome linked to the essential part of the P4 chromosome have been isolated by in vivo as well as in vitro recombination. These hybrids express gene Q and grow in the presence of a P2 helper even if defective in gene Q.     

Studies on some enzymes relevant to citric acid accumulation by Aspergillus niger

Enzymatic studies have been performed on a local strain of Aspergillus niger to find a correlation with citric acid accumulation. The activity of aconitase [aconitate hydratase, citrate(isocitrate) hydrolyase, EC 4.2.1.3] and isocitrate dehydrogenase (NADP⁺) [threo-d_s-isocitrate:NADP⁺ oxidoreductase (decarboxylating) EC 1.1.1.42] decreased after 4 days whereas that of citrate synthase [citrate oxaloacetate-lyase (pro-3S-CH₂COO^?→acetylCoA), EC 4.1.3.7] did so after 8 days, when citric acid accumulation in the medium reached a maximum (45.9 mg ml^?1). In vitro studies with mycelial cell-free extracts demonstrated inhibition of citrate synthase activity by sodium azide and potassium ferricyanide on both the 4th and 8th days. Aconitase was inhibited by sodium arsenate, sodium fluoride, iodoacetic acid and potassium ferricyanide only on the 4th day. Isocitrate dehydrogenase (NADP⁺) activity on the 4th and 8th days was inhibited by iodoacetic acid but was stimulated by potassium ferricyanide. The possible existence of isozyme species of these enzymes is discussed.     

Protein transfer from isoelectric focusing gels: The native blot

The nonelectrophoretic transfer of proteins from thin (0.5 mm) isoelectric focusing gels to nitrocellulose was complete in 1 h. Blotting was bidirectional with 60% of the protein transferred to the top blot and the remaining 40% to the bottom blot. The use of nondenaturing transfer buffers permits proteins to be blotted in the native state. Immunological determinants are preserved and enzyme activity can be detected on the blots.     

Isolation of a cloned DNA segment containing a ribosomal protein gene of Drosophila melanogaster

A Drosophila genomic DNA library in the vector Charon 4 was screened using cDNA derived from the small (6S-12S) poly(A)+ mRNA of 2-6-h-old Drosophila embryos. This fraction of mRNA is enriched for ribosomal protein-coding sequences. The selected recombinants were hybridized to total mRNA under conditions which allowed for isolation of homologous mRNAs. The mRNA from these RNA/DNA hybrids was eluted and translated in vitro. The translation products were analyzed by one- and two-dimensional electrophoresis with authentic ribosomal proteins as standards. One cloned DNA segment was found to contain a ribosomal protein gene, and a sequence which hybridizes strongly to at least 5 other ribosomal protein mRNAs.     

The Contribution of Citrate to the Synthesis of Acetyl Units in Synaptosomes of Developing Rat Brain

Abstract: The activities of pyruvate dehydrogenase, citrate synthase, and choline acetyltransferase in rat brain synaptosomes increased during on-togenesis by 3 and 14 times, respectively. Activity of ATP-citrate lyase decreased by 26% during the same period. Pyruvate consumption by synapto-somes from 1-day-old animals was 40% lower than that found in older rats; however, citrate efflux from intrasynaptosomal mitochondria in immature synaptosomes was over twice as high as that in mature ones. The rates of production of synaptoplasmic acetyl-CoA, ATP-citrate lyase were 1.03, 1.40, and 0.49 nmol/min/mg protein in 1-, 10-day-old, and adult rats, respectively. 3-Bromopyruvate (0.5 m M ) inhibited pyruvate consumption by 70% and caused a complete block of citrate utilization by citrate lyase in every age group. Parameters of citrate metabolism in cerebellar synaptosomes were the same as those in cerebral ones. These data indicate that production of acetyl-CoA. from citrate in synaptoplasm may be regulated either by adaptative, age-dependent changes in permeability and carrier capacity of the mitochondrial membrane or by the inhibition of synthesis of intramitochondrial acetyl-CoA. ATP-citrate lyase activity is not a rate-limiting factor in this process. Metabolic fluxes of pyruvate to cytoplasmic citrate and acetyl-CoA. are presumably the same in both cholinergic and noncholinergic nerve endings. The significance of citrate release from intrasynaptosomal mitochondria as a regulatory step in acetylcholine synthesis is discussed.