ColE1 hybrid plasmids containing Escherichia coli genes involved in the biosynthesis of glutamate and glutamine

The Clarke-Carbon bank of Escherichia coli strains carrying ColE1 hybrid plasmids was screened for complementation of gdh, gltB, and glnA mutations affecting nitrogen metabolism in E. coli. Plasmids which complemented each one of these mutations were isolated. In every case, the plasmids conferred to otherwise mutant cells the capacity to synthesize the corresponding wild-type enzymes: glutamate dehydrogenase, glutamate synthase, and glutamine synthetase (GS), respectively. For three representative plasmids, endonuclease restriction maps were constructed. One of the plasmids, pACR1, which complemented glnA mutations, including the glnA21::Tn5 insertion, was deemed to carry the glnA⁺ allele. GS synthesis by pACR1 $\text{glnA}{}^{\mathrm{+}}\text{glnA20}$ heterozygous merodiploids was subjected to repression by growth on 15 mm NH₄⁺ and had a twofold high derepressed level than wild-type (glnA⁺) haploid cells when grown on 0.5 mm NH₄⁺ or on glutamate as only nitrogen sources. The presence of glutamine as sole nitrogen source promoted repressed GS synthesis in the $\text{glnA}{}^{\mathrm{+}}\text{glnA20}$ merodiploids. By contrast, glutamine allowed almost fully derepressed synthesis of GS in glnA⁺ haploid cells.     

On the conformational dependence of the proton chemical shifts in nucleosides and nucleotides III. Proton chemical shifts of 5′-nucleotides as a function of different conformational parameters

The variation of the chemical shift of the protons of 5′-UMP and 5′-AMP is calculated as a function of χ_CN, ψ and ? torsion angles. The shift of H8 of 5′-AMP and H6 of 5′-UMP is found to be very sensitive to the value of χ_CN. For the anti conformations the shift of these protons is more sensitive to the value of the rotation about CS′-05′ than about C4′-CS′. For the protons of the ribose the calculations show that for the C2′-endo pucker H3′ and H2′ undergo the largest chemical shift variations when ? and ψ vary. The calculated variations are considered in relation with the role of the conformation of the nucleotides in the chemical shift variation between mono and polynucleotides and between the different helical structures of polynucleotides.     

DIASTOLIC OPENING OF THE AORTIC VALVE (AV) IN A CASE OF AORTIC INSUFFICIENCY DUE TO AV FENESTRATION

A patient with severe aortic insufficiency due to fenestration of the non-coronary aortic valve leaflet is described. A preoperative echocardiogram demonstrated early closure of the mitral valve and early diastolic separation of the aortic valve leaflets. These findings disappeared after partial surgical correction and subsequent hemodynamic improvement. Premature opening of the aortic valve is common in severe aortic insufficiency.     

Aggregation of mono- and dinucleosomes into chromatin-like fibers

Three classes of chicken erythrocyte chromatin particles differing in their content of lysine-rich histones and/or spacer DNA have been studied in order to determine their ability to aggregate into complexes resembling those observed in native chromatin. The complexes have been obtained in the presence of MgCl₂ and NaCl and studied by electron microscopy. Mononucleosomes, containing spacer DNA and histones H1 and H5, give rise to thick (about 70 nm) ellipsoidal particles in the presence of 0.5 mM MgCl₂. These particles are disrupted by the addition of small amounts of NaCl (5–20 mM). On the other hand in 0.5 mM MgCl₂ dinucleosomes give rise to regular fibrous complexes of about 40 nm in diameter which are very similar to native chromatin fibers. These complexes are much more stable when NaCl is added. We conclude that for the stability of nucleosomal aggregates, similar to native chromatin fibers, a continuity of DNA structure is not required, but the presence of divalent cations, spacer DNA and lysine-rich histones is essential.     

Mutations affecting the Shine-Dalgarno sequences of the untranslated region of the Escherichia coli gltBDF operon.

Individual mutations which affected each of the two Shine-Dalgarno sequences at the 5' untranslated region of the gltB gene of Escherichia coli were characterized. They were isolated in plasmids carrying a gltB'-'lacZ protein fusion preceded by the regulatory region of the gltBDF operon. Subcloning and nucleotide sequencing of approximately 1,206 bp of DNA encompassing the gltBDF regulatory region showed that the mutations affected the first base at each of the two identical Shine-Dalgarno sequences, SD1 and SD2, located 40 and 8 bases, respectively, upstream from the putative gltB open reading frame. Only mutation gltB2r227, an adenine in place of a guanine, affecting the first base of SD2, lowered beta-galactosidase expression significantly, i.e., about fivefold. The results suggest that SD2 is the preferred functional site at which ribosomes initiate gltB mRNA translation.     

Enhancement of Endogenous Release of Glutamate and γ-Aminobutyric Acid from Hippocampus CA1 Slices After In Vivo Long-Term Potentiation

The effect of long-term potentiation (LTP) on endogenous amino acid release from rat hippocampus slices was studied. LTP was induced in vivo by application of a tetanus (200 Hz, 200 ms) to the Schaffer collateral fibers in unanesthetized rats. Endogenous release of glutamate and gamma-aminobutyric acid (GABA) was investigated 60 min after tetanization in CA1 subslices of potentiated and control rats. No significant effects of LTP were observed in basal and K(+)-induced Ca(2+)-independent release components of these amino acids. In contrast, K(+)-induced Ca(2+)-dependent release of both glutamate and GABA increased approximately 100% in slices from potentiated rats. No differences were observed in total content of glutamate and GABA between the subslices from control and LTP animals. These results suggest a persistent increase in the recruitment of the presynaptic vesicular pool of glutamate and GABA during LTP.     

Phospholipid turnover during phagocytosis in human polymorphonuclear leucocytes

We have previously observed that the phagocytosis of zymosan particles coated with complement by human polymorphonuclear leucocytes is accompanied by a time- and dose-dependent inhibition of phosphatidylcholine synthesis by transmethylation [García Gil, Alonso, Sánchez Crespo & Mato (1981) Biochem. Biophys. Res. Commun. 101, 740–748]. The present studies show that phosphatidylcholine synthesis by a cholinephosphotransferase reaction is enhanced, up to 3-fold, during phagocytosis by polymorphonuclear cells. This effect was tested by both measuring the incorporation of radioactivity into phosphatidylcholine in cells labelled with [Me-¹⁴C]choline, and by assaying the activity of CDP-choline:diacylglycerol cholinephosphotransferase. The time course of CDP-choline:diacylglycerol cholinephosphotransferase activation by zymosan mirrors the inhibition of phospholipid methyltransferase activity previously reported. The extent of incorporation of radioactivity into phosphatidylcholine induced by various doses of zymosan correlates with the physiological response of the cells to this stimulus. This effect was specific for phosphatidylcholine, and phosphatidyl-ethanolamine turnover was not affected by zymosan. The purpose of this enhanced phosphatidylcholine synthesis is not to provide phospholipid molecules rich in arachidonic acid. The present studies show that about 80% of the arachidonic acid generated in response to zymosan derives from phosphatidylinositol. A transient accumulation of arachidonoyldiacylglycerol has also been observed, which indicates that a phospholipase C is responsible, at least in part, for the generation of arachidonic acid. Finally, isobutylmethylxanthine and quinacrine, inhibitors of phosphatidylinositol turnover, inhibit both arachidonic acid generation and phagocytosis, indicating a function for this pathway during this process.     

Role of H-2 antigens in the host response to methylcholanthrene-induced tumors

H-2 loss variant sublines of a sarcoma (M-AS), induced by methylcholanthrene in an (A × A.SW)F₁ mouse, were used to study the role of the MHC products in the recognition of MC-TSTA. The two reciprocal variant sublines (M-A and M-S) were found to express the TSTA of the original tumor as shown by cross-reactions in graft rejection experiments performed in (A × A.SW)F₁ mice. In the A/Sn and A.SW mice the presence of the reciprocal parental H-2 antigens on the immunizing cells decreased the response against the tumor antigens. An admixture of lymphocytes derived from hyperimmune mice inhibited the outgrowth of the tumor cells. The growth inhibition was mediated by T cells and was H-2 restricted. Cells derived from hyperimmune syngeneic mice inhibited the outgrowth of the variant subline used for immunization but had no effect on the reciprocal variant subline.     

Tyrosine transport by membrane vesicles isolated from rat brain

Tyrosine uptake by membrane vesicles derived from rat brain has been investigated. The uptake is dependent on an Na⁺ gradient ([$\text{Na}{}^{\mathrm{+}}\text{]}\text{outside}\text{> [}\text{Na}{}^{\mathrm{+}}\text{]}\text{inside}$). The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. The process is stimulated by a membrane potential (negative inside) as demonstrated by the effect of the ionophores valinomycin and carbonyl cyanide $\text{m-}\text{chlorophenylhydrazone}$ and anions with different permeabilities. Kinetic data show that tyrosine is accumulated by two systems with different affinities. Tyrosine uptake is inhibited by the presence of phenylalanine and tryptophan.