Nuclear magnetic resonance study of the impact of ribose 2′-O-methylation on the aqueous solution conformation of cytidylyl-(3′ → 5′)-cytidine

In order to obtain information about the conformational characteristics at the nearestneighbor level in the 2′-O-methylated region of t-RNA, as well as in the bizarre 5′-terminus of eucaryotic mRNA, a detailed nuclear magnetic resonance study of 2′-O-methyl-cytidylyl-(3′ → 5′)-cytidine (CmpC) was conducted. Proton spectra were recorded at 270 MHz in the Fourier mode in D₂O solutions, 0.01M, pD 7.3 in the temperature range 5–80°C. Complete accurate sets of nmr parameters were derived for each of the nucleotidyl units by a combination of homo-nuclear decouplings and simulation iteration methods. The data were translated into conformational parameters using procedures developed in earlier studies from these laboratories. It is shown that the ribofuranose ring exists at a ²E ? ³E equilibrium with clear preference [(75–80)%] for the ³E mode. The C(4′)-C(5′) and C(5′)-O(5′) bonds form a stable conformational network with outspoken preference for conformers in which Ψ₁, Ψ₂ ? 60° and ?₂ ? 180°. The orientation of the 3′-phosphate and 2′-O-methyl groups is such that ?₁′ ? 210° and ?″ ? 60°. The phosphodiester bonds are flexible and shift trends for base, H(1′), and H(5″) suggest the existence of a conformational blend of right-handed stack (g^?g^?), left-handed stack (g⁺g⁺), and unstacked arrays (tg^? and tg⁺). Elevation of temperature perturbs the ²E ? ³E equilibrium accompanied with modest depopulation of ψ₁, ψ₂ ? 60° and ?₂ ? 180° conformers. The major effect of elevation of temperature is in the increase of unstacked arrays at the expense of g^?g^? and g⁺g⁺ conformers. The shift trend of Cmp-H(3′) with temperature shows that torsional variation about O(3′)-P is facilitated by increase in temperature and the preferred rotamer about O(3′)-P in the unstacked form is t (ω₁′ = 180°). A detailed comparison of the aqueous solution conformations of CpC and CmpC reveals that 2′-O-methylation causes: (i) a reduction in the magnitude of _χ1; (ii) an increase in the population of ³E pucker at the 3′-nucleotidyl unit; and (iii) modest perturbations in the O(3′)-P and P-O(5′) bond conformations. Comparison of the aqueous solution conformations of AmpA and CmpC makes clear that the conformational properties of pyrimidine-pyrimidine and purine-purine dimers which carry a 2′-O-methylated 3′-nucleotidyl unit are significantly different.     

Endogene Synthese kurzlebiger Messenger-RNS in der Oocyte vonDysdercus intermedius Dist. nach Abschluß der Vitellogenese

Zusammenfassung In den Oocyten des telotroph-meroistischen Ovars vonDysdercus intermedius Dist. findet während der Endphase der Oogenese, 4–14 h vor der Eiablage, eine Synthese von nichtribosomaler RNS statt. Eine in vivo-Markierung dieser RNS läßt sich erreichen, wenn radioaktive RNS-Vorstufen einem Nucleotidpool zugeführt werden, der im Ooplasma vor der Chorionbildung angelegt wird.Diese vor der Eiablage gebildete RNS zeichnet sich durch einen hohen Turnover aus. Sie erscheint zunächst in Form einer hochmolekularen Vorstufe und wird im Verlauf weniger Stunden in kleinere, nichtribosomale Moleküle mit S-Werten zwischen 30 und 5 umgewandelt. Im frisch abgelegten Ei fehlen RNS-Spezies, die dieser endogenen Oocytensynthese entstammen; es sind nur noch ihre Degradationsprodukte, die sich innerhalb der Nucleotidfraktion ansammeln, nachweisbar. Die endogen synthetisierte RNS wird demnach im Gegensatz zu der in den Nährzellen synthetisierten und im Ei in stabiler Form gespeicherten RNS nicht für einen Bedarf während der Embryogenese konserviert.Die endogen synthetisierte RNS zeichnet sich durch einen hohen Poly (A)-Gehalt aus; 57% hybridisieren mit an Glasfaserfiltern immobilisiertem Poly(U). Wenige Stunden vor der Eiablage findet man kurzlebige oocytäre RNS-Moleküle an Polysomen assoziiert. Die Inkubation dieser Polysomen in einem in vitro-Proteinsynthese-System liefert Polypeptide, deren Auftrennung am SDS-Polyacrylamid-Gel ein charakteristisches Bandenspektrum ergibt. Die Molekulargewichte der 4 Hauptbanden liegen bei 65000, 48000, 44000, und 40000. Keines dieser Proteine ist mit einem Chorionprotein identisch.Die Kurzlebigkeit, der relativ hohe Poly (A)-Gehalt sowie die Fähigkeit, die Proteinsynthese sowohl in vivo als auch in vitro zu aktivieren, spricht dafür, daß die spät-oocytär gebildete heterogene Population von RNS-Molekülen mRNS-Komponenten enthält.Bei Frl. Heidrun Greipel bedanken wir uns für die ausgezeichnète Assistenz.     

Early events in calcium-induced liposome fusion

Calcium-induced interaction of liposomes composed of pure phosphatidylserine (PS) has been studied using a rapid-mixing, rapid-freeze device. Freeze-fracture electron microscopy of this material revealed that liposomes react very rapidly after addition of calcium ions. After only 10 ms (the resolution of the technique) vesicle fusion was apparent. At the same time, however, vesicles also collapsed, and appeared as aggregates of flattened membranes. This may explain controversies which have arisen over vesicle fusion studied with more indirect methods.     

The distribution of C-arachidonic acid in hamster lungs is sensitive to quinacrine

Isolated hamster lungs were labelled with ¹⁴C-arachidonic acid. When the lungs were ventillated with a respirator only a small amount of radioactivity was released to the perfusion effluent. This release was not changed significantly by pulmonary infusion of quicacrine (0.5 mM), a known inhibitor of phospholipase A₂. After the perfusion about 75% of the radioactivity in the lungs was in phospholipids, mainly in phosphatidylcholine, phosphatidylethanolamine and phosphatidylinostil and to a lesser degree in phosphatidylserine and phosphatidic acid. About one fourth of the radioactivity was in neutral lipids (tri- and diacylglycerols) and as free unmetabolized ¹⁴C-arachiodonic acid. Pulmonary infusion of quinacrine increased the amount of radioactivity in diacylglycerols and phosphatidylinositol but had no effect on that in phosphatidylcholine, phosphatidylserine, phosphatidic acid and triacylglycerols. The amount of radioactivity in phosphatidylethanolamine was decreased by quinacrine and increased in the vicinity of an unidentified phospholipid-quinacrine complex. The present study indicates that the distribution of ¹⁴C-arachidonic acid in hamster lung lipids is sensitive to quinacrine. The detected changes can, however, not be explained by an overall inhibition of phospholipase A₂ activities.     

Localization of vimentin,the nonspecific intermediate filament protein,in embryonal glia and in early differentiating neurons: In vivo and in vitro immunofluorescence study of the rat embryo with vimentin and neurofilament antisera

Antisera raised against vimentin, the protein subunit of nonspecific intermediate-sized filaments (IFs), were used in conjunction with neurofilament (NF) antisera to study the early development of neurons and glia in the rat embryo. Vimentin-positive fibers spanning the entire thickness of the neural tube including the cerebral vesicles were first observed on Day 12, concomitant with the appearance of NF protein in more confined areas (anterolateral regions of spinal cord and brain stem; motor roots emerging from the NF-positive areas). From Day 15 onwards vimentin and NF antisera selectively decorated glia and neurons, respectively, both in vivo and in vitro. Before Day 15 it appeared that NF-positive structures also stained with antivimentin in cryostat sections. In vitro experiments confirmed the presence of vimentin in early differentiating neurons. NF-positive cells were observed which also reacted with antivimentin in cultures obtained from 13- and 14-day embryos, but not later in development. Most neurons in these cultures became vimentin negative after 2–3 days in vitro.     

Delineation of type-specific regions on the envelope glycoproteins of human T cell leukemia viruses.

Two different approaches were used to map the type-specific regions on human T cell leukemia virus (HTLV) envelope glycoproteins. 1) Antibody reactivities of polymerase chain reaction-confirmed HTLV-I or HTLV-II carriers' sera were analyzed by Western blot assay with seven recombinant proteins containing different regions of HTLV-I or HTLV-II envelope proteins. 2) Rabbit antibodies elicited by nine HTLV-I Env synthetic peptides were used to react with the native HTLV envelope glycoproteins in an antibody-dependent cellular cytotoxicity (ADCC) assay. The results of the Western blot analysis showed that RP-B2, which contains amino acid residues 166 to 213 from HTLV-II exterior glycoprotein, was specifically reactive with 90.6% (48 of 53) of the HTLV-II carriers' sera but not with any of the HTLV-I carriers' serum (0 of 71). In contrast, RP-B, which contains amino acid residues 166 to 229 from HTLV-I exterior glycoprotein, was reactive with 85.1% (114 of 134) of the HTLV-I carriers' sera but not with any HTLV-II carrier serum (0 of 62). Furthermore, anti-HTLV-I Env synthetic peptide antibody-mediated ADCC identified several distinguishing HTLV-I ADCC epitopes in the middle region (amino acid residues 177 to 257) of the HTLV-I exterior glycoprotein. Therefore, HTLV type-specific epitopes reside mainly in a 69-amino acid sequence bounded by two cysteine residues (amino acids 157 and 225 for HTLV-I and 153 and 221 for HTLV-II), in the middle region of the exterior envelope glycoproteins.     

Inhibition of hepatic proteolysis by insulin. Role of hormone-induced alterations of the cellular K+ balance

1. Proteolysis was measured as [3H]leucine release from isolated perfused livers from rats, which had been labeled in vivo by an intraperitoneal injection of [3H]leucine about 16 h prior to the perfusion experiment. In livers from fed rats, insulin (35 nM) inhibited [3H]leucine release by 24.5 +/- 1.3% (n = 15) and led to an amiloride-sensitive, bumetanide-sensitive and furosemide-sensitive net K+ uptake of 5.53 +/- 0.31 mumol.g-1 (n = 15). Both the insulin effects on net K+ uptake and on [3H]leucine release were diminished by about 65% or 55% in presence of furosemide (0.1 mM) or bumetanide (5 microM), respectively. The insulin-induced net K+ uptake was virtually abolished in the presence of amiloride (1 mM) plus furosemide (0.1 mM). 2. In perfused livers from 24-h-starved rats, both the insulin-stimulated net K+ uptake and the insulin-induced inhibition of [3H]leucine release were about 80% lower than observed in experiments with livers from fed rats. The insulin effects on K+ balance and [3H]leucine release were not significantly influenced in the presence of glycine (2 mM), although glycine itself inhibited [3H]leucine release by 30.3 +/- 0.3% (n = 4) and 13.8 +/- 1.2% (n = 5) in livers from starved and fed rats, respectively. When livers from fed rats were preswollen by hypoosmotic perfusion (225 mOsmol.l-1), both the insulin-induced net K+ uptake and the inhibition of [3H]leucine release were diminished by 50-60%. 3. During inhibition of [3H]leucine release by insulin, further addition of glucagon (100 nM) led to a marked net K+ release from the liver (3.82 +/- 0.24 mumol.g-1), which was accompanied by stimulation of [3H]leucine release by 16.4 +/- 4.6% (n = 4). 4. Ba2+ (1 mM) infusion led to a net K+ uptake by the liver of 3.2 +/- 0.2 mumol.g-1 (n = 4) and simultaneously inhibited [3H]leucine release by 12.4 +/- 1.7% (n = 4). 5. There was a close relationship between the Ba2+ or insulin-induced net K+ uptake and the degree of inhibition of [3H]leucine release, even when the K+ response to insulin was modulated by bumetanide, furosemide, glucagon, hypotonic or glycine-induced cell swelling or the nutritional state. 6. The data suggest that the insulin-induced net K+ uptake involves activation of both NaCl/KCl cotransport and Na+/H+ exchange.(ABSTRACT TRUNCATED AT 400 WORDS)     

The organization of the gene for the human cerebroside sulfate activator protein

The organization of 14 exons covering 97% of the cDNA sequence of human cerebroside sulfate activator protein precursor has been determined from two overlapping EMBL-4 human genomic clones extending over 17kb. All exons and exon/intron splice junctions and five introns were sequenced. Exon 8 consists of only 9 bp and is involved in alternative splicing which generates three different mRNAs of cerebroside sulfate activator precursor.