Changes in brain components during the development of mice homozygous for the locus “dwarf” (dw)1,5

Brain composition and developmental changes were investigated in mice homozygous for the locus dwarf, and characterized by a reduced level of growth hormone, thyroid stimulating hormone, and prolactin, and by secondary hypothyroidism. The difference in adult brain weight (–32%) between the dwarf and the normal mice was not found to parallel the difference in body weight (–71%), whereas the differences in the weight of the liver (–79%) and that of the kidney (–75%) did. Several biochemical parameters of brain development were assayed in dwarf and normal mice between the ages of 15 and 210 days. Levels of cerebrosides, sulfatides, gangliosides, phospholipids, cholesterol, protein, and RNA (per gram wet weight) were the same for the dwarf and the controls, but the net difference in total brain DNA was less than the net total brain RNA difference (–11% vs. –27%). Total brain lipids (absolute quantities) were the same at 15 days. The difference was –37% by the 50th day, and remained constant thereafter. No change in the specific activity of 2,3-cyclic nucleotide 3-phosphohydrolase or 3-phosphoadenosine-5-phosphosulfate: galactocerebroside sulfotransferase was observed. These data suggest that the regulation of the development of brain structures is maintained, but the level of the synthesis of the various brain constituents is reduced in proportion to the brain weight. The development of the dwarf brain seems to proceed harmoniously.Abbreviations used PAPS 3-phosphoadenosine-5-phosphosulfate - PAPS-CST 3-phosphoadenosine-5-phosphosulfate:galactocerebroside sulfotransferase - CNP 2, 3-cyclic nucleotide 3-phosphohydrolase - Neu NAc N-acetylneuraminic acid This paper is part of the Doctorat d'Etat thesis of L. L. Sarliève.     

Age Dependence of the Level ofcopia Retrotransposon Expression in the Testes of Drosophila melanogaster

Expression of the lacZ reporter gene controlled by various deletion derivatives of the regulatory region of the copia retrotransposon was studied in the testes of adult transgenic males of the Drosophila melanogaster y ¹ w ^67c23(2) strain at the age of 3, 6–9, 12–15, 18–21, and 24–27 days. When the construct contained the full-length regulatory region, which included the 5-long terminal repeat (LTR) and the 5-untranslated region (UTR), expression was the lowest in males aged 12–15 days and the highest in males aged 3 or 24–27 days. A similar V-shaped age dependence was previously observed for the copia transposition rate and RNA content in a strain with a high rate of copia transposition. Thus, the V-shaped age dependence of expression, which is unusual for Drosophila, proved to be characteristic of copiaregardless of its transposition rate. Deletion of the 5 or 3 end of the LTR, but not of the UTR, changed the age dependence of the level of reporter gene expression. In this case, expression was the highest in 3-day-old males and gradually decreased with age, as typical for many Drosophila genes. It was assumed that the 5- and 3-terminal regions of the copiaLTR contain regulatory elements responsible for the V-shaped age dependence of expression, while the expression level depends to a greater extent on the regulatory elements of UTR.     

Oligomerization of deoxynucleoside-bisphosphate dimers: Template and linkage specificity

Evidence is presented that a poly(U) template selectively favors the oligomerization of the activated, 3–5 pyrophosphate-linked dimer pdAppdAp, in comparison with the 3–3 and 5–5 linked dimers. In the absence of poly(U), the 5–5linked dimer is the most reactive, and chains are formed which are more than 60 monomer units in length.Nucleic Acid-Like Structures V. For the previous paper in this series see Visscher and Schwartz (1988).     

(2′–5′) Oligoadenylate synthetase in the maturation of rabbit reticulocytes

Summary The (2–5) oligoadenylate synthetase normally found in interferon-treated cells has also been detected in considerable amounts in normal rabbit reticulocytes not exposed to interferon. The activity of this enzyme has been followed during the development of the reticulocytes to erythrocytes.A high level was found just after the formation of reticulocytes and this activity decayed with a half-life of about 3 days. In lymphocytes the (2–5) oligoadenylate synthetase was found to stay at a constant level, which indicates the absence of interferon in the plasma.     

Observation of a distinct transition in the mode of interconversion of ring pucker conformers in non-crystalline d-ribose-2'-d from 2H NMR spin-alignment

Internal motions of d-ribose selectively ²H-labeled at the 2 position were measured using solid state ²H NMR experiments. A sample of d-ribose-2 -d was prepared in a hydrated, non-crystalline state to eliminate effects of crystal-packing. Between temperatures of –74 and –60°C the C2–H2 bond was observed to undergo two kinds of motions which were similar to those of C2–H2/H2 found previously in crystalline deoxythymidine (Hiyama et al. (1989) J. Am. Chem. Soc., 111, 8609–8613): (1) Nanosecond motion of small angular displacement with an apparent activation energy of 3.6 ± 0.7 kcal mol^–1, and (2) millisecond to microsecond motion of large amplitude with an apparent activation energy 4 kcal mol^–1. At –74°C, the slow, large-amplitude motion was best characterized as a two-site jump with a correlation time on the millisecond time scale, whereas at –60°C it was diffusive on the microsecond time scale. The slow, large-amplitude motions of the C2–H2 bond are most likely from interconversions between C2-endo and C3-endo by way of the O4-endo conformation, whereas the fast, small-amplitude motions are probably librations of the C2–H2 bond within the C2-endo and C3-endo potential energy minima.     

Effects of midazolam on the contraction and relaxation of segments of thoracic aorta stripped of endothelium and stimulated by adrenaline – experimental study in rabbits

To evaluate the effects of midazolam on the angiokinesis of segments of rabbits' thoracic aorta stripped of endothelium and stimulated by adrenaline.Two groups of aortic rings removed from albinic rabbits anesthetized with thiopental were used (Group I – 6 animals; Group II – 12 animals), stripped of endothelium, studied in an organ chamber, perfused by Krebs-Henseleit solution. The groups were stimulated by adrenaline, recording the maximum contraction and dT/dt at 12, 36, 60 and 120. When the plateau phase was reached, the vessel was washed with perfusion solution, recording relaxation at 2, 4 and 6. When the base values were reached, Group I underwent a new adrenergic stimulus; and Group II was stimulated with midazolam and then with adrenaline, and the same values were recorded. T test was applied as a statistical analysis when two variables were studied. When studying more than two variables the Anova test was used, supplemented by the Tuckey test.Group I did not show any significant difference between the two stimuli. Group II – the midazolam significantly reduced the maximum contraction induced by adrenaline (83.01 ± 4.11%) (p < 0.01). The dT/dt was reduced at 12 (57.06 ± 8.47%), and also at 36 (70.59 ± 5.26%). There was no significance at 60 and 120 (p < 0.01).The relaxation increased significantly at all measurements – at 2-adrenaline 39.31 ± 9.60%; adrenaline/midazolam: 44.06 ± 9.62% (p < 0.05). At 4-adrenaline: 53.08 ± 8.3%; adrenaline/midazolam: 61.68 ± 8.50% (p < 0.01). At 6-adrenaline: 76.26 ± 5.45%; adrenaline/midazolam: 84.20 ± 7.96% (p < 0.01).Midazolam significantly reduced the maximum contraction obtained by the adrenergic stimulus as well as the dT/dt in the initial phases of contraction. The relaxation speed also increased.     

Sugar conformation of a stereospecific 2′-R or 2′-S deuterium-labeled DNA decamer studied with proton-proton J coupling constants

The sugar conformation of a DNA decamer was studied with proton-proton ³J coupling constants. Two samples, one comprising stereospecifically labeled 2-R-²H for all residues and the other 2-S-²H, were prepared by the method of Kawashima et al. [J. Org. Chem. (1995) 60, 6980–6986; Nucleosides Nucleotides (1995) 14, 333–336], the deuterium labeling being highly stereospecific 99% for all 2-²H, 98% for 2-²H of A, C, and T, and 93% for 2-²H of G). The ³J values of all H1-H2 and H1-H2 pairs, and several H2-H3 and H2-H3 pairs were determined by line fitting of 1D spectra with 0.1–0.2 Hz precision. The observed J coupling constants were explained by the rigid sugar conformation model, and the sugar conformations were found to be between C3-exo and C2-endo with _m values of 26° to 44°, except for the second and 3 terminal residues C2 and C10. For the C2 and C10 residues, the lower fraction of S-type conformation was estimated from J_H1H2 and J_H1H2 values. For C10, the N–S two-site jump model or Gaussian distribution of the torsion angle model could explain the observed J values, and 68% S-type conformation or C1-exo conformation with 27° distribution was obtained, respectively. The differences between these two motional models are discussed based on a simple simulation of J-coupling constants.     

Influence of the xyloadenosine analogue of 2′,5′-oligoriboadenylate on poly(A)-specific, 2′,5′-oligoriboadenylate degrading 2′,3′-exoribonuclease and further enzymes involved in poly(A)(+)mRNA metabolism

The homogeneous poly(A)-specific 2,3-exoribonuclease from calf thymus gland, which cleaves both 3,5-and 2,5-linked oligoriboadenylates, does not degrade (xyloA2'p)₂ xyloA, the xylofuranosyladenosine analogue of the 2-5A core. This oligonucleotide, which is supposed to enter intact cells rapidly, was found to possess an increased stability and an enhanced antiherpesvirus activity compared to the natural (A2'p)₂A (Eppstein, D. A., Barnett, J. W., Marsh, Y. V., Gosselin, G. and Imbach, J.-L. (1983) Nature 302, 723–724). The poly(A) anabolic enzyme, poly(A) polymerase (Mn²⁺-dependent), from the same source, which is initiated by (A3'p)₂A and its higher oligomers, does not accept 2–5A core and its xyloadenosine analogue as primer. Both oligonucleotides exert no influence on endoribonuclease IV and on the integrity of the poly(A)-ribonucleoprotein complex.Abbreviations 2-5A ppp(A2'p)_nA(n2). 5-triphospho-oligo [(2–5)adenylyl]adenosine - 2-5A core (A2'p)₂A, adenylyl(2–5) adenylyl(2–5)adenosine - xyto 2-tA core (xyloA2'p)₂ xyloA, xyloadenylyl(2–5)xyloadenylyl(2–5)xyloadenosine The other abbreviations are according to the recommendations of the Commission on Biochemical Nomenclature, see Europ. J. Biochem.15 (1970) 203–208.     

Photoreactive fatty acid analogues that bind to the rat liver fatty-acid binding protein: 11-(5′-azido-salicylamido)-undecanoic acid derivatives

Summary Photoreactive probes for the hydrophobic pocket of the liver fatty acid-binding protein, 11-(5-azido-salicylamido)-undecanoic acid (5 ASU) and its acetyl ester (Ac5 ASU), were synthesized and their interaction with the protein was assessed. Fatty acid-binding proteins are closely related proteins which are abundantly expressed in tissues with active lipid metabolism. A simple model that assumes that the protein possesses a single kind of sites fitted the binding of radioiodinated 5 ASU to L-FABP satisfactorily. The apparent dissociation constant, 1.34×10^–7 M, evidenced a slightly higher affinity than that reported for C16–C20 fatty acids. Consistent with the binding curve, 5 ASU effectively competed with palmitic acid for the hydrophobic sites and the effect was nearly complete for concentrations of 1 gmM; oleic acid, in turn, displaced the radiolabelled probe. Irradiation at 366 nm of¹²⁵I-5 ASU bound to L-FABP caused the covalent cross-linking of the reagent. The amount of radioactivity covalently bound reached a maximum after 2 min thus agreeing with the photo-activation kinetics of the unlabelled compound that evidenced a t_1/2 of 31.1 sec. The yield with which probes bound to L-FABP became covalently linked to the protein, appraised after SDS-PAGE of irradiated samples, was estimated as 23 and 26 per cent for 5 ASU and Ac5 ASU respectively. In turn, irradiation of L-FABP incubated with 5ASU or Ac5 ASU resulted in the irreversible loss of about one fourth its ability to bind palmitic acid. Both results, taken together, suggested that the derivatives are linked to the protein through the sites for fatty acids. When cross-linking of¹²⁵I-5 ASU was performed after incubation with delipidated cytosol and products were analyzed by SDS-PAGE, a band was visualized in a position similar to that of purified L-FABP.Abbreviations FABP Fatty Acid-Binding Protein - L-FABP Hepatic FABP - I-FABP Intestinal FABP - C-FABP Cardiac FABP - 5 ASU-11 (5-azido-salicylamido)-undecanoic acid - Ac5 ASU-11 (O-acetyl-5-azido-salicylamido)-undecanoic acid     

Pre-moult foraging trips and moult locations of Emperor penguins at the Mawson Coast

The movements of nine breeding adult emperor penguins Aptenodytes forsteri from two colonies, Auster (67° 23S 64° 04E) and Taylor Glacier (67° 28S 60° 54E), were determined by satellite telemetry on their pre-moult foraging trips. While preparing for their annual moult the penguins travelled for 22–38 days and reached distances of up to 618 km from the colony. Six of the nine tracked penguins were followed to three different moult locations all to the west of their breeding colonies and near other known emperor penguins colonies, such as Kloa Point (66°38S, 59°23E) and Fold Island (67°17S, 59°23E). Sea-ice conditions changed throughout the tracking period; as the birds travelled north the sea-ice contracted south.     

A study on relations of vegetation,climate and soils in Shanxi province,China

Relationships of vegetation, climate and soils in Shanxi plateau wereanalyzed by use of Canonical Correspondence Analysis (CCA). Shanxi province,located at 34°35–40°43 N, 110°15–114°33 E, was divided into a series of rectangular districts of30 latitude by 20 longitude. Areas of vegetation formations and soil types ineach district were measured carefully using fine grain on the vegetation andsoil maps of Shanxi. Climatic data were mean values of 25 years records in eachdistrict. Three data matrices of climate, vegetation and soil were combined byCCA. The results showed that the distribution of vegetation is closely relatedto the variety of climates and to soils distribution.     

AMP synthesis in aqueous solution of adenosine and phosphorus pentoxide

Possible formation of a P₄O₁₀ molecule in magma, the stability of the molecule in hydrous volcanic gas at high temperatures and a possible prebiotic phosphate cycle were discussed in relation to chemical evolution. To demonstrate the utility of phosphorus pentoxide as a phosphorylating agent, aqueous solutions of adenosine (0.02M) and phosphorus pentoxide (0.2M) were incubated at 37°C for 5 months. The pH of the solutions was adjusted every day or every few days to each fixed value (9.0, 10.5, 11.5, 12.5) with 10 N NaOH. The HPLC analysis showed the formation of 2-AMP, 3-AMP, 5-AMP, cyclic (2–3)-AMP and cyclic (3–5)-AMP. The main components of the products were 2- and 3-AMP, though cyclic (2–3)-AMP was the main component in the early period of the incubation at pH 9.0. The yields (conversion rate of adenosine to AMPs) were increased almost linearly with the incubation time for 5 months in the case of pH 9.0. The final yields were about 3% (pH 9.0), 6% (pH 9.0, 1 M NaCl), 5% (pH 9.0, 0.01 M CaCl₂, 0.01 M MgCl₂), 7% (pH 9.0, 0.5 M NaCl, 0.01 M CaCl₂, 0.01 M MgCl₂), 9% (pH 9.0, 1 M NaCl, 0.01 M CaCl₂, 0.01 M MgCl₂), 32% (pH 10.5), 43% (pH 11.5), 35% (pH 12.5).     

Identification of the site of translational frameshifting required for production of the transposase encoded by insertion sequence IS 1.

Summary Previous genetic analyses indicated that translational frameshifting in the –1 direction occurs within the run of six adenines in the sequence 5-TTAAAAAACTC-3 at nucleotide positions 305–315 in IS 1, where the two out-of-phase reading frames insA and B-insB overlap, to produce transposase with a polypeptide segment Leu-Lys-Lys-Leu at residues 84–87. IS 1 mutants with a 1 by insertion, which encode mutant transposases with an amino acid substitution within the polypeptide segment at residues 84–87, did not efficiently mediate cointegration, except for an IS 1 mutant which encodes a mutant transposase with a Leu-Arg-Lys-Leu segment instead of Leu-LysLys-Leu. An IS 1 mutant with the DNA segment 5-CTTAAAAACTC-3 at positions 305–315 carrying the termination codon TAA in the B-insB reading frame could still mediate cointegration, indicating that codon AAA for Lys corresponding to second, third and fourth positions in the run of adenines is the site of frameshifting. The -galactosidase activity specified by several IS 1- lacZ fusion plasmids, in which B-insB is in-frame with lacZ, showed that the region 292–377 is sufficient for frameshifting. The protein produced by frameshifting from the IS 1-lacZ plasmid in fact contained the polypeptide segment Leu - Lys - Lys - Leu encoded by the DNA segment 5-TTAAAAAACTC-3, indicating that –1 frameshifting does occur within the run of adenines.     

Analysis of 5′-upstream non-coding region of FI-carboxymethyl cellulase gene from Aspergillus aculeatus

The 5-upstream non-coding region of an FI-carboxymethyl cellulase (CMCase) gene of Aspergillus aculeatus No. F-50 was obtained and sequenced up to –777 nucleotide in pCMG23. The 5-upstream region of different lengths were constructed and fused to a reporter gene (Escherichia coli lacZ) in pAN923-42BD, and the resulted constructs were introduced into the A. nidulans. The -galactosidase activities of the transformant with 5-upstream fragment of larger than 319 bp were expressed by the induction, but that of 109 bp drastically decreased to the basal level. This suggests that the region between –109 and –319 of the 5-upstream non-coding region is involved in the regulation of the FI-CMCase expression.     

Oligomerization of uridine phosphorimidazolides on montmorillonite: A model for the prebiotic synthesis of rna on minerals

The 5-phosphorimidazolide of uridine reacts on Na⁺-montmorillonite 22A in aqueous solution to give oligomers as long as 7 mers. The maximum chain length increases to 9 mers and the overall oligomer yield increases when 9:1 ImpU, A⁵ ppA mixtures react under the same conditions. The oligomer yield and maximum chain length decreases with the structure of the added pyrophosphate in the order A⁵ ppA>A⁵ ppU>U⁵ ppU. Structure analysis of individual oligomer fractions was performed by selective enzymatic hydrolyses followed by HPLC analysis of the products. The regioselectivity for 3,5-bond formation is 80–90% in the 9:1 ImpU, A⁵ ppA reaction, a percentage comparable to that observed in the 9:1 ImpA, A⁵ ppA reaction. Oligomerization of ImpU is inhibited by addition of dA⁵ ppdA, and MeppA. No oligomers containing A⁵ ppU were products of the 9:1 ImpU, A⁵ ppA reaction, a finding consistent with the simple addition of the ImpU to the A⁵ ppA and not the rearrangement of an ImpU-A⁵ ppA adduct. Concentrations of lysine or arginine which were close to that of the ImpU did not inhibit oligomer formation. Treatment of Na⁺-montmorillonite with 1 M arginine yielded arginine-montmorillonite, an amino acid-mineral adduct which did not catalyze ImpU oligomerization. Neither the 4–9 mers formed in the 9:1 ImpU, A⁵ ppA reaction nor the 4–9 mers formed by the base hydrolysis of poly(U) served as templates for the formation of oligo(A)s.     

Regioselective preparation of 2′, 3′-di-O-acylribonucleosides carrying lipophilic acyl groups through a lipase-catalysed alcoholysis

Candida antarctica B lipase-catalysed alcoholysis of 2, 3, 5-tri-O-hexanoyluridine (1a), 2, 3, 5-tri-O-dodecanoyluridine (1b), 2, 3, 5-tri-O-hexanoylinosine (1c) and 2, 3, 5-tri-O-dodecanoylinosine (1d) proceeded regioselectively to produce the corresponding 2, 3-di-O-acylribonucleosides 2a–d, providing a simple and efficient access to these new lipophilic compounds. Contrasting to the alcoholysis, enzymatic hydrolysis of 1a–d using different enzymes and experimental conditions did not proceed regioselectively.     

Preparation of 5′-GMP-rich yeast extracts from spent brewer's yeast

Spent brewer's yeast was autolysed and used as a raw material for the preparation of 5-GMP-rich yeast extracts. Malt rootlets were used as a source of 5-phosphodiesterase. The crude enzyme was extracted from malt rootlets and pretreated to inactivate 5-nucleotidase. The optimum pretreatment conditions were heating at 65 °C for 30 min or 70 °C for 7 min. The effects of autolysis time, phosphodiesterase concentration and incubation period on 5-GMP content were examined. The suitable autolysis time was 8 h. The preferable enzyme treatment period was in the range of 8–14 h. Longer autolysis and enzyme incubation periods caused a decrease in the 5-GMP content from 0.7–0.9% (w/w) to 0.2–0.4% (w/w). The 5-GMP content in extracts from debittered and non-debittered yeast was similar. The highest 5-GMP content in yeast extract was 0.93% (w/w), obtained with a phosphodiesterase concentration of 1.6unit/ml of yeast extract (5% solids content).     

Montmorillonite Catalysis of 30–50 Mer Oligonucleotides: Laboratory Demonstration of Potential Steps in the Origin of the RNA World

Elongation of the primer ³²pdA(pdA)₈pA proceeds by thereaction of the 5-phosphorimidazolides of adenosine and uridine in the presence of montmorillonite clay. Daily addition of the activated nucleotides for up to 14 days results in theformation of 40–50 mers using the 5-phosphorimidazolide of adenosine (ImpA) and 25–30 mers using the 5-phosphorimidazolide of uridine (ImpU). The limitation on thelengths of the chains formed is not due to the inhibitors formedsince the same chain lengths were formed using 2–3 times the amount of montmorillonite catalyst. The shorter oligomers formedby the addition of U monomers is not due to its greater rate ofdecomposition since it was found that both the A and the U adducts decompose at about the same rates. Alkaline phosphatase hydrolysis studies revealed that some of the oligomers are cappedat the 5-end to form, with ImpA,Ap³²pdA(pdA)₈pA(pA)_n. The extent of capping depends on the reaction time and the purine or pyrimidine base inthe activated mononucleotide. Hydrolysis with ribonuclease T₂ followed by alkaline phosphatase determined the sites ofthe 3, 5- and 2, 5-phosphodiester bonding to the primer. The potential significance of the mineral catalyzed formation of 50 mer oligonucleotides to the origin of life basedon RNA (the RNA world scenario) is discussed.     

A novel PH-CT-COSY methodology for measuring JPH coupling constants in unlabeled nucleic acids. Application to HIV-2 TAR RNA

A quantitative analysis of J_PH scalar couplings in nucleic acids is difficult due to small couplings to phosphorus, the extreme overlap of the sugar protons and the fast relaxation of the spins involved in the magnetization transfer. Here we present a new methodology that relies on heteronuclear Constant Time Correlation Spectroscopy (CT-COSY). The three vicinal ³J_PH3, ³J_PH5 and ³J_PH5 scalar couplings can be obtained by monitoring the intensity decay of the P_i-H3_{i – 1} peak as a function of the constant time T in a 2D correlation map. The advantage of the new method resides in the possibility of measuring the two ³J_PH5 and ³J_PH5 scalar couplings even in the presence of overlapped H5/H5 resonances, since the quantitative information is extracted from the intensity decay of the P-H3 peak. Moreover, the relaxation of the H3 proton is considerably slower than that of the H5/H5 geminal protons and the commonly populated conformations of the phosphate backbone are associated with large ³J_PH3 couplings and relatively small ³J_{PH5 / H5}. These two facts lead to optimal signal-to-noise ratio for the P-H3 correlation compared to the P-H5/H5 correlation.The heteronuclear CT-COSY experiment is suitable for oligonucleotides in the 10–15 kDa molecular mass range and has been applied to the 30mer HIV-2 TAR RNA. The methodology presented here can be used to measure P-H dipolar couplings (D_PH) as well. We will present qualitative results for the measurement of P-H_base and P-H2 dipolar couplings in the HIV-2 TAR RNA and will discuss the reasons that so far precluded the quantification of the D_PHs for the 30mer RNA.