Synthetic approaches to 6-substituted 9-(3-azido-3,4-dideoxy-β-d,l-erythro-pentopyranosyl)purines

Methyl 3-azido-2-O-benzoyl-3,4-dideoxy-β-dl-erythro-pentopyranoside (6) was synthesized through two routes in five steps from methyl 2,3-anhydro-4-deoxy-β-dl-erythro-pentopyranoside (1). The first route proceeded via selective azide displacement of the 3-tosyloxy group of methyl 4-deoxy-2,3-di-O-tosyl-α-dl-threo-pentopyranoside, followed by detosylation and benzoylation. The second route consisted, with a better overall yield, in the azide displacement of the mesyloxy group of methyl O-benzoyl-4-deoxy-3-O-methylsulfonyl-α-dl-threo-pentopyranoside (10), obtained by benzylate opening of 1, followed by benzoylation, debenzylation, and mesylation. Compound 6 was transformed into its glycosyl chloride, further treated by 6-chloropurine to give the nucleoside 9-(3-azido-2-O-benzoyl-3,4-dideoxy-β-dl-erythro-pentopyranosyl)-6-chloropurine (13). When treated with propanolic ammonia, 13 yielded 9-(3-azido-3,4-dideoxy-β-dl-erythro-pentopyranosyl)adenine.     

Enzymatic conversion of guanosine 3'' adjacent to the anticodon of yeast tRNAPhe to N1-methylguanosine and the wye nucleoside: dependence on the anticodon sequence.

N1-Methylguanosine (m1G) or wye nucleoside (Y) are found 3' adjacent to the anticodon (position 37) of eukaryotic tRNAPhe. The biosynthesis of these two modified nucleosides has been investigated. The importance of the type of nucleosides in the anticodon of yeast tRNAPhe on the potentiality of this tRNA to be a substrate for the corresponding maturation enzyme has also been studied. This involved microinjection into Xenopus laevis oocytes and incubation in a yeast extract of restructured yeast tRNAPhe in which the anticodon GmAA and the 3' adjacent Y nucleoside were substituted by various tetranucleotides ending with a guanosine. The results obtained by oocyte microinjection indicate: that all the restructured yeast tRNAsPhe are efficient substrates for the tRNA (guanosine-37 N1)methyltransferase. This means that the anticodon sequence is not critical for the tRNA recognition by this enzyme; in contrast, for Y nucleoside biosynthesis, the anticodon sequence GAA is an absolute requirement; the conversion of G-37 into Y-37 nucleoside is a multienzymatic process in which m1G-37 is the first obligatory intermediate; all the corresponding enzymes are cytoplasmic. In a crude yeast extract, restructured yeast tRNAPhe with G-37 is efficiently modified only into m1G-37; the corresponding enzyme is a S-adenosyl-L-methionine-dependent tRNA methyltransferase. The pure Escherichia coli tRNA (guanosine-37 N1) methyltransferase is unable to modify the guanosine-37 of yeast tRNAPhe.     

The in vivo stability, maturation and aminoacylation of anticodon-substituted Escherichia coli initiator methionine tRNAs

We have constructed eight anticodon-modified Escherichia coli initiator methionine (fMet) tRNAs by insertion of synthetic ribotrinucleotides between two fragments ('half molecules') derived from the initiator tRNA. The trinucleotides, namely CAU (the normal anticodon), CAA, CAC, CAG, GAA, GAC, GAG and GAU, were joined to the 5' and 3' tRNA fragments with T4 RNA ligase. The strategy of reconstruction permitted the insertion of radioactive 32P label between nucleotides 36 and 37. tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes, and the following properties were evaluated: the stability of these eubacterial tRNA variants in the eukaryotic oocytes; the enzymatic modification of the adenosine at position 37 (3' adjacent to the anticodon) and aminoacylation of the chimeric tRNAs by endogenous oocyte aminoacyl-tRNA synthetases. In contrast to other variants, the two RNAs having CAU and GAU anticodons were stable and underwent quantitative modification at A-37. These results show that the enzyme responsible for the modification of A-37 to N-[N-(9-beta-D-ribofuranosylpurine-6-yl)carbamoyl]threonine (t6A) is present in the cytoplasm of oocytes and is very sensitive to the anticodon environment of the tRNA. Also, these same GAU and CAU anticodon-containing tRNAs are fully aminoacylated with the heterologous oocyte aminoacyl-tRNA synthetases in vivo. During the course of this work we developed a generally applicable assay for the aminoacylation of femtomole amounts of labelled tRNAs.     

On the primacy of primordial RNA

The ability of RNA to catalyze biochemical reactions is used to develop a self-consistent picture of how a primordial RNA could have given rise to the necessary factors and processes of early life forms. Essential to this proposal is the impact of RNA structural domains, "selected" by thermodynamic criteria, on the structure of early proteins (exons) and the assembly of functional complexes. Based on this analysis, the chronological appearance of informational molecules follows the order: primordial RNA, proteins whose structures are determined by primordial RNA sequences and finally DNA.     

Effects of phospholipases,proteases and neuraminidase on γ-hydroxybutyrate binding sites

Summary -Hydroxybutyric acid (GHB) is a natural compound of mammalian brain synthesized from GABA. The characteristics of its synthesis, transport, release, distribution and turnover, in addition to the presence of a high affinity binding site for this substance in brain are in favor of a modulator role for GHB. The effects of hydrolytic enzymes on the specific binding capacity of GHB have been studied in the present work. Phospholipases A₂ and C, neuraminidase and Pronase markedly decrease GHB binding to crude synaptosomal membranes from rat brain. This effect is time and enzyme concentration dependent. Trypsin, under the conditions employed, is less active. The inhibitory effects of phospholipases is correlated with phospholipid hydrolysis. Lysophospholipids, in the absence of bovine fatty acid free serum albumin partially inhibit GHB binding. The action of neuraminidase has been followed by sialic acid release and modifications of the ganglioside profile. The effects of phospholipase C and of neuraminidase are completely different to those on GABA binding sites. These results represent further data concerning the molecular existence of specific GHB binding sites on rat brain membranes.Abbreviations GHB -hydroxybutyrate - LPC L--lysophosphatidylcholine - LPE Lysophosphatidylethanolamine - PC Phosphatidylcholine - PE Phosphatidylethanolamine - BSA Bovine Serum Albumin     

Peptide-N 4-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity could explain the occurrence of extracellular xylomannosides in a plant cell suspension

We have previously isolated mannoside and xylomannoside oligosaccharides with one or two terminal reducingN-acetylglucosamine residues from the extracellular medium of white campion (Silene alba) suspension culture. We have now demonstrated the presence of peptide-N ⁴-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity in cell extracts as well in the culture medium that could explain the production of those compounds. An additional xylomannoside, (GlcNAc)Man₃(Xyl)GlcNAc(Fuc)GlcNAc, was characterized, and¹H- and¹³C-NMR assignments for the oligosaccharide Man₃(Xyl)GlcNAc(Fuc)GlcNAc were obtained using homonuclear and heteronuclear spectroscopy (COSY).Abbreviations Endo endo--N-acetylglucosaminidase - Fuc fucose - GlcNAc N-acetylglucosamine - Man mannose - NMR nuclear magnetic resonance - PNGase peptide-N ⁴-(N-acetylglucosaminyl)asparagine amidase - Xyl xylose     

Effects of the Introduction of Agrobacterium tumefaciens T-DNA ipt Gene in Nicotiana tabacum L. cv. Petit Havana SR1 Plant Cells

Transformation of tobacco leaf discs with the ‘cytokinin’ipt gene yielded several transgenic callus tissue lines, respectiveto the kind of ipt construction present in the A. tumefacienscointegrates. Those calli containing an active ipt gene wereable to grow hormone-autotrophically and showed an increasedendogenous cytokinin level in comparison with controls. Analysisof endogenous IAA level did not allow any quantitative correlationwith the cytokinin content. However, a minimal level of auxinseems to be necessary to obtain hormone-autotrophic growth.Exogenously supplied NAA significantly reduced the endogenouscytokinin content without modifying growth characteristics. The varying chlorophyll content in the different callus lineselicited the study of the ultrastructure of the plastids. Thecontrols contained small plastids, often filled with starchor accumulated vesicles that did not allow observation of theinternal membrane system. The ‘Pssu-ipt’ line, havinga higher cytokinin content, showed plastids with an internalmembrane system consisting of stroma and grana thylakoids, butthis structure was lost during subculture. Swollen thylakoidsappeared, the amount of starch was reduced and vesicles wereaccumulating. (Received November 15, 1990; Accepted March 4, 1991)     

Parenteral supplementation with zinc in surgical patients corrects postoperative serum-zinc drop

Zinc has been known for a long time to facilitate wound healing. But, so far, supplementation trials in patients treated by major severity surgery gave either partial or controversial results. In a double-blind, randomized study including 30 patients, we show that zinc supplements (30 mg/d for 3 d) administered by a drip correct postoperative drop of serum zinc, that this correction concerns the available part of serum zinc (i.e., zinc that is bound to compounds other than alpha-2 macroglobulin in serum), and that this supplementation can improve clinical wound healing. Possible influence of increased urinary output after the intervention is discussed, and we found that serum cortisol remains stable when zinc/albumin ratio is stable, and increases sharply when the same ratio drops. Cortisol, therefore, seems to play a major role in zinc redistribution after surgery.