All pyruvylated galactose in Schizosaccharomyces pombe N-glycans is present in the terminal disaccharide, 4, 6-O-[(R)-(1- carboxyethylidine)]-Galbeta1,3Galalpha1-

The large N-linked oligosaccharides released from Schizosaccharomyces pombe by endo-beta-N-acetylglucosaminidase H were examined to determine how the negatively chargedpyruvylated galactoses present (Gemmill,T.R., and Trimble,R.B., 1996, J. Biol. Chem ., 271, 25945-25949) were attached to the oligosaccharide chains. Binding of biotinylated human serum amyloid P and peanut agglutinin to native and depyruvylated S.pombe glycoproteins, respectively, indicated that the pyruvylated epitope was likely to be in the beta configuration. Examination by high- field 1H NMR of whole glycans and a disaccharide fragment released from them on partial acid hydrolysis showed that the pyruvylated galactose species was in fact beta1,3-linked to a second galactose, and this occurred an average of five to six times on nominal Gal57Man64GlcNAc N- glycans. The pyruvate-2,(4,6)Gal-beta1,3Gal epitope is chemically similar to acetaldehyde-Galbeta1,3Gal groups found on the glycoproteins from Paramyxovirus-infected bovine kidney cells (Prehm, P., Scheid,A. and Choppin,P.W. ,1979, J. Biol. Chem ., 254, 9669-9677). The 1:1 stoichiometry between pyruvate and beta-linked galactose in these S.pombe glycans indicates that either pyruvate addition to terminal beta1,3Gal is highly efficient or that pyruvylated Gal is transferred en bloc to alpha1,2-linked Gal residues in theN-linked chains. In contradiction to many galactomannan-producing fungi, which add substantial amounts of Gal in the furanose form to their glycoproteins, all detectable Gal in the large S.pombe galactomannans is in the pyranose form, as found in higher eukaryotes. The current work shows that the S.pombe outer chain structure is a poly-alpha1,6Man backbone 2- O-substituted with either Gal or the pyruvylated galactobiose and contains little alpha1,2-linked or 2-O-substituted Man. This is in contrast to the S. cerevisiae outer chain, which is poly-alpha1,6Man substituted with alpha1,2-linked Man sidechains (Ballou,C.E. ,1990, Methods Enzymol , 185, 440-470).      

Glutamine: A Suitable Nitrogen Source for Enhanced Shoot Multiplication in Cucumis sativus L.

Shoot tip explants of cucumber (Cucumis sativus L. cv. Poinsett 76) were cultured in vitro on Murashige-Skoog medium with L-glutamine, ammonium nitrate, adenine sulphate, asparagine, ammonium succinate, potassium nitrate and sodium nitrate as the nitrogen sources along with optimal concentration of 0.044 mM benzyladenine to study their effects on in vitro morphogenesis. The explants grown with 0.068 mM L-glutamine displayed the highest culture response (74.6 %) and greatest shoot number per explant (13.6) at the end of two subcultures. The explants cultured with other nitrogen sources resulted in low culture frequency and low number of shoots per explant accompanied by basal callusing and necrosis.     

Metabolomics of plant saponins: bioprospecting triterpene glycoside diversity with respect to mammalian cell targets

One of the goals of cancer chemotherapy and prevention is the discovery of compounds that are relatively selective to tumor cells and, therefore, have reduced effects on normal cell growth. In previously published studies, it was shown that certain triterpene saponins (called avicins) from a desert tree, Acacia victoriae, are selectively toxic to tumor cells at very low doses (IC(50): 0.2 microg/mL for Jurkat cells). To extend this research to human clinical studies, we needed to find a reliable supply of avicins and have developed a transformed "hairy root" culture as a means of biomass production. Protocols were optimized for A. victoriae micropropagation; after a boiling water treatment, A. victoriae seeds were maintained under in vitro conditions on defined media. Embryo-axis explants from shoot tips were removed and infected with Agrobacterium rhizogenes Conn (R 1000) for hairy root induction. Plasmid integration was confirmed by PCR analysis with a primer set for a segment of the rol B gene. Culture conditions have been optimized for root biomass production, and various inducers have been investigated for enhancement of avicin production. Hairy root cultures were compared with intact pod tissue from field-grown sources for avicin content following partial purification of triterpene glycosides and HPLC separation of the secondary metabolites. From bioassays of the collected HPLC fractions, we have identified putative triterpene "metabolic clusters" with enhanced activity against tumor cells. This now provides a system for both production of clinical trial lots of active samples, but also a means to correlate structure of individual triterpene glycosides with specific cellular target activity in mammalian cells.     

In Vivo Antiviral Activity of 5-Amino-1-Methyl-3-β-D-Ribofuranosyl-Pyrazolo[4,3-d]Pyrimidin-7(6H)-One and Related Guanosine Analogues Prepared from Formycin

Abstract

A short and simple synthesis of 5-amino-3-β-D-ribofuranosylpyrazolo[4,3-d]pyrimidin-7(6H)-one, (7) was achieved from 7-amino-5-chloro-3-β-D-ribofuranosylpyrazolo[4,3-d]pyrimidine (5), in two steps, first deamination of 5 with NOCI, followed by amination of 6 with MeOH/NH₃. Also, an efficient synthesis of 5-amino-1(or 2)-methyl-3-β-D-ribofuranosylpyrazolo[4,3-d]pyrimidin-7(6H)-one was accomplished from the corresponding 1 (or 2)-methyloxoformycin B in four steps by a sequence consisting of (i) 2′,3′,5′ acetylation with AC₂O, (ii) 5,7-chlorination with PhP(O)Cl₂, (iii) selective hydrolysis of the 7-chloro group with aqueous Na₂CO₃, (iv) followed by amination of the 5-chloro group with MeOH/NH₃. Single crystal X-ray analysis off 11 confirmed position 7 as the site of selective hydrolysis with Na₂CO₃. The three guanosine C-nucleosides prepared were evaluated for their ability to inhibit certain RNA and DNA viral replication in vitro and Semliki Forest virus infection in vivo. Only 5-amino-1-methyl-3-β-D-ribofuranosylpyrazolo[4,3-d]pyrimidin-7(6H)-one (13) provided protection (67% survivors, compared to 0% for placebo controls) against a lethal dose of Semliki Forest virus infection in mice. The antiviral effect of 13 is believed to be due to the enhancement of the host immune function.     

Synergistic Effect of 5-Nitro-2′-deoxyuridine with Ganciclovir Against Human Cytomegalovirus In Vitro

Abstract

In this paper we describe a practical synthesis of 5-nitro-2′-deoxyuridine (4) and 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-nitrouracil (11). These compounds were then evaluated for their ability to inhibit the growth of human cytomegalovirus (HCMV, strain AD169) in MRC-5 cells using a plaque reduction assay. Compound 11 was unable to inhibit the growth of HCMV at the highest concentration tested (100 μg/mL). However, compound 4 (5-NO₂-dU) exhibited marginal activity against HCMV in vitro in a dose-dependent manner with a 50% inhibitory concentrations (IC₅₀) of 1 to 5 μg/mL. Combinations of 5-NO₂-dU with ganciclovir synergistically inhibited HCMV induced cell killing in culture.