Transformation of 2,2′-Bimorphine to the Novel Compounds 10-α-S-Monohydroxy-2,2′-Bimorphine and 10,10′-α,α′-S,S′-Dihydroxy-2,2′-Bimorphine by Cylindrocarpon didymum

Whole-cell suspensions of Cylindrocarpon didymum were observed to transform 2,2′-bimorphine to the compounds 10-α-S-monohydroxy-2,2′-bimorphine and 10,10′-α,α′-S,S′-dihydroxy-2,2′-bimorphine. Mass spectrometry and ¹H nuclear magnetic resonance spectroscopy confirmed the identities of these new morphine alkaloids.     

Microbial Reduction of 1,3-Dioxo-2-Methyl-2-(3′-Oxo-6′-Carbomethoxyhexyl)-Cyclopentane to Form 1β-Hydroxy-3-Oxo-2β-Methyl-2α-(3′-Oxo-6′-Carbomethoxy-hexyl)-Cyclopentane, an Intermediate for Steroid Total Synthesis

The rate and extent of stereoselective reduction of 1,3-dioxo-2-methyl-2-(3′-oxo-6′-carbomethoxyhexyl)-cyclopentane to form the 1β-hydroxy-2β-methyl isomer by cultures of Schizosaccharomyces pombe ATCC 2476 was dramatically increased by addition to the fermentation of certain α,β-unsaturated ketones and allyl alcohol.     

Relationship of Adenosine 3′,5′-Monophosphate to Growth and Metabolism of Tetrahymena pyriformis

The concentration of adenosine 3',5'-monophosphate (cyclic AMP) and the activity of adenylate cyclase were determined for the first time in conjuncation with cyclic 3',5'-nucleotide phosphodiesterase (phosphodiesterase) during the growth cycle of Tetrahymena pyriformis. High levels of cyclic AMP observed during early exponential and late stationary phases were associated with elevated adenylate cyclase and decreased phosphodiesterase activities. Adenylate cyclase and cyclic AMP were decreased and phosphodiesterase was increased in cells grown in glucose-supplemented medium. In contrast to findings in mammalian liver, cyclic AMP was decreased during active gluconeogenesis in Tetrahymena. This suggests a different modulation of carbohydrate metabolism in the two species. The results illustrate that both the content of cyclic AMP and its action as a regulatory agent in Tetrahymena are uniquely suited to the metabolism of this organism.     

Uncoupling of Protein and Ribonucleic Acid Synthesis by 5′,5′,5′-Trifluoroleucine in Salmonella typhimurium

The addition of 5',5',5'-trifluoroleucine (fluoroleucine) to leucine auxotrophs of Salmonella typhimurium permitted protein but not ribonucleic acid (RNA) synthesis to continue after leucine depletion. The uncoupling of the formation of these macromolecules by fluoroleucine was apparent if RNA and protein synthesis was measured either by the uptake of radioactive precursors or by direct chemical determinations. The analogue did not appear to be an inhibitor of RNA formation, since it was as effective as leucine in permitting RNA synthesis in a leucine auxotroph upon the addition of small amounts of chloramphenicol. In contrast to these data, fluoroleucine allowed continued protein and RNA formation in a leucine auxotroph of Escherichia coli strain W. In addition, contrary to the results obtained with S. typhimurium, the analogue replaced leucine for repression of the leucine bio-synthetic enzymes as well as the isoleucine-valine enzymes. We propose that these ambivalent effects of fluoroleucine on repression and RNA and protein synthesis in the two strains are due to differences in the ability of the analogue to attach to the various species of leucine transfer RNA.     

The Crystal and Molecular Structure of 5-Fluoro-2′-Deoxy-β-Uridine

The crystal structure of 5-fluoro-2′-deoxy-β-uridine (FUDR) has been solved and the details of the molecular structure determined by x-ray analysis. The bond lengths and angles have been measured and are compared with the dimensions found for calcium thymidylate and adenylic acid. In all three compounds the bonds formed by the nitrogen atom involved in the glycosidic bond are not coplanar. Furthermore, it is found that in FUDR the C2′ carbon atom is out of the plane of the ribose ring. The possible effect of these observations on nucleic acid structures is discussed.     

Metabolism of Adenosine 3′,5′-Cyclic Monophosphate and Induction of Fruiting Bodies in Coprinus macrorhizus

The adenyl cyclase and phosphodiesterase metabolizing adenosine 3',5'-cyclic monophosphate (cyclic AMP) were detected in mycelia of strains of Coprinus macrorhizus which form fruiting bodies, but not in those of strains which do not form fruiting bodies. The adenyl cyclase synthesized cyclic AMP from adenosine triphosphate. The phosphodiesterase degr[UNK]ded cyclic AMP to adenosine-5'-monophosphate and was inhibited by adenosine-3'-monophosphate, theophylline, and caffeine. The strains which form fruiting bodies incorporated and metabolized cyclic AMP, but strains which do not form fruiting bodies did not. The possible participation of cyclic AMP in the induction of fruiting bodies is discussed.     

Haloperoxidases: Enzymatic Synthesis of α,β-Halohydrins from Gaseous Alkenes

The enzymatic synthesis of α,β-halohydrins from gaseous alkenes is described. The enzymatic reaction required an alkene, a halide ion, dilute hydrogen peroxide, and a haloperoxidase enzyme. A wide range of gaseous alkenes were suitable for this reaction, including those containing isolated, conjugated, and cumulative carbon-carbon double bonds. Chlorohydrins, bromohydrins, and iodohydrins could be formed. The combining of this enzymatic synthesis with a previously described enzymatic synthesis of epoxides from α,β-halohydrins provides an alternate pathway, other than the well-known enzymatic direct epoxidation pathway, from alkene to an epoxide.     

β-β′ Subunits of Ribonucleic Acid Polymerase in Episome-Free Minicells of Escherichia coli

Episome-free minicells of Escherichia coli, previously shown to lack ribonucleic acid polymerase activity, do contain the beta-beta' subunits of the polymerase.     

Release of the β-Galactosidase-Synthesizing System from Ultraviolet Catabolite Repression by Cyclic 3′,5′-Adenosine Monophosphate, Dark Repair, Photoreactivation, and Cold Treatment

Recovery from the inhibitory effect of ultraviolet irradiation on the induced synthesis of beta-galactosidase was studied in Escherichia coli B/r. When irradiated cells (520 ergs/mm(2) at 254 nm) were induced and incubated in minimal medium supplemented with Casamino Acids (conditions of catabolite repression), the ability to form enzyme was greatly reduced for about 100 min and then recovery began. The inhibition observed immediately after ultraviolet irradiation was partially reversed by cyclic 3',5'-adenosine monophosphate (cyclic AMP) or by photoreactivation treatment. Inhibition was reduced if the cells were given cold treatment (5 C) before or during irradiation; the kinetics of induced enzyme formation in each case were similar to those of irradiated cells receiving cyclic AMP. These kinetics suggest that the cold treatments, like cyclic AMP, cause the release of the beta-galactosidase-synthesizing system from catabolite repression. When irradiated cells were incubated for various times before cyclic AMP or photoreactivation treatment, some reversal of the inhibition of induced enzyme formation was obtained, but by 100 min the treatments were ineffective. Because 100 min was also the time at which dark recovery of enzyme formation began, the recovery process was interpreted to be the result of completion of DNA repair, which, in turn, released the beta-galactosidase-synthesizing system from catabolite repression.     

Production of Epoxides from α,β-Halohydrins by Flavobacterium sp

The relative activity of Flavobacterium whole cells on the enzymatic synthesis of epoxides from α,β-chlorohydrins, -bromohydrins, and -iodohydrins is described.     

The Protein Phosphatase 2A Regulatory Subunits B′β and B′δ Mediate Sustained TrkA Neurotrophin Receptor Autophosphorylation and Neuronal Differentiation

Nerve growth factor (NGF) is critical for the differentiation and maintenance of neurons in the peripheral and central nervous system. Sustained autophosphorylation of the TrkA receptor tyrosine kinase and long-lasting activation of downstream kinase cascades are hallmarks of NGF signaling, yet our knowledge of the molecular mechanisms underlying prolonged TrkA activity is incomplete. Protein phosphatase 2A (PP2A) is a heterotrimeric Ser/Thr phosphatase composed of a scaffolding, catalytic, and regulatory subunit (B, B′, and B" gene families). Here, we employ a combination of pharmacological inhibitors, regulatory subunit overexpression, PP2A scaffold subunit exchange, and RNA interference to show that PP2A containing B′ family regulatory subunits participates in sustained NGF signaling in PC12 cells. Specifically, two neuron-enriched regulatory subunits, B′β and B′δ, recruit PP2A into a complex with TrkA to dephosphorylate the NGF receptor on Ser/Thr residues and to potentiate its intrinsic Tyr kinase activity. Acting at the receptor level, PP2A/ B′β and B′δ enhance NGF (but not epidermal growth factor or fibroblast growth factor) signaling through the Akt and Ras-mitogen-activated protein kinase cascades and promote neuritogenesis and differentiation of PC12 cells. Thus, select PP2A heterotrimers oppose desensitization of the TrkA receptor tyrosine kinase, perhaps through dephosphorylation of inhibitory Ser/Thr phosphorylation sites on the receptor itself, to maintain neurotrophin-mediated developmental and survival signaling.     

Cyclic Adenosine 3′,5′-Monophosphate in Escherichia coli

The concentration of cyclic adenosine 3',5'-monophosphate (c-AMP) in Escherichia coli growing on different sources of carbon was studied. Cultures utilizing a source of carbon that supported growth relatively poorly had consistently higher concentrations of c-AMP than did cultures utilizing sugars that supported rapid growth. This relationship was also observed in strains defective in c-AMP phosphodiesterase and simultaneously resistant to catabolite repression; in such strains the c-AMP concentration was slightly higher for several sources of carbon tested. Cultures continued to synthesize c-AMP and secreted it into the medium, under conditions that brought about an inhibition of the intracellular accumulation of the cyclic nucleotide. Transient repression of the synthesis of beta-galactosidase was not associated with an abrupt decrease in the cellular concentration of c-AMP.     

Regulation of Human Immunodeficiency Virus Replication by 2′,5′-Oligoadenylate-Dependent RNase L

Activation of RNase L by 2′,5′-linked oligoadenylates (2-5A) is one of the antiviral pathways of interferon action. To determine the involvement of the 2-5A system in the control of human immunodeficiency virus type 1 (HIV-1) replication, a segment of the HIV-1 nef gene was replaced with human RNase L cDNA. HIV-1 provirus containing sense orientation RNase L cDNA caused increased expression of RNase L and 500- to 1,000-fold inhibition of virus replication in Jurkat cells for a period of about 2 weeks. Subsequently, a partial deletion of the RNase L cDNA which coincided with increases in virus production occurred. The anti-HIV activity of RNase L correlated with decreases in HIV-1 RNA and with an acceleration in cell death accompanied by DNA fragmentation. Replication of HIV-1 encoding RNase L was also transiently suppressed in peripheral blood lymphocytes (PBL). In contrast, recombinant HIV containing reverse orientation RNase L cDNA caused decreased levels of RNase L, increases in HIV yields, and reductions in the anti-HIV effect of alpha interferon in PBL and in Jurkat cells. To obtain constitutive and continuous expression of RNase L cDNA, Jurkat cells were cotransfected with HIV-1 proviral DNA and with plasmid containing a cytomegalovirus promoter driving expression of RNase L cDNA. The RNase L plasmid suppressed HIV-1 replication by eightfold, while an antisense RNase L construct enhanced virus production by twofold. These findings demonstrate that RNase L can severely impair HIV replication and suggest involvement of the 2-5A system in the anti-HIV effect of alpha interferon.     

Adenosine 3′,5′-Cyclic Monophosphate-Deficient Mutants of Vibrio cholerae

Two adenosine 3',5'-cyclic monophosphate (AMP)-deficient mutants of Vibrio cholerae (biotype El Tor) were successfully isolated by nitrosoguanidine treatment followed by pencillin screening for pleiotropic sugar-negative clones. Exogenous cyclic AMP is required for the fermentation of sucrose, trehalose, fructose, maltose, and mannose but not of glucose, as well as for the formation of normal flagella and specific somatic antigens. A striking characteristic of the mutants is their growth behavior at higher temperatures. They cannot grow on TCBS selective plates at 37 C or higher unless they are provided with a supply of exogenous cyclic AMP, although they are capable of producing colonies on the same medium, even without cyclic AMP, at temperatures lower than 30 C. Since the mutants are converted to spheroplasts, spindle forms, and spiral filaments in cyclic AMP-free media at 37 C, and this phenomenon is stopped by the addition of cyclic AMP or a combination of 20% sucrose and 0.2% magnesium chloride, it is assumed that cyclic AMP is essential for the synthesis of the cell wall of V. cholerae at higher temperatures.     

A bridged nucleic acid, 2′,4′-BNACOC: synthesis of fully modified oligonucleotides bearing thymine, 5-methylcytosine, adenine and guanine 2′,4′-BNACOC monomers and RNA-selective nucleic-acid recognition

Recently, we synthesized pyrimidine derivatives of the 2′-O,4′-C-methylenoxymethylene-bridged nucleic-acid (2′,4′-BNA^COC) monomer, the sugar conformation of which is restricted in N-type conformation by a seven-membered bridged structure. Oligonucleotides (BNA^COC) containing this monomer show high affinity with complementary single-stranded RNA and significant resistance to nuclease degradation. Here, BNA^COC consisting of 2′,4′-BNA^COC monomers bearing all four bases, namely thymine, 5-methylcytosine, adenine and guanine was efficiently synthesized and properties of duplexes containing the 2′,4′-BNA^COC monomers were investigated by UV melting experiments and circular dichroism (CD) spectroscopy. The UV melting curve analyses showed that the BNA^COC/BNA^COC duplex possessed excellent thermal stability and that the BNA^COC increased thermal stability with a complementary RNA strand. On the other hand, BNA^COC/DNA heteroduplexes showed almost the same thermal stability as RNA/DNA heteroduplexes. Furthermore, mismatched sequence studies showed that BNA^COC generally improved the sequence selectivity with Watson–Crick base-pairing compared to the corresponding natural DNA and RNA. A CD spectroscopic analysis indicated that the BNA^COC formed duplexes with complementary DNA and RNA in a manner similar to natural RNA.