The brain in vivo expresses the 2',3'-cAMP-adenosine pathway

Although multiple biochemical pathways produce adenosine, studies suggest that the 2',3'-cAMP-adenosine pathway (2',3'-cAMP→2'-AMP/3'-AMP→adenosine) contributes to adenosine production in some cells/tissues/organs. To determine whether the 2',3'-cAMP-adenosine pathway exists in vivo in the brain, we delivered to the brain (gray matter and white matter separately) via the inflow perfusate of a microdialysis probe either 2',3'-cAMP, 3',5'-cAMP, 2'-AMP, 3'-AMP, or 5'-AMP and measured the recovered metabolites in the microdialysis outflow perfusate with mass spectrometry. In both gray and white matter, 2',3'-cAMP increased 2'-AMP, 3'-AMP and adenosine, and 3',5'-cAMP increased 5'-AMP and adenosine. In both brain regions, 2'-AMP, 3-AMP and 5'-AMP were converted to adenosine. Microdialysis experiments in 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) wild-type mice demonstrated that traumatic brain injury (controlled cortical impact model) activated the brain 2',3'-cAMP-adenosine pathway; similar experiments in CNPase knockout mice indicated that CNPase was involved in the metabolism of endogenous 2',3'-cAMP to 2'-AMP and to adenosine. In CSF from traumatic brain injury patients, 2',3'-cAMP was significantly increased in the initial 12 h after injury and strongly correlated with CSF levels of 2'-AMP, 3'-AMP, adenosine and inosine. We conclude that in vivo, 2',3'-cAMP is converted to 2'-AMP/3'-AMP, and these AMPs are metabolized to adenosine. This pathway exists endogenously in both mice and humans.     

Mammalian 2',3' cyclic nucleotide phosphodiesterase (CNP) can function as a tRNA splicing enzyme in vivo

Yeast and plant tRNA splicing entails discrete healing and sealing steps catalyzed by a tRNA ligase that converts the 2',3' cyclic phosphate and 5'-OH termini of the broken tRNA exons to 3'-OH/2'-PO4 and 5'-PO4 ends, respectively, then joins the ends to yield a 2'-PO4, 3'-5' phosphodiester splice junction. The junction 2'-PO4 is removed by a tRNA phosphotransferase, Tpt1. Animal cells have two potential tRNA repair pathways: a yeast-like system plus a distinctive mechanism, also present in archaea, in which the 2',3' cyclic phosphate and 5'-OH termini are ligated directly. Here we report that a mammalian 2',3' cyclic nucleotide phosphodiesterase (CNP) can perform the essential 3' end-healing steps of tRNA splicing in yeast and thereby complement growth of strains bearing lethal or temperature-sensitive mutations in the tRNA ligase 3' end-healing domain. Although this is the first evidence of an RNA processing function in vivo for the mammalian CNP protein, it seems unlikely that the yeast-like pathway is responsible for animal tRNA splicing, insofar as neither CNP nor Tpt1 is essential in mice.     

1-[2-Hydroxy-3-octadecan-1'-oate]propyl-2'',2'',5'',5''-tetramethyl pyrolidine-N-oxyl-3''-carboxylate as a potential spin probe for membrane structure studies

The synthesis of a new minimum steric perturbing proxyl nitroxide, which is a derivative of glycerol and contains a stearic acid moiety, has been carried out. Its localization in model membrane L-alpha-dipalmitoyl phosphatidyl choline (DPPC) was ascertained with the help of ESR, DSC, 1H and 31P NMR techniques. The nitroxide was used for detecting the changes in the phase transition temperature of the model membranes in the presence and absence of drugs. The permeation of the vasodilating drug epinephrine has also been studied using this spin label. The results prove the potential applicability of the new spin probe in the spin labeling of biomembranes.     

(2'-5')An-dependent endoribonuclease: enzyme levels are regulated by IFN beta, IFN gamma, and cell culture conditions

The levels of a (2'-5')An-dependent endonuclease (RNase L) were determined in extracts prepared from murine L cells and Ehrlich ascites tumor (EAT) cells by measuring specific binding of protein to a labeled derivative of (2'-5')An, (2'-5')A3[32P]pCp. RNase L levels were found to depend both on interferon (IFN) treatment and on cell growth conditions. Treatment of murine L cells and EAT cells with 100-2,000 IRU IFN beta or IFN gamma resulted in a similar 2-4-fold increase in the levels of RNase L when cells were present at low density. The levels of RNase L were also shown to increase 2-3-fold as cells approached saturation density. Serum-starved cells also displayed relatively high levels of RNase L. RNase L levels in cells maintained at high cell density did not change appreciably following treatment with IFN beta or IFN gamma. Regulation of RNase L levels by cell growth conditions as well as by IFN beta or IFN gamma treatment suggests that RNase L may play an important role in regulating the levels of cellular mRNAs as well as acting to degrade viral RNAs.     

A pterocarpan and two isoflavans from alfalfa

(−)6aR,11aR-Dihydro-3-hydroxy-9,10-dimethoxy-6H-benzofuro[3,2c] [1]-benzopyran (10-methoxymedicarpin), (+)-(2,3,4,-trimethoxyphenyl)-2,3-dihydro-7-hydroxy-4H-1-benzopyran (7-hydroxy-2′,3′,4′-trimethoxyisoflavan) and (+)-(2,3,4-trimethoxy-5-hydroxyphenyl)-2,3-dihydro-7-hydroxy-4H-1-benzopyran (7,5′-dihydroxy-2′,3′,4′-trimethoxyisoflavan) were isolated for the first time from dried Medicago sativa hay. Structural assignments were based on ¹H NMR and mass spectra, X-ray crystallography, and optical rotations.     

On the discovery of interferon-inducible, double-stranded RNA activated enzymes: the 2'-5'oligoadenylate synthetases and the protein kinase PKR

The demonstration that double-stranded (ds) RNA inhibits protein synthesis in cell-free systems prepared from interferon-treated cells, lead to the discovery of the two interferon-induced, dsRNA-dependent enzymes: the serine/threonine protein kinase that is referred to as PKR and the 2′,5′-oligoadenylate synthetase (2′,5′-OAS), which converts ATP to 2′,5′-linked oligoadenylates with the unusual 2′-5′ instead of 3′-5′ phosphodiesterase bond. We raised monoclonal and polyclonal antibodies against human PKR and the two larger forms of the 2′,5′-OAS. Such specific antibodies proved to be indispensable for the detailed characterization of these enzyme and the cloning of cDNAs corresponding to the human PKR and the 69–71 and 100 kDa forms of the 2′,5′-OAS. When activated by dsRNA, PKR becomes autophosphorylated and catalyzes phosphorylation of the protein synthesis initiation factor eIF2, whereas the 2′-5′OAS forms 2′,5′-oligoadenylates that activate the latent endoribonuclease, the RNAse L. By inhibiting initiation of protein synthesis or by degrading RNA, these enzymes play key roles in two independent pathways that regulate overall protein synthesis and the mechanism of the antiviral action of interferon. In addition, these enzymes are now shown to regulate other cellular events, such as gene induction, normal control of cell growth, differentiation and apoptosis.     

Cyclic guanosine 3',5'-monophosphate and phosphodiesterase activity in mitogen-stimulated human lymphocytes.

The cyclic adenosine 3′,5′-monophosphate (cyclic AMP) phosphodiesterase from human leukemic lymphocytes differes from the normal cell enzyme in having a much higher activity and a loss of inhibition by cyclic guanosine 3′,5′-monophosphate (cyclic GMP). In an effort to determine the mechanism of these alterations, we have studied this enzyme in a model system, lectin-stimulated normal human lymphocytes. Following stimulation of cells with concanavalin A (con A) the enzyme activity gradually becomes altered, until it fully resembles the phosphodiesterase found in leukemic lymphocytes. The changes in the enzyme parallel cell proliferation as measured by increases in thymidine incorporation into DNA. The addition of a guanylate cyclase inhibitor preparation from the bitter melon prevents both the changes in the phosphodiesterase and the thymidine incorporation into DNA. This blockage can be partially reversed by addition of 8-bromo cyclic guanosine 3′,5′-monophosphate (8-bromo cyclic GMP) to the con A-stimulated normal lymphocytes. These results indicate a possible role of cyclic GMP in a growth related alteration of cyclic AMP phosphodiesterase.     

An experimental and theoretical approach to 5alpha-cholestan-6-spiro-1',2',4'-triazolidine-3'-one

The 5alpha-cholestan-6-one semicarbazone (1) on reaction with hydrogen peroxide at 0 degrees C affords selectively 5alpha-cholestan-6-spiro-1',2',4'-triazolidine-3'-one. (2) The structural assignment of the product was confirmed on the basis of its elemental, analytical and spectral analysis. The Hartree-Fock method using 6-31G* basis set was employed in order to explore the reaction mechanism. The results of the computational study show that the reaction proceeds through two radical intermediates formation. The different characteristics involved during the reaction were explained, firstly, the lower energy conformation of each molecule using total energy, hardness and dipole moment, and secondly, the explanation of the free radical mechanism, using frontier molecular orbital (FMO) theory, encoded electrostatic potential, spin electronic density and atomic charges. The localization of highest occupied molecular orbital (HOMO) or alpha-HOMO, lowest unoccupied molecular orbital (LUMO) or alpha-LUMO and the flow of atomic charges are in good agreement to support the present mechanism of the reaction. Stability and feasibility of all the optimized structures were supported by their respective fundamental frequencies and energy minima.     

Dual-acting agents that possess reversing resistance and anticancer activities: Design, synthesis, MES-SA/Dx5 cell assay, and SAR of Benzyl 1,2,3,5,11,11a-hexahydro-3,3-dimethyl-1-oxo-6H-imidazo[3',4':1,2]pyridin[3,4-b]indol-2-substitutedacetates

Based on the structural analysis of fumitremorgin C (FTC), imidazoline and β-carboline amino acid benzylester, 14 novel 2-substitutedtetracyclic derivatives of tetrahydrocarboline 4a–n were prepared. We demonstrated that the exposure of MES-SA/Dx5 cells to some of 4a–n resulted in significant reduction of resistance of the cells against doxorubicin. This reduced resistance was accompanied by lowering of IC₅₀ value to doxorubicin from 1.55 ± 0.26 μmol/L to 0.33 ± 0.05 μmol/L for 2-(2-butyl)-derivative 4c, to 1.03 ± 0.22 μmol/L for 2-methyl-derivative 4d, to 0.46 ± 0.04 μmol/L for 2-benzyl-derivative 4f, to 0.98 ± 0.25 μmol/L for 2-indole-3-yl-methyl-derivative 4h, to 0.36 ± 0.03 μmol/L for 2-benzyloxycarbonylmethyl-derivative 4i, to 0.77 ± 0.08 μmol/L for 2-benzyloxycarbonylethyl-derivative 4j, and to 0.77 ± 0.08 μmol/L for 2-benzyloxycarbonylamino-n-butyl-derivative 4l. Proliferation assays of 4a–n indicated 4c,f,i,j were able to inhibit the proliferation of doxorubicin resistant MES-SA/Dx5 cells. The SAR analysis revealed that the benzylester form and the tetracyclic structure of 4a–n were critical for both sensitizing doxorubicin and the cellular anti-proliferative effect.     

Validation of a high-performance liquid chromatographic-mass spectrometric method for the measurement of 5-chloro-2′,3′-dideoxy-3′-fluorouridine (935U83) in human plasma

An isocratic reversed-phase LC-MS method for measuring concentrations of 5-chloro-2′,3′-dideoxy-3′-fluorouridine (935U83; I) directly and its 5′-glucuronide metabolite (5-chloro-2′,3′-dideoxy-5′-O-β- -glucopyranuronosyl-3′-fluorouridine) indirectly in human plasma was developed, validated, and applied to a Phase I clinical study. The pyrimidine nucleoside, I, was extracted from human plasma by using anionic solid-phase extraction. The concentration of the glucuronide conjugate was determined from the difference between the molar concentration of I in a sample hydrolyzed with β-glucuronidase and the nonhydrolyzed sample. Recovery of I from human plasma averaged 90%. The bias of the assay for I ranged from −5.5 to 7.1% during the validation and from −6.0 to 1.4% during application of the assay to the Phase I single-dose escalation study. The intra- and inter-day precision was less than 8% for I and its glucuronide conjugate. The lower and upper limits of quantitation for a 50-μl sample were 4 ng/ml and 3000 ng/ml, respectively. No significant endogenous interferences were noted in human plasma obtained from drug-free volunteers nor from predose samples of HIV-infected patients.     

Glucose repression of the inducible catabolic pathway for N-acetylglucosamine in yeast.

In order to investigate the mechanism of glucose repression of the N-acetylglucosamine metabolic enzymes in $\text{Candida}\text{albicans}$, an obligatory aerobic yeast, the activities of the following inducible enzymes were assayed: the N-acetylglucosamine uptake, N-acetylglucosamine kinase and glucosamine-6-phosphate deaminase. In the presence of glucose or other sugars e.g. succinate and glycerol, synthesis of these enzymes took place at a normal rate, suggesting that the hexose produces no catabolite repression in this organism. On the contrary, strong inhibition by glucose was observed on the activities of N-acetylglucosamine uptake and deaminase in N-acetylglucosamine-grown cells of $\text{Saccharomyces}\text{cerevisiae}$, a facultative aerobe. From the results, it is concluded that “glucose effect” or catabolite repression is absent in $\text{Candida}\text{albicans}$, a pathogenic strain of yeast.     

Isolation and X-ray structure determination of a neolignan from Clerodendron inerme seeds

A new neolignan, 5,8-epoxy-6,7-dimethyl 2′,3′,2″,3″-dimethylene dioxy-4′,1″-dimethoxy-1,2:3,4-dibenzo-1,3-cyclooctadiene, from the petrol extract of Clerodendron inerme seeds, was characterized by spectroscopic and X-ray crystallographic methods. This compound makes up ca 5% by wt of the seeds.     

Synthesis and biological evaluation of fatty acyl ester derivatives of 2',3'-didehydro-2',3'-dideoxythymidine

A number of 5′-O-fatty acyl derivatives of 2′,3′-didehydro-2′,3′-dideoxythymidine (stavudine, d4T) were synthesized and evaluated for anti-HIV activities against cell-free and cell-associated virus, cellular cytotoxicity, and cellular uptake studies. The conjugates were found to be more potent than d4T. Among these conjugates, 5′-O-12-azidododecanoyl derivative of d4T (2), displaying EC₅₀ = 3.1-22.4 μM, showed 4- to 9-fold higher activities than d4T against cell-free and cell-associated virus. Cellular uptake studies were conducted on CCRF-CEM cell line using 5(6)-carboxyfluorescein derivatives of d4T attached through β-alanine (9) or 12-aminododecanoic acid (10) as linkers. The fluorescein-substituted analog of d4T with long chain length (10) showed 12- to 15-fold higher cellular uptake profile than the corresponding analog with short chain length (9). These studies reveal that conjugation of fatty acids to d4T enhances the cellular uptake and anti-HIV activity of stavudine.