Lysine 2,3-aminomutase. Support for a mechanism of hydrogen transfer involving S-adenosylmethionine

The conversion of L-lysine to L-beta-lysine is catalyzed by lysine 2,3-aminomutase. The reaction involves the interchange of the 2-amino group of lysine with a hydrogen at carbon 3. As such the reaction is formally analogous to adenosylcobalamin-dependent rearrangements. However, the enzyme does not contain and is not activated by this coenzyme. Instead it contains iron and pyridoxal phosphate and is activated by S-adenosylmethionine. Earlier experiments implicated adenosyl-C-5' of S-adenosylmethionine in the hydrogen transfer mechanism, apparently in a role similar or analogous to that of adenosyl moiety of adenosylcobalamin in the B12-dependent rearrangements. The question of whether both hydrogens or only one hydrogen at adenosyl-C-5' participate in the hydrogen-transfer process has been addressed by carrying out the lysine 2,3-aminomutase reaction with S-[5'-3H] adenosylmethionine in the presence of 10 times its molar concentration of enzyme. Under these conditions all of the tritium appeared in lysine and beta-lysine, showing that C-5'-hydrogens participate. To determine whether hydrogen transfer is compulsorily intermolecular and intramolecular, various molar ratios of [3,3-2H2]lysine and unlabeled lysine were submitted to the action of lysine 2,3-aminomutase under conditions in which 10-15% conversion to beta-lysine occurred. Mass spectral analysis of the beta-lysine for monodeutero and dideutero species showed conclusively that hydrogen transfer is both intramolecular and intermolecular. The results quantitatively support our postulate that activation of the enzyme involves a transformation of S-adenosylmethionine into a form that promotes the generation of an adenosyl-5' free radical, which abstracts hydrogen from lysine to form 5'-deoxyadenosine as an intermediate.     

Nicotine and cannabinoids as adjuncts to neuroleptics in the treatment of Tourette syndrome and other motor disorders

Animal studies suggest nicotine and cannabinoids may significantly enhance the therapeutic value of neuroleptics in motor disorders. This was recently demonstrated in humans by the finding that chewing nicotine gum produced striking relief from tics and other symptoms of Tourette syndrome not controlled by neuroleptic treatment alone. It appears that the use of nicotine or cannabinoids may greatly improve the clinical response to neuroleptics in motor disorders.     

Amino acid-specific ADP-ribosylation: structural characterization and chemical differentiation of ADP-ribose-cysteine adducts formed nonenzymatically and in a pertussis toxin-catalyzed reaction.

ADP-ribosylation is a posttranslational modification of proteins by amino acid-specific ADP-ribosyltransferases. Both pertussis toxin and eukaryotic enzymes ADP-ribosylate cysteine residues in proteins and also, it has been suggested, free cysteine. Analysis of the reaction mechanisms of cysteine-specific ADP-ribosyltransferases revealed that free ADP-ribose combined nonenzymatically with cysteine. L- and D-cysteine, L-cysteine methyl ester, and cysteamine reacted with ADP-ribose, but alanine, serine, lysine, arginine, N-acetyl-L-cysteine, 2-mercaptoethanol, dithiothreitol, and glutathione did not. The 1H NMR spectrum of the product, along with the requirement for both free sulfhydryl and amino groups of cysteine, suggested that the reaction produced a thiazolidine linkage. ADP-ribosylthiazolidine was labile to hydroxylamine and mercuric ion, unlike the ADP-ribosylcysteine formed by pertussis toxin and NAD in guanine nucleotide-binding (G-) proteins, which is labile to mercuric ion but stable in hydroxylamine. In the absence of G-proteins but in the presence of NAD and cysteine, pertussis toxin generated a hydroxylamine-sensitive product, suggesting that a free ADP-ribose intermediate, expected to be formed by the NADase activity of the toxin, reacted with cysteine. Chemical analysis, or the use of alternative thiol acceptors lacking a free amine, is necessary to distinguish the enzymatic formation of ADP-ribosylcysteine from nonenzymatic formation of ADP-ribosylthiazolidine, thereby differentiating putative NAD:cysteine ADP-ribosyltransferases from NAD glycohydrolases.     

Chloride-induced Ca2+ Release from the Sarcoplasmic Reticulum of Chemically Skinned Rabbit Psoas Fibers and Isolated Vesicles of Terminal Cisternae

There is increasing evidence that Ca²⁺ release from sarcoplasmic reticulum (SR) of mammalian skeletal muscle is regulated or modified by several factors including ionic composition of the myoplasm. We have studied the effect of Cl⁻ on the release of Ca²⁺ from the SR of rabbit skeletal muscle in both skinned psoas fibers and in isolated terminal cisternae vesicles. Ca²⁺ release from the SR in skinned fibers was inferred from increases in isometric tension and the amount of release was assessed by integrating the area under each tension transient. Ca²⁺ release from isolated SR was measured by rapid filtration of vesicles passively loaded with ⁴⁵Ca²⁺. Ca²⁺ release from SR was stimulated in both preparations by exposure to a solution containing 191 mm choline-Cl, following pre-equilibration in Ca²⁺-loading solution that had propionate as the major anion. Controls using saponin (50 μg/ml), indicated that the release of Ca²⁺ was due to direct action of Cl⁻ on the SR rather than via depolarization of T-tubules. Procaine (10 mm) totally blocked Cl⁻- and caffeine-elicited tension transients recorded using loading and release solutions having ([Na⁺] + [K⁺]) × [Cl⁻] product of 6487.69 mm ² and 12361.52 mm ², respectively, and blocked 60% of Ca²⁺ release in isolated SR vesicles. Surprisingly, procaine had only a minor effect on tension transients elicited by Cl⁻ and caffeine together. The data from both preparations suggests that Cl⁻ induces a relatively small amount of Ca²⁺ release from the SR by activating receptors other than RYR-1. In addition, Cl⁻ may increase the Ca²⁺ sensitivity of RYR-1, which would then allow the small initial release of Ca²⁺ to facilitate further release of Ca²⁺ from the SR by Ca²⁺-induced Ca²⁺ release. Received: 6 February 1996/Revised: 17 July 1996     

Patterns of viral replication correlate with outcome in simian immunodeficiency virus (SIV)-infected macaques: effect of prior immunization with a trivalent SIV vaccine in modified vaccinia virus Ankara.

The dynamics of plasma viremia were explored in a group of 12 simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta) that had received prior immunization with either nonrecombinant or trivalent (gag-pol, env) SIV-recombinant vaccinia viruses. Three distinct patterns of viral replication observed during and following primary viremia accounted for significant differences in survival times. High-level primary plasma viremia with subsequently increasing viremia was associated with rapid progression to AIDS (n = 2). A high-level primary plasma virus load with a transient decline and subsequent progressive increase in viremia in the post-acute phase of infection was associated with progression to AIDS within a year (n = 6). Low levels of primary plasma viremia followed by sustained restriction of virus replication were associated with maintenance of normal lymphocyte subsets and intact lymphoid architecture (n = 4), reminiscent of the profile observed in human immunodeficiency virus type 1-infected long-term nonprogressors. Three of four macaques that showed this pattern had been immunized with an SIV recombinant derived from the attenuated vaccinia virus, modified vaccinia virus Ankara. These data link the dynamics and extent of virus replication to disease course and suggest that sustained suppression of virus promotes long-term, asymptomatic survival of SIV-infected macaques. These findings also suggest that vaccine modulation of host immunity may have profound beneficial effects on the subsequent disease course, even if sterilizing immunity is not achieved.     

Folding, assembly, and intracellular trafficking of the human immunodeficiency virus type 1 envelope glycoprotein analyzed with monoclonal antibodies recognizing maturational intermediates.

Monoclonal antibodies (MAbs) that bind linear or conformational epitopes on monomeric or oligomeric human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins were screened for their recognition of maturational intermediates. On the basis of reactivities with gp160 at different times after pulse-labeling, the MAbs were sorted into groups that exhibited binding which was immediate and constant, immediate but transient, delayed, late, or very late. This grouping was consistent with the selectivity of the MAbs for structural features of gp160. Thus, a MAb to the V3 loop reacted with envelope proteins at all times, in accord with the relative conformational independence and accessibility of the epitope. Several MAbs that preferentially react with monomeric gp160 exhibited diminished binding after the pulse. A 10-min tag occurred before gp160 reacted with conformational MAbs that inhibited CD4 binding. The availability of epitopes for other conformational MAbs, including some that react equally with monomeric and oligomeric gp160 and some that react better with oligomeric forms, was half-maximal in 30 min and closely followed the kinetics of gp160 oligomerization. Remarkably, there was a 1- to 2-h delay before gp160 reacted with stringent oligomer-specific MAbs. After 4 h, approximately 20% of the gp160 was recognized by these MAbs. Epitopes recognized by monomerspecific or CD4-blocking MAbs but not by oligomer-dependent MAbs were present on gp160 molecules associated with the molecular chaperone BiP/GRP78. MAbs with a preference for monomers reacted with recombinant or HIV-1 envelope proteins in the endoplasmic reticulum, whereas the oligomer-specific MAbs recognized them in the Golgi complex. Additional information regarding gp160 maturation and intracellular trafficking was obtained by using brefeldin A, dithiothreitol, and a low temperature.     

Temporal changes in the level of infestation of Simulium ornatum Meigen (complex) (Simuliidae: Diptera) larvae by the endosymbiotic fungus Harpella melusinae Lichtwardt (Harpellales: Trichomycetes)

The fungus Harpella melusinae (Harpellales: Trichomycetes) is obligately associated with the midguts of larval Simuliidae (Diptera). The level of infestation of a population of Simulium ornatum by H. melusinae was monitored at a stream in Hampshire, England. Significant temporal changes in the level of infestation were recorded during monthly and weekly collections; a twenty-fold increase being recorded over a nine-day period. Possible mechanisms by which these changes occur are discussed.     

Competition during colonization vs competition after colonization in disturbed environments: A metapopulation approach

How two species interact during and after colonization influences which of them will be present in each stage of succession. In the tolerance model of ecological succession in a patchy environment, empty patches can be colonized by any species, but the ability to tolerate reduced resource levels determines which species will exclude the other. Here, we analyze a meta-population model of the possible roles of competition in colonization and succession, using non-linear Markov chains as a mathematical framework. Different kinds of competition affect the final equilibrial, abundances of the species involved in qualitatively different ways. An explicit criterion is given to determine which interactions have stronger effects on the final equilibrial levels of the weaker, species. Precise conditions are stated for the co-existence of both species. Both species are more likely to co-exist in the presence of an intermediate disturbance frequency.