Synthesis of some new analogs of the immunoadjuvant glycopeptide MDP (N-acetyl-muramyl-L-alanyl-D-isoglutamine)

In our continued efforts to elucidate the relationship between the structure and the immunoadjuvant, antiinfectious or mitogenic activity of N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP), we report the synthesis of 11 new analogs. Five of them present some modifications in the N-acetyl-muramyl moiety.     

Thapsigargin inhibits contraction and Ca2+ transient in cardiac cells by specific inhibition of the sarcoplasmic reticulum Ca2+ pump.

Regulation of the level of ionized calcium, [Ca2+]i, is critical for its use as an important intracellular signal. In cardiac and skeletal muscle the control of fluctuations of [Ca2+]i depend on sarcolemmal and sarcoplasmic reticulum ion channels and transporters. We have investigated the sesquiterpine lactone, thapsigargin (TG), because of its reported action to alter cellular calcium regulation in diverse cell types, including striated muscle cells. We have combined biochemical and physiological methods at the cellular level to determine the site of action of this agent, its specificity, and its cellular effects. Using a patch-clamp method in whole cell configuration while measuring [Ca2+]i with Indo-1 salt, we find that TG (100 nM) largely blocks the contraction and the [Ca2+]i transient in rat ventricular myocytes. Analysis of these data indicate that no sarcolemmal current or transport system is directly altered by TG, although indirect [Ca2+]i-dependent processes are affected. In permeabilized myocytes, TG blocked oxalate-stimulated calcium uptake (half-maximal effect at 10 nM) into the SR. However, TG (100 microM) had no effect on Ca(2+)-induced Ca(2+)-release in purified muscle (ryanodine-receptor enriched) vesicles while clearly blocking Ca(2+)-ATPase activity in purified (longitudinal SR) vesicles. We conclude that in striated muscle TG markedly alters calcium metabolism and thus alters contractile function only by its direct action on the Ca(2+)-ATPase.     

Substitution of Tyr254 with Phe at the active site of flavocytochrome b2: consequences on catalysis of lactate dehydrogenation

A role for Tyr254 in L-lactate dehydrogenation catalyzed by flavocytochrome b2 has recently been proposed on the basis of the known active-site structure and of studies that had suggested a mechanism involving the initial formation of a lactate carbanion [Lederer, F., & Mathews, F.S. (1987) in Flavins and Flavoproteins, Proceedings of the Ninth International Symposium, Atlanta, GA, 1987 (Edmondson, D.E., & McCormick, D.B., Eds.) pp 133-142, Walter de Gruyter, Berlin]. This role is now examined after replacement of Tyr254 with phenylalanine. The kcat is decreased about 40-fold, Km for lactate appears unchanged, and the mainly rate-limiting step is still alpha-hydrogen abstraction, as judged from the steady-state deuterium isotope effect. Modeling studies with lactate introduced into the active site indicate two possible substrate conformations with different hydrogen-bonding partners for the substrate hydroxyl. If the hydrogen bond is formed with Tyr254, as was initially postulated, the mechanism must involve removal by His373 of the C2 hydrogen, with carbanion formation. If, in the absence of the Tyr254 phenol group, the hydrogen bond is formed with His373 N3, the substrate is positioned in such a way that the reaction must proceed by hydride transfer. Therefore the mechanism of the Y254F enzyme was investigated so as to distinguish between the two mechanistic possibilities. 2-Hydroxy-3-butynoate behaves with the mutant as a suicide reagent, as with the wild-type enzyme. Similarly, the mutant protein also catalyzes the reduction and the dehydrohalogenation of bromopyruvate under transhydrogenation conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+/Calmodulin-dependent Protein Kinase IIα (αCaMKII) Controls the Activity of the Dopamine Transporter: IMPLICATIONS FOR ANGELMAN SYNDROME

The dopamine transporter (DAT) is a crucial regulator of dopaminergic neurotransmission, controlling the length and brevity of dopaminergic signaling. DAT is also the primary target of psychostimulant drugs such as cocaine and amphetamines. Conversely, methylphenidate and amphetamine are both used clinically in the treatment of attention-deficit hyperactivity disorder and narcolepsy. The action of amphetamines, which induce transport reversal, relies primarily on the ionic composition of the intra- and extracellular milieus. Recent findings suggest that DAT interacting proteins may also play a significant role in the modulation of reverse dopamine transport. The pharmacological inhibition of the serine/threonine kinase αCaMKII attenuates amphetamine-triggered DAT-mediated 1-methyl-4-phenylpyridinium (MPP(+)) efflux. More importantly, αCaMKII has also been shown to bind DAT in vitro and is therefore believed to be an important player within the DAT interactome. Herein, we show that αCaMKII co-immunoprecipitates with DAT in mouse striatal synaptosomes. Mice, which lack αCaMKII or which express a permanently self-inhibited αCaMKII (αCaMKII(T305D)), exhibit significantly reduced amphetamine-triggered DAT-mediated MPP(+) efflux. Additionally, we investigated mice that mimic a neurogenetic disease known as Angelman syndrome. These mice possess reduced αCaMKII activity. Angelman syndrome mice demonstrated an impaired DAT efflux function, which was comparable with that of the αCaMKII mutant mice, indicating that DAT-mediated dopaminergic signaling is affected in Angelman syndrome.     

Comparative proteomics of human monkeypox and vaccinia intracellular mature and extracellular enveloped virions.

Orthopoxviruses are among the largest and most complex of the animal viruses. In response to the recent emergence of monkeypox in Africa and the threat of smallpox bioterrorism, two orthopoxviruses with different pathogenic potentials, human monkeypox virus and vaccinia virus, were proteomically compared with the goal of identifying proteins required for pathogenesis. Orthopoxviruses were grown in HeLa cells to two different viral forms (intracellular mature virus and extracellular enveloped virus), purified by sucrose gradient ultracentrifugation, denatured using RapiGest surfactant, and digested with trypsin. Unfractionated samples and strong cation exchange HPLC fractions were analyzed by high-resolution reversed-phase nano-LC-MS/MS, and analyses of the MS/MS spectra using SEQUEST and X! Tandem resulted in the confident identification of hundreds of monkeypox, vaccinia, and copurified host-cell proteins. The unfractionated samples were additionally analyzed by LC-MS using an LTQ-Orbitrap, and the accurate mass and elution time tag approach was used to perform quantitative comparisons. Possible pathophysiological roles of differentially abundant Orthopoxvirus proteins are discussed. Data, processed results, and protocols are available at http://www.proteomicsresource.org/.