Phosphocreatinine, a high-energy phosphate in muscle, spontaneously forms phosphocreatine and creatinine under physiological conditions

Phosphocreatinine undergoes the following spontaneous simultaneous reactions at pH 7.4 (0.02 M sodium phosphate and 120 mM KCl) and 38 degrees C. (Formula: see text) The first order rate constants are 0.046 h-1 (ka) and 0.048 h-1 (kb). There is a major effect of pH on the reactions such that at pH values higher than 7.4 phosphocreatine production predominates, while at pH values less than 7.4 creatinine is the major product. This along with titration data showing apparent pK values of about 3.0 and 7.5 for phosphocreatinine suggest that the dianionic form of phosphocreatinine is involved in the conversion to phosphocreatine, whereas the monoanionic form is exclusively converted to creatinine. Possible mechanisms to account for the reactivity of phosphocreatinine are discussed. Several lines of evidence suggest that the apparent Keq for phosphocreatine formation from phosphocreatinine is about 300 at pH 9.0 and about 70 at pH 7.0, and the delta G0' (pH 7.0) is-2.6 kcal/mol. The delta G0' (pH 7.0) for the hydrolysis of the phosphoryl bond in phosphocreatinine is-12.8 kcal/mol. The phosphocreatinine content of rabbit white skeletal muscle was measured to be 0.05 mumol/g, which is 0.4% of the phosphocreatine content. The in vitro experiments suggest that phosphohydrolysis of phosphocreatinine can account for a creatinine formation equal to 0.5% of the phosphocreatine content/day. We conclude that it is likely that a substantial fraction of the in vivo creatinine production from phosphocreatine goes through the novel high energy phosphate, phosphocreatinine, as an intermediate.     

3-Phenyl-5-isothiazole carboxamides with potent mGluR1 antagonist activity

The disclosed 3-phenyl-5-isothiazole carboxamides are potent allosteric antagonists of mGluR1 with generally good selectivity relative to the related group 1 receptor mGluR5. Pharmacokinetic properties of a member of this series (1R,2R)-N-(3-(4-methoxyphenyl)-4-methylisothiazol-5-yl)-2-methylcyclopropanecarboxamide (14) are good, showing acceptable plasma and brain exposure after oral dosing. Oral administration of isothiazole 14 gave robust activity in the formalin model of persistent pain which correlated with CNS receptor occupancy.     

Evaluation of the RAPID ID 32A system for the identification of Bacteroides fragilis and related organisms

S.A. JENKINS, D.B. DRUCKER, M.G.L. KEANEY AND L.A. GANGULI. 1991. This study evaluated the ability of a rapid identification system for anaerobic bacteria. ATB 32A, now renamed RAPID ID 32A (API-bioMérieux UK Ltd., Basingstoke), to identify accurately 74 strains of the 'B. fragilis group'. ATB 32A identified correctly 78.4% of strains to species level, without supplemental tests. The percentage of strains identified to species level rose to 94.6% when a supplementary test (advised by bioMérieux) for catalase production was used to differentiate between Bacteroides ovatus and Bacteroides uniformis. RAPID ID 32A is a rapid, accurate method for the identification of members of the 'B. fragilis group' isolated within a routine clinical laboratory.     

Weaponry,size, and sex ratio affect spatial distribution within small and large groups of the maritime earwig (Anisolabis maritima)

Dispersion patterns within a group can reveal important aspects about social interactions and sexual selection within a species. We examined the distribution patterns of the maritime earwig (Anisolabis maritima), an insect well suited for studies of aggression, sociality, and sexual selection since both sexes live in close proximity and possess weaponry in the form of sexually dimorphic pincers. To examine intra‐ and intersexual interactions within small groups, we conducted trials with three earwigs with limited access to shelters. In single‐sex trios, we found that both males and females exhibited strong size‐based intrasexual aggression, as larger individuals were less likely to be excluded from shelters; however, males were more likely to cohabitate than females. In mixed‐sex trios, we found that both males and females preferred smaller opposite‐sex partners, and cohabitation patterns indicate that both sex‐ and size‐based differences in aggression can influence overall spatial distribution. We also examined larger single‐sex and mixed‐sex groups of 18 earwigs to determine whether they had random, uniform, or clumped distributions. Similar to previous field observations, males tended to form aggregations, whereas females were distributed uniformly, a pattern indicative of territoriality. Mixed‐sex groups, on the other hand, were uniform during nocturnal periods of high activity but then become clumped after settling into more stable daytime positions. Overall, our results suggest that females have high levels of aggression regardless of the social context, whereas males alter their aggressive behavior in the presence of females.     

Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups: RESISTANCE OF α2-8-LINKED N-GLYCOLYLNEURAMINIC ACID TO ENZYMATIC CLEAVAGE

The sialic acid (Sia) N-acetylneuraminic acid (Neu5Ac) and its hydroxylated derivative N-glycolylneuraminic acid (Neu5Gc) differ by one oxygen atom. CMP-Neu5Gc is synthesized from CMP-Neu5Ac, with Neu5Gc representing a highly variable fraction of total Sias in various tissues and among different species. The exception may be the brain, where Neu5Ac is abundant and Neu5Gc is reported to be rare. Here, we confirm this unusual pattern and its evolutionary conservation in additional samples from various species, concluding that brain Neu5Gc expression has been maintained at extremely low levels over hundreds of millions of years of vertebrate evolution. Most explanations for this pattern do not require maintaining neural Neu5Gc at such low levels. We hypothesized that resistance of α2-8-linked Neu5Gc to vertebrate sialidases is the detrimental effect requiring the relative absence of Neu5Gc from brain. This linkage is prominent in polysialic acid (polySia), a molecule with critical roles in vertebrate neural development. We show that Neu5Gc is incorporated into neural polySia and does not cause in vitro toxicity. Synthetic polymers of Neu5Ac and Neu5Gc showed that mammalian and bacterial sialidases are much less able to hydrolyze α2-8-linked Neu5Gc at the nonreducing terminus. Notably, this difference was not seen with acid-catalyzed hydrolysis of polySias. Molecular dynamics modeling indicates that differences in the three-dimensional conformation of terminal saccharides may partly explain reduced enzymatic activity. In keeping with this, polymers of N-propionylneuraminic acid are sensitive to sialidases. Resistance of Neu5Gc-containing polySia to sialidases provides a potential explanation for the rarity of Neu5Gc in the vertebrate brain.     

T cell-tropic HIV gp120 mediates CD4 and CD8 cell chemotaxis through CXCR4 independent of CD4: implications for HIV pathogenesis

HIV entry is determined by one or more chemokine receptors. T cell-tropic viruses bind CXCR4, whereas macrophage-tropic viruses use CCR5 and other CCRs. Infection with CXCR4 and CCR5-tropic HIV requires initial binding to CD4, and chemotaxis induced by the CCR5-tropic envelope has been reported to be strictly dependent on CD4 binding. We demonstrate that, in contrast to CD4-dependent gp120 signaling via CCR5, envelope signaling through CXCR4 is CD4 independent, inducing chemotaxis of both CD4 and CD8 T cells. Signaling by virus or soluble envelope through CXCR4 may affect pathogenesis by attracting and activating target and effector cells.     

Glucagon receptor-mediated activation of Gs is accompanied by subunit dissociation

The effect of the glucagon receptor on the activation of the stimulatory GTP-binding protein of adenylyl cyclase (Gs) in the native rat liver membrane environment was studied. The activated state of Gs was assessed by its ability to reconstitute the cyc- S49 cell membrane adenylyl cyclase. The Gs protein was activated by saturating concentrations of guanosine 5'-thiotriphosphate (GTP gamma S) or guanyl-5'-yl imidodiphosphate in a hormone-dependent manner at 0.4 mM Mg2+ in native membranes or in membranes that had been treated with 1 mM N-ethylmaleimide to eliminate the catalytic activity of adenylyl cyclase. At 50 mM Mg2+, Gs was fully activated by GTP gamma S in the absence of hormone. The unactivated Gs protein migrates around 4 S, whereas activated Gs migrates around 2 S on sucrose density gradients. When pure Gs is analyzed on sucrose density gradients, it is found that the unactivated protein migrates at 4.1 S. Gs was activated by saturating concentrations of GTP gamma S and Mg2+, and the alpha subunit of Gs was chromatographically purified. The resolved alpha subunit of Gs that is capable of stimulating the cyc- adenylyl cyclase migrates at 2.1 S. From these data, we conclude that activation of Gs results in the dissociation of this protein in the membrane environment and that the hormone-occupied receptor promotes this dissociation process under conditions where Mg2+ ions are limiting.     

Cell shape and negative links in regulatory motifs together control spatial information flow in signaling networks

The role of cell size and shape in controlling local intracellular signaling reactions, and how this spatial information originates and is propagated, is not well understood. We have used partial differential equations to model the flow of spatial information from the beta-adrenergic receptor to MAPK1,2 through the cAMP/PKA/B-Raf/MAPK1,2 network in neurons using real geometries. The numerical simulations indicated that cell shape controls the dynamics of local biochemical activity of signal-modulated negative regulators, such as phosphodiesterases and protein phosphatases within regulatory loops to determine the size of microdomains of activated signaling components. The model prediction that negative regulators control the flow of spatial information to downstream components was verified experimentally in rat hippocampal slices. These results suggest a mechanism by which cellular geometry, the presence of regulatory loops with negative regulators, and key reaction rates all together control spatial information transfer and microdomain characteristics within cells.     

Purification and characterization of a 34 kDa antioxidant protein (β-turmerin) from turmeric (Curcuma longa) waste grits

β-Turmerin from turmeric (Curcuma longa) waste grits obtained after extraction of curcumin was purified by successive gel permeation chromatography. Homogeneity of β-turmerin was confirmed by its movement as single band both in SDS-PAGE and as well as in native (basic) PAGE. The apparent molecular mass is 34 kDa by SDS-PAGE. It is more hydrophobic protein and showed sharp single peak in RP-HPLC with retention time of 62.17 min. It is a glycoprotein as it shows the presence of amino sugars up to 0.021 gm%. In three different model systems i.e., linolenic acid micelles, erythrocyte membrane systems and liposomes, β-turmerin at 0.125 μM offered 70%, 64%, and 60% inhibition of lipid peroxidation, which is 3200 times more efficient than the standard antioxidants BHA (400 μM) and α-tocopherol (400 μM). β-turmerin inhibited diene–triene and tetraene conjugation up to 54%, 72% and 47%, respectively. β-turmerin also effectively scavenges hydroxyl radicals when compared to BHA and α-tocopherol. β-turmerin (2.5 μM) further inhibited the activation of PMNL mediated by fMLP up to the extent of 75%, where as standards BHA (400 μM) and mannitol (10 μM) inhibited the same to 65% and 55%, respectively. At 0.125 μM dose β-turmerin prevented t-BOOH induced cell death at all time intervals. In addition to the above properties, it is non-toxic to lymphocytes as it did not affect the viability of cells. The mechanism of antioxidant action of β-turmerin could probably be by counteracting/quenching of reactive oxygen species (ROS). We report the purification and characterization of β-turmerin (34 kDa), a potent antioxidant protein from turmeric waste grits.