Anomeric and other substrate specificity studies with myo-inositol-1-P synthase

L-myo-Inositol-1-phosphate synthase has been found to have at least a 5-fold preference for the beta-anomer of its natural substrate D-Glc-6-P. The alpha-anomer appears to be an inhibitor of the reaction and may be converted to product as well. As well as showing an enzymatic preference for the equatorial C-1 hydroxyl of D-Glc-6-P, our results suggest that it is the pyranose form of D-Glc-6-P that binds to the enzyme and that ring-opening is an enzymatic step. We have also found D-2-dGlc-6-P, D-2-F-2-dGlc-6-P, and D-Man-6-P each to be both competitive inhibitors and substrates that are converted to inositol phosphates by the synthase. D-Allose-6-P is a weak inhibitor of the enzyme, but not a substrate. D-Gal-6-P is neither substrate nor inhibitor. Thus the specificity of the synthase with respect to single position epimers of D-Glc-6-P increases in the order C1 less than C2 much less than C3 less than C4.     

Effect of Feed Restriction on Performance and Postprandial Nutrient Metabolism in Pigs Co-Infected with Mycoplasma hyopneumoniae and Swine Influenza Virus

As nutritional status and inflammation are strongly connected, feeding and nutritional strategies could be effective to improve the ability of pigs to cope with disease. The aims of this study were to investigate the impact of a feed restriction on the ability of pigs to resist and be tolerant to a coinfection with Mycoplasma hyopneumoniae (Mhp) and the European H1N1 swine influenza virus, and the consequences for nutrient metabolism, with a focus on amino acids. Two groups of specific pathogen-free pigs were inoculated with Mhp and H1N1 21 days apart. One group was fed ad libitum, the other group was subjected to a two-week 40% feed restriction starting one week before H1N1 infection. The two respective mock control groups were included. Three days post-H1N1 infection, 200 g of feed was given to pigs previously fasted overnight and serial blood samples were taken over 4 hours to measure plasma nutrient concentrations. Throughout the study, clinical signs were observed and pathogens were detected in nasal swabs and lung tissues. Feed-restricted pigs presented shorter hyperthermia and a positive mean weight gain over the 3 days post-H1N1 infection whereas animals fed ad libitum lost weight. Both infection and feed restriction reduced postprandial glucose concentrations, indicating changes in glucose metabolism. Post-prandial plasma concentrations of the essential amino acids histidine, arginine and threonine were lower in co-infected pigs suggesting a greater use of those amino acids for metabolic purposes associated with the immune response. Altogether, these results indicate that modifying feeding practices could help to prepare animals to overcome an influenza infection. Connections with metabolism changes are discussed.     

Nitroimidazole conjugates of bis(thiosemicarbazonato)Cu(II) - Potential combination agents for the PET imaging of hypoxia

Combination agents comprising two different pharmacophores with the same biological target have the potential to show additive or synergistic activity. Bis(thiosemicarbazonato)copper(II) complexes (e.g. ⁶⁴Cu-ATSM) and nitroimidazoles (e.g. ¹⁸F-MISO) are classes of tracer used for the delineation of tumor hypoxia by positron emission tomography (PET). Three nitroimidazole-bis(thiosemicarbazonato)copper(II) conjugates were produced in order to investigate their potential as combination hypoxia imaging agents. Two were derived from the known bifunctional bis(thiosemicarbazone) H₂ATSM/A and the third from the new precursor diacetyl-2-(4-N-methyl-3-thiosemicarbazone)-3-(4-N-ethylamino-3-thiosemicarbazone) - H₂ATSM/en. Oxygen-dependent uptake studies were performed using the ⁶⁴Cu radiolabelled complexes in EMT6 carcinoma cells. All the complexes displayed appreciable hypoxia selectivity, with the nitroimidazole conjugates displaying greater selectivity than a simple propyl derivative used as a control. Participation of the nitroimidazole group in the trapping mechanism is indicated by the increased hypoxic uptake of the 2- vs. the 4-substituted ⁶⁴Cu-ATSM/A derivatives. The 2-nitroimidazole derivative of ⁶⁴Cu-ATSM/en demonstrated superior hypoxia selectivity to ⁶⁴Cu-ATSM over the range of oxygen concentrations tested. Biodistribution of the radiolabelled 2-nitroimidazole conjugates was carried out in EMT6 tumor-bearing mice. The complexes showed significantly different uptake trends in comparison to each other and previously studied Cu-ATSM derivatives. Uptake of the Cu-ATSM/en conjugate in non-target organs was considerably lower than for derivatives based on Cu-ATSM/A.     

The existence of an optimal range of cytosolic free calcium for insulin-stimulated glucose transport in rat adipocytes

We have examined the effects of extracellular and intracellular Ca2+ concentrations upon basal and insulin-stimulated 2-deoxyglucose uptake in isolated rat adipocytes. In the absence of extracellular Ca2+, both basal and insulin-stimulated glucose uptake were significantly reduced. Insulin-stimulated glucose transport was optimal at 1 and 2 mM Ca2+. Further increases in extracellular Ca2+ concentration (3 mM) significantly diminished insulin-stimulated glucose uptake. When intracellular Ca2+ concentrations were augmented by ionomycin (1 microM), insulin-stimulated glucose uptake was significantly reduced at extracellular Ca2+ concentrations of 2 and 3 mM. The levels of intracellular free Ca2+ concentrations were then measured with Ca2+ indicator fura-2. The correlation between the levels of intracellular free Ca2+ and the magnitude of insulin-stimulated glucose uptake revealed that the optimal effect of insulin is observed at Ca2+ levels between 140 and 370 nM. At both extremes outside of this window, both low and high levels of intracellular Ca2+ result in diminished cellular responsiveness to insulin. These data suggest that intracellular calcium concentrations may exert a dual role in the regulation of cellular sensitivity to insulin. First, there must exist a minimal concentration of intracellular calcium to promote insulin action. Second, increased levels of intracellular calcium may provide a critical signal for diminution of insulin action.     

Microcalorimetric assay of acetylcholinesterase

Comparative assays were made in a spectrophotometer and a microcalorimeter for the reaction between acetylcholinesterase (EC 3.1.1.7) and acetylthiocholine. The rate of light absorbance change and the rate of heat flow were measured from similar and simultaneous reactions in spectrophotometer and microcalorimeter, respectively. At the enzyme activity levels studied, i.e., 0.05–0.15 I.U. in calorimetry and 1–4 I.U. in spectrophotometry, the reaction rates were linear and showed first-order kinetics. A highly significant positive correlation was seen between the two methods (r = 0.997). More importantly, spectrophotometric assay with acetylthiocholine (which utilized a secondary reaction with chromagen, dithiobisnitrobenzoic acid) stood in highly significant positive correlation with calorimetric assays (which did not require a chromagen) either with the same substrate (r = 0.976) or with acetylcholine (r = 0.900). It appears that microcalorimetry can be used in preference to spectrophotometry for enzyme kinetic studies to overcome the complexity of reaction mixture and interference problems and with the advantage of using natural substrates.     

Identification of a rice RNA- and microtubule-binding protein as the multifunctional protein, a peroxisomal enzyme involved in the beta -oxidation of fatty acids.

The control of subcellular mRNA localization and translation is often mediated by protein factors that are directly or indirectly associated with the cytoskeleton. We report the identification and characterization of a rice seed protein that possesses both RNA and microtubule binding activities. In vitro UV cross-linking assays indicated that this protein binds to all mRNA sequences tested, although there was evidence for preferential binding to RNAs that contained A-C nucleotide sequence motifs. The protein was purified to homogeneity using a two-step procedure, and amino acid sequencing identified it as the multifunctional protein (MFP), a peroxisomal enzyme known to possess a number of activities involved in the beta-oxidation of fatty acids. The recombinant version of this rice MFP binds to RNA in UV cross-linking and gel mobility shift experiments, co-sediments specifically with microtubules, and possesses at least two enzymatic activities involved in peroxisomal fatty acid beta-oxidation. Taken together these data suggest that MFP has an important role in mRNA physiology in the cytoplasm, perhaps in regulating the localization or translation of mRNAs through an interaction with microtubules, in addition to its peroxisomal function.     

Crystal structure of human PACRG in complex with MEIG1 reveals roles in axoneme formation and tubulin binding

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