Isolation of K88 Antigen Variants (ab,ac, ad) from Porcine Enterotoxigenic Escherichia coli Belonging to Different Serotypes

Fimbriae isolation by means of thermal shock was applied to fifteen K88-positive (three K88ab, nine K88ac and three K88ad) Escherichia coli reference strains belonging to serotypes O8:K87, O32, O45, O138:K81, O141:K85, O147:K89, O149:K91, and O157, as well as to ten K88-positive enterotoxigenic strains isolated from porcine diarrhea in Spain, all of them belonging to the O149 serogroup. Fimbriae were removed from the bacterial cells by thermal shock at 60 C and then precipitated using ammonium sulfate. The final amount of K88 antigen and the purification degree were not related to the serogroup of the bacteria or to the antigen variant but were related to the buffer used for isolation. Phosphate buffer containing urea was shown to be more effective than Tris-HCl for isolation of K88 antigen. The molecular weights by SDS-PAGE for K88ab, K88ac, and K88ad were 28.5, 29.2, and 31.0 kDa, respectively. All enterotoxigenic E. coli strains isolated in Spain showed the K88ac variant.     

Crystallization,characterization, and preliminary crystallographic studies of mitochondrial carbamoyl phosphate synthetase I of Rana catesbeiana

Carbamoyl phosphate synthetase I (ammonia; E C 6.3.4.16) was purified from the liver of Rana catesbeiana (bullfrog). Crystals of the protein have been obtained at 22°C by the hanging drop vapor diffusion technique, with polyethylene glycol as precipitant. Tetragonal crystals of about 0.3 × 0.3 × 0.7 mm diffract at room temperature to at least 3.5 Å using a conventional source and are stable to X-radiation for about 12 h. Therefore, these crystals are suitablefor high resolution studies. The space group is P4₁2₁2 (or its enantiomorph P4₃2₁2), with unit cell dimensions a = b = 291.6 Å and c = 189.4 Å. Density packing considerations areconsistent with the presence of 4-6 monomers (M_r of the monomer, 160,000) in the asymmetric unit. Amino-terminal sequence of the enzyme and of a chymotryptic fragment of 73.7 kDa containing the COOH-terminus has been obtained. The extensive sequence identity with rat and human carbamoyl phosphate synthetase I indicates the relevance for mammals of structural data obtained with the frog enzyme. © 1995 Wiley-Liss, Inc.     

Growth and cell wall changes in rice coleoptiles

A fractionation of non-cellulosic sugars of Oryza sativa L. coleoptile cell walls was carried out and the composition of each fraction was studied during coleoptile growth.Percentages of fractions extracted with boiling water and with oxalate (pectic substances) were almost constant throughout development. An increase in the K II hemicellulosic fraction (extracted with 24% KOH) content, and a decrease in the K I hemicellulosic fraction (extracted with 10% KOH) were detected, when coleoptile growth finished.The percentage of glucose content in the K I hemicellulosic fraction was highest in young coleoptiles and lowest in old ones. Furthermore, a highly significant linear relationship between amounts of glucose and growth rate was obtained, while a inverse relationship between the amount of xylose and arabinose and growth rate was attained.Abreviations GLC gas liquid chromatography - IAA indole-3-acetic-acid - TFA trifluoroacetic acid - T_o minimum stress-relaxation time     

Enhancement by Cytidine of Membrane Phospholipid Synthesis

Cytidine, as cytidine 5'-diphosphate choline, is a major precursor in the synthesis of phosphatidylcholine in cell membranes. In the present study, we examined the relationships between extracellular levels of cytidine, the conversion of [14C]choline to [14C]phosphatidylcholine, and the net syntheses of phosphatidylcholine and phosphatidylethanolamine by PC12 cells. The rate at which cytidine (as [3H]cytidine) was incorporated into the PC12 cells followed normal Michaelis-Menten kinetics (Km = 5 microM; Vmax = 12 x 10(-3) mmol/mg of protein/min) when the cytidine concentrations in the medium were below 50 microM; at higher concentrations, intracellular [3H]cytidine nucleotide levels increased linearly. Once inside the cell, cytidine was converted mainly into cytidine triphosphate. In pulse-chase experiments, addition of cytidine to the medium caused a time- and dose-dependent increase (by up to 30%) in the incorporation of [14C]choline into membrane [14C]-phosphatidylcholine. When the PC12 cells were supplemented with both cytidine and choline for 14 h, small but significant elevations (p less than 0.05) were observed in their absolute contents of membrane phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, all increasing by 10-15% relative to their levels in cells incubated with choline alone. Exogenous cytidine, acting via cytidine triphosphate, can thus affect the synthesis and levels of cell membrane phospholipids.     

THDP17 Decreases Ammonia Production through Glutaminase Inhibition. A New Drug for Hepatic Encephalopathy Therapy

Ammonia production is implicated in the pathogenesis of hepatic encephalopathy (HE), being intestinal glutaminase activity the main source for ammonia. Management of ammonia formation can be effective in HE treatment by lowering intestinal ammonia production. The use of glutaminase inhibitors represents one way to achieve this goal. In this work, we have performed a search for specific inhibitors that could decrease glutaminase activity by screening two different groups of compounds: i) a group integrated by a diverse, highly pure small molecule compounds derived from thiourea ranging from 200 to 800 Daltons; and ii) a group integrated by commonly use compounds in the treatment of HE. Results shown that THDP-17 (10 µM), a thiourea derivate product, could inhibit the intestinal glutaminase activity (57.4±6.7%). Inhibitory effect was tissue dependent, ranging from 40±5.5% to 80±7.8% in an uncompetitive manner, showing V_max and K_m values of 384.62 µmol min⁻¹, 13.62 mM with THDP-17 10 µM, respectively. This compound also decreased the glutaminase activity in Caco-2 cell cultures, showing a reduction of ammonia and glutamate production, compared to control cultures. Therefore, the THDP-17 compound could be a good candidate for HE management, by lowering ammonia production.     

NLRP3 inflammasome suppression improves longevity and prevents cardiac aging in male mice

While NLRP3‐inflammasome has been implicated in cardiovascular diseases, its role in physiological cardiac aging is largely unknown. During aging, many alterations occur in the organism, which are associated with progressive impairment of metabolic pathways related to insulin resistance, autophagy dysfunction, and inflammation. Here, we investigated the molecular mechanisms through which NLRP3 inhibition may attenuate cardiac aging. Ablation of NLRP3‐inflammasome protected mice from age‐related increased insulin sensitivity, reduced IGF‐1 and leptin/adiponectin ratio levels, and reduced cardiac damage with protection of the prolongation of the age‐dependent PR interval, which is associated with atrial fibrillation by cardiovascular aging and reduced telomere shortening. Furthermore, old NLRP3 KO mice showed an inhibition of the PI3K/AKT/mTOR pathway and autophagy improvement, compared with old wild mice and preserved Nampt‐mediated NAD⁺ levels with increased SIRT1 protein expression. These findings suggest that suppression of NLRP3 prevented many age‐associated changes in the heart, preserved cardiac function of aged mice and increased lifespan.