A heterologous system for detecting eukaryotic enzymes which synthesize pseudouridine in transfer ribonucleic acids

tRNA pseudouridylation activities have been detected in embryonic mouse cell fractions and in extracts from HeLa, mouse L-cell and baby hamster kidney (BHK) cell lines. These activities were identified by the use of heterologous reaction systems, with tRNA from hisT strains of Salmonella typhimurium as substrate. hisT mutants are defective for an enzyme that forms psi residues in the anticodon region of many tRNAs and accumulate undermodified species of tRNA. The pseudouridylation activity from BHK cells has been examined in detail and quantitated by a modified tritium release assay (Cortese, R., Kammen, H.O., Spengler, S.J., and Ames, B.N. (1974) J. Biol. Chem. 249, 1103-1108). Maximal rates of tritium release required a suitable cationic environment (optimally, a combination of Mg2+ and NH4+) and a thiol reductant. The activity was totally inhibited in the presence of thiol-reactive reagents, such as 5,5'-dithiobis(2-nitrobenzoic acid) and p-chloromercuribenzoate. A major portion of this 3H release activity was associated with psi modification reactions. This conclusion stems from the following observations: (a) BHK extracts preferentially catalyzed a release of 3H from hisT [5-3H]tRNA, rather than from similarly labeled wild type tRNA; (b) this activity was specific for protons attached to C5 of the pyrimidine rings; no release of 3H was obtained with hisT or wild type [6-3H]tRNA as substrate; (c) the reaction products of hisT tRNA with BHK enzyme were examined by reverse phase column chromatography of tRNAPhe isoacceptors on RPC-5 columns. The enzyme modified both of the principal isoacceptors of hisT tRNAPhe to an equal extent, yielding products indistinguishable from wild type tRNAPhe. Significant levels of 3H release were obtained by the action of enzyme on wild type [5-3H]tRNA, even after gel filtration of the enzyme. This suggests that the enzyme may be able to hypermodify certain species of wild type S. typhimurium tRNA. The activities for wild type tRNA and hisT tRNA appeared to be associated with the same enzyme.     

Evidence for active and passive urate transport in the rat proximal tubule

Studies utilizing the technique of simultaneous microperfusion of peritubular capillaries and tubular lumen of the proximal tubule of the rat were performed to determine if the absorption of urate was an active transport process and to determine the passive permeability coefficient for urate. When radioactive urate of equal specific activity and concentration was present in both perfusion solutions, the ratio of collected to initial concentrations of urate in the luminal perfusate (CO/CI) was 0.71 +/- 0.02. This gradient was higher than that predicted at equilibrium from the electrical potential difference determined in the in vitro perfused rabbit proximal tubule. The addition of para-chloromercuribenzoate (PCMB) to both solutions resulted in a significantly higher CO/CI of 0.90 +/- 0.02. This latter value is closer to the value predicted at electrochemical equilibrium. In separate studies, the unidirectional fluxes of urate were determined in the presence of PCMB. The calculated passive permeability coefficient averaged approximately 0.94 pmol . min-1 . mm-1 . mM-1 and was equal in both directions. These results indicate that in the rat proximal tubule urate absorption is an active transport process. In addition, there exists a passive permeation pathway for urate movement out of and into the proximal tubule.     

The three isoforms of transforming growth factor-beta co-stimulate rat T cells and inhibit lymphocyte apoptosis

We examined the therapeutic effect of cefluprenam (CFLP) on the polymicrobial urinary tract infection associated with infectious stones as compared to that of ceftazidime (CAZ), using the experimental polymicrobial urinary tract infection caused by Proteus mirabilis (P. mirabilis), Pseudomonas aeruginosa (P. aeruginosa) and Enterococcus faecalis (E. faecalis). In order to form bladder stones in rats, a sterile zinc ring was implanted as a foreign body, followed by inoculating P. mirabilis E05106 transurethrelly. Thereafter, P. aeruginosa E030033 and E. faecalis 966 were inoculated according to the same way. CFLP or CAZ was administered intravenously twice a day for 5 days. The therapeutic effect was evaluated by the eradication of these bacteria from the urine, the kidney and stones, inhibition of growth of stones, and also a value of BUN. The urinary excretion rates of CFLP and CAZ was also determined (20 mg/kg). In result, CFLP significantly more effective in the eradication of P. mirabilitis, P. aeruginosa and E. faecalis from the urine, the kidney and stones than the control than the control (p < 0.05). Furthermore, CFLP significantly more effective in the eradication of P. aeruginosa from the urine and also E. facalis from the urine and stones (p < 0.05). Meanwhile, CAZ significantly more effective in the eradication of P. mirabilis from the urine, the kidney and stones and also P. aeraginosa from the kidney and stones than the control. However, CAZ did not show eradicative effect on E. faecalis. The urinary excretion rates of CFLP and CAZ at rats were 59.3% and 59.4%, respectively, within 8 hrs after administration, showing a similar excretion pattern. CFLP exhibited the prominent therapeutic effect on polymicrobial urinary tract infection associated with infectious stones caused by P. mirabilis, P. aeruginosa and E. faecalls. On the basis of these results, it has been strongly suggested that CFLP is highly beneficial in the management of intractable polymicrobial urinary tract infectious in clinic.     

Comparative study on Inclusion compounds of 4-Biphenylacetic acid with β-Cyclodextrin, Hydroxypropylated-β-Cyclodextrins, and methylated-β-Cyclodextrins

The inclusion behavior of Hydroxypropyl-β-Cyclodextrin (HP-β-Cyd) and of methylated-β-Cyclodextrins, heptakis-(2,6-di-O-methyl)-β-Cyclodextrin (DM-β-Cyd) and heptakis-(2,3,6-tri-O-methyl)-β-Cyclodextrin (TM-β-Cyd), in solution and solid state was compared with that of natural β-Cyclodextrin (β-Cyd) using an anti-inflammatory drug, 4-biphenylacetic acid (BPAA), as a guest molecule. The solubility of BPAA with β-Cyd and β-Cyd derivatives in aqueous solution were determined. Stability constants were calculated by phase solubility method at various pH values and temperatures. The formation of inclusion complexes with β-Cyd and β-Cyd derivatives in the solid slate were confirmed by infrared spectroscopy, differential scanning calorimetry and X-Ray diffractometry, and in the liquid phase by ultraviolet spectroscopy, circular dichroism and NMR studies. Dissolution rate and “in vitro” release of BPAA from complexes were examined. The results obtained suggest that DM-β-Cyd is more effective than other β-Cyclodextrins in improving the pharmaceutical properties of BPAA.     

Determination of ultratrace levels of selenium in fruit and vegetable samples grown and consumed in Portugal

The selenium content in fruit and vegetable samples from two regions in Portugal were analysed using hydride generation atomic fluorescence spectrometry (HG-AFS) and radiochemical nuclear activation analysis (RNAA) – two analytical methods with very low limits of detection. The lower detection limits of HG-AFS, 3 μg kg⁻¹ and 8 μg kg⁻¹ (according to conditions used for digestion), and for RNAA, 10 μg kg⁻¹, meant that it was possible to determine selenium in samples previously analysed using the replicate sample instrumental nuclear activation analysis (RSINAA) with a higher detection limit associated.     

Views on contentious practices in dairy farming: The case of early cow-calf separation

The public has become increasingly interested in the welfare of food animals, but the food animal industries possess few mechanisms for public engagement. Here we present results from a web-based forum designed to allow stakeholders to share views on controversial issues in dairying. In response to the question “Should dairy calves be separated from the cow within the first few hours after birth?” participants were able to indicate “yes,” “no,” or “neutral” and either write a reason in support of their view or select reasons provided by other participants. Four independent groups of participants were recruited (a total of 163 people); 31% said they had no involvement in the dairy industry; the remaining 69% (with some involvement in the industry) were students or teachers (33%), animal advocates (13%), producers (11%), veterinarians (9%) and other dairy industry professionals (3%). Overall, little consensus existed among participants across groups; 44% chose “yes,” 48% “no,” and 9% “neutral.” Responses varied with demographics, with opposition to early separation higher among females, animal advocates, and those with no involvement with the dairy industry. A fifth group was recruited at a dairy industry conference (an additional 28 participants); 46% chose “yes,” 32% “no,” and 21% “neutral.” Across all 5 groups, opponents and supporters often referenced similar issues in the reasons they provided. Opponents of early separation contended that it is emotionally stressful for the calf and cow, it compromises calf and cow health, it is unnatural, and the industry can and should accommodate cow-calf pairs. In contrast, supporters of early separation reasoned that emotional distress is minimized by separating before bonds develop, that it promotes calf and cow health, and that the industry is limited in its ability to accommodate cow-calf pairs. These results illustrate the potential of web-based forums to identify areas of agreement and conflict among stakeholders, providing a basis for the development of practices that address shared concerns.     

VOLATILE NITROGEN-CONTAINING COMPOUNDS GENERATED FROM MAILLARD REACTIONS UNDER SIMULATED DEEP-FAT FRYING CONDITIONS

A total of 29 volatile nitrogen-containing compounds were identified from model systems containing glutamine, glutamic acid, asparagine and aspartic acid, respectively, with glucose under simulated deep-fat frying conditions in corn oil. Alkylpyrazines were the most important flavor compound generated. Glutamine, which released free ammonia easily under this condition gave the highest yield of alkylpyrazines. The profiles of pyrazines produced by each amino acid were significantly different. In a glutamine system, 2-(2-furyl)pyrazine, 2-(2-furyl)- 5-methylpyrazine and 2-(2-furyl)-6-methylpyrazine were the major compounds generated.     

Dried Soy Tofu Powder Effects on Frankfurters and Pork Sausage Patties

Regular frankfurters treated with tofu powder had decreased fat with no significant differences in color, texture, or overall acceptability compared to a control by sensory analysis. Lean frankfurters with tofu powder had lower moisture and color, better texture, and overall acceptability with no flavor differences from controls. Except for fracturability, there were no differences in texture-related parameters by Texture Profile Analysis (TPA) between regular and control frankfurters but treated lean frankfurters had improved texture. Lean pork sausages treated with tofu powder were lower in fat, higher in protein, and moisture with no differences in sensory attributes compared to controls.