Deoxyribonuclease activities in Myxococcus coralloides D

Myxococcus coralloides D produced cell-bound deoxyribonucleases (DNases) during the exponential phase of growth in liquid medium. DNase activity was much higher than that detected in other myxobacterial strains and was fractionated into three different peaks by filtration through Sephadex G-200. The DNases were named G, M and P. The optimum temperatures were 37 degrees C, 33 degrees C and 25 degrees C respectively, although high activities were recorded over the temperature range 20-45 degrees C. The pH range of high activity was between 6.0 and 9.0, with an optimum for each DNase at 8.0. DNases M and P were strongly inhibited by low concentrations of NaCl, but activity of DNase G was less affected by NaCl. The three activities required divalent metal ions as cofactors (especially Mg2+ and Mn2+); however, other metal ions (Fe2+, Ni2+, Zn2+) were inhibitors. The molecular weights were estimated by gel filtration chromatography and SDS-PAGE as 44 kDa (DNase G), 49 kDa (DNase M) and 39 kDa (DNase P).     

ms2i6A deficiency enhances proofreading in translation.

The hypermodified base 2-methylthio-N6-isopentenyladenosine (ms2i6A) at position 37 occurs frequently in tRNAs that read codons starting with uridine. Here we have studied how ms2i6A affects the accuracy of poly(U) translation in vitro. Deficiency leads to a higher rejection rate of tRNA4(Leu) by more aggressive proofreading on the wild-type ribosome, but with the initial selection step unchanged. Our data indicate that ms2i6A has no effect on codon-anticodon interactions on wild-type ribosomes as long as aminoacyl-tRNA is in ternary complex with EF-Tu and GTP. ms2i6A deficiency in the cognate poly(U) reader tRNA(Phe) leads to increased misreading when the near-cognate competitor tRNA4(Leu) is wild-type. ms2i6A deficiency in tRNA4(Leu) gives a decreased error level in competition with wild-type tRNA(Phe).     

Fibroblastlike primary cells from human colon adenocarcinoma explants: Collagen biosynthesis

Summary Fibroblastlike primary cells have been obtained from human colon adenocarcinoma explants. Such cells disappear during cell culture and thus have not been previously studied. These cells have a number of altered phenotypic characteristics: a) morphology; b) growth behavior and adherence to culture substrate (they required 3 h for 90% attachment and only presented a flattened morphology 40 h after platting); and c) collagen metabolism. Increased protein biosynthesis (about double than control colon-derived fibroblasts) and maintained ability for collagen biosynthesis have been observed for the tumor-associated fibroblastlike cells. Thus, the collagen to noncollagenous proteins ratio was decreased for these cells. They exhibited an altered type I:type III collagen (5:1 instead of 3:1 in colon fibroblasts) and procollagen (2:1 against 5:1 in colon fibroblasts) ratios as well as a decreased secretion of collagen with an abnormal deposition of procollagens in the cell layer. These studies show a permanent phenotypic alteration in the tumor-associated fibroblastlike cells.     

Comparison Between Different Assays for Superoxide Dismutase-Like Activity

The direct and indirect methods for assaying the superoxide dismutase activity of a compound are compared. With the use of a direct method. the mechanism of the catalysis of O₂-dismutation by the tested compound can be determined. while with the indirect method it cannot. and this may lead to misinterpretation of the results. Assuming that the catalysis occurs via the 'ping-pong' mechanism, both the direct and indirect methods are limited to the determination of values of k_cat ≥ 10⁵M^-1s^-1 and k_cat ≥ 3 × 10⁶M^-1s^-1. respectively. Moreover, many side reactions may occur with the indirect method which may interfere with the measurements. Nevertheless. the indirect method approximates better the in vivo conditions than the direct method, and a tested compound that has high SOD activity using a direct method and low SOD activity using an indirect method. will most probably be a poor SOD mimic in vivo.     

Diurnal periodicity in Daphnia reproduction

The diurnal periodicity of reproduction in a reservoir population of Daphnia galeata Sars was tested by two methods (egg age distribution analyses and direct observation of hatching). No significant diurnal synchronization of reproduction in the natural cladoceran population was found. A conspicuous shortage of older embryonic stages was parallely revealed by analyses of diurnal changes of egg age distribution. Suggested selective feeding of fish on gravid females of Daphnia brooding embryos with black eye pigmentation is discussed.     

Rat erythrocyte glycophorins can be isolated by the lithium diiodosalicylate method used for other glycophorins.

1. The lithium diiodosalicylate/phenol method, widely employed for the isolation of membrane sialoglycoproteins (glycophorins) from mammalian erythrocytes, was applied for the first time to the purification of homologous glycoproteins from rat erythrocyte membranes. 2. The resulting preparations showed to be composed of four components, fractionated on SDS-PAGE. All four were positive for periodic acid-Schiff's reagent stain, the two largest of them being major. 3. Isolated rat glycophorins accounted for 60% of the ghost sialic acid and 1.5% of their protein. The presence of O-acetyl groups was confirmed in one-third of the sialic acid residues. 4. The molecular masses of the four glycophorin components were determined by a method which takes into account the anomalous mobility of glycoproteins on SDS-electrophoresis. Estimated values thus obtained for the actual molecular masses were 74, 32, 25 and 17 kDa.     

In vitro studies on the growth of Shigella sonnei by Lactobacillus casei and Lact. acidophilus.

The inhibitory effect of lactobacilli on growth of Shigella sonnei was studied. The effect was not due to pH alone, as addition of hydrochloric, lactic or acetic acids to culture media did not inhibit the normal growth of the shigellas. The degree of inhibition was measured by disc assay and showed that the inhibitory substance(s) can be extracellular and diffusible, varying the degrees of inhibition depending on the media tested. When broth was inoculated with mixed cultures of Lactobacillus and Shigella strains, the inhibition began at 6 h and the death phase at 9 h. The higher inhibition was produced by the mixture of lactobacilli (35.5 +/- 2.5% at 6 h culture, 57.4 +/- 1.9% at 9 h and 91.2 +/- 1.2% at 14 h). The degree of inhibition was higher when the relationship pathogen : lactobacilli was 1:10(3). The specific growth rate of lactobacilli and shigella was different in pure or mixed cultures. When the lactobacillus alone was grown for 12 h and the shigellas then added, the numbers of shigellas began to decrease immediately at 37 degrees C. This work shows that the Lactobacillus strains employed in fermented milk can be used to inhibit the growth of Sh. sonnei.     

Substrate specificity and properties of the aryl-alcohol oxidase from the ligninolytic fungus Pleurotus eryngii.

The production in a 5-1 fermenter of the extracellular enzymes laccase and aryl-alcohol oxidase by the fungus Pleurotus eryngii was studied. The latter enzyme has been purified 50-fold by Sephacryl S-200 and Mono Q chromatography. Purified aryl-alcohol oxidase is a unique flavoprotein with 15% carbohydrate content, a molecular mass of 72.6 kDa (SDS/PAGE) and a pI of 3.9. The enzyme presents wide specificity, showing activity on benzyl, cinnamyl, naphthyl and aliphatic unsaturated alcohols. Neither activity nor inhibition of veratryl alcohol oxidation was found with saturated alcohols, but competitive inhibition was produced by aromatic compounds which were not aryl-alcohol oxidase substrates, such as phenol or 3-phenyl-1-propanol. From these results, it was apparent that a double bond conjugated with a primary alcohol is necessary for substrate recognition by aryl-alcohol oxidase, and that activity is increased by the presence of additional conjugated double bonds and electron donor groups. Both affinity and maximal velocity during enzymic oxidation of methoxybenzyl alcohols were affected in a similar way by ring substituents, increasing from benzyl alcohol (Km = 0.84 mM, Vmax = 52 U/mg) to 4-methoxybenzyl alcohol (Km = 0.04 mM, Vmax = 208 U/mg). Aryl-alcohol oxidase presents also a low oxidase activity with aromatic aldehydes, but the highest activity was found in the presence of electron-withdrawing groups.     

Biochemical ethanol effects affected by a non-steroidal anti-inflammatory drug.

The non-steroidal anti-inflammatory drug, piroxicam, prevents the hepatic increase of triacylglycerols and malondialdehyde resulting from the acute intoxication of rats with ethanol. In addition, in the intoxicated rats, piroxicam consistently produces a decrease in the levels of blood ethanol in comparison with control animals. It is suggested that the anti-inflammatory compound stimulates ethanol oxidation.     

Use of whey for production of exocellular polysaccharide by a mutant strain ofXanthomonas campestris

Growth and kinetics of the production of exocellular polysaccharide was studied in a mutant strain ofXanthomonas campestris lac ⁺ during cultivation in a submerged culture in a medium containing whey. The maximum production of the polymer was observed at the initial stage of the stationary growth phase of the culture. The mean production yield was about 1.4%. The results were comparable with those obtained during cultivation on a lactose medium. Translated by Č. Novotny