Single atom modification (O-->S) of tRNA confers ribosome binding. (1/2673)

Escherichia coli tRNALysSUU, as well as human tRNALys3SUU, has 2-thiouridine derivatives at wobble position 34 (s2U*34). Unlike the native tRNALysSUU, the full-length, unmodified transcript of human tRNALys3UUU and the unmodified tRNALys3UUU anticodon stem/loop (ASLLys3UUU) did not bind AAA- or AAG-programmed ribosomes. In contrast, the completely unmodified yeast tRNAPhe anticodon stem/loop (ASLPheGAA) had an affinity (Kd = 136+/-49 nM) similar to that of native yeast tRNAPheGmAA (Kd = 103+/-19 nM). We have found that the single, site-specific substitution of s2U34 for U34 to produce the modified ASLLysSUU was sufficient to restore ribosomal binding. The modified ASLLysSUU bound the ribosome with an affinity (Kd = 176+/-62 nM) comparable to that of native tRNALysSUU (Kd = 70+/-7 nM). Furthermore, in binding to the ribosome, the modified ASLLys3SUU produced the same 16S P-site tRNA footprint as did native E. coli tRNALysSUU, yeast tRNAPheGmAA, and the unmodified ASLPheGAA. The unmodified ASLLys3UUU had no footprint at all. Investigations of thermal stability and structure monitored by UV spectroscopy and NMR showed that the dynamic conformation of the loop of modified ASLLys3SUU was different from that of the unmodified ASLLysUUU, whereas the stems were isomorphous. Based on these and other data, we conclude that s2U34 in tRNALysSUU and in other s2U34-containing tRNAs is critical for generating an anticodon conformation that leads to effective codon interaction in all organisms. This is the first example of a single atom substitution (U34-->s2U34) that confers the property of ribosomal binding on an otherwise inactive tRNA.  (+info)

Decolorization and detoxification of extraction-stage effluent from chlorine bleaching of kraft pulp by Rhizopus oryzae. (2/2673)

Rhizopus oryzae, a zygomycete, was found to decolorize, dechlorinate, and detoxify bleach plant effluent at lower cosubstrate concentrations than the basidiomycetes previously investigated. With glucose at 1 g/liter, this fungus removed 92 to 95% of the color, 50% of the chemical oxygen demand, 72% of the adsorbable organic halide, and 37% of the extractable organic halide in 24 h at temperatures of 25 to 45 degrees C and a pH of 3 to 5. Even without added cosubstrate the fungus removed up to 78% of the color. Monomeric chlorinated aromatic compounds were removed almost completely, and toxicity to zebra fish was eliminated. The fungal mycelium could be immobilized in polyurethane foam and used repeatedly to treat batches of effluent. The residue after treatment was not further improved by exposure to fresh R. oryzae mycelium.  (+info)

Identification and characterization of alkenyl hydrolase (lysoplasmalogenase) in microsomes and identification of a plasmalogen-active phospholipase A2 in cytosol of small intestinal epithelium. (3/2673)

A lysoplasmalogenase (EC 3.3.2.2; EC 3.3.2.5) that liberates free aldehyde from 1-alk-1'-enyl-sn-glycero-3-phospho-ethanolamine or -choline (lysoplasmalogen) was identified and characterized in rat gastrointestinal tract epithelial cells. Glycerophosphoethanolamine was produced in the reaction in equimolar amounts with the free aldehyde. The microsomal membrane associated enzyme was present throughout the length of the small intestines, with the highest activity in the jejunum and proximal ileum. The rate of alkenyl ether bond hydrolysis was dependent on the concentrations of microsomal protein and substrate, and was linear with respect to time. The enzyme hydrolyzed both ethanolamine- and choline-lysoplasmalogens with similar affinities; the Km values were 40 and 66 microM, respectively. The enzyme had no activity with 1-alk-1'-enyl-2-acyl-sn-glycero-3-phospho-ethanolamine or -choline (intact plasmalogen), thus indicating enzyme specificity for a free hydroxyl group at the sn-2 position. The specific activities were 70 nmol/min/mg protein and 57 nmol/min/mg protein, respectively, for ethanolamine- and choline-lysoplasmalogen. The pH optimum was between 6.8 and 7.4. The enzyme required no known cofactors and was not affected by low mM levels of Ca2+, Mg2+, EDTA, or EGTA. The detergents, Triton X-100, deoxycholate, and octyl glucoside inhibited the enzyme. The chemical and physical properties of the lysoplasmalogenase were very similar to those of the enzyme in liver and brain microsomes. In developmental studies the specific activities of the small intestinal and liver enzymes increased markedly, 11.1- and 3.4-fold, respectively, in the first approximately 40 days of postnatal life. A plasmalogen-active phospholipase A2 activity was identified in the cytosol of the small intestines (3.3 nmol/min/mg protein) and liver (0.3 nmol/min/mg protein) using a novel coupled enzyme assay with microsomal lysoplasmalogenase as the coupling enzyme.  (+info)

Formation of 4-hydroxy-2-nonenal-modified proteins in ischemic rat heart. (4/2673)

4-Hydroxy-2-nonenal (HNE) is a major lipid peroxidation product formed during oxidative stress. Because of its reactivity with nucleophilic compounds, particularly metabolites and proteins containing thiol groups, HNE is cytotoxic. The aim of this study was to assess the extent and time course for the formation of HNE-modified proteins during ischemia and ischemia plus reperfusion in isolated rat hearts. With an antibody to HNE-Cys/His/Lys and densitometry of Western blots, we quantified the amount of HNE-protein adduct in the heart. By taking biopsies from single hearts (n = 5) at various times (0, 5, 10, 15, 20, 35, and 40 min) after onset of zero-flow global ischemia, we showed a progressive, time-dependent increase (which peaked after 30 min) in HNE-mediated modification of a discrete number of proteins. In studies with individual hearts (n = 4/group), control aerobic perfusion (70 min) resulted in a very low level (296 arbitrary units) of HNE-protein adduct formation; by contrast, after 30-min ischemia HNE-adduct content increased by >50-fold (15,356 units, P < 0.05). In other studies (n = 4/group), administration of N-(2-mercaptopropionyl)glycine (MPG, 1 mM) to the heart for 5 min immediately before 30-min ischemia reduced HNE-protein adduct formation during ischemia by approximately 75%. In studies (n = 4/group) that included reperfusion of hearts after 5, 10, 15, or 30 min of ischemia, there was no further increase in the extent of HNE-protein adduct formation over that seen with ischemia alone. Similarly, in experiments with MPG, reperfusion did not significantly influence the tissue content of HNE-protein adduct. Western immunoblot results were confirmed in studies using in situ immunofluorescent localization of HNE-protein in cryosections. In conclusion, ischemia causes a major increase in HNE-protein adduct that would be expected to reflect a toxic sequence of events that might act to compromise tissue survival during ischemia and recovery on reperfusion.  (+info)

Conversion of (+/-)-synephrine into p-hydroxyphenylacetaldehyde by Arthrobacter synephrinum. A novel enzymic reaction. (5/2673)

A partically purified enzyme from Arthrobacter synephrinum was found to catalyse the conversion of (+/-)-synphrine into p-hydroxyphrenylacetaldehyde and methylamine. The enzyme is highly specific for synephrine and is distinctly different from monoamine oxidase.  (+info)

Hyperglycemia causes oxidative stress in pancreatic beta-cells of GK rats, a model of type 2 diabetes. (6/2673)

Reactive oxygen species are involved in a diversity of biological phenomena such as inflammation, carcinogenesis, aging, and atherosclerosis. We and other investigators have shown that the level of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker for oxidative stress, is increased in either the urine or the mononuclear cells of the blood of type 2 diabetic patients. However, the association between type 2 diabetes and oxidative stress in the pancreatic beta-cells has not been previously described. We measured the levels of 8-OHdG and 4-hydroxy-2-nonenal (HNE)-modified proteins in the pancreatic beta-cells of GK rats, a model of nonobese type 2 diabetes. Quantitative immunohistochemical analyses with specific antibodies revealed higher levels of 8-OHdG and HNE-modified proteins in the pancreatic beta-cells of GK rats than in the control Wistar rats, with the levels increasing proportionally with age and fibrosis of the pancreatic islets. We further investigated whether the levels of 8-OHdG and HNE-modified proteins would be modified in the pancreatic beta-cells of GK rats fed with 30% sucrose solution or 50 ppm of voglibose (alpha-glucosidase inhibitor). In the GK rats, the levels of 8-OHdG and HNE-modified proteins, as well as islet fibrosis, were increased by sucrose treatment but reduced by voglibose treatment. These results indicate that the pancreatic beta-cells of GK rats are oxidatively stressed, and that chronic hyperglycemia might be responsible for the oxidative stress observed in the pancreatic beta-cells.  (+info)

Formation of lipoxygenase-pathway-derived aldehydes in barley leaves upon methyl jasmonate treatment. (7/2673)

In barley leaves, the application of jasmonates leads to dramatic alterations of gene expression. Among the up-regulated gene products lipoxygenases occur abundantly. Here, at least four of them were identified as 13-lipoxygenases exhibiting acidic pH optima between pH 5.0 and 6.5. (13S,9Z,11E,15Z)-13-hydroxy-9,11,15-octadecatrienoic acid was found to be the main endogenous lipoxygenase-derived polyenoic fatty acid derivative indicating 13-lipoxygenase activity in vivo. Moreover, upon methyl jasmonate treatment > 78% of the fatty acid hydroperoxides are metabolized by hydroperoxide lyase activity resulting in the endogenous occurrence of volatile aldehydes. (2E)-4-Hydroxy-2-hexenal, hexanal and (3Z)- plus (2E)-hexenal were identified as 2,4-dinitro-phenylhydrazones using HPLC and identification was confirmed by GC/MS analysis. This is the first proof that (2E)-4-hydroxy-2-hexenal is formed in plants under physiological conditions. Quantification of (2E)-4-hydroxy-2-hexenal, hexanal and hexenals upon methyl jasmonate treatment of barley leaf segments revealed that hexenals were the major aldehydes peaking at 24 h after methyl jasmonate treatment. Their endogenous content increased from 1.6 nmol.g-1 fresh weight to 45 nmol.g-1 fresh weight in methyl-jasmonate-treated leaf segments, whereas (2E)-4-hydroxy-2-hexenal, peaking at 48 h of methyl jasmonate treatment increased from 9 to 15 nmol.g-1 fresh weight. Similar to the hexenals, hexanal reached its maximal amount 24 h after methyl jasmonate treatment, but increased from 0.6 to 3.0 nmol.g-1 fresh weight. In addition to the classical leaf aldehydes, (2E)-4-hydroxy-2-hexenal was detected, thereby raising the question of whether it functions in the degradation of chloroplast membrane constituents, which takes place after methyl jasmonate treatment.  (+info)

Disinfection of upper gastrointestinal fibreoptic endoscopy equipment: an evaluation of a cetrimide chlorhexidine solution and glutaraldehyde. (8/2673)

There is little information available on the bacteriological contamination of upper gastrointestinal fibreoptic endoscopes during routine use and the effects of 'disinfecting solutions'. A bacteriological evaluation was therefore made of cleaning an endoscope and its ancillary equipment with (1) water, (2) an aqueous solution of 1% cetrimide with 0.1% chlorhexidine, and (3) activated aqueous 2% glutaraldehyde. All equipment, but particularly the endoscope itself, was found to be heavily contaminated after use with a wide variety of organisms of which 53% were Gram positive. Cleaning the endoscope and ancillary equipment with water and the cetrimide/chlorhexidine solution alone or in combination was inadequate to produce disinfection but immersion in glutaraldehyde for two minutes consistently produced sterile cultures with our sampling technique. A rapid and simple method for disinfection of endoscopic equipment is therefore recommended and we think this is especially suitable for busy endoscopy units.  (+info)

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