Reduction of methylglyoxal in Escherichia coli K12 by an aldehyde reductase and alcohol dehydrogenase

Two enzymes, one NADPH-dependent and another NADH-dependent which catalyze the reduction of methylglyoxal to acetol have been isolated and substantially purified from crude extracts of Escherichia coli K₁₂ cells. Substrate specificity and formation of acetol as the reaction product by both the enzymes, reversibility of NADH-dependent enzyme with alcohols as substrates and inhibitor study with NADPH-dependent enzyme indicate that NADPH-dependent and NADH-dependent enzymes are identical with an aldehyde reductase (EC 1.1.1.2) and alcohol dehydrogenase (EC 1.1.1.1) respectively. The K_m for methylglyoxal have been determined to be 0.77 mM for NADPH-dependent and 3.8 mM for NADH-dependent enzyme. Stoichiometrically equimolar amount of acetol is formed from methylglyoxal by both NADPH- and NADH-dependent enzymes. In phosphate buffer, both the enzymes are active in the pH range of 5.8–6.6 with no sharp pH optimum. Molecular weight of both the enzymes were found to be 100,000 ± 3,000 by gel filtration on a Sephacryl S-200 column. Both NADPH- and NADH-dependent enzymes are sensitive to sulfhydryl group reagents.     

α-phenyl-tert-butyl-nitrone inhibits free radical release in brain concussion

Traumatic brain injury (TBI) is one of the important causes of mortality and morbidity. The pathogenesis of the underlying brain dysfunction is poorly understood. Recent data have suggested that oxygen free radicals play a key role in the primary and secondary processes of acute TBI. We report direct electron spin resonance (ESR) evidence of hydroxyl (·OH) radical generation in closed-head injury of rats. Moderate brain concussion was produced by controlled and reproducible mechanical, fixed, closed-head injury. A cortical cup was placed over one cerebral hemisphere within 20 min of the concussion, perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent pyridyl-N-oxide-tert-butyl nitrone (POBN, 100 mM), and superfusate samples collected at 10 min intervals for a duration up to 130 min post brain trauma. In addition, POBN was administered systematically (50 mg/kg body wt.) 10 min pretrauma and 20 min posttrauma to improve our ability to detect free radicals. ESR analysis of the superfusate samples revealed six line spectra (α_N = 15.4 and α^β_H = 2.5 G) characteristic of POBN-OH radical adducts, the intensity of which peaked 40 min posttrauma. The signal was undetectable after 120 min. Administration of α-phenyl-tert-butyl-nitrone (PBN), a spin adduct forming agent systemically (100 mg/kg body wt. IP 10 min prior to concussion) alone or along with topical PBN (100 mM PBN in aCSF),6significantly (P< 0.001) attenuated the ESR signal, suggesting its possible role in the treatment of TBI.     

Alpha-Phenyl-Tert-Butyl-Nitrone (PBN) Attenuates Hydroxyl Radical Production During Ischemia-Reperfusion Injury of Rat Brain: An EPR Study

-phenyl-tert-butyl-nitrone (PBN) a spin adduct forming agent is believed to have a protective action in ischemia-reperfusion injury of brain by forming adducts of oxygen free radicals including ±OH radical. Electron paramagnetic resonance (EPR) has been used to both detect and monitor the time course of oxygen free radical formation in the in vivo rat cerebral cortex. Cortical cups were placed over both cerebral hemispheres of methoxyflurane anesthetized rats prepared for four vessel occlusion-evoked cerebral ischemia. Prior to the onset of sample collection, both cups were perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent -(4-pyridyl-1-oxide)-N-tert butylnitrone (POBN 100 mM) for 20 min. In addition 50 mg/kg BW of POBN was administered intraperitoneally (IP) 20 min prior to ischemia in order to improve our ability to detect free radical adducts. Cup fluid was subsequently replaced every 15 min during ischemia and every 10 min during reperfusion with fresh POBN containing CSF and the collected cortical superfusates were analyzed for radical adducts by EPR spectroscopy. After a basal 10 min collection, cerebral ischemia was induced for 15 or 30 min (confirmed by EEG flattening) followed by a 90 min reperfusion. -OH radical adducts (characterized by six line EPR spectra) were detected during ischemia and 90 min reperfusion. No adduct was detected in the basal sample or after 90 min of reperfusion. Similar results were obtained when diethylenetriaminepenta-acetic acid (100 μM; DETAPAC) a chelating agent was included in the artificial CSF. Systemic administration of PBN (100 mg/kg BW) produced a significant attenuation of radical adduct during reperfusion. A combination of systemic and topical PBN (100 mM) was required to suppress -OH radical adduct formation during ischemia as well as reperfusion. PBN free radical adducts were detected in EPR spectra of the lipid extracts of PBN treated rat brains subjected to ischemia/reperfusion. Thus this study suggests that PBN's protective action in cerebral ischemia/reperfusion injury is related to its ability to prevent a cascade of free radical generation by forming spin adducts.     

The int genes of bacteriophages P22 and λ are regulated by different mechanisms

Bacteriophage P22 and λ are related bacteriophages with similar gene organizations. In λ the cII-dependent P_I promoter is responsible for λint gene expression. The only apparent counterpart to P_I in P22 is oriented in the opposite direction, and cannot transcribe the P22 int gene. We show that this promoter, called P_al, is active both in vivo and in vitro, and is dependent upon the P22 cII-like gene, called c1. We have also determined the DNA sequence of a 3.3 kb segment that closes the gap between previously reported sequences to give a continuous sequence between the P22 p_L promoter and the int gene. The newly determined sequence is densely packed with genes from the p_L direction, and the proteins predicted by the sequence show excellent correlation with the proteins mapped by Youderian and Susskind in 1980. However, the sequence contains no apparent genes in the opposite (p_al) direction, and no additional binding motifs for the P22 c1 protein. We conclude that int gene expression in P22 is regulated by a different mechanism than in λ.     

Vibrational circular dichroism of polypeptides. III. Film studies of several alpha-helical and beta-sheet polypeptides

Vibrational CD (VCD) of amides A, I, and II vibrations of a variety of polypeptide films have been measured. VCD of films of α-helical and β-sheet structures are compared in the three regions. Reproducible spectra could only be obtained for thin films free of orientation dependence. The sign and band shape of the VCD of films are not always the same as that in solution. However, the magnitude of the observed VCD seems to correlate with the secondary structure such that α-helical molecules typically have much larger Δε/ε values than do β-sheet molecules. The possibility of interference by artifacts owing to light-scattering effects is discussed.     

Water relations in three species of theConvolvulaceae in the Indian arid zone

The relationship of water deficit, actual water content, area, water loss and stomatal behaviour in leaves of different insertion levels of three members of Indian arid zoneConvolvulaceae—Merremia aegyptia, M. dissecta andIpomoea pes-caprae were measured simultaneously in the month of May, 1974, when field conditions were extremely adverse for growth. The two former species loose far more water than the latter. The values measured varied considerably with the leaf insertion levels and they reversed inI. pes-caprae when compared withMerremia species.