Characteristics and N-terminal amino acid sequence of a manganese peroxidase purified from Lentinula edodes cultures grown on a commercial wood substrate

Summary Extracellular culture filtrates from ligninolytic cultures of the lignin-degrading basidiomycete Lentinula (syn. Lentinus) edodes (Berk.) Pegler contained one major peroxidase when grown on a commercial oak-wood substrate. The peroxidase was purified by polyethylenimine clarification, anion-exchange chromatography, and hydrophobic-interaction HPLC. The enzyme (MnP1) was a heme-iron protein with an apparent molecular weight of 44 600 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and an isoelectric point of pH 3.2. The native enzyme had an absorption maximum at 407 nm, which shifted to 420 nm upon H₂O₂ addition. The pyridine-hemochrome-absorption spectrum indicated that one heme group was present per enzyme as protoporphyrin IX. N-terminal amino acid sequencing showed that MnP1 had higher sequence homology with manganese peroxidases than with lignin peroxidases reported from Phanerochaete chrysosporium. L. edodes MnP1 was capable of oxidizing lignin and lignin-model compounds in the presence of manganese and H₂O₂.On leave from the Department of Biochemistry, University of Otago, P. O. Box 56, Dunedin, New Zealand.Research carried out while a visiting scientist at the USDA Forest Products Laboratory from the National Chemistry Laboratory, Pune, India 41 1008 Offprint requests to: I. T. Forrester     

Psychophysiological responsivity on a laboratory stress task: Methodological implications for a stress-muscle hyperactivity pain model

A stress-muscle hyperactivity-pain (SMP) model has been proposed to explain the etiology of certain musculoskeletal pain disorders. According to this model, subjects should show physiological arousal during periods of stress relative to periods of rest. In a test of this prediction, 31 subjects performed a reaction time task that has been used in previous laboratory studies. Multiple psychophysiological variables were monitored during initial and final 10-minute baselines, during performance on nine 2-minute reaction time tasks, and during 36-second rest intervals following each of the 2-minute tasks. Results showed small but statistically significant differences generally supporting the SMP model when masseter EMG was averaged over time periods of 12 seconds to 2 minutes, but not when masseter EMG was averaged over 10- to 18-minute blocks. These results demonstrated the importance of carefully selecting time intervals for analysis. Additional analyses that compared TMD with symptom-free subjects revealed small differences in EMG that supported the SMP model. Analyses of EMG over shorter time intervals also showed, however, that masseter EMG increased during the 36-second rest interval following performance on a 2-minute stress task; this result suggested that a modification of the SMP model may be necessary.This research was supported in part by Grant 2 S06RR08038-17 funded by the National Institutes of Health.     

Elevated uridine nucleotide pools in fluorouracil/fluorouridine resistant mutants ofNocardia lactamdurans

Summary Selection of spontaneous mutants ofNocardia lactamdurans MA2908 for resistance to 5-fluorouracil results in the simultaneous development of resistance to 5-fluorouridine. The resulting mutants fall into four distinct classes based on the amount of uracil accumulating in fermentation broths. An additional characteristic of these mutants is a reduction in the ability to incorporate exogenous uracil into nucleic acids even though transport and conversion to the nucleotide level appears normal. Finally, production of efrotomycin is increased in these mutants in both chemically defined and complex fermentation media to levels equivalent to those of MA4820, the first productivity mutant isolated in a conventional strain improvement program. Resistance development and uracil excretion are adequately explained by an elevation of the intracellular uridine nucleotide pool, in particular UMP. The role of the uridine necleotides in the efrotomycin fermentation is unknown.     

Identification of consensus patterns in unaligned DNA sequences known to be functionally related

We have developed a method for identifying consensus patternsin a set of unaligned DNA sequences known to bind a common proteinor to have some other common biochemical function. The methodis based on a tnatrix representation of binding site patterns.Each row of the matrix represents one of the four possible bases,each column represents one of the positions of the binding siteand each element is determined by the frequency the indicatedbase occurs at the indicated position. The goal of the methodis to find the most significant matrix-i.e. the one with thelowest probability of occurring by chance-out of all the matricesthat can be formed from the set of related sequences. The reliabilityof the method improves with the number of sequences, while thetime required increases only linearly with the number of sequences.To test this method, we analysed 11 DNA sequences containingpromoters regulated by the Escherichia coli LexA protein. Thematrices we' found were consistent with the known consensussequence, and could distinguish the generally accepted LexAbinding sites from other DNA sequences. Received on November 6, 1989; accepted on December 20, 1989     

Cold responses of Arabidopsis mutants impaired in freezing tolerance

Mutants of Arabidopsis thaliana L. (Heynh), characterized asdeficient in their freezing tolerance after cold acclimation,were surveyed for some of the normal responses to cold exposure.In foliar tissue, the coldinducibility of three proteins, thelevels of sucrose and glucose, the fatty acyl composition oflipids, and the accumulation of anthocyanin was examined. Fourmutations (sfr3, sfr4, sfr6, and sfr7) reduced or eliminatedthe accumulation of anthocyanin during cold acclimation. Onemutation (sfr4) prevented the normally cold-induced elevationof sucrose and glucose levels; both sfr4 and another mutation(sfr7) affected fatty acid composition after (and only after)cold acclimation. On the other hand mutations sfr1, sfr2 andsfr5 did not differ significantly from the wild type in anyof the parameters tested, suggesting that they have other, perhapshighly specific, effects on lowtemperature responses. Key words: Arabidopsis thaliana, cold acclimation, freezing tolerance, mutation     

Regional Reductions of Transketolase in Thiamine-Deficient Rat Brain

Abstract: Thiamine deficiency impairs oxidative metabolism and causes metabolic encephalopathy. An early reduction in transketolase (TK) activity may be an important pathogenic event. To assess the role of TK, we have delineated the regional/cellular distribution of TK protein and mRNA in adult rat brain in pyrithiamine-induced thiamine deficiency. TK activity declined in both vulnerable and spared regions. Immunoblots showed a parallel reduction of TK protein. With a few exceptions, immunocytochemistry indicated an overall decline of TK immunoreactivity and the decrease was not specific to vulnerable areas. In contrast to the pronounced, general decline of TK protein, in situ hybridization revealed a regional decrease of 0–25% of TK mRNA in thiamine deficiency. Northern blots indicated a similar level of TK mRNA in whole brain in thiamine deficiency. These results show that the decline of TK activity results from a proportional decrease of TK protein, and the deficiency may be due to an instability of TK protein or an inhibition of TK mRNA translation. The lack of correlation of the distribution, and the absence of specific alteration, of TK in affected regions suggest that the reduced TK may not be linked directly to selective vulnerability in thiamine deficiency.     

Chronic In Vivo Sodium Azide Infusion Induces Selective and Stable Inhibition of Cytochrome c Oxidase

Abstract: The effect of chronic subcutaneous infusion of sodium azide on the activity of mitochondrial respiratory chain enzymes was investigated in Sprague-Dawley rats. Treatment with ∼1 mg/kg/h sodium azide induced chronic, partial inhibition of cytochrome c oxidase, whereas the activities of respiratory complexes I and III were not significantly affected. The inhibition of cytochrome c oxidase was evident by 7 days after infusion began, and the effect was stable for at least 3 weeks. The selectivity of azide for cytochrome c oxidase is discussed in the context of other findings of azide effects on enzymes. The results of the present study indicate that the sodium azide infusion paradigm described here provides a useful tool for the evaluation of selective and stable cytochrome oxidase inhibition in vivo.     

Global Brain Ischemia and Reperfusion: Modifications in Eukaryotic Initiation Factors Associated with Inhibition of Translation Initiation

Abstract: We used in vitro translation and antibodies against phosphoserine and the eukaryotic initiation factors eIF-4E, eIF-4G, and eIF-2α to examine the effects of global brain ischemia and reperfusion on translation initiation and its regulation in a rat model of 10 min of cardiac arrest followed by resuscitation and 90 min of reperfusion. Translation reactions were performed on postmitochondrial supernatants from brain homogenates with and without aurintricarboxylic acid to separate incorporation due to run-off from incorporation due to peptide synthesis initiated in vitro. The rate of leucine incorporation due to in vitro-initiated protein synthesis in normal forebrain homogenates was ∼0.4 fmol of leucine/min/µg of protein and was unaffected by 10 min of cardiac arrest, but 90 min of reperfusion reduced this rate 83%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blots of these homogenates showed that neither 10 min of global brain ischemia nor 90 min of reperfusion induced significant alterations in the quantity or serine phosphorylation of eIF-4E. However, we observed in all 90-min-reperfused samples eIF-4G fragments that also bound eIF-4E. The amount of eIF-2α was not altered by ischemia or reperfusion, and immunoblotting after isoelectric focusing did not detect serine-phosphorylated eIF-2α in normal samples or in those obtained after ischemia without reperfusion. However, serine-phosphorylated eIF-2α was uniformly present after 90 min of reperfusion and represented 24 ± 3% of the eIF-2α in these samples. The serine phosphorylation of eIF-2α and partial fragmentation of eIF-4G observed after 90 min of reperfusion offer an explanation for the inhibition of protein synthesis.     

Non-reciprocal cross-incompatibility in Trichogramma deion

In non-reciprocal cross-incompatibility (NRCI), the crossing of a female of a strain A with a male of a strain B results in hybrid offspring, whereas the reciprocal cross produces few or no hybrids. Only females are of hybrid origin in Hymenoptera because they arise from fertilized eggs; males arise from unfertilized (haploid) eggs. Crosses between many strains of Trichogramma deion showed some degree of NRCI. Crosses between a T. deion culture collected in Seven Pines, California (SVP) with one from Marysville, California (MRY) showed an extreme form of NRCI in which practically no female offspring was produced when MRY females were crossed with SVP males. The reciprocal cross produced a close to normal proportion of female and male offspring. Detailed studied of this cross indicated that 1) the female offspring produced in the compatible interstrain cross were not the result of parthenogenesis but were true hybrids, 2) the incompatible interstrain cross did not produce female offspring because fertilized eggs died during development, 3) the death of these eggs could not be prevented by either antibiotic or temperature treatment, 4) cytoplasmically inherited factors causing NRCI could be discounted because backcrossed females with the genome of MRY and the cytoplasm of SVP, exhibit the NRCI relationship characteristic of their genome. Therefore the NRCI between these strains appears to be caused by a modification coded for by the nuclear genes of MRY that results in incompatibility when SVP sperm fertilizes MRY eggs. In addition the level of incompatibility in crosses between the SVP females and MRY males is temperature sensitive, the higher the rearing temperature the lower the level of compatibility.