PubMed Health⌕ Search

Biomedical subjects

M M Shah

Publications and source records attributed to M M Shah.

At least 19 recordsLinked to original sources
PubMed2004

Construction and evaluation of cDNA libraries for large-scale expressed sequence tag sequencing in wheat (Triticum aestivum L.).

A total of 37 original cDNA libraries and 9 derivative libraries enriched for rare sequences were produced from Chinese Spring wheat (Triticum aestivum L.), five other hexaploid wheat genotypes (Cheyenne, Brevor, TAM W101, BH1146, Butte 86), tetraploid durum wheat (T. turgidum L.), diploid wheat (T. monococcum L.), and two other diploid members of the grass tribe Triticeae (Aegilops speltoides Tausch and Secale cereale L.). The emphasis in the choice of plant materials for library construction was reproductive development subjected to environmental factors that ultimately affect grain quality and yield, but roots and other tissues were also included. Partial cDNA expressed sequence tags (ESTs) were examined by various measures to assess the quality of these libraries. All ESTs were processed to remove cloning system sequences and contaminants and then assembled using CAP3. Following these processing steps, this assembly yielded 101,107 sequences derived from 89,043 clones, which defined 16,740 contigs and 33,213 singletons, a total of 49,953 "unigenes." Analysis of the distribution of these unigenes among the libraries led to the conclusion that the enrichment methods were effective in reducing the most abundant unigenes and to the observation that the most diverse libraries were from tissues exposed to environmental stresses including heat, drought, salinity, or low temperature.

Expressed Sequence Tags↗
PubMed2002

Molecular correlates of the M-current in cultured rat hippocampal neurons.

M-type K(+) currents (I(K(M))) play a key role in regulating neuronal excitability. In sympathetic neurons, M-channels are thought to be composed of a heteromeric assembly of KCNQ2 and KCNQ3 K(+) channel subunits. Here, we have tried to identify the KCNQ subunits that are involved in the generation of I(K(M)) in hippocampal pyramidal neurons cultured from 5- to 7-day-old rats. RT-PCR of either CA1 or CA3 regions revealed the presence of KCNQ2, KCNQ3, KCNQ4 and KCNQ5 subunits. Single-cell PCR of dissociated hippocampal pyramidal neurons gave detectable signals for only KCNQ2, KCNQ3 and KCNQ5; where tested, most also expressed mRNA for the vesicular glutamate transporter VGLUT1. Staining for KCNQ2 and KCNQ5 protein showed punctate fluorescence on both the somata and dendrites of hippocampal neurons. Staining for KCNQ3 was diffusely distributed whereas KCNQ4 was undetectable. In perforated patch recordings, linopirdine, a specific M-channel blocker, fully inhibited I(K(M)) with an IC(50) of 3.6 +/- 1.5 microM. In 70 % of these cells, TEA fully suppressed I(K(M)) with an IC(50) of 0.7 +/- 0.1 mM. In the remaining cells, TEA maximally reduced I(K(M)) by only 59.7 +/- 5.2 % with an IC(50) of 1.4 +/- 0.3 mM; residual I(K(M)) was abolished by linopirdine. Our data suggest that KCNQ2, KCNQ3 and KCNQ5 subunits contribute to I(K(M)) in these neurons and that the variations in TEA sensitivity may reflect differential expression of KCNQ2, KCNQ3 and KCNQ5 subunits.

Animals↗
PubMed2001

Identification of connexin43 (alpha1) gap junction gene mutations in patients with hypoplastic left heart syndrome by denaturing gradient gel electrophoresis (DGGE).

Gap junction channels formed by the connexin43 protein are considered to play crucial roles in development and function because they allow the direct cell-to-cell exchange of molecules that mediate multiple signaling events. Previous results have shown that connexin43 channels are intricately gated by phosphorylation and that disruption of this regulation gives rise to severe heart malformations and defects of laterality in human, chick and frog. Here we report the identification of connexin43 gene mutations that represent a minor population of connexin43 alleles, which could be reliably detected by using denaturing gradient gel electrophoresis (DGGE) to visualize normal and mutant DNAs that were separately sequenced. In contrast, sequencing of total PCR products without DGGE-pre-selection failed to consistently identify these mutations. Forty-six controls and 20 heart transplant recipients were examined in this study. In the latter group, 14 children had hypoplastic left heart syndrome (HLHS) in which connexin43 gene defects were detected in eight. The remaining six transplant patients with HLHS and all controls showed no defects. All eight HLHS children with gene defects had the same four substitutions: two that were silent polymorphisms, and two that were missense, replacing arginine codons at positions 362 and 376 with codons for glutamines. All four of these substitutions are identical to the nucleotide sequence of the connexin43 pseudogene, suggesting the possibility of an illicit recombination. A breakpoint region was identified 5' to the mutation site in a 63bp domain that is 100% identical in the gene and pseudogene. Results from in vitro phosphorylation indicate that the absence of arginines 362 and 376 completely abolishes phosphorylation in the connexin43 channel regulation domain suggesting a possible mechanism for the pathologies associated with HLHS.

Base Sequence↗
PubMed2001

Clotrimazole analogues: effective blockers of the slow afterhyperpolarization in cultured rat hippocampal pyramidal neurones.

1. The pharmacology of the slow afterhyperpolarization (sAHP) was studied in cultured rat hippocampal pyramidal neurones. 2. Clotrimazole, its in vivo metabolite, 2-chlorophenyl-bisphenyl-methanol (CBM) and the novel analogues, UCL 1880 and UCL 2027, inhibited the sI(AHP) with similar IC50s (1-2 microM). 3. Clotrimazole and CBM also inhibited the high voltage-activated (HVA) Ca2+ current in pyramidal neurones with IC50s of 4.7 microM and 2.2 microM respectively. UCL 1880 was a less effective Ca2+ channel blocker, reducing the HVA Ca2+ current by 50% at 10 microM. At concentrations up to 10 microM, UCL 2027 had no effect on the Ca2+ current, indicating that its effects on the sI(AHP) were independent of Ca2+ channel block. 4. Clotrimazole also inhibited both the outward holding current (IC50=2.8 microM) present at a potential of -50 mV and the apamin-sensitive medium AHP (mAHP; IC50 approximately amp;10 microM). The other clotrimazole analogues tested had smaller effects on these two currents. The present work also shows that 100 nM UCL 1848, an inhibitor of apamin-sensitive conductances, abolishes the mAHP. 5. Currents were recorded from HEK293 cells transfected with hSK1 and rSK2. The SK currents were very sensitive to inhibition by UCL 1848 but were not significantly reduced by the sI(AHP) inhibitor, UCL 2027 (10 microM). 10 microM UCL 1880 reduced the hSK1 current by 40%. 6. UCL 2027 appears to be the first relatively selective blocker of the sAHP to be described. Furthermore, the ability of UCL 2027 to block the sAHP with minimal effect on SK1 channel activity questions the role of this channel in the sAHP.

Action Potentials↗
PubMed2001

A spontaneously arising mutation in connexin32 with repeated passage of FRTL-5 cells coincides with increased growth rate and reduced thyroxine release.

In this study we examine changes in the cellular properties of FRTL-5 cells as a function of passage number, with particular emphasis on gap junction expression, karyotype, morphology, growth rate and thyroxine (T(4)) release. Early passage FRTL-5 follicular cells transfer dye through gap junctions from injected cell(s) to third-order neighboring cells and beyond within their respective follicles and have immuno-detectable connexin32 (Cx32) type gap junctional plaques in their lateral contacting plasma membranes. By contrast, FRTL-5 cells established as monolayers, or as follicles from cultures passed more than 15 times, did not transfer microinjected Lucifer Yellow dye to contiguous neighboring cells and did not express any immuno-detectable rat thyroid specific connexins (Cx43, Cx32 or Cx26). Western blots confirmed that total, membrane and cytosolic Cx32 protein was present only in early pass follicular cultures. To better understand the passage-dependent loss of Cx32 expression, RT-PCR primers were made to the most unique sequences of the rat Cx32 molecule, the cytoplasmic and carboxyl-terminal regions. These primers were used to screen FRTL-5 RNA from cultures of various passage numbers. The results revealed that later passage cultures had a single base deletion in the middle of the Cx32 cytoplasmic loop region at nucleotide position 378. This base deletion was in the middle position of the codon for amino acid 116, which is normally a CAC (histidine) but read with the frame shift was a CCC (proline). The four amino acids that followed this deletion were also altered with the fourth one becoming UAA, the ochre translation stop codon. This premature stopping of translation resulted in a truncation of 60% of the protein, which included the remaining cytoplasmic loop, third and fourth transmembrane regions and the carboxyl-terminus. The later passage cultures did not produce a carboxyl-terminal RT-PCR product, indicating that the mRNA was also truncated. These regions of the Cx32 molecule contain the sequences and epitopes to which probes and antibodies are directed, and as such alterations of these regions with repeated passage explains reports by others that FRTL-5 cells do not express Cx32, and implies that cultures used for these assessments were passed more than 15 times. To determine if genetic or epigenetic abnormalities existed in FRTL-5 cells we performed chromosome spreads from various passage cultures. FRTL-5 cells have been reported to be diploid and more recently non-diploid; however, we found them to be fully tetraploid. This tetraploidy appears to be unstable in that later passes are tetraploid plus two or three extra chromosomes. There were no obvious translocations, breaks or large-scale interstitial deletions of any chromosomes in the FRTL-5 cultures tested. As FRTL-5 cells were repeatedly passed their morphology changed. Monolayer areas spread from beneath the follicles, and the follicles became flattened in appearance. These physical changes were coincident with dramatically increased growth rates. Early cultures (passed 3-12 times) divided on average every 49+/-1 h, whereas later passes (passes 20-25) divided every 28+/-3 h. To correlate these changes with a measure of thyroid function we assayed T(4) output. Early passage follicular cultures incubated for 6 h with sodium iodide, released on average 5.27+/- 0.33 ng/ml of T(4)/100 follicles. Later passes, or early passes treated with heptanol to down-regulate Cx32, released an average of 3.84+/-0.50 ng/ml of T(4)/100 follicles. There was a 27% difference in T(4) release between early follicular cultures, that were coupled by Cx32, and late or down-regulated early follicular cultures, that were uncoupled (P<0.0001). Collectively, the physical changes documented in this study were coincident with the loss of functional Cx32. This implies a relationship between the loss of intercellular communication and changes in morphogenic appearance, growth rate and reduced thyroid function and supports the previously postulated, tumor-suppressor role for Cx32. FRTL-5 cultures from low passage numbers are an excellent model of primary thyroid cells. However, many reports in the literature ascribe features to FRTL-5 cells that are mutually inconsistent. These differences may be resolved in the future by addressing the passage number and the conditional differences of the cultures being studied.

Amino Acid Sequence↗
PubMed1999

Further studies on periodic limb movement disorder and restless legs syndrome in children with attention-deficit hyperactivity disorder.

Fourteen consecutive children who were newly diagnosed with attention-deficit hyperactivity disorder (ADHD) and who had never been exposed to stimulants and 10 control children without ADHD underwent polysomnographic studies to quantify Periodic Limb Movements in Sleep (PLMS) and arousals. Parents commonly gave both false-negative and false-positive reports of PLMS in their children, and a sleep study was necessary to confirm their presence or absence. The prevalence of PLMS on polysomnography was higher in the children with ADHD than in the control subjects. Nine of 14 (64%) children with ADHD had PLMS at a rate of >5 per hour of sleep compared with none of the control children (p <0.0015). Three of 14 children with ADHD (21%) had PLMS at a rate of >20 per hour of sleep. Many of the PLMS in the children with ADHD were associated with arousals. Historical sleep times were less for children with ADHD. The children with ADHD who had PLMS chronically got 43 minutes less sleep at home than the control subjects (p = 0.0091). All nine children with ADHD who had a PLMS index of >5 per hour of sleep had a long-standing clinical history of sleep onset problems (>30 minutes) and/or maintenance problems (more than two full awakenings nightly) thus meeting the criteria for Periodic Limb Movement Disorder (PLMD). None of the control children had a clinical history of sleep onset or maintenance problems. The parents of the children with ADHD were more likely to have restless legs syndrome (RLS) than the parents of the control children. Twenty-five of 28 biologic parents of the children with ADHD and all of the biologic parents of the control children were reached for interview. Eight of twenty-five parents of the children with ADHD (32%) had symptoms of RLS as opposed to none of the control parents (p = 0.011). PLMS may directly lead to symptoms of ADHD through the mechanism of sleep disruption. Alternative explanations for the association between ADHD and RLS/PLMS are that they are genetically linked, they share a common dopaminergic deficit, or both.

Arousal↗
PubMed1996

Elimination of nitrite from the explosive 2,4,6-trinitrophenylmethylnitramine (tetryl) catalyzed by ferredoxin NADP oxidoreductase from spinach.

Nitroreductase enzymes generally catalyze the reduction of nitroaromatic compounds to the corresponding amines. In contrast, ferredoxin NADP oxidoreductase (FNR), glutathione reductase, xanthine oxidase, and cytochrome c reductase catalyze the NADPH dependent elimination of the nitramine nitro group from 2,4,6-trinitrophenylmethylnitramine to form N-methylpicramide (NMP). Nitrite elimination was inhibited under aerobic conditions. Our results suggest that under aerobic conditions, tetryl is enzymatically reduced to the nitroanion radical which is then involved in the reduction of molecular oxygen. Under anaerobic conditions, the radical is reduced to NMP and nitrite is eliminated.

Anaerobiosis↗
PubMed1996

Effects of perinatal zidovudine on hematopoiesis: a comparison of effects on progenitors from human fetuses versus mothers.

OBJECTIVE: To compare the effects of zidovudine (ZDV) on clonal maturation of hematopoietic progenitors obtained from the bone marrow of women of child-bearing age with its effects on progenitors obtained from the marrow, liver and blood of fetuses. We also sought to determine whether the adverse effects of ZDV on fetal hematopoiesis resulted exclusively from an action on progenitors, or also involved the inhibition of the production of hematopoietic growth factors. PARTICIPANTS: Hematopoietic progenitors were obtained from bone-marrow aspirates of seven women of child-bearing age, from the bone marrow and liver of seven mid-trimester abortuses, and from the umbilical cord blood of seven term infants. METHODS: We added increasing concentrations of ZDV to clonal assays of hematopoietic progenitors, after which we assayed clonal maturation of progenitors, and counted the number of erythrocytes per erythroid clone and the number of neutrophils per granulocytic clone. Light-density cell fractions and enriched CD34+ progenitor fractions were studied. In other studies we determined the effect of increasing concentrations of ZDV on production of granulocyte colony-stimulating factor (G-CSF) protein [enzyme-linked immunosorbent assay (ELISA)] and mRNA by fetal and maternal monocytes, and on production of erythropoietin protein (ELISA) and mRNA by Hep3B cells. RESULTS: Mature erythroid progenitors were the most sensitive to the adverse effects of ZDV on clonal maturation, and multipotent progenitors were the most resistant. Erythroid progenitors from all fetal and neonatal sources were more sensitive to the effect of ZDV than those from the bone marrow of adult women. The inhibitory effects were explained by an action on CD34+ cells; no effect was observed on production of G-CSF or erythropoietin. CONCLUSIONS: We speculate that the low hematocrits of neonates delivered after antenatal ZDV treatment are due to reduced clonal maturation of erythroid progenitors, and that fetal erythroid progenitors are inhibited to a greater extent than maternal progenitors.

Adult↗
PubMed1995

Mutations of the Connexin43 gap-junction gene in patients with heart malformations and defects of laterality.

BACKGROUND: Gap junctions are thought to have a crucial role in the synchronized contraction of the heart and in embryonic development. Connexin43, the major protein of gap junctions in the heart, is targeted by several protein kinases that regulate myocardial cell-cell coupling. We hypothesized that mutations altering sites critical to this regulation would lead to functional or developmental abnormalities of the heart. METHODS: Connexin43 DNA from 25 normal subjects and 30 children with a variety of congenital heart diseases was amplified by the polymerase chain reaction and sequenced. Mutant DNA was expressed in cell culture and examined for its effect on the regulation of cell-cell communication. RESULTS: The 25 normal subjects and 23 of the 30 children with heart disease had no amino acid substitutions in connexin43. All six children with syndromes that included complex heart malformations had substitutions of one or more phosphorylatable serine or threonine residues. Four of these children had two independent mutations, suggesting an autosomal recessive disorder. Five of these children had substitutions of proline for serine at position 364. A seventh child, with a different heart condition, also had a point mutation in connexin43. Transfected cells expressing the Ser364Pro mutant connexin43 sequence showed abnormalities in the regulation of cell-cell communication, as compared with cells expressing normal connexin43. CONCLUSIONS: Mutations in the connexin43 gap-junction gene, which lead to abnormally regulated cell-cell communication, are associated with visceroatrial heterotaxia.

Adolescent↗
PubMed1995

Reduced cell-cell communication in a spontaneous murine model of autoimmune thyroid disease.

MRL-lpr/lpr mice manifest a systemic lupus-like autoimmune disease. As part of this syndrome, the mice spontaneously develop autoimmune thyroiditis, which is morphologically and biochemically similar to human autoimmune thyroiditis. In this study we investigated whether thyroid tissue obtained from sites of chronic inflammation had altered gap junctional communication. Fresh tissue sections revealed that thyroid from the nondiseased mice (MRL-(+)/+) had connexins (Cx) localized to the plasma membrane at points of thyroid cell-cell contact. In contrast, the Cx in diseased mouse (MRL-lpr/lpr) thyroid tissue were not localized to the plasma membrane, and the fluorescent intensity was reduced for Cx43 and Cx26. Northern analysis confirmed that murine thyroid tissue expressed messenger RNA for these Cx. However, the diseased tissue expressed lower levels of Cx32 and Cx26 messenger RNA. The infiltrating cells and their biologically active products present in the diseased thyroid tissue may mediate the reduced Cx expression and aberrant gap junctional assembly. We established primary thyrocyte cultures to determine whether these differences persisted when the inflammatory factors were removed. The nondiseased thyroid cells were communication competent, with fluorescent dye transfer proceeding from the injected cell to primary contacts (95%) and to second and third order neighboring cells in 75% of the trials. Thyroid cells from the diseased mice were communication incompetent, in that 80% of microinjections failed to result in dye transfer to cells in direct contact. Immunocytochemistry indicated that the functional coupling in the normal mouse thyroid cells was associated with Cx43 located in the plasma membrane as assembled gap junctional plaques. The communication-deficient diseased thyroid cells had internalized Cx43 predominantly localized to perinuclear regions of the cells. Collectively, these data document altered Cx-protein distribution in the autoimmune diseased thyroid. The diseased thyroid tissue was devoid of plasma membrane identifiable gap junctions and deficient in intercellular communication. Culturing removed the inflammatory mediators; however, the disease cells retained their communication incompetence. These results suggest that if this deficiency was initiated by components of the inflammation process, then protracted changes must have occurred so that the continued presence of these factors was no longer required to sustain this difference.

Animals↗
PubMed1993

Iodide as the mediator for the reductive reactions of peroxidases.

Lignin peroxidase H2 (LiPH2) from the white rot fungus Phanerochaete chrysosporium catalyzed the reduction of cytochrome c, nitro blue tetrazolium, ferric iron, molecular oxygen, and triiodide in a reaction mixture containing LiPH2, H2O2, EDTA, and iodide. Activity followed first order kinetics with respect to EDTA concentration. The reductive activity observed with LiPH2 using iodide as the mediator was comparable to that obtained using a variety of other free radical mediators such as veratryl alcohol, 1,4-dimethoxybenzene, and 1,2,3- and 1,2,4-trimethoxybenzene. EDTA-derived radicals were detected by ESR spin trapping upon incubation of LiPH2 with H2O2, iodide, and EDTA. Reduction activity was also observed using other peroxidases such as lactoperoxidase, horseradish peroxidase, and myeloperoxidase. For the reduction activity of LiPH2, it is proposed that the oxidation of EDTA is mediated by the iodide radical, and the reduction of various electron acceptors is mediated by EDTA radicals. The inhibition of reduction activity at higher concentrations of iodide might be due to the combination of iodide radicals to form I2 which forms a stable triiodide complex by reacting with excess iodide.

Basidiomycota↗
PubMed1993

Reduction of CCl4 to the trichloromethyl radical by lignin peroxidase H2 from Phanerochaete chrysosporium.

We were able to spin trap the trichloromethyl radical with PBN in a reaction mixture containing lignin peroxidase H2, H2O2, CCl4, veratryl alcohol and EDTA. Upon removal of CCl4, EDTA radicals were detected. Based on a previously reported study (Shah et al., 1992, J. Biol. Chem. 267, 21564-21569), we propose that the oxidation of EDTA to EDTA derived radicals is mediated by the veratryl alcohol cation radical, and the reduction of CCl4 to the trichloromethyl radical is caused by the EDTA derived radicals. The novel finding here is that CCl4, which is neither a substrate for the enzyme nor a good reductant, is degraded via free radicals generated by lignin peroxidases under reducing conditions.

Carbon Tetrachloride↗
PubMed1993

Veratryl alcohol-dependent production of molecular oxygen by lignin peroxidase.

Veratryl alcohol- and H2O2-dependent production of oxygen by lignin peroxidase isozyme H2 (LiPH2) from Phanerochaete chrysosporium was investigated. Veratryl alcohol oxidation by LiPH2 decreased with increasing concentrations of H2O2 while oxygen evolution increased. The absorption spectrum of the LiPH2 in these experiments indicated that it was in the compound II state. We propose that O2 production results from the one electron oxidation of H2O2 by the veratryl alcohol cation radical to yield superoxide, as the addition of superoxide dismutase stimulated oxygen production. It has been reported previously that oxygen is consumed in reaction mixtures containing lignin peroxidase, H2O2, veratryl alcohol, and oxalate (Popp, J. L., Kalyanaraman, B., and Kirk, T.K. (1990) Biochemistry 29, 10475-10480). In the presence of oxalate, we observed oxygen consumption that was dependent on the H2O2 concentration. The ability of other methoxybenzenes to mediate oxygen production appeared to be related to their redox potential. It was concluded that cation radicals can oxidize H2O2 by one electron which results in the production of superoxide and the evolution of molecular oxygen. Thus, the rates of LiPH2-catalyzed O2 consumption or O2 production are dependent on the relative concentrations of H2O2 and oxalate.

Agaricales↗
PubMed1993

Oxidation of halides by peroxidases and their subsequent reductions.

The iodide oxidase activity and iodide-dependent pseudocatalatic activity of lignin peroxidase H2, an extracellular enzyme of the white rot fungus Phanerochaete chrysosporium, was inhibited by EDTA. The inhibition of iodide oxidase activity by EDTA was reversed at higher concentrations of iodide. Similar results were observed with a number of peroxidases. On further investigation, it was found that EDTA was decarboxylated in a reaction mixture containing a peroxidase, iodide, H2O2, and EDTA. EDTA was also decarboxylated by hypoiodite, a possible intermediate during oxidation of iodide by peroxidases. Iodide-dependent pseudocatalatic activity was increased with an increase in the concentration of H2O2 and inhibited at higher concentrations of iodide. EDTA was also oxidized by horseradish peroxidase, lactoperoxidase, and myeloperoxidase using iodide or bromide as a mediator. However, only myeloperoxidase was able to decarboxylate EDTA using chloride as a mediator. It is proposed that halide is oxidized to hypohalite by peroxidases. The hypohalite is then reduced by EDTA, H2O2, or halide. Reduction is associated with the decarboxylation of EDTA, oxidation of H2O2 to molecular oxygen, or oxidation of halide.

Agaricales↗
PubMed1993

Reductive activity of a manganese-dependent peroxidase from Phanerochaete chrysosporium.

A manganese-dependent peroxidase (MnP) from Phanerochaete chrysosporium catalyzed the reduction of cytochrome c in a reaction mixture containing H2O2, Mn(II)-tartrate, and p-hydroquinone. Electron spin resonance studies have shown that the hydroquinone-dependent reductive activity of MnP is due to the benzosemiquinone formed upon the one-electron oxidation of p-hydroquinone by Mn(III)-tartrate, which is formed upon the oxidation of Mn(II) by MnP. The reductive activity increased linearly with an increase in the concentration of p-hydroquinone. The reductive activity was also observed using other hydroquinones such as methylhydroquinone, 2,5-dimethylhydroquinone, and trimethylhydroquinone. The apparent Km values for Mn(II) and H2O2 for the hydroquinone-dependent reductive activity were similar to those for oxidative reactions of MnP. A stoichiometry study showed that about 1.5 mol of cytochrome c was reduced per mole of H2O2 consumed. The stoichiometry decreased with an increase in the concentration of H2O2. The optimal pH for the reductive activity was 5.0, approximately the physiological pH of the fungus. The reduction of cytochrome c was also observed using a quinone and cellobiose:quinone oxidoreductase isolated from the extracellular medium of the fungus.

Basidiomycota↗
PubMed1992

Production of hydroxyl radical by lignin peroxidase from Phanerochaete chrysosporium.

The mechanism for the production of hydroxyl radical by lignin peroxidase from the white rot fungus Phanerochaete chrysosporium was investigated. Ferric iron reduction was demonstrated in reaction mixtures containing lignin peroxidase isozyme H2 (LiPH2), H2O2, veratryl alcohol, oxalate, ferric chloride, and 1,10-phenanthroline. The rate of iron reduction was dependent on the concentration of oxalate and was inhibited by the addition of superoxide dismutase. The addition of ferric iron inhibited oxygen consumption in reaction mixtures containing LiPH2, H2O2, veratryl alcohol, and oxalate. Thus, the reduction of ferric iron was thought to be dependent on the LiPH2-catalyzed production of superoxide in which veratryl alcohol and oxalate serve as electron mediators. Oxalate production and degradation in nutrient nitrogen-limited cultures of P. chrysosporium was also studied. The concentration of oxalate in these cultures decreased during the period in which maximum lignin peroxidase activity (veratryl alcohol oxidation) was detected. Electron spin resonance studies using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide were used to obtain evidence for the production of the hydroxyl radical in reaction mixtures containing LiPH2, H2O2, veratryl alcohol, EDTA, and ferric chloride. It was concluded that the white rot fungus might produce hydroxyl radical via a mechanism that includes the secondary metabolites veratryl alcohol and oxalate. Such a mechanism may contribute to the ability of this fungus to degrade environmental pollutants.

Agaricales↗