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Biomedical subjects

T M Wood

Publications and source records attributed to T M Wood.

At least 19 recordsLinked to original sources
PubMed2000

Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1alpha during hypoxia: a mechanism of O2 sensing.

During hypoxia, hypoxia-inducible factor-1alpha (HIF-1alpha) is required for induction of a variety of genes including erythropoietin and vascular endothelial growth factor. Hypoxia increases mitochondrial reactive oxygen species (ROS) generation at Complex III, which causes accumulation of HIF-1alpha protein responsible for initiating expression of a luciferase reporter construct under the control of a hypoxic response element. This response is lost in cells depleted of mitochondrial DNA (rho(0) cells). Overexpression of catalase abolishes hypoxic response element-luciferase expression during hypoxia. Exogenous H(2)O(2) stabilizes HIF-1alpha protein during normoxia and activates luciferase expression in wild-type and rho(0) cells. Isolated mitochondria increase ROS generation during hypoxia, as does the bacterium Paracoccus denitrificans. These findings reveal that mitochondria-derived ROS are both required and sufficient to initiate HIF-1alpha stabilization during hypoxia.

Androstadienes↗
PubMed1999

Prediction of performance on the RCMP physical ability requirement evaluation.

The Royal Canadian Mounted Police use the Physical Ability Requirement Evaluation (PARE) for screening applicants. The purposes of this investigation were to identify those field tests of physical fitness that were associated with PARE performance and determine which most accurately classified successful and unsuccessful PARE performers. The participants were 27 female and 21 male volunteers. Testing included measures of aerobic power, anaerobic power, agility, muscular strength, muscular endurance, and body composition. Multiple regression analysis revealed a three-variable model for males (70-lb bench press, standing long jump, and agility) explaining 79% of the variability in PARE time, whereas a one-variable model (agility) explained 43% of the variability for females. Analysis of the classification accuracy of the males' data was prohibited because 91% of the males passed the PARE. Classification accuracy of the females' data, using logistic regression, produced a two-variable model (agility, 1.5-mile endurance run) with 93% overall classification accuracy.

Adult↗
PubMed1996

Arabinoxylan-degrading enzyme system of the fungus Aspergillus awamori: purification and properties of an alpha-L-arabinofuranosidase.

An alpha-L-arabinofuranosidase produced by the fungus Aspergillus awamori had a molecular mass of approximately 64 kDa on sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) and was optimally active at pH 4.6 and 50 degrees C. The enzyme, which chromatographed as a single component on SDS-PAGE, appeared to consist of two isoenzymes of pI 3.6 and 3.2. Acting in isolation, the alpha-L-arabinofuranosidase had only a very limited capacity to release L-arabinose (less than 11%) directly from arabinoxylans that had been extracted from a number of plant cell wall preparations using 18% alkali, but a much higher proportion of the L-arabinose (46%) was released from a wheat straw arabinoxylan that had been isolated by steam treatment. There was a marked synergistic effect between the alpha-L-arabinofuranosidase and an endo-(1 --> 4)-beta-D-xylanase produced by A. awamori in both the rate and extent of the release of L-arabinose from both oat straw and wheat straw arabinoxylans, suggesting that L-arabinose-substituted oligosaccharides generated by the endoxylanase action were better substrates for enzyme action. A novel property of the alpha-L-arabinofuranosidase was its capacity to release a substantial proportion (42%) of feruloyl L-arabinose from intact wheat straw arabinoxylan. The concerted action of the alpha-L-arabinofuranosidase and endoxylanase released 71% of the feruloyl L-arabinose and 69% of the p-coumaroyl L-arabinose substituents from wheat straw arabinoxylan.

Arabinose↗
PubMed1995

D-xylan-degrading enzyme system from the fungus Phanerochaete chrysosporium: isolation and partial characterisation of an alpha-(4-O-methyl)-D-glucuronidase.

A number of fungi were screened for their capacities to produce extracellular alpha-(4-O-methyl)-D-glucuronidase. Of those tested, Phanerochaete chrysosporium ATCC 24725 produced the enzyme in greatest yield. The single alpha-(4-O-methyl)-D-glucuronidase produced by this fungus was purified by a series of chromatographic methods involving anion exchange, hydrophobic interaction and chromatofocusing. Isolated in this way, the enzyme had an apparent molecular mass of 112 kDa in sodium dodecyl sulphate polyacrylamide gels, and a pI of 4.6 when determined by isoelectric focusing in polyacrylamide gels. The enzyme was optimally active at pH 3.5, but showed significant activity over the pH range 3-5. In the absence of substrate the enzyme was inactivated at pH 3.5 in 2 h at 50 degree C: at pH 5.0 it retained 42% of its activity for 24 h at this temperature. The enzyme showed little activity on glucuronoxylan polysaccharides, but some short-chain xylo-oligosaccharides which were substituted with alpha-linked 4-O-methyl-D-glucopyranosyl uronic acid attached to the 2-position of the non-reducing D-xylopyranosyl residue were readily hydrolysed. There were marked synergistic effects apparent in the release of 4-O-methyl-D-glucopyranosyl uronic acid from various glucuronoxylans when the alpha-(4-O-methyl)-D-glucuronidase was acting in concert with endo-(1-->4)-beta-D-xylanase, and with beta-D-xylosidase and/or an alpha-L-arabinofuranosidase.

Basidiomycota↗
PubMed1995

The cellulase system of the anaerobic rumen fungus Neocallimastix frontalis: studies on the properties of fractions rich in endo-(1-->4)-beta-D-glucanase activity.

Seven fractions rich in endoglucanase activity were separated from the extracellular cellulase system of the anaerobic rumen fungus Neocallimastix frontalis. The fractions (ES1, ES3, ES2U1, ES2U2, ES2U4, ES2U3C1 and ES2U3C2) were separated from each other and from a fraction that could solubilize crystalline cellulose (the so-called crystalline-cellulose-solubilizing component, CCSC) by the sequential use of differential adsorption on the microcrystalline cellulose Avicel, gel filtration and affinity chromatography on concanavalin-A-Sepharose. The molecular masses of the endoglucanase fractions, when determined by gel filtration, were 64, 30, 61, 113, 17, 38 and 93 kDa respectively. Each enzyme degraded carboxymethylcellulose and was rich in activity to cellulose swollen in phosphoric acid to break the hydrogen bonding: cellobiose, cellotriose and cellotetraose were released in differing proportions. Each fraction showed a characteristic gradient when the capacity of each enzyme to increase the fluidity of a solution of carboxymethylcellulose was plotted against the increase in reducing power of the solution. Although neither endoglucanase fraction, acting in isolation, could degrade crystalline cellulose, three of the fractions (ES1, ES3 and ES2U1) could act synergistically with the CCSC fraction in this regard. Remarkably, the same three fractions also acted in synergism with the cellobiohydrolases (CBH I and CBH II) of the aerobic fungus Penicillium pinophilum in degrading crystalline cellulose, but only when both cellobiohydrolase enzymes were present in the solution along with any one of the three endoglucanases. These observations support the conclusion that the mechanism of action of the cellulase system of N. frontalis in degrading crystalline cellulose may be similar to that operating in the aerobic fungi.

Anaerobiosis↗
PubMed1995

Studies on the capacity of the cellulase of the anaerobic rumen fungus Piromonas communis P to degrade hydrogen bond-ordered cellulose.

The anaerobic rumen fungus Piromonas communis, when cultured on cotton fibre as the carbon source, produces an extracellular cellulase that is capable of solubilizing "crystalline" hydrogen-bond-ordered cellulose, in the form of the cotton fibre, at a rate that is greater than that of any other cellulases reported in the literature hitherto. The cell-free culture fluid is also very rich in xylan-degrading enzymes. The activity towards crystalline cellulose resides in a high-molecular-mass (approximately 700-1000 kDa) component (so-called crystalline-cellulose-solubilizing component, CCSC) that comprises endo (1-->4)-beta-D-glucanase (carboxymethylcellulase), beta-D-glucosidase and another enzyme that appears to be important for the breakdown of hydrogen-bond-ordered cellulose. The CCSC is associated with only a small amount of the endo(1-->4)-beta-D-glucanase (1.9%), beta-D-glucosidase (0.7%) and protein (0.5%) found in the crude cell-free cellulase preparation. The CCSC, unlike the bulk of the endo(1-->4)-beta-D-glucanase and beta-D-glucosidase, is very strongly absorbed on the microcrystalline cellulose, Avicel.

Anaerobiosis↗
PubMed1995

Alpha-(4-O-methyl)-D-glucuronidase activity produced by the rumen anaerobic fungus Piromonas communis: a study of selected properties.

The rumen anaerobic fungus Piromonas communis, unlike the rumen anaerobic fungi Neocallimastix frontalis and Neocallimastix patriciarum, produced extracellular alpha-(4-O-methyl)-D-glucuronidase when grown in cultures containing filter-paper, barley straw, birchwood xylan or birchwood sawdust as carbon source. The highest concentration of enzyme was produced in cultures containing birchwood sawdust. The aldobiouronic acid O-alpha-(4-O-methyl-D-glucopyranosyluronic acid)-(1-->2)-D-xylopyranose (MeGlcAXyl) was the best substrate of those tested: the aldotriouronic acid O-alpha-(4-O-methyl-D-glucopyranosyluronic acid (1-->2)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (MeGlcAXyl2) and the aldotetraouronic acid O-alpha-(4-O-methyl-D-glucopyranosyluronic acid)-(1-->2)-O-beta-D- xylopyranosyl-(1-->4)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (MeGlcAXyl3) were also attacked but the rate fell as the degree of polymerisation increased. When the same substituted xylo-oligosaccharides were reduced to the corresponding alditols the enzyme activity disappeared. Similarly, p-nitrophenyl-alpha-D-glucuronide was not a substrate. Remarkably, the relative rates of attack shown by the alpha-(4-O-methyl)-D-glucuronidase on the aldouronic acids and on xylans extracted from birchwood, oat spelts and oat straw differed according to the carbon source used to produce the enzyme. The alpha-(4-O-methyl)-D-glucuronidase had a pH optimum of 5.5 and a temperature optimum of 50 degrees C. On gel filtration the enzyme was shown to be associated with proteins covering the range 100-300 kDa, but a major peak of activity in the column effluent appeared to have a molecular mass of 103 kDa.

Anaerobiosis↗
PubMed1993

Mode of action, kinetic properties and physicochemical characterization of two different domains of a bifunctional (1-->4)-beta-D-xylanase from Ruminococcus flavefaciens expressed separately in Escherichia coli.

Two catalytic domains, A and C, of xylanase A (XYLA) from Ruminococcus flavefaciens were expressed separately as truncated gene products from lacZ fusions in Escherichia coli. The fusion products, referred to respectively as XYLA-A1 and XYLA-C2, were purified to homogeneity by anion-exchange chromatography and chromatofocusing. XYLA-A1 was isoelectric at pH 5.0 and had a molecular mass of 30 kDa, whereas XYLA-C2 had a pI of 5.4 and a molecular mass of 44 kDa. The catalytic activity shown by both domains was optimal at 50 degrees C, but XYLA-A1 was more sensitive than XYLA-C2 to temperatures higher than the optimum. XYLA-A1 showed a higher sensitivity to pH than XYLA-C2. The enzyme activity of both domains was completely inactivated in the presence of copper or silver ions and partially inactivated by iron or zinc ions. Neither domain was active on xylo-oligosaccharides shorter than xylopentaose: the rate of degradation of longer xylo-oligosaccharides (degree of polymerization 5-10) increased as the chain length increased. Analysis of the products of hydrolysis of xylo-oligosaccharides and xylan (arabinoxylan) polysaccharide showed that the two domains differed in their modes of action: xylobiose was the shortest product of the hydrolysis. With oat spelt xylan as substrate, XYLA-A1 activity was apparently restricted to regions where xylopyranosyl residues did not carry arabinofuranosyl substituents, whereas XYLA-C2 was able to release hetero-oligosaccharides carrying arabinofuranosyl residues. Neither domain was able to release arabinose from oat spelt xylan.

Animals↗
PubMed1993

A modular esterase from Pseudomonas fluorescens subsp. cellulosa contains a non-catalytic cellulose-binding domain.

The 5' regions of genes xynB and xynC, coding for a xylanase and arabinofuranosidase respectively, are identical and are reiterated four times within the Pseudomonas fluorescens subsp. cellulosa genome. To isolate further copies of the reiterated xynB/C 5' region, a genomic library of Ps. fluorescens subsp. cellulosa DNA was screened with a probe constructed from the conserved region of xynB. DNA from one phage which hybridized to the probe, but not to sequences upstream or downstream of the reiterated xynB/C locus, was subcloned into pMTL22p to construct pFG1. The recombinant plasmid expressed a protein in Escherichia coli, designated esterase XYLD, of M(r) 58,500 which bound to cellulose but not to xylan. XYLD hydrolysed aryl esters, released acetate groups from acetylxylan and liberated 4-hydroxy-3-methoxycinnamic acid from destarched wheat bran. The nucleotide sequence of the XYLD-encoding gene, xynD, revealed an open reading frame of 1752 bp which directed the synthesis of a protein of M(r) 60,589. The 5' 817 bp of xynD and the amino acid sequence between residues 37 and 311 of XYLD were almost identical with the corresponding regions of xynB and xynC and their encoded proteins XYLB and XYLC. Truncated derivatives of XYLD lacking the N-terminal conserved sequence retained the capacity to hydrolyse ester linkages, but did not bind cellulose. Expression of truncated derivatives of xynD, comprising the 5' 817 bp sequence, encoded a non-catalytic polypeptide that bound cellulose. These data indicate that XYLD has a modular structure comprising of a N-terminal cellulose-binding domain and a C-terminal catalytic domain.

Amino Acid Sequence↗
PubMed1993

Purification and characterisation of a beta-D-xylosidase from the anaerobic rumen fungus Neocallimastix frontalis.

A beta-D-xylosidase from the anaerobic rumen fungus Neocallimastix frontalis was purified by anion-exchange and gel filtration chromatography. The enzyme was isoelectrically homogeneous and had an isoelectric point of pH 4.6. The apparent molecular mass calculated by gel filtration was 150,000 Da. Under denaturing conditions, the enzyme appeared as a dimer composed of two polypeptides with molecular masses of 83,000 and 53,000 Da. The pH and temperature optimum were 6.4 and 37 degrees C, respectively: the activity was very sensitive to temperature. The enzyme was inhibited by copper, silver and zinc ions, EDTA and SDS, and was stimulated by calcium and magnesium ions. It was competitively inhibited by D-xylose with an apparent Ki of 3.98 mM. The beta-D-xylosidase exhibited hydrolytic activity on xylobiose and xylo-oligosaccharides of dp up to 7: the specific activities and maximum velocities decreased as the chain length increased. Analysis of the products of hydrolysis by HPLC indicated a typical exo-action. A mixture of beta-D-xylosidase and a xylanase acted synergistically in producing high reducing sugar values, using a xylan from oat spelts.

Animals↗
PubMed1992

Fungal cellulases.

Explore the source record for details and available documents.

Carbohydrate Sequence↗
PubMed1990

Study of the mode of action and site-specificity of the endo-(1----4)-beta-D-glucanases of the fungus Penicillium pinophilum with normal, 1-3H-labelled, reduced and chromogenic cello-oligosaccharides.

The modes of action of the five major endo-(1----4)-beta-D-glucanases (I, II, III, IV and V) purified from Penicillium pinophilum cellulase were compared by h.p.l.c. analysis, with normal, 1-3H-labelled and reduced cello-oligosaccharides and 4-methylumbelliferyl glycosides as substrates. Significant differences were observed in the preferred site of cleavage even when substrates with the same number of glycosidic bonds were compared. Thus, although endoglucanase I was unable to attack normal cello-oligosaccharides shorter than degree of polymerization 6, it hydrolysed reduced cellopentaose to yield cellotriose and cellobi-itol, and it produced cellotriose and 4-methylumbelliferyl glucoside from 4-methylumbelliferyl cellotetraoside. Endoglucanase IV hydrolysed [1-3H]cellotriose but did not attack either cellotri-itol or 4-methylumbelliferyl cellobioside. These and other anomalous results indicated clearly that modification of the reducing glycosyl residue on the cello-oligosaccharides induces in an apparent change in the mode of action of the endoglucanases. It is suggested that, although cello-oligosaccharide derivatives are useful for differentiating and classifying endoglucanases, conclusions on the mechanism of cellulase action resulting from these measurements should be treated cautiously. Unequivocal information on the mode of endoglucanase action on cello-oligosaccharides was obtained with radiolabelled cello-oligosaccharides of degree of polymerization 3 to 5. Indications that transglycosylation was a property of the endoglucanases were particularly evident with the 4-methylumbelliferyl cello-oligosaccharides. Turnover numbers for hydrolysis of the umbelliferyl cello-oligosaccharides were calculated, and these, along with the other analytical data collected on the products of hydrolysis of the normal, reduced and radiolabelled cello-oligosaccharides, suggested that the various endoglucanases had different roles to play in the overall hydrolysis of cellulose to sugars small enough to be transported through the cell membrane.

Bacterial Proteins↗
PubMed1990

Calcium-binding affinity and calcium-enhanced activity of Clostridium thermocellum endoglucanase D.

Clostridium thermocellum endoglucanase D (EC 3.2.1.4: EGD), which is encoded by the celD gene, was found to bind Ca2+ with an association constant of 2.03 x 10(6) M-1. Ca2+ stimulated the activity of EGD towards swollen Avicel by 2-fold. In the presence of Ca2+, the Kd of the enzyme towards p-nitrophenyl-beta-D-cellobioside and carboxymethylcellulose was decreased by 4-fold. Furthermore, Ca2+ increased the half-life of the enzyme at 75 degrees C from 13 to 47 min. Since the 3' sequence of celD encodes a duplicated region sharing similarities with the Ca2+-binding site of several Ca2+-binding proteins, a deleted clone was constructed and used to purify a truncated form of the enzyme which no longer contained the duplicated region. The truncated enzyme was very similar to EGD expressed from the intact gene with respect to activity, Ca2(+)-binding kinetics and Ca2+ effects on substrate binding and thermostability. Thus the latter parameters do not appear to be mediated through the duplicated conserved region.

Amino Acid Sequence↗
PubMed1990

The predictive validity of a neuropsychological screening measure.

This study examined the efficacy of a neuropsychological screening measure in discriminating between neurologically impaired and nonimpaired subjects. It also examined the ability of this screening measure correctly to classify impaired subjects according to right and left hemisphere involvement. The results showed that some 96% of subjects could be correctly classified as impaired or nonimpaired. The measure was also found correctly to identify the hemisphere involved in 95% of the impaired cases. The value and limitations of neuropsychological screening instruments was discussed in terms of portability and ease of administration.

Adult↗
PubMed1989

Fungal cellulase systems. Comparison of the specificities of the cellobiohydrolases isolated from Penicillium pinophilum and Trichoderma reesei.

Reaction patterns for the hydrolysis of chromophoric glycosides from cello-oligosaccharides and lactose by the cellobiohydrolases (CBH I and CBH II) purified from Trichoderma reesei and Penicillium pinophilum were determined. They coincide with those found for the parent unsubstituted sugars. CBH I enzyme from both organisms attacks these substrates in a random manner. Turnover numbers are, however, low and do not increase appreciably as a function of the degree of polymerization of the substrates. The active-site topology of the CBH I from T. reesei was further probed by equilibrium binding experiments with cellobiose, cellotriose, lactose and some of their derivatives. These point to a single interaction site (ABC), spatially restricted as deduced from the apparent independency of the thermodynamic parameters. It appears that the putative subsite A can accommodate a galactopyranosyl or glucopyranosyl group, and subsite B a glucopyranosyl group, whereas in subsite C either a glucopyranosyl or a chromophoric group can be bound, scission occurring between subsites B and C. The apparent kinetic parameters (turnover numbers) for the hydrolysis of cello-oligosaccharides (and their derivatives) by the CBH II type enzyme increase as a function of chain length, indicative of an extended binding site (A-F). Its architecture allows for specific binding of beta-(1----4)-glucopyranosyl groups in subsites A, B and C. Binding of a chromophore in subsite C produces a non-hydrolysable complex. The thermodynamic interaction parameters of some ligands common to both type of enzyme were compared: these substantiate the conclusions reached above.

Cellulose 1,4-beta-Cellobiosidase↗