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J H Clarke

Publications and source records attributed to J H Clarke.

At least 37 records · Page 2Linked to original sources
PubMed1996

Evidence that linker sequences and cellulose-binding domains enhance the activity of hemicellulases against complex substrates.

Xylanase A (XYLA) and arabinofuranosidase C (XYLC) from Pseudomonas fluorescens subsp. cellulosa are modular enzymes consisting of discrete cellulose-binding domains (CBDs) and catalytic domains joined by serine-rich linker sequences. To evaluate the role of the CBDs and interdomain regions, the capacity of full-length and truncated derivatives of the two enzymes, lacking either the linker sequences or CBDs, to hydrolyse a range of substrates, and bind to cellulose, was determined. Removal of the CBDs did not affect either the activity of XYLA or XYLC against soluble arabinoxylan. Similarly, deletion of the linker sequences did not alter the affinity of the enzymes for cellulose or their activity against soluble substrates, even when bound to cellulose via the CBDs. Truncated derivatives of XYLA lacking either the linker sequences or the CBD were less active against xylan contained in cellulose-hemicellulose complexes, compared with the full-length xylanase. Similarly, removal of the CBD from XYLC diminished the activity of the enzyme (XYLC''') against plant-cell-wall material containing highly substituted arabinoxylan. The role of CBDs and linker sequences in the catalytic activity of hemicellulases against the plant cell wall is discussed.

Binding Sites↗
PubMed1996

A modular xylanase from mesophilic Cellulomonas fimi contains the same cellulose-binding and thermostabilizing domains as xylanases from thermophilic bacteria.

The xynC gene from mesophilic Cellulomonas fimi encodes a large 125 kDa modular xylanase (XYLC), consisting of six distinct functional domains. In addition to a single Family 10 catalytic domain, XYLC contains a domain homologous with the nodulation protein, NodB, from nitrogen-fixing bacteria and thermostabilizing and cellulose-binding domains found previously only in xylanases from thermophilic bacteria.

Actinomycetales↗
PubMed1996

Interleukin-1 stimulates rapid release of cytokine-induced neutrophil chemoattractant (CINC) in rat lungs.

We found that intratracheal insufflation of interleukin-1 alpha (IL-1) in rats rapidly increased lung lavage cytokine-induced neutrophil chemoattractant (CINC) concentrations, lung tissue myeloperoxidase (MPO) activity, and lung lavage neutrophil counts, and that CINC elevation preceded the migration of neutrophils into the lung. Further, we found that bolus CINC insufflation increased CINC concentrations in plasma, and we found that alveolar macrophages (AM) in lung tissue selections or AM recovered by lavage from rats given IL-1 intratracheally stained positively for CINC by immunohistochemistry. In addition, incubating rat AM with increasing doses of IL-1 in vitro progressively increased CINC concentrations in the culture medium. Our results suggest that the potent neutrophil chemoattractant CINC is rapidly produced and released by rat AM following challenge with IL-1 in vivo or in vitro, and support the hypothesis that CINC is an important mediator in the development of pulmonary inflammation in the rat.

Animals↗
PubMed1996

Do the non-catalytic polysaccharide-binding domains and linker regions enhance the biobleaching properties of modular xylanases?

Xylanase A (XylA) from Pseudomonas fluorescens subsp. cellulosa consists of an N-terminal non-catalytic cellulose-binding domain joined to a functionally independent C-terminal catalytic domain by a sequence rich in serine residues. Xylanase D (XylD) from Cellulomonas fimi also exhibits a modular structure comprising an N-terminal catalytic domain linked to an internal non-catalytic xylan-binding domain and a C-terminal cellulose-binding domain. To determine the importance of the non-catalytic polysaccharide-binding domains and linker sequences of XylA and XylD in relation to their capacity to hydrolyse pulp xylan and enhance bleachability, purified full-length and modified derivatives of both enzymes were incubated with a hardwood kraft pulp. Deletion of the cellulose-binding domain or linker region from XylA decreased the activity of the enzyme against pulp xylan, but had no significant effect on the capacity of the enzyme to facilitate delignification and reduce pulp kappa number. While full-length and truncated forms of XylD, lacking either the cellulose-binding or the cellulose- and xylan-binding domains, were equally effective in hydrolysing pulp xylan, enzyme derivatives containing a polysaccharide-binding domain were marginally more efficient in reducing pulp kappa number. The reduction in kappa number elicited by full-length and isolated catalytic domains of XylA and XylD was reflected in an increase in the brightness of paper handsheets derived from pretreated pulps. Thus, the polysaccharide-binding domains of XylA and XylD did not appear to confer any advantage in terms of the ability of the enzymes to improve pulp bleachability. However, XylA and XylD, which belong to different glycosyl hydrolase families, differed in their ability to hydrolyse pulp xylan and facilitate the delignification of kraft pulp.

Actinomycetales↗
PubMed1996

Molecular dynamics simulations of valinomycin and its potassium complex in homogeneous solvents.

Molecular dynamics simulations of kalinomycin (VM) and its potassium complex in water and in a Lennard Jones solvent are reported. In agreement with experimental evidence the structure of K(+)-VM in nonpolar solution is similar to the solid state structure whereas the structure of uncomplexed VM is not. In water uncomplexed VM retains the Lac and HyV faces (which are lost in nonpolar solution) and shows some similarity with the solid-state structure obtained by crystallization from dimethyl sulfoxide (DMSO). However, also in agreement with spectroscopic data a dynamic equilibrium between a set of conformers is established in both solvents. Our model reproduces the experimental dipole moment (3.6 D) of VM in nonpolar solution. We also observed the spontaneous decomplexation of K(+)-VM in water, with the ion passing through the HyV face in preference to the Lac face. Water attack was observed through both faces. The time scale for all conformational transitions is of the order of 100 ps, with structural changes associated with the (de)-complexation reaction controlled by the ring dihedrals in the vicinity of the L-lactic acid residues. Global structural functions, radial distribution functions, and VM ring dihedral analysis are presented, along with an analysis of the decomplexation event.

Algorithms↗
PubMed1996

Teaching clinical hypnosis in U.S. and Canadian dental schools.

This paper reports the results of a 1995 survey of the teaching of hypnosis in U.S. and Canadian dental schools. Responses were received from 50 schools (78%). The results are compared with prior surveys by Parrish (1973) and Simpson, Dedmon, Logan and Jakobsen (1980). The number of complete courses in hypnosis increased from 1973 to 1980 but has remained static since then. The number of other courses in which a one to two hour introduction to hypnosis is presented increased from 18% in 1980 to 30% in 1995. However, during approximately that same period (1982-1995) dentist membership in A.S.C.H. dropped from 14.9% to 4.4%. Some commentary on this phenomenon and needed future studies are offered.

Canada↗
PubMed1995

Novel cellulose-binding domains, NodB homologues and conserved modular architecture in xylanases from the aerobic soil bacteria Pseudomonas fluorescens subsp. cellulosa and Cellvibrio mixtus.

To test the hypothesis that selective pressure has led to the retention of cellulose-binding domains (CBDs) by hemicellulase enzymes from aerobic bacteria, four new xylanase (xyn) genes from two cellulolytic soil bacteria, Pseudomonas fluorescens subsp. cellulosa and Cellvibrio mixtus, have been isolated and sequenced. Pseudomonas genes xynE and xynF encoded modular xylanases (XYLE and XYLF) with predicted M(r) values of 68,600 and 65000 respectively. XYLE contained a glycosyl hydrolase family 11 catalytic domain at its N-terminus, followed by three other domains; the second of these exhibited sequence identity with NodB from rhizobia. The C-terminal domain (40 residues) exhibited significant sequence identity with a non-catalytic domain of previously unknown function, conserved in all the cellulases and one of the hemicellulases previously characterized from the pseudomonad, and was shown to function as a CBD when fused to the reporter protein glutathione-S-transferase. XYLF contained a C-terminal glycosyl hydrolase family 10 catalytic domain and a novel CBD at its N-terminus. C. mixtus genes xynA and xynB exhibited substantial sequence identity with xynE and xynF respectively, and encoded modular xylanases with the same molecular architecture and, by inference, the same functional properties. In the absence of extensive cross-hybridization between other multiple cel (cellulase) and xyn genes from P. fluorescens subsp. cellulosa and genomic DNA from C. mixtus, similarity between the two pairs of xylanases may indicate a recent transfer of genes between the two bacteria.

Amidohydrolases↗
PubMed1995

QTL analysis of flowering time in Arabidopsis thaliana.

Quantitative trait loci (QTL) analyses based on restriction fragment length polymorphism maps have been used to resolve the genetic control of flowering time in a cross between two Arabidopsis thaliana ecotypes H51 and Landsberg erecta, differing widely in flowering time. Five quantitative trait loci affecting flowering time were identified in this cross (RLN1-5), four of which are located in regions containing mutations or loci previously identified as conferring a late-flowering phenotype. One of these loci is coincident with the FRI locus identified as the major determinant for late flowering and vernalization responsiveness in the Arabidopsis ecotype Stockholm. RLN5, which maps to the lower half of chromosome five (between markers mi69 and m233), only affected flowering time significantly under short day conditions following a vernalization period. The late-flowering phenotype of H51 compared to Landsberg erecta was due to alleles conferring late flowering at only two of the five loci. At the three other loci, H51 possessed alleles conferring early flowering in comparison to those of Landsberg erecta. Combinations of alleles conferring early and late flowering from both parents accounted for the transgressive segregation of flowering time observed within the F2 population. Three QTL, RLN1, RLN2 and RLN3 displayed significant genotype-by-environment interactions for flowering time. A significant interaction between alleles at RLN3 and RLN4 was detected.

Alleles↗
PubMed1994

Mapping FRI, a locus controlling flowering time and vernalization response in Arabidopsis thaliana.

Two loci FRI (FRIGIDA) and KRY (KRYOPHILA) have previously been identified as having major influences on the flowering time of the late-flowering, vernalization-responsive Arabidopsis ecotype, Stockholm. We report here on the mapping and subsequent analysis of these two loci. FRI was mapped to the top of chromosome 4 between markers w122 and m506, using restriction fragment length polymorphism (RFLP) analysis. Due to lack of segregation in of the late-flowering phenotype under the environmental conditions used, KRY could only be localized, by "subtractive genotyping", to chromosome 5 or part of chromosome 3. The map position of FRI indicates that it is not allelic to any of the late-flowering loci identified by mutagenesis of the early-flowering ecotype Landsberg erecta. The late-flowering phenotype conferred by the Stockholm allele of FRI is modified (towards earlier flowering) by Landsberg erecta alleles at an unknown number of loci, perhaps accounting for the absence of fri mutations among mutant lines recovered in Landsberg erecta.

Alleles↗
PubMed1994

Evidence for a general role for high-affinity non-catalytic cellulose binding domains in microbial plant cell wall hydrolases.

Cellulases expressed by Cellulomonas fimi consist of a catalytic domain and a discrete non-catalytic cellulose-binding domain (CBD). To establish whether CBDs are common features of plant cell-wall hydrolases from C. fimi, the molecular architecture of xylanase D (XYLD) from this bacterium was investigated. The gene encoding XYLD, designated xynD, consisted of an open reading frame of 1936 bp encoding a protein of M(r) 68,000. The deduced primary sequence of XYLD was confirmed by the size (64 kDa) and N-terminal sequence of the purified recombinant xylanase. Biochemical analysis of the purified enzyme revealed that XYLD is an endoacting xylanase which displays no detectable activity against polysaccharides other than xylan. The predicted primary structure of XYLD comprised an N-terminal signal peptide followed by a 190-residue domain that exhibited significant homology to Family-G xylanases. Truncated derivatives of xynD, encoding the N-terminal 193 amino acids of mature XYLD directed the synthesis of a functional xylanase, confirming that the 190-residue N-terminal sequence constitutes the catalytic domain. The remainder of the enzyme consisted of two approximately 90-residue domains, which exhibited extensive homology with each other, and limited sequence identity with CBDs from other polysaccharide hydrolases. Between the two putative CBDs is a 197-amino-acid sequence that exhibits substantial homology with Rhizobium NodB proteins. The four discrete domains in XYLD were separated by either threonine/proline-or novel glycine-rich linker regions. Although full-length XYLD adsorbed to cellulose, truncated derivatives of the enzyme lacking the C-terminal CBD hydrolysed xylan but did not bind to cellulose.(ABSTRACT TRUNCATED AT 250 WORDS)

Actinomycetales↗
PubMed1993

The short loop between epidermal growth factor-like domains 4 and 5 is critical for human thrombomodulin function.

Thrombomodulin (TM) is a cofactor for activation of protein C by thrombin. We showed that 80-90% of this cofactor activity is lost by oxidation of Met388, located within the short interdomain loop between epidermal growth factor-like domains 4 and 5 (Glaser, C. B., Morser, J., Clarke, J. H., Blasko, E., McLean, K., Kuhn, I., Chang, R.-J., Lin, J.-H., Vilander, L., Andrews, W. H., and Light, D. R. (1992) J. Clin. Invest. 90, 2565-2573). For each of the 3 amino acids of the loop, site-specific mutants are described in which, 1) all possible single amino acid substitutions are made, 2) deletions are made, or 3) alanine is inserted adjacent to each residue of the loop. Most substitutions within the loop (38/57) result in a > 50% decrease in cofactor activity, while changes in the length of this region result in > 90% loss of activity. Only the Met388-->Leu mutant has higher cofactor activity (2-fold) than wild-type TM. A number of soluble and full-length TM analogs with the Met388-->Leu substitution are improved thrombin cofactors, whether produced in bacteria, insect, or mammalian cells. Detailed kinetic analysis of a soluble TM analog consisting of the six EGF-like domains secreted from insect cells shows that the enhanced activity of the Met388-->Leu mutant results from an increased catalytic efficiency (kcat/Km). This enhancement is maximal at physiological concentrations of calcium. The loss of activity following Met388 oxidation in the wild-type protein is the result of both decreased binding to thrombin (Kd effect) and a decreased interaction of the TM.thrombin complex with protein C (Km effect). We demonstrate the critical role of this interdomain loop in the biological anticoagulant properties of TM.

Amino Acid Sequence↗
PubMed1992

Oxidation of a specific methionine in thrombomodulin by activated neutrophil products blocks cofactor activity. A potential rapid mechanism for modulation of coagulation.

Endothelial thrombomodulin (TM) plays a critical role in hemostasis as a cofactor for thrombin-dependent formation of activated protein C, a potent anticoagulant. Chloramine T, H2O2, or hypochlorous acid generated from H2O2 by myeloperoxidase rapidly destroy 75-90% of TM cofactor activity. Activated PMN, the primary in vivo source of biological oxidants, also rapidly inactivate TM. Oxidation of TM by PMN is inhibited by diphenylene iodonium, an inhibitor of NADPH oxidase. Both Met291 and Met388 in the six epidermal growth factor-like repeat domain are oxidized; however, only substitutions of Met388 lead to TM analogues that resist oxidative inactivation. We suggest that in inflamed tissues activated PMN may inactivate TM and demonstrate further evidence of the interaction between the inflammatory process and induction of thrombotic potential.

Adult↗