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Biomedical subjects

R A Warren

Publications and source records attributed to R A Warren.

At least 109 records · Page 6Linked to original sources
PubMed1991

Unusual sequence organization in CenB, an inverting endoglucanase from Cellulomonas fimi.

The nucleotide sequence of the cenB gene was determined and used to deduce the amino acid sequence of endoglucanase B (CenB) of Cellulomonas fimi. CenB comprises 1,012 amino acids and has a molecular weight of 105,905. The polypeptide is divided by so-called linker sequences rich in proline and hydroxyamino acids into five domains: a catalytic domain of 607 amino acids at the N terminus, followed by three repeats of 98 amino acids each which are greater than 60% identical, and a C-terminal domain of 101 amino acids which is 50% identical to the cellulose-binding domains of C. fimi cellulases Cex and CenA. A deletion mutant of the cenB gene encodes a polypeptide lacking the C-terminal 333 amino acids of CenB. The truncated polypeptide is catalytically active and, like intact CenB, binds to cellulose, suggesting that CenB has a second cellulose-binding site. The sequence of amino acids 1 to 461 of CenB is 35% identical, with a further 15% similarity, to that of a cellulase from avocado, which places CenB in cellulase family E. CenB releases mostly cellobiose and cellotetraose from cellohexaose. Like CenA, CenB hydrolyzes the beta-1,4-glucosidic bond with inversion of the anomeric configuration. The pH optimum for CenB is 8.5, and that for CenA is 7.5.

Amino Acid Sequence↗
PubMed1991

Multiple domains in endoglucanase B (CenB) from Cellulomonas fimi: functions and relatedness to domains in other polypeptides.

Endoglucanase B (CenB) from the bacterium Cellulomonas fimi is divided into five discrete domains by linker sequences rich in proline and hydroxyamino acids (A. Meinke, C. Braun, N. R. Gilkes, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, J. Bacteriol. 173:308-314, 1991). The catalytic domain of 608 amino acids is at the N terminus. The sequence of the first 477 amino acids in the catalytic domain is related to the sequences of cellulases in family E, which includes procaryotic and eucaryotic enzymes. The sequence of the last 131 amino acids of the catalytic domain is related to sequences present in a number of cellulases from different families. The catalytic domain alone can bind to cellulose, and this binding is mediated at least in part by the C-terminal 131 amino acids. Deletion of these 131 amino acids reduces but does not eliminate activity. The catalytic domain is followed by three domains which are repeats of a 98-amino-acid sequence. The repeats are approximately 50% identical to two repeats of 95 amino acids in a chitinase from Bacillus circulans which are related to fibronectin type III repeats (T. Watanabe, K. Suzuki, K. Oyanagi, K. Ohnishi, and H. Tanaka, J. Biol. Chem. 265:15659-15665, 1990). The C-terminal domain of 101 amino acids is related to sequences, present in a number of bacterial cellulases and xylanases from different families, which form cellulose-binding domains (CBDs). It functions as a CBD when fused to a heterologous polypeptide. Cells of Escherichia coli expressing the wild-type cenB gene accumulate both native CenB and a stable proteolytic fragment of 41 kDa comprising the three repeats and the C-terminal CBD. The 41-kDa polypeptide binds to cellulose but lacks enzymatic activity.

Amino Acid Sequence↗
PubMed1991

Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families.

Several types of domain occur in beta-1, 4-glycanases. The best characterized of these are the catalytic domains and the cellulose-binding domains. The domains may be joined by linker sequences rich in proline or hydroxyamino acids or both. Some of the enzymes contain repeated sequences up to 150 amino acids in length. The enzymes can be grouped into families on the basis of sequence similarities between the catalytic domains. There are sequence similarities between the cellulose-binding domains, of which two types have been identified, and also between some domains of unknown function. The beta-1, 4-glycanases appear to have arisen by the shuffling of a relatively small number of progenitor sequences.

Amino Acid Sequence↗
PubMed1991

Bacterial cellulose-binding domain-like sequences in eucaryotic polypeptides.

The catalytic domain of endoglucanase II of Trichoderma reesei and the spore germination-specific polypeptide 270-11 of Dictyostelium discoideum contain amino acid sequences which share identity with the sequences of the cellulose-binding domains of several bacterial beta-1,4-glycanases. This is the first report of the presence of such sequences in eucaryotic polypeptides.

Amino Acid Sequence↗
PubMed1990

The tertiary structure of a bacterial cellulase determined by small-angle X-ray-scattering analysis.

CenA from Cellulomonas fimi is a beta-1,4-endoglucanase that binds tightly to cellulose. X-ray-scattering analyses show that the enzyme is tadpole-shaped: the previously identified catalytic and cellulose-binding domains comprise the head and tail respectively. It appears that this structural and functional organization is common to several cellulases from bacteria and fungi.

Actinomycetales↗
PubMed1990

Electrophysiological studies of acetylcholine and the role of the basal forebrain in the somatosensory cortex of the cat. I. Cortical neurons excited by glutamate.

1. Microelectrodes attached to iontophoretic pipettes were used to isolate 410 single neurons in the primary somatosensory cortex of halothane-anesthetized cats. Basal forebrain (BF) stimulation, when paired with pulses of iontophoretically administered glutamate, affected the responsiveness in 24 (54%) of 39 neurons; 17 were facilitated, and seven were inhibited. Five minutes after BF stimulation the average response for a sample of 20 cells was enhanced by 45% (+/- 19). All but one of the effects lasted as long as the cell was studied, often greater than 1 h. 2. When atropine was administered while the BF was stimulated during glutamate excitation, 7 of 16 cells were enhanced, but the average increase was only 16% (+/- 15) for a sample of 15 cells. After the atropine had dissipated, four cells were enhanced by the BF stimulus. In three of these the enhancement had been blocked previously by atropine. 3. BF stimulation had effects similar to iontophoretically administered acetylcholine (ACh), but the effects appeared more frequently with BF stimulation than they had with acetylcholine administration. 4. We propose that the enhanced neuronal responsiveness is due to the release of acetylcholine by cortical terminals of cholinergic neurons located in the BF. The BF stimulus may be more effective than acetylcholine administration because corticopetal cholinergic fibers may end in the immediate vicinity of receptors responsible for long-term changes in membrane permeability.

Acetylcholine↗
PubMed1990

Electrophysiological studies of acetylcholine and the role of the basal forebrain in the somatosensory cortex of the cat. II. Cortical neurons excited by somatic stimuli.

1. Of the sample of 322 neurons located in somatosensory cortex and tested for their responsiveness to somatic stimulation, 91 (28%) responded to stimuli applied to the skin. The majority were located in the middle cortical layers. Each of the cells subjected to tests with glutamate and acetylcholine (ACh) was rapidly adapting to cutaneous stimuli, giving a response at the onset of skin indentation and sometimes after the stimulus withdrawal. 2. Of the 30 cells tested by pairing basal forebrain (BF) stimulation with cutaneous stimulation. 18 (60%) displayed enhanced responses to the same cutaneous stimulus after the pairing. These effects lasted for greater than 5 min in 17 cases, persisting for as long as the cell was studied, sometimes greater than 1 h. 3. The enhanced responsiveness to cutaneous stimuli could not be reversed by atropine, but in each of the 11 cells where atropine was administered while the BF stimulus was paired with the skin stimulus, the pairing produced no enhancement. 4. We conclude that pairing a BF stimulus with a cutaneous stimulus leads to long-term facilitation of the responsiveness of the cortical neuron subjected to this treatment and that this effect is mediated by the release of acetylcholine from BF cholinergic neurons that act on muscarinic receptors found on neurons in the somatosensory cortex.

Acetylcholine↗
PubMed1990

Ready-Stitch: a new device for the closure of skin wounds.

Ready-Stitch (Med United Ltd., London, UK) is a new wound closure device that may have considerable advantages over conventional sutures in closing skin wounds. A preliminary study was carried out to ascertain the most appropriate surgical self-adhesive tape to use in the construction of Ready-Stitch. Twenty-four skin wounds were then closed with either Ready-Stitch or conventional sutures. Ready-Stitch was much preferred by patients and staff alike, produced good cosmetic results, and may reduce the incidence of wound infection and the likelihood of needle-stick injury in hospital staff.

Adhesives↗
PubMed1989

Structural and functional analysis of a bacterial cellulase by proteolysis.

CenA is an endo-beta 1,4-glucanase from the cellulolytic bacterium Cellulomonas fimi. It is a bifunctional enzyme comprising an amino-terminal cellulose-binding domain and a carboxyl-terminal catalytic domain joined by a short sequence of prolyl and threonyl residues (the Pro-Thr box). Additional structural and functional information was revealed by a detailed analysis of the products generated by proteolytic cleavage of a nonglycosylated form of CenA. An extracellular C. fimi protease attacked nonglycosylated CenA at the junctions between the Pro-Thr box and the two functional domains. A stable "core" peptide (p30), corresponding to the catalytic domain, remained after extensive proteolysis. p30 was resistant to further attack even in the presence of 2-mercaptoethanol plus urea or dithiothreitol, but treatment in the presence of sodium dodecyl sulfate allowed complete fragmentation to small peptides. Stable peptides, identical, or closely related to p30, were generated by alpha-chymotrypsin or papain. These results indicated that the catalytic domain adopts a tightly folded conformation affording protection from proteolytic attack. In contrast, the cellulose-binding domain showed a relatively loose conformation. Progressive proteolytic truncation from the amino terminus was apparent during incubation with alpha-chymotrypsin or papain, or with C. fimi protease under reducing conditions. Affinity for cellulose was retained by products missing up to 64 amino-terminal amino acids. The remaining carboxyl-proximal region of the cellulose-binding domain with affinity (47 amino acids) contained sequences highly conserved in analogous domains from other bacterial endo-beta 1,4-glucanases. By analogy with other systems, the properties of the Pro-Thr box are consistent with an elongated conformation. The results of this investigation suggest that CenA has a tertiary structure which resembles that of certain fungal cellulases.

Amino Acid Sequence↗
PubMed1989

Fusion to an endoglucanase allows alkaline phosphatase to bind to cellulose.

Endoglucanase CenA of Cellulomonas fimi comprises an N-terminal cellulose-binding domain and a C-terminal catalytic domain joined together by a sequence of 23 proline and threonine residues (the Pro-Thr box). The domains function independently when separated by proteolysis. TnphoA has been used to generate cenA'-'phoA fusions. CenA'-'PhoA fusion polypeptides which contain the entire cellulose-binding domain of CenA bind to cellulose, allowing their purification from periplasmic extracts in a single, facile step. This result has implications for purification or immobilisation of chimeric proteins on a cheap cellulose matrix.

Actinomycetales↗
PubMed1989

Purification and characterization of endoglucanase C of Cellulomonas fimi, cloning of the gene, and analysis of in vivo transcripts of the gene.

Two nonglycosylated endoglucanases which bind to Sephadex were purified from culture supernatants of Cellulomonas fimi grown on microcrystalline cellulose. Their Mrs were 120,000 and 130,000. The N-terminal amino acid sequences of the enzymes were identical, suggesting that the enzymes were related. A DNA fragment encoding this N-terminal sequence was cloned in Escherichia coli. The nucleotide sequence corresponding to the N-terminal amino acid sequence was preceded by a sequence encoding a typical leader peptide. Transcripts hybridizing to the cloned fragment were detected in total RNA isolated from C. fimi cells grown on carboxymethyl cellulose but not from cells grown on glycerol or glucose. Transcription started at a cluster of sites 53 to 59 nucleotides upstream of a GUG translation initiation codon and terminated at either of two closely spaced C residues immediately downstream of a region of potential secondary structure. The size of the transcript was approximately 3.5 kilobases, sufficient to encode a polypeptide of 130 kilodaltons. The 130-kilodalton polypeptide is designated endoglucanase C (CenC), and the gene encoding it is designated cenC.

Amino Acid Sequence↗
PubMed1988

Precise excision of the cellulose binding domains from two Cellulomonas fimi cellulases by a homologous protease and the effect on catalysis.

An endo-beta-1,4-glucanase (CenA) and an exo-beta-1,4-glucanase (Cex) were prepared from Escherichia coli expressing recombinant DNA of the cellulolytic bacterium Cellulomonas fimi. Purification was facilitated by the high affinities of these enzymes for cellulose. An extracellular C. fimi protease cleaved both enzymes in vivo in a highly specific manner. The affinity of the parent enzyme for cellulose was contained independently in an amino-terminal fragment (p20) of CenA and a carboxyl-terminal fragment (p8) of Cex. These fragments contained homologous amino acid sequences which were proposed to comprise cellulose binding domains. Corresponding fragments, p30 from CenA and p35 from Cex, which were unable to bind to cellulose, contained catalytic domains. In both enzymes, the two functional domains were joined by a hinge region consisting solely of prolyl and threonyl residues. The binding domain was excised from CenA by proteolytic cleavage immediately adjacent to the carboxyl terminus of this hinge. Cex was cleaved at an exactly analogous site. p30 and p35 retained several of the catalytic functions of their parent enzymes. However, p30 was less active than intact CenA against microcrystalline cellulose implying a critical role for the binding domain of CenA in the hydrolysis of crystalline substrate.

Actinomycetales↗
PubMed1988

Mallet finger: a trial of two splints.

Over a period of twelve months, 116 cases of mallet finger were allocated randomly to treatment with either a Stack or Abouna splint. The two splints were equally effective, producing a cure or a significant improvement in approximately 50% of cases. However, the Stack splint was much preferred by the patients, who found it more comfortable, more robust and easier to keep clean.

Adult↗
PubMed1988

Mallet finger: comparison between operative and conservative management in those cases failing to be cured by splintage.

Sixty-nine patients with mallet finger who failed to be cured by a period of splintage were offered either tenodermodesis or Kirschner wire fixation of the DIP joint. Eleven (16%) accepted the offer and eight of these were significantly improved. Of those patients declining surgery, 30 were available for review after a minimum period of six months; of these 13 (43%) had undergone a significant spontaneous improvement.

Bone Wires↗
PubMed1988

The microvascular anatomy of the distal digital extensor tendon.

The circulation of six hands from four human cadavers was washed out, the hand then being injected with contrast material to provide 24 fingers for analysis under the dissecting and light microscopes. The microvascular anatomy of the distal part of the extensor tendon was outlined and is described, with particular emphasis on the fine vessels within the tendon and on the tendon surface. An area of deficient blood supply was shown within the tendon which may have implications in the aetiology and treatment of mallet finger.

Cadaver↗
PubMed1988

Expression in Escherichia coli of the Cellulomonas fimi Structural Gene for Endoglucanase B.

Endoglucanase B (EB) of Cellulomonas fimi has an M(r) of 110,000 when it is produced in Escherichia coli. The level of expression of the cenB gene (encoding EB) was significantly increased by replacing its normal transcriptional and translational regulatory signals with those of the E. coli lac operon. EB was purified to homogeneity from the periplasmic fraction of E. coli in one step by affinity chromatography on microcrystalline cellulose (Avicel). Alignment of the NH(2)-terminal amino acid sequence with the partial nucleotide sequence of a fragment of C. fimi DNA showed that EB is preceded by a putative signal polypeptide of 33 amino acids. The signal peptide functions and is processed correctly in E. coli, even when its first 15 amino acids are replaced by the first 7 amino acids of beta-galactosidase. The intact EB polypeptide is not required for enzymatic activity. Active polypeptides with M(r)s of 95,000 and 82,000 also appear in E. coli, and a deletion mutant of cenB encodes an active polypeptide with an M(r) of 72,000.

Journal Article↗