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Biomedical subjects

J Knowles

Publications and source records attributed to J Knowles.

At least 37 records · Page 2Linked to original sources
PubMed1994

The precursor of the Streptomyces R61 DD-peptidase containing a C-terminal extension is inactive.

The Streptomyces R61 DD-peptidase gene encodes a 26-residue C-terminal extension which is not found in the mature protein. When the gene was expressed in Escherichia coli, the extension was not cleaved and the precursor protein was not enzymatically active. It also reacted with penicillins significantly more slowly than the mature protein. The introduction of a 'stop' codon after that corresponding to the C-terminal residue of the mature protein resulted in the production of an active protein in the periplasm of E. coli.

Amino Acid Sequence↗
PubMed1994

Knee function after patellectomy. A 12- to 48-year follow-up.

We describe 83 knees (69 patients) which had had patellectomy for anterior knee pain (52), patellofemoral osteoarthritis (25) or comminuted fractures (6) between 1942 and 1978. The patients were questioned about their symptoms and the function of the operated knee 14 to 50 years after operation. In the group with anterior knee pain, 76% achieved good results and were satisfied with the operation. Only 54% of the osteoarthritis group had satisfactory relief of pain and most had progressive deterioration of function. Sixteen patients who had had unilateral patellectomy were assessed by dynamometry, ultrasound and radiography. The average quadriceps muscle power was 60% of that on the normal side although two patients had stronger muscles in their operated than in their unoperated legs.

Adult↗
PubMed1993

Inhibition of axonal growth by SNAP-25 antisense oligonucleotides in vitro and in vivo.

Axonal elongation and the transformation of growth cones to synaptic terminals are major steps of brain development and the molecular mechanisms involved form the basis of the correct wiring of the nervous system. The same mechanisms may also contribute to the remodelling of nerve terminals that occurs in the adult brain, as a morphological substrate to memory and learning. We have investigated the function of the nerve terminal protein SNAP-25 (ref. 2) during development. We report here that SNAP-25 is expressed in axonal growth cones during late stages of elongation and that selective inhibition of SNAP-25 expression prevents neurite elongation by rat cortical neurons and PC-12 cells in vitro and by amacrine cells of the developing chick retina in vivo. These results demonstrate that SNAP-25 plays a key role in axonal growth. They also suggest that high levels of SNAP-25 expression in specific areas of the adult brain may contribute to nerve terminal plasticity.

Animals↗
PubMed1993

Open reduction-fixation of mandibular subcondylar fractures. A review.

From 1973 to 1990, 392 mandibular subcondylar fractures were treated at the University of California, Davis, by the Otolaryngology Department. Of these, 17% were handled by open reduction and internal fixation. Twenty-one patients from this group were located for follow-up at an average interval of 64 months. Retrospective review shows the operation to be safe, with few complications and no permanent sequelae. Patient examination often revealed abnormalities of occlusion and mandibular function; however, these objective findings did not correlate well with patients' relative lack of subjective complaints. An 86% incidence of roentgenographic evidence of condylar disease after open reduction and internal fixation was found. We question the long-term efficacy of open reduction and internal fixation in restoring fracture alignment and maintaining mandibular height given the high rate (86%) of condylar disease in our patient population.

Adolescent↗
PubMed1993

Development of a degradable composite for orthopaedic use: mechanical evaluation of an hydroxyapatite-polyhydroxybutyrate composite material.

This study evaluates the mechanical properties of a composite material comprising polyhydroxybutyrate with hydroxyapatite added in proportions varying from 0 to 50%. Among the three methods of production, injection moulding was found to result in the most satisfactory mechanical properties. The tensile and compressive strength and the modulus of elasticity of composite produced in this way fell within the range for fresh human bone from different anatomical sites. With the additional advantages of biocompatibility, biodegradability and the potential for piezoelectric stimulation of new local bone formation, it was concluded that the injection-moulded composite material has considerable potential for use in orthopaedic surgery, both as a material to construct certain orthopaedic implants and as an alternative to corticocancellous bone graft.

Biocompatible Materials↗
PubMed1991

Genetic engineering of Trichoderma to produce strains with novel cellulase profiles.

Genetic engineering has been used to modify the proportion of different cellulases produced by a hypercellulolytic Trichoderma reesei mutant strain. A general expression vector, pAMH110, containing the promoter and terminator sequences of the strongly expressed main cellobiohydrolase 1 (cbh1) gene was used to overexpress a cDNA coding for EGI, the major endoglucanase (1,4,beta-D-glucan glucanohydrolase, EC 3.2.1.4). An in vitro modified cbh1 cDNA, incapable of coding for active enzyme, was used to inactivate the major cellobiohydrolase (1,4-beta-D-glucan cellobiohydrolase, EC 3.2.1.91) gene. In this way, new strains producing elevated amounts of the specific endoglucanase 1 (EGI) and/or lacking the major cellobiohydrolase (CBHI) were produced, and these have been further characterized.

Base Sequence↗
PubMed1990

Genetic homogeneity between acute and chronic forms of spinal muscular atrophy.

The childhood-onset spinal muscular atrophies (SMAs) describe a heterogeneous group of disorders that selectively affect the alpha motoneuron. We have shown that chronic childhood-onset SMA (SMA II and III) maps to a single locus on chromosome 5q. Acute SMA (SMA Type I/Werdnig-Hoffmann/severe/infantile) is the main cause of heritable infant mortality. Mapping the acute SMA locus by conventional methods is complicated by the rapidly fatal course of the disease and its recessive mode of inheritance. We present here the typing of four inbred acute-SMA families with DNA markers on chromosome 5q and analysis of these together with acute families from our previous study to demonstrate genetic homogeneity between the acute and chronic forms of SMA. The data indicate that the acute SMA locus maps to chromosome 5q11.2-13.3. Two families seem unlinked to 5q markers, raising the possibility of genetic heterogeneity or disease misclassification within the acute and chronic family sets.

Acute Disease↗
PubMed1990

Recombinant brewer's yeast strains suitable for accelerated brewing.

Four brewer's yeast strains carrying the alpha-ald gene of Klebsiella terrigena (ex. Aerobacter aerogenes) or of Enterobacter aerogenes on autonomously replicating plasmids were constructed. The alpha-ald genes were linked either to the ADC1 promoter or to the PGK1 promoter of yeast Saccharomyces cerevisiae. In pilot scale brewing (50 l) with three of these recombinant yeasts the formation of diacetyl in beer was so low during fermentation that lagering was not required. All other brewing properties of the strains were unaffected and the quality of finished beers was as good as that of finished beer prepared with the control strain. The total process time of beer production could therefore be reduced to 2 weeks, in contrast to about 5 weeks required in the conventional process.

Base Sequence↗
PubMed1990

Site-directed mutagenesis of a thermostable alpha-amylase from Bacillus stearothermophilus: putative role of three conserved residues.

The relationship between structure, activity, and stability of the thermostable Bacillus stearothermophilus alpha-amylase was studied by site-directed mutagenesis of the three most conserved residues. Mutation of His-238 to Asp involved in Ca2+ and substrate binding reduced the specific activity and thermal stability, but did not affect the pH and temperature optima. Replacement of Asp-331 by Glu in the active site caused almost total inactivation. Interestingly, in prolonged incubation this mutant enzyme showed an altered end-product profile by liberating only maltose and maltotriose. Conservative mutation of the conserved Arg-232 by Lys, for which no function has yet been proposed, resulted in lowered specific activity: around 12% of the parental enzyme. This mutant enzyme had a wider pH range but about the same temperature optimum and thermal stability as the wild-type enzyme. Results obtained with different mutants were interpreted by computer aided molecular modeling.

Amino Acid Sequence↗
PubMed1989

Crystallization of the core protein of cellobiohydrolase II from Trichoderma reesei.

Single crystals of the core protein of the cellulase cellobiohydrolase II have been grown in polyethylene glycol 6000 with the hanging drop method. Successful crystallization occurred only when 82 amino acids were removed from the N terminus by papain cleavage. Crystals belong to the space group P2(1) and have cell constants a = 49.1 A, b = 75.8 A, c = 92.9 A, beta = 103.2. The diffraction pattern extends to better than 2.0 A.

Cellulose 1,4-beta-Cellobiosidase↗
PubMed1989

Determination of the three-dimensional solution structure of the C-terminal domain of cellobiohydrolase I from Trichoderma reesei. A study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing.

The solution structure of a synthetic 36-residue polypeptide comprising the C-terminal cellulose binding domain of cellobiohydrolase I (CT-CBH I) from Trichoderma reesei was investigated by nuclear magnetic resonance (NMR) spectroscopy. The 1H NMR spectrum was completely assigned in a sequential manner by two-dimensional NMR techniques. A large number of stereospecific assignments for beta-methylene protons, as well as ranges for the phi, psi, and chi 1 torsion angles, were obtained on the basis of sequential and intraresidue nuclear Overhauser enhancement (NOE) and coupling constant data in combination with a conformational data base search. The structure calculations were carried out in an iterative manner by using the hybrid distance geometry-dynamical simulated annealing method. This involved computing a series of initial structures from a subset of the experimental data in order to resolve ambiguities in the assignments of some NOE cross-peaks arising from chemical shift degeneracy. Additionally, this permitted us to extend the stereospecific assignments to the alpha-methylene protons of glycine using information on phi torsion angles derived from the initial structure calculations. The final experimental data set consisted of 554 interproton distance restraints, 24 restraints for 12 hydrogen bonds, and 33 phi, 24 psi, and 25 chi 1 torsion angle restraints. CT-CBH I has two disulfide bridges whose pairing was previously unknown. Analysis of structures calculated with all three possible combinations of disulfide bonds, as well as without disulfide bonds, indicated that the correct disulfide bridge pairing was 8-25 and 19-35. Forty-one structures were computed with the 8-25 and 19-35 disulfide bridges, and the average atomic rms difference between the individual structures and the mean structure obtained by averaging their coordinates was 0.33 +/- 0.04 A for the backbone atoms and 0.52 +/- 0.06 A for all atoms. The protein has a wedgelike shape with an amphiphilic character, one face being predominantly hydrophilic and the other mainly hydrophobic. The principal element of secondary structure is made up of an irregular triple-stranded antiparallel beta-sheet composed of residues 5-9 (beta 1), 24-28 (beta 2), and 33-36 (beta 3) in which strand beta 3 is hydrogen bonded to the other two strands.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1989

Detection of feline calicivirus antigens in the joints of infected cats.

Twelve specific pathogen free cats were used to investigate the role of calicivirus in causing lameness. These were divided into two groups each of six cats; one group of cats had previously been vaccinated, the other had not. Three cats in each group were given live vaccine virus (F9 related) by the subcutaneous route and two in each group were challenged intranasally with field virus (A4), either four or seven days before euthanasia. The other two cats were controls. Virus was isolated from the oropharynx of five cats and the conjunctiva of a single cat. Four of these cats had been given the field virus and two the vaccine strain; the latter two cats had been previously immunised and had high circulating neutralising antibodies to calicivirus. No virus was isolated from the joints of any cat but immunofluorescence examination revealed viral antigens within the synovial macrophages of 14 joints from five cats, three having been given the field virus and two the vaccine virus seven days before euthanasia. Immunofluorescence also demonstrated the presence of immunoglobulin and complement within synovial macrophages suggesting that the virus was in the form of an immune complex. No lameness was reported in any cat and the synovial histological changes were minimal.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Isolation and characterization of the 3-phosphoglycerate kinase gene (pgk) from the filamentous fungus Trichoderma reesei.

The 3-phosphoglycerate kinase gene (pgk) from Trichoderma reesei was isolated by hybridization with the corresponding Saccharomyces cerevisiae PGK gene. The 1,545 nt long nucleotide sequence of the cloned gene codes for a 416 amino acid protein. The coding sequence contains two introns of 219 and 75 nt, respectively, at positions identical to those corresponding genes from the other filamentous fungi Aspergillus nidulans and Penicillum chrysogenum. This gene codes for two mRNAs of about 1.65 kb and 1.85 kb. The PGK protein of Trichoderma shows extensive homology to the PGKs of other fungi A. nidulans (77%), P. chrysogenum (73%) and Saccharomyces cerevisiae (69%). However, the PGKs of the two other filamentous fungi, A. nidulans and P. chrysogenum, seem to be more closely related to each other than to the T. reesei enzyme.

Amino Acid Sequence↗
PubMed1988

Studies of the cellulolytic system of Trichoderma reesei QM 9414. Analysis of domain function in two cellobiohydrolases by limited proteolysis.

Limited action of papain on the native forms of two cellobiohydrolases (CBH) from Trichoderma reesei (CBH I, 65 kDa, and CBH II, 58 kDa) leads to the isolation of the respective core fragments (56 kDa and 45 kDa) which are fully active on small, soluble substrates, but have a strongly reduced activity (respectively 10% and 50% of the initial value) on microcrystalline cellulose (Avicel). By partial sequencing at the C terminus of the CBH I core and at the N terminus of the CBH II core the papain cleavage sites have been assigned in the primary structures (at about residue 431 and 82 respectively). This limited action of papain on the native enzymes indicates the presence of hinge regions linking the core to these terminal glycopeptides. The latter conserved sequences appear either at the C or N terminus of several cellulolytic enzymes from Trichoderma reesei [Teeri et al. (1987) Gene 51, 43-52]. The specific activities of the intact enzymes and their cores on two forms of insoluble cellulose (crystalline, amorphous) differentiate the CBH I and CBH II in terms of adsorption and catalytic properties. Distinct functions can be attributed to the terminal peptides: for intact CBH II the N-terminal region contributes in the binding onto both cellulose types; the homologous C-terminal peptide in CBH I, however, only affects the interaction with microcrystalline cellulose. It could be inferred that CBH I and its core bind preferentially to crystalline regions. This seems to be corroborated by the results of CBH I/CBH II synergism experiments.

Amino Acid Sequence↗
PubMed1987

Construction of cDNA libraries by blunt-end ligation: high-frequency cloning of long cDNAs from filamentous fungi.

A simplified cDNA synthesis and cloning method, suitable for efficient generation of cDNA libraries at frequencies up to 10(6) clones/micrograms mRNA, is described. Routine synthesis of transcripts of well over 4 kb is facilitated by the use of high-quality RNA template isolated from materials rich in RNases. Laborious cloning steps, like tailing or addition of linkers, can be omitted by the use of efficient blunt-end ligation to plasmid vectors, and rapid verification as well as characterization of the clones is possible by double-stranded plasmid sequencing. Using this method we have constructed several cDNA libraries of different filamentous fungi and show here the synthesis and cloning of cDNA copies larger than 1.8 kb corresponding to three Trichoderma reesei cellulases.

Bacteriophage lambda↗
PubMed1987

A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei.

An efficient transformation system for the cellulolytic filamentous fungus Trichoderma reesei has been developed. Transformation was obtained with plasmid carrying the dominant selectable marker amdS or the argB gene of Aspergillus nidulans, which was found to complement the respective argB mutation of T. reesei. The transformation frequency can be up to 600 transformants per microgram of transforming DNA. The efficiency of co-transformation with unselected DNA was high (approx. 80%). The transforming DNA was found to be integrated at several different locations, often in multiple tandem copies in the T. reesei genome. In addition, the Escherichia coli beta-galactosidase was expressed in T. reesei in enzymatically active form from the A. nidulans gpd promoter.

Amidohydrolases↗
PubMed1987

Homologous domains in Trichoderma reesei cellulolytic enzymes: gene sequence and expression of cellobiohydrolase II.

Fungal cellobiohydrolases are unique enzymes capable of degrading highly ordered crystalline cellulose. We present here the isolation and complete sequence analysis of the chromosomal and cDNA copies of the structural gene (cbh2) coding for one of the major cellobiohydrolases (CBH II) of Trichoderma reesei. We also present data on expression of the cbh2 gene and show that the transcription start points of the cbh2 gene are heterogeneous and are located 32 to 52 bp downstream from a putative TATA box. The derived CBH II protein sequence is 471 amino acids long and the coding region is interrupted by three short introns. Most of the CBH II protein bears no apparent resemblance to CBH I and endoglucanase I. However, a short region of extensive homology is found in all Trichoderma cellulases characterized so far, suggesting that this region is important for cellulose hydrolysis. The implications of this information with regard to the evolution of fungal cellulase genes and the enzymology of cellulose hydrolysis are discussed.

Amino Acid Sequence↗