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Biomedical subjects

M J Kershaw

Publications and source records attributed to M J Kershaw.

13 recordsLinked to original sources
PubMed2001

Complementation of the Magnaporthe grisea deltacpkA mutation by the Blumeria graminis PKA-c gene: functional genetic analysis of an obligate plant pathogen.

Obligate plant-pathogenic fungi have proved extremely difficult to characterize with molecular genetics because they cannot be cultured away from host plants and only can be manipulated experimentally in limited circumstances. Previously, in order to characterize signal transduction processes during infection-related development of the powdery mildew fungus Blumeria graminis (syn. Erysiphe graminis) f. sp. hordei, we described a gene similar to the catalytic subunit of cyclic AMP-dependent protein kinase A (here renamed Bka1). Functional characterization of this gene has been achieved by expression in a deltacpkA mutant of the nonobligate pathogen Magnaporthe grisea. This nonpathogenic M. grisea deltacpkA mutant displays delayed and incomplete appressorium development, suggesting a role for PKA-c in the signal transduction processes that control the maturation of infection cells. Transformation of the deltacpkA mutant with the mildew Bka1 open reading frame, controlled by the M. grisea MPG1 promoter, restored pathogenicity and appressorium maturation kinetics. The results provide, to our knowledge, the first functional genetic analysis of pathogenicity in an obligate pathogen and highlight the remarkable conservation of signaling components regulating infection-related development in pathogenic fungi.

Archaeal Proteins↗
PubMed1998

Complementation of the mpg1 mutant phenotype in Magnaporthe grisea reveals functional relationships between fungal hydrophobins.

The functional relationship between fungal hydrophobins was studied by complementation analysis of an mpg1(-) gene disruption mutant in Magnaporthe grisea. MPG1 encodes a hydrophobin required for full pathogenicity of the fungus, efficient elaboration of its infection structures and conidial rodlet protein production. Seven heterologous hydrophobin genes were selected which play distinct roles in conidiogenesis, fruit body development, aerial hyphae formation and infection structure elaboration in diverse fungal species. Each hydrophobin was introduced into an mpg1(-) mutant by transformation. Only one hydrophobin gene, SC1 from Schizophyllum commune, was able partially to complement mpg1(-) mutant phenotypes when regulated by its own promoter. In contrast, six of the transformants expressing hydrophobin genes controlled by the MPG1 promoter (SC1 and SC4 from S.commune, rodA and dewA from Aspergillus nidulans, EAS from Neurospora crassa and ssgA from Metarhizium anisopliae) could partially complement each of the diverse functions of MPG1. Complementation was always associated with partial restoration of a rodlet protein layer, characteristic of the particular hydrophobin being expressed, and with hydrophobin surface assembly during infection structure formation. This provides the first genetic evidence that diverse hydrophobin-encoding genes encode functionally related proteins and suggests that, although very diverse in amino acid sequence, the hydrophobins constitute a closely related group of morphogenetic proteins.

Ascomycota↗
PubMed1998

Hydrophobins and repellents: proteins with fundamental roles in fungal morphogenesis.

Fungal hydrophobins are secreted proteins which react to interfaces between fungal cell walls and the air or between fungal cell walls and solid surfaces. They have been shown to be important in many morphogenetic processes, including sporulation, fruit body development, and infection structure formation. Hydrophobins form hydrophobic surface layers by self-assembly of secreted protein monomers in response to the environment. This process results in amphipathic polymers of interwoven rodlets on surfaces of fungal aerial structures and hyphal aggregations. Hydrophobin self-assembly is also involved in attachment of hyphae to hydrophobic surfaces and this may act as a conformational cue for certain developmental processes. Although hydrophobins appear to be ubiquitous among fungal taxa, a second class of fungal protein with very different biochemical characteristics could fulfill a similar role. These proteins, called repellents, have been identified in only one fungal species so far, but clearly help to make aerial hyphae hydrophobic. The functional similarities between hydrophobins and repellents highlight the importance of aerial development to the fungal lifestyle.

Amino Acid Sequence↗
PubMed1996

Cloning and sequence analysis of an intron-containing domain from a peptide synthetase-encoding gene of the entomopathogenic fungus Metarhizium anisopliae.

A gene encoding a putative peptide synthetase has been cloned and partially sequenced from the filamentous fungus, Metarhizium anisopliae. The deduced amino acid sequence of one entire domain and the following spacer is typical of fungal peptide synthetases, showing good conservation of the six expected core sequences. There are two introns within this region, the first interrupting core 5 (RLDLTDIE) of the domain and the second in a conserved area of the spacer region.

Amino Acid Sequence↗
PubMed1987

A single autoantigen in Goodpasture's syndrome identified by a monoclonal antibody to human glomerular basement membrane.

A mouse monoclonal antibody (P1) to the autoantigenic component of human glomerular basement membrane (GBM) was used to study the immunochemistry and tissue distribution of the Goodpasture antigen and the specificity of the human autoimmune response in Goodpasture's syndrome (anti-GBM disease). In solid phase assays, monoclonal antibody P1 bound to collagenase-solubilized human GBM (the ligand used in assays for human autoantibody), but not to other biochemically defined components of basement membrane. On Western blotting, P1 bound to the same 6 bands in solubilized GBM (between 26 and 58 kilodaltons with major bands at 26 and 54 kilodaltons) that were recognized by sera from all 42 patients studied with anti-GBM disease. Preincubation with sera from 8/8 patients blocked the subsequent binding of P1 from 83 to 89% on densitometer scanning of the Western blot; and preincubation with P1 blocked the binding of sera from 6/6 patients from 58 to 89%. Indirect immunofluorescence and immunoperoxidase studies revealed that the pattern of binding of P1 was identical to that of antibody eluted from the kidneys of a patient with Goodpasture's syndrome; there was linear binding to GBM, Bowman's capsule, and distal tubular basement membrane. In addition, P1 bound to basement membranes in lung and choroid plexus, and to membranes of the lens capsule, choroid, and retina of the eye and cochlea, but not to other organs studied. It is concluded that there is a single major autoantigenic component of human GBM (the Goodpasture antigen), which is present on fragments of different molecular weight in the collagenase digest. This antigen is distributed throughout well-defined basement membranes known to be involved in both Goodpasture's and Alport's syndromes. Human anti-GBM antibodies bind to the same (or closely related) determinants which are recognized by P1, demonstrating that the autoimmune response in Goodpasture's syndrome is of highly restricted specificity.

Anti-Glomerular Basement Membrane Disease↗
PubMed1986

The Goodpasture antigen in Alport's syndrome: studies with a monoclonal antibody.

A mouse monoclonal antibody (MCA-P1), which recognizes an antigenic determinant in human glomerular basement membrane against which autoantibodies are directed in Goodpasture's syndrome, was used in indirect immunofluorescence studies to investigate glomerular basement membrane structure in Alport's syndrome. We found reduced or absent binding of MCA-P1 to glomerular and distal tubular basement membranes in renal biopsy tissue from ten patients with Alport's syndrome. Antiglomerular basement membrane antibody eluted from the kidneys of a patient who had died from Goodpasture's syndrome was used to confirm these findings. In contrast, there was bright linear fluorescence of MCA-P1 on glomerular and tubular basement membranes of normal renal material and renal biopsy tissue obtained from patients with a variety of glomerulonephritides. These results suggest an abnormality or a variable quantity of the immunoreactive autoantigen in the glomerular basement membrane of patients with Alport's syndrome. Furthermore, MCA-P1 may be of value in the diagnostic interpretation of renal biopsies from patients with familial nephritis.

Anti-Glomerular Basement Membrane Disease↗
PubMed1986

Hyperamylasemia following methyl alcohol intoxication. Source and significance.

Methyl alcohol intoxication has been reported to cause hyperamylasemia and pancreatitis. We describe a patient with severe, nonfatal methyl alcohol intoxication who had a rise in serum amylase activity with the level peaked on the second hospital day at tenfold the upper limit of normal. However, isoamylase analysis showed that this striking hyperamylasemia was due to salivary-type amylase. Furthermore, the serum lipase activity remained entirely normal during the peak amylase elevation. Thus, in cases of methyl alcohol intoxication, as in other clinical situations, hyperamylasemia, even when striking, should not be equated with pancreatitis. More specific laboratory tests for pancreatitis should be used before embarking on extensive investigations of the pancreas.

Acute Disease↗
PubMed1984

Automated kinetic assay for lactate dehydrogenase isoenzyme 1 by centrifugal analysis after alkaline inactivation.

Lactate dehydrogenase isoenzyme 1 (LD1, EC 1.1.1.27) has been used widely as a marker for myocardial infarction, and many analytical methods for it have been developed, most of which are relatively labor intensive. On the basis of a recent report by Takizawa et al. (Clin Chem 29: 1941-1945, 1983) describing the marked stability of LD1 in buffered alkaline solutions, and with use of a centrifugal analyzer, we have developed a fully automated assay for LD1. Results with this method are precise (between-day SD = 2.1 U/L), vary linearly with LD1 activity to 600 U/L, and correlate well with LD1 as determined by immunological (Roche Isomune-LD) and agarose electrophoretic methods (r = 0.985 and 0.986, respectively). Furthermore, the method is easy and convenient, and should provide a substantial savings in labor and reagent costs to laboratories currently determining LD1 by electrophoretic or immunologic methods.

Autoanalysis↗
PubMed1984

Direct analysis for urinary protein with biuret reagent, with use of urine ultrafiltrate blanking: comparison with a manual biuret method involving trichloroacetic acid precipitation.

We describe a method for measuring urinary protein with a centrifugal analyzer. Biuret reagent is used, and blanking with an ultrafiltrate of urine eliminates interferences from the nonprotein, biuret-positive chromogens in urine. We compare results by this new method with those by a manual method in which trichloroacetic acid precipitation and biuret reagent are used. The new method shows good precision and excellent correlation (r = 0.997) with the manual method. The ease and convenience of this assay should make this a useful method for the routine clinical laboratory.

Anti-Bacterial Agents↗