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Biomedical subjects

M J Perry

Publications and source records attributed to M J Perry.

At least 91 records · Page 5Linked to original sources
PubMed1993

Localization and activity of transglutaminase, a retinoid-inducible protein, in developing rat spinal cord.

The distribution of the retinoid-inducible enzyme, tissue transglutaminase (tTG) in developing rat spinal cord was determined by enzyme assay and immunocytochemistry. tTG activity was at its highest in the forebrain in late foetal development. In hindbrain and spinal cord, elevated activity persisted until after birth. In spinal cord only, a second peak of activity occurred during the first week post partum (P3). tTG was associated with both the cytosolic and particulate tissue fractions throughout spinal cord development, but the particulate component was more prominent in the early postnatal period. tTG was more concentrated during this period in the ventral horn, where the particulate-associated enzyme activity was highest. In spinal cord at 3 days post partum, particulate tTG could be solubilized with lubrol-PX, dithiothreitol and potassium thiocyanate. Both soluble and particulate-associated tTG coeluted with guinea-pig liver transglutaminase C by DEAE-sephacel chromatography. The first peak of tTG activity during late foetal life coincided with the transient localization of the enzyme by immunocytochemistry in vascular endothelia throughout the spinal cord. The second peak of activity at 3 days post partum, by which time vascular immunoreactivity was absent, coincided with the occurrence of small numbers of intensely immunoreactive motor neurones in the ventral horn. Immunoreactive motor neurones were seen predominantly at two levels: the lower thoracic segments and lumbar enlargement. The abnormal appearance of many immunoreactive neurones suggested degenerative changes were occurring. tTG was also present in central canal cluster cells from birth onwards. No neuronal immunoreactivity was seen throughout foetal development. A proportion of motor neurones prepared from E15 spinal cord and grown in coculture with spinal cord astrocytes, were immunoreactive for tTG. All immunoreactive neurones showed signs of degeneration. Addition of myotube-conditioned medium (a source of cholinergic differentiation factor, CDF) reduced the proportion of tTG-immunoreactive neurons in the cultures. Schwann cell-conditioned medium (a source of ciliary neurotrophic factor, CNTF) had a similar but less potent effect on the numbers of immunoreactive neurones. The possibility that tTG is a marker for late, but not early-phase programmed cell death in the developing rat spinal cord is discussed in the light of a proposed role for tTG in the mechanism of natural cell death by apoptosis.

Animals↗
PubMed1993

Cellular transglutaminases in neural development.

Enzymes of the transglutaminase family catalyze the Ca(2+)-dependent covalent cross-linking of peptide-bound glutamine residues of proteins and glycoproteins to the epsilon-amino group of lysine residues to create inter- or intramolecular isopeptide bonds. Transglutaminases can also covalently link a variety of primary amines to peptide-bound glutamine residues giving rise to two possibilities; firstly, where the primary amine has two or more amino groups, further catalysis can result in the formation of cross-linked bridges between glutamine residues, and secondly, where the primary amine is a monoamine, glutamine residues are rendered inert to further modification. The products are therefore in the main, homo- or heterodimers, or extensive, metabolically-stable multimeric complexes or matrices. Ca(2+)-dependent transglutaminase activity is present in the mammalian peripheral and central nervous systems and transglutaminase-catalyzed cross-linking of endogenous substrates has been demonstrated in neurons of Aplysia and the mammalian brain. Transglutaminase activity increases in the brain during development, principally owing to the increasing preponderance of glial cell activity. In a few regions including the cerebellar cortex, activity is also high in early development. Cellular transglutaminases occur widely in differentiating cells and tissues in mammals, with more than one transglutaminase frequently associated with a single cell type. The primary protein sequences of three cellular transglutaminases have been fully determined in different species, together with that of a mammalian protein homologue (band 4.2) which shares extensive sequence homologies with transglutaminases, but lacks the active site cysteine residue. The upstream sequences of two mammalian cellular transglutaminase genes (C and K) contain numerous regulatory sites, and an invertebrate transglutaminase, annulin, is spatially regulated within homeodomains. Multiple molecular forms of transglutaminase C and possibly other cellular transglutaminases exist in mammalian brain. The emerging picture is one of a family of cytosolic and membrane-bound proteins central to several regulatory pathways whose functions is to stabilize the cellular and intercellular superstructure in growing organisms. The targeted formation of glu-lys isopeptide bonds between proteins is central to this function. Cytoskeletal proteins, membrane-associated receptors, enzymes in signal transduction pathways and extracellular glycoproteins are candidate substrates as are polyamines, but few cellular proteins have been identified as components of naturally-occurring covalently-bonded matrices. Transglutaminases participate in the programme of neuronal differentiation in some but not all classes of neurone. Both neuronal and non-neuronal expression of transglutaminases may be important for guidance of migrating neurons or growth cones and sustainment of cell shape and coordinates during development.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1991

Neurofilament immunoreactivity in populations of rat primary afferent neurons: a quantitative study of phosphorylated and non-phosphorylated subunits.

Neurofilament subunits in rat dorsal root ganglion (DRG) neurons were examined using five antibodies: NFH, RT97 and NFHP- recognise the 200 kDa subunit (NF200); NFH recognises both phosphorylated and non-phosphorylated forms of NF200 whereas RT97 and NFHP- are specific for the phosphorylated and non-phosphorylated forms respectively; 155 and anti-68 kD recognise the 155 kDa and 68 kDa subunits respectively. All the antibodies apart from NFHP- distinguished between the two populations of neurons corresponding to the light (L) and small dark (SD) cell types as previously shown for RT97. This demonstrates that L and SD neurons contain different levels of neurofilament and that the ability to discriminate between them is not unique to the antibody RT97. It is also evident that DRG neurons contain neurofilament composed of all three subunits. Since NFH and RT97, but not NFHP-, distinguished between the two populations, it appears that it is the presence of the phosphorylated form of NF200 that provides the basis for discrimination between the two cell types. After dephosphorylation of the neurofilament, NFHP- also discriminated between the two populations, indicating that there is more NF200 regardless of phosphorylation state in the L neurons. Observations made from unfixed DRGs indicate that all neurons contain some neurofilament and the neurofilament rich and neurofilament poor populations were also apparent. The use of colchicine apparently caused a small increase in neurofilament levels in at least some perikarya, presumably due to its blocking effect on axoplasmic transport. This caused some SD neurons to become neurofilament rich. We conclude that L neurons contain more neurofilament than SD neurons since both cell types contain non-phosphorylated NF200, but the L neurons also contain a much greater amount of the phosphorylated form.

Animals↗
PubMed1991

Populations of rat spinal primary afferent neurons with choleragenoid binding compared with those labelled by markers for neurofilament and carbohydrate groups: a quantitative immunocytochemical study.

A quantitative comparison was made of the population of L5 dorsal root ganglion neurons that bind choleragenoid and the population of neurons labelled by RT97, an anti-neurofilament antibody, SSEA3, an antibody to a globoseries carbohydrate group and 1B2, an antibody to a lactoseries carbohydrate group. Neurons with choleragenoid binding formed a population of 37% of all the cells and 94% of these were also immunoreactive to RT97. The choleragenoid-and RT97-positive groups of cells showed the same size distribution. Two small populations of cells existed which were choleragenoid negative/RT97 positive and choleragenoid positive/RT97 negative. SSEA3 immunoreactivity was found in 8% of the dorsal root ganglion neurons which were of all sizes. The 1B2 immunoreactivity was found in 32% of the dorsal root ganglion cells, the majority of which were in the small size range. More than half of the SSEA3-positive cells but only a few of the 1B2-positive neurons were labelled for choleragenoid. These results demonstrate that choleragenoid labels the majority of the light (RT97-positive) cell population and that light cells unlabelled by choleragenoid are small in number and spread across a wide size range.

Animals↗
PubMed1988

Monoclonal antibodies to crosslinked fibrin degradation products (XL-FDP). I. Characterization and preliminary evaluation in plasma.

Monoclonal antibodies (mabs) were raised against X-oligomers, the earliest soluble fragments released from crosslinked fibrin (XL-FN), by the action of plasmin. Two of the mabs (NIBn 52 and NIBn 123) were monospecific for X-oligomers in that they showed no binding to fibrinogen, the plasmic fragments of fibrinogen (D and E) and non-crosslinked fibrin (X, Y, D and E), or the terminal digestion product of XL-FN, fragment DD-E. One other mab (NIBn 178) was panspecific for X-oligomers in that it exhibited a weak affinity for fibrinogen. The mabs were used to develop a two-site immunoradiometric assay (IRMA) and an enzyme-linked immunospecific assay (ELISA) which permitted the specific measurement of X-oligomers directly in plasma, rather than in serum. This immunoassay is a true assay of fibrinolysis as distinct from fibrinogenolysis and may be a potential aid in the diagnosis and evaluation of thrombosis. In preliminary studies, the assay detected low levels of X-oligomers in normal plasma and elevated levels in patients with disseminated intravascular coagulation.

Antibodies, Monoclonal↗
PubMed1987

Bad lights stops play.

The visual performance of cricket batsmen is simulated over a wide range of ambient illumination. When illumination falls to the level at which they usually stop batting (the gloom level) a rapid increase in reaction time commences. It is also the level at which office and car sidelights are turned on and the sensation of "gloom" occurs.

Adult↗
PubMed1986

Plasma levels of fibrin fragments in men with myocardial infarction.

Degradation products of cross-linked fibrin were measured in the plasma of 30 men who had been discharged from hospital following a myocardial infarct and in 30 age-matched controls selected from a general practice list. Levels were significantly higher in cases than controls, and the direction of the differences was highly consistent in case control pairs. These findings may represent increased thrombogenesis, increased fibrinolysis, or both, in persons who have suffered myocardial infarction.

Adult↗
PubMed1985

Unreliability of current serum fibrin degradation product (FDP) assays.

Previously, assays of fibrin-fibrinogen degradation products (FDP) had to be performed on serum samples. However, monoclonal antibodies (Mabs) are now available which permit the measurement of FDP directly in plasma. We have employed two Mabs, one monospecific for FDP originating from crosslinked fibrin and another panspecific for the FDP fraction, to determine normal FDP levels in plasma and serum. The monospecific Mab gave a value of 40 ng FDP/ml in plasma and 10 ng/ml in serum, while the serum level of FDP recorded using the panspecific Mab was greater than 1000 ng/ml, at all the concentrations of thrombin employed. Similarly, when a solution of purified fibrinogen was treated with thrombin, the concentration of FDP present in the clot supernatant was greater than 1000 ng/ml when assayed using the panspecific Mab. Thus during serum preparation as much as 75% of the native FDP is incorporated into the clot while in excess of 1000 ng/ml of laboratory generated FDP, probably incompletely polymerized fibrin, is measured using panspecific antisera. These data indicate that current FDP assays using polyclonal antibodies are not a reliable reflection of the FDP level generated in vivo. The use of FDP-specific Mabs which do not react with fibrinogen is recommended for future FDP assays performed directly on plasma.

Antibodies, Monoclonal↗
PubMed1984

Peptide heterogeneity in a preparation of synthetic fibrinopeptide B.

A commercially available preparation of synthetic human fibrinopeptide B (FpB) was shown by hplc to contain two chromatographically distinct peaks, one of which was identical to FpB. Our results suggest that the contaminant peptide (FpB-2), which represented approximately 43% of the total peptide composition, is FpB containing an alpha-aminosuccinimide (Asc) residue. This Asc residue probably arose as a result of the cyclization of 5Asp-6Asn during either the coupling or deprotection reactions. FpB-2 was rapidly hydrolysed by carboxypeptidase B to des-Arg-FpB-2. It was stable under acidic conditions but in dilute alkali was converted to equimolar amounts of FpB and FpB containing beta-Asp at residue 5. Since it has been suggested that 5Asp-6Asn is a major immunorecognition site in FpB, our observations emphasize the need to establish the purity of synthetic FpB preparations destined for use in the immunoassay of either FpB or des-Arg-FpB.

Amino Acids↗
PubMed1984

Single shot intrasplenic immunization: an advantageous procedure for production of monoclonal antibodies specific for human fibrin fragments.

We describe the use of the single shot intrasplenic immunization technique as a particularly effective procedure for the production of specific monoclonal antibodies against a high molecular weight antigen. We found that with this technique several different, completely specific monoclonal antibodies could be produced against high molecular weight crosslinked fibrin degradation products. These results contrasted with those obtained using conventional multidose immunization, which only produced monoclonal antibodies that were cross-reactive with fibrinogen and/or noncrosslinked fibrin degradation products.

Animals↗
PubMed1984

Aspergillic acids produced by mixed cultures of Aspergillus flavus and Aspergillus nidulans.

A mixed culture of Aspergillus nidulans (GH79) and Aspergillus flavus (CMI 91019B) produced two antibiotics, designated VI and VII, which were not elaborated when either fungus was grown alone. Chemical and spectroscopic analysis of VI, the major component, indicated that this compound was identical to hydroxyaspergillic acid. The minor component, VII, was produced in too low a yield for its identity to be established. However, partial characterization suggests that this antibiotic also belongs to the aspergillic acid group of mycotoxins.

Anti-Bacterial Agents↗
PubMed1983

Fusidane antibiotics produced by dermatophytes.

Isolates of Microsporum canis, Microsporum gypseum and Epidermophyton floccosum were observed to produce antibacterial activities under cross-resistance to fusidic acid. The activity from E. floccosum was shown to be due to fusidic acid, diketofusidic acid and 3-ketofusidic acid. Possible contributions of these antibiotics to microbial interaction during dermatophytosis is discussed.

Anti-Bacterial Agents↗
PubMed1980

Immunofluorescent Assay for the Marine Ammonium-Oxidizing Bacterium Nitrosococcus oceanus.

Nitrification is one of the important microbiological transformations of nitrogen in the ocean. Traditional enrichment-culture methods for enumerating the autotrophic bacteria which oxidize ammonium to nitrite are very time consuming (months) and are believed to seriously underestimate natural abundances. A fluorescent-antibody assay for a marine ammonium-oxidizing bacterium was developed to provide a rapid and direct means of identifying these microorganisms. Antibodies to Nitrosococcus oceanus were prepared and tested against pure cultures of marine, freshwater, and soil ammonium oxidizers and against bacteria from natural seawater samples. Cell counts of culture samples determined by the fluorescent-antibody assay agreed with hemacytometer and acridine orange counts. Our results demonstrated that the immunofluorescent assay is a powerful tool for the detection of Nitrosococcus in the marine environment.

Journal Article↗
PubMedDate not supplied

Surface expression and metabolic half-life of AMPA receptors in cultured rat cerebellar granule cells.

The surface expression and metabolic turnover time of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors are important factors in determining the involvement of these proteins in synaptic function. We used the membrane-impermeant chemical crosslinking reagent BS3 and subsequent immunoblot analysis to show that a large proportion of each of the AMPA receptor subunits GluR1-4 is surface expressed in cultured rat cerebellar granule cells. The apparent molecular mass of the individual subunits was 105 kDa and the surface expressed crosslinked complex was 530 kDa for each of the antibodies tested. These results are consistent with functional ionotropic glutamate receptors being pentameric subunit assemblies. We investigated the time course of AMPA receptor surface expression using the membrane-impermeant biotinylating agent NHS-SS-biotin. The half-life of surface expressed AMPA receptors was found to be 30 h. To estimate the mean degradation rate of AMPA receptors and total trichloracetic acid (TCA)-precipitable protein we used [35S]methionine/cysteine pulse-chase labelling. The half-life of AMPA receptors immunoprecipitated with anti-GluR1 antibody was approximately 48 h and the half-life of total TCA-precipitable protein from the same samples was 37 h.

Animals↗