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Biomedical subjects

J R McDermott

Publications and source records attributed to J R McDermott.

At least 19 recordsLinked to original sources
PubMed2005

Immune control of food intake: enteroendocrine cells are regulated by CD4+ T lymphocytes during small intestinal inflammation.

BACKGROUND AND AIMS: Gastrointestinal inflammation reduces food intake but the biological mechanisms explaining suppressed feeding during inflammation are unknown. We have used a model of upper gut infection (Trichinella spiralis in the mouse) to study the effect of inflammation on food intake, and explored the role of a key enteroendocrine cell (EEC) in the regulation of feeding by the immune response. METHODS: Food intake of NIH mice infected with the intestinal nematode Trichinella spiralis was measured. Duodenal cholecystokinin (CCK) cells were counted. Plasma CCK was measured. Infected mice were treated with a specific CCK1 receptor antagonist, and food intake reassessed. The influence of the immune response on food intake and CCK was mechanistically examined by treating mice with CD4 or mast cell neutralising antibodies. The role of the T helper 2 response was further explored in mice genetically deficient for interleukin (IL)-4, IL-13, or IL-4Ralpha (receptor alpha subunit). RESULTS: Food intake of infected mice was significantly reduced at the temporal peak of intestinal inflammation. CCK expressing EEC were upregulated in infected mice, and plasma CCK levels were increased. A CCK1 receptor antagonist restored the food intake of infected mice to a significant degree. Furthermore, suppression of food intake was completely abolished in the absence of CD4+ T lymphocytes or IL-4Ralpha. CONCLUSIONS: The data show for the first time that intestinal inflammation results in reduced food intake due to upregulation of CCK. Moreover, following infection, food intake and CCK expressing cells are under the specific control of CD4+ T-cells, via release of IL-4 and IL-13.

Animals↗
PubMed2001

Leucocyte recruitment during enteric nematode infection.

Resolution of infection with the intestinal nematode Trichinella spiralis depends on the host mounting a T helper 2 (Th2) response. It is known that both mast cells and T cells play a crucial role. We have previously shown that efficient migration of mast cells to the gut during infection depends on their expression of the integrin beta 7. beta 7 forms a heterodimer complex with either alpha E or alpha 4 integrin chains, alpha E beta 7 binding to E-cadherin expressed by epithelial cells and alpha 4 beta 7 binding to mucosal addressin cell adhesion molecule (MAdCAM-1) on the endothelium. We were interested to know whether dysfunctional mast cell localization to the gut in the absence of beta 7 was due to the failure of alpha 4 beta 7 to bind to MAdCAM-1 or the failure of alpha E beta 7 to bind to E-cadherin. We used blocking monoclonal antibodies against alpha E (M290) or alpha 4 (PS2) or beta 7 (HB293) during T. spiralis infection of C57BL/6 mice and found that all antibody treatments reduced mastocytosis. In contrast, none of the antibody treatments prevented the migration of CD3(+) T cells into the intestine. These results indicate that during inflammation (a) there is integrin redundancy for lymphocytes but not for mast cells and (b) both alpha E beta 7 and alpha 4 beta 7 are crucial either for the entry of mast cells into the gut or for their maturation once they have entered.

Animals↗
PubMed2000

Beta7 integrin-deficient mice: delayed leukocyte recruitment and attenuated protective immunity in the small intestine during enteric helminth infection.

The ontogeny and function of gut-associated-lymphoid tissue is known to be critically dependent on the beta7 integrin subfamily. We have investigated the development of intestinal inflammation and pathogen-specific protective immunity to enteric helminth infection in beta7 integrin knockout (KO) mice. During Trichinella spiralis infection of the small intestine there was a significant delay and reduction in the magnitude of intestinal eosinophilia and mastocytosis in the absence of P7 integrin, resulting in impaired host protection. Aberrant distribution of mast cells was also observed in the small intestine of infected KO mice. Adoptive transfer of primed wild-type mesenteric lymph node cells into T. spiralis-infected beta7 KO mice did not restore the intestinal mast cell response, suggesting that the defect in intestinal mastocytosis is due to the absence of beta7 expression on this population rather than an indirect consequence of reduced T cell numbers. In contrast, no impairment in leukocyte recruitment or protection against Trichuris muris infection of the large intestine was observed in KO mice. Taken together the data provide the first description of reduced leukocyte homing and attenuated protective immunity against helminth infection in beta7 KO mice. Furthermore, these results suggest that beta7 integrin-independent adhesion molecule interactions are deployed in the large but not small intestine during intestinal inflammation.

Animals↗
PubMed1997

Degradation of Alzheimer's beta-amyloid protein by human and rat brain peptidases: involvement of insulin-degrading enzyme.

We examined the degradation of Alzheimer's beta-amyloid protein (1-40) by soluble and synaptic membrane fractions from post mortem human and fresh rat brain using HPLC. Most of the activity at neutral pH was in the soluble fraction. The activity was thiol and metal dependent, with a similar inhibition profile to insulin-degrading enzyme. Immunoprecipitation of insulin-degrading enzyme from the human soluble fraction using a monoclonal antibody removed over 85% of the beta-amyloid protein degrading activity. Thus insulin-degrading enzyme is the main soluble beta-amyloid degrading enzyme at neutral pH in human brain. The highest beta-amyloid protein degrading activity in the soluble fractions occurred between pH 4-5, and this activity was inhibited by pepstatin, implicating an aspartyl protease. Synaptic membranes had much lower beta-amyloid protein degrading activity than the soluble fraction. EDTA (2mM) caused over 85% inhibition of the degrading activity but inhibitors of endopeptidases -24.11, -24.15, -24.16, angiotensin converting enzyme, aminopeptidases, and carboxypeptidases had little or no effect.

Alzheimer Disease↗
PubMed1996

Degradation of Alzheimer's beta-amyloid protein by human cathepsin D.

The aim of the study was to identify and characterize human brain peptidases capable of degrading Alzheimer's beta-amyloid protein. Synthetic beta-amyloid protein (1-40) was rapidly degraded by a human brain soluble fraction, optimum activity occurring at around pH4. Pepstatin totally inhibited the activity showing that an aspartyl protease was responsible. HPLC separation and identification of the degradation products showed that the L34-M35 bond was the primary site of cleavage followed by hydrolysis of the F19-F20 and F20-A21 bonds. The major lysosomal aspartyl protease, cathepsin D, hydrolysed beta-amyloid protein with the same pH profile, inhibitor sensitivity and bond specificity as the activity present in human brain soluble fraction. We suggest that cathepsin D may play an important role in regulating brain concentrations of beta-amyloid protein (1-40).

Amino Acid Sequence↗
PubMed1995

Copper binding to the N-terminal tandem repeat region of mammalian and avian prion protein: structural studies using synthetic peptides.

Using CD spectroscopy we have investigated the effect of Cu2+ on the secondary structure of synthetic peptides Octa4 and Hexa4 corresponding to tetra-repeats of the octapeptide of mammalian PrP and the hexapeptide of chicken PrP. In addition, fluorescence spectroscopy was used to estimate the dissociation constants (Kd), of Cu2+ binding by both peptides. Both peptides exhibited unusual CD spectra, complicated by the high proportion of aromatic residues, revealing little secondary structure in aqueous solution. Addition of Cu2+ to Hexa4 induced an increase in random coil to resemble Octa4. The fluorescence of both peptides was quenched by Cu2+ and this was used to calculate Kd's of 6.7 microM for Octa4 and 4.5 microM for Hexa4. Other divalent cations showed lesser effects on the fluorescence of the peptides.

Amino Acid Sequence↗
PubMed1995

Copper binding to the N-terminal tandem repeat regions of mammalian and avian prion protein.

Mammalian prion protein (PrP) is a normal cellular protein (PrPc) which through post-translational modification produces the infectious prion protein (PrPsc). We have shown, using mass spectrometry, that synthetic peptides containing three or four copies of an octapeptide repeat sequence (PHGGGWGQ), found in a highly conserved N-terminal domain of PrP, preferentially bind copper over other metals. Peptides from the analogous region of chicken PrP, which contains an N-terminal repeat domain of the hexapeptide (NPGYPH), showed similar specificity for copper binding. In addition, gel filtration chromatography demonstrated concentration dependent binding of copper to the mammalian tetra repeat PrP peptide. These results suggest that PrP may be a copper binding protein in vivo.

Amino Acid Sequence↗
PubMed1993

Purification and characterization of tripeptidylpeptidase-II from post-mortem human brain.

A soluble tripeptidylaminopeptidase has been isolated from human post-mortem cerebral cortex by anion exchange, hydrophobic interaction and size-exclusion chromatography. From gel filtration studies the active enzyme can exist in both high molecular weight (M(r) > 10(6) and smaller forms. The enzyme hydrolyses Ala-Ala-Phe-7-amido-4-methylcoumarin with a pH optimum of around 7.5 and Km of 148 microM. It did not hydrolyse N-succinyl-Ala-Ala-Phe-7-amido-4-methylcoumarin, aminoacyl- or dipeptidyl-7-amido-methylcoumarins and was not inhibited by bestatin. The enzyme was inhibited by phenylmethylsulphonyl-fluoride, 3,4-dichloroisocoumarin, N-hydroxymercuriphenyl-sulphonic acid and N-ethylmaleimide showing that its activity is serine and cysteine dependent. The purified enzyme released tripeptides from several naturally occurring neuropeptides with quite broad specificity. Cholecystokinin octapeptide, angiotensin III and neurokinin A were the most rapidly hydrolysed. Peptides with Pro residues around the point of cleavage were not hydrolysed.

Amino Acid Sequence↗
PubMed1992

Human brain peptidase activity with the specificity to generate the N-terminus of the Alzheimer beta-amyloid protein from its precursor.

The synthetic peptide acetyl-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-NH2, which spans the cleavage site (Met-Asp) required to generate the N-terminus of the Alzheimer beta-amyloid protein from its precursor, was used to search for human brain peptidases which may be involved in this potentially amyloidogenic process. In both soluble and particulate fractions from human brain the primary cleavage point of the peptide was the Met-Asp bond. Purification and characterization of the activity from the soluble fraction showed that it was metalloendopeptidase 24.15 (EC3.4.24.15). This enzyme is therefore a candidate for the generation of the N-terminus of beta-amyloid protein from its precursor.

Alzheimer Disease↗
PubMed1991

The processing of Alzheimer A4/beta-amyloid protein precursor: identification of a human brain metallopeptidase which cleaves -Lys-Leu- in a model peptide.

A search for human brain peptidases with the specificity to cleave the 695 residue A4/beta amyloid precursor protein within the -Gln-Lys-Leu- (611-613) sequence was carried out using carbobenzoxy-Gln-Lys-Leu-p-nitroanilide as substrate. A metalloendopeptidase was identified in the soluble fraction of post mortem human cerebral cortex which cleaves the substrate at the Lys-Leu bond. The enzyme was partially purified by anion exchange and size exclusion chromatography; it has a Mr of approximately 105-120 kda, is inhibited by EDTA but can be reactivated by Mn++ ions, and has maximum activity between pH 6.8 and 8.

Alzheimer Disease↗
PubMed1991

Human brain leucyl aminopeptidase: isolation, characterization and specificity against some neuropeptides.

In order to obtain a greater understanding of the role of aminopeptidases in the degradation of peptides and proteins in the nervous system, we have isolated and characterized leucyl aminopeptidase (EC 3.4.11.1) from human cerebral cortex and studied its action on some physiologically important neuropeptides. The enzyme has a low specificity constant for the hydrolysis of Leu-7-amido-4-methylcoumarin (69s-1M-1) but the peptides Tyr-Gly-Gly and Tyr-Gly-Gly-Phe-Leu (Leu5-enkephalin) were much better substrates (specificity constants 8300 and 18050s -1M-1 respectively). Optimum activity for the degradation of Leu-enkephalin was obtained at pH10.5 in the presence of 5mM-Mn++. A sharp drop in specificity constant occurred with increasing chain length in the series Leu-enkephalin, dynorphin 1-8, 1-10 and 1-13, suggesting that the enzyme functions only as an oligopeptidase. Other neuropeptides were poor substrates (cholecystokinin octapeptide, angiotensin-I) or not hydrolysed at all (somatostatin, Arg8-vasopressin).

Amino Acid Sequence↗
PubMed1991

Multicatalytic, high-Mr endopeptidase from postmortem human brain.

The main high molecular weight (650K) multicatalytic endopeptidase has been purified from postmortem human cerebral cortex. As in other tissues and species, this enzyme is composed of several subunits of 24-31K and has three distinct catalytic activities, as shown by the hydrolysis of the fluorogenic tripeptide substrates glutaryl-Gly-Gly-Phe-7-amido-4-methylcoumarin, benzyloxycarboxyl-Gly-Gly-Arg-7-amido-4-methylcoumarin, and benzyloxycarboxyl-Leu-Leu-Glu-2-naphthylamide with hydrophobic (Phe), basic (Arg), and acidic (Glu) residues in the P1 position, respectively. These activities are distinguishable by their differential sensitivity to peptidase inhibitors. The enzyme hydrolysed neuropeptides at pH 7.4 at multiple sites with widely differing rates, ranging from 113 nmol/min/mg for substance-P, down to 2 nmol/min/mg for bradykinin. The enzyme also had proteinase activity as shown by the hydrolysis of casein. For the hydrolysis of the Tyr5-Gly6 bond in luteinizing hormone-releasing hormone, the Km was 0.95 mM and the specificity constant (kcat/Km) was 4.7 X 10(3) M-1 s-1. The bond specificity of the enzyme at neutral pH was determined by identifying the degradation products of 15 naturally occurring peptide sequences. The bonds most susceptible to hydrolysis had a hydrophobic residue at P1 and either a small (e.g., -Gly or -NH2) or hydrophobic residue at P'1. Hydrolysis of -Glu-X bonds (most notably in neuropeptide Y) and the Arg6-Arg7 bond in dynorphin peptides was also seen. Thus the three activities identified with fluorogenic substrates appear to be expressed against oligopeptides.

Brain↗
PubMed1988

Amino acid neurotransmitter levels in gliomas and their relationship to the incidence of epilepsy.

The cause of epilepsy due to gliomas is not known. Explanations that is due to mass effect, infiltration and site of the tumour appear insufficient. We have investigated the possibility that epilepsy due to gliomas is caused by interference with normal GABA and glutamate uptake and metabolism in the surrounding cortex. Analysis of human glioma biopsy specimens for the amino acid neurotransmitters and glutamine has shown that gliomas associated with epilepsy have a higher concentration of glutamine. This may be of importance since an elevated concentration of glutamine has been shown to be associated with the onset and severity of cobalt-induced epilepsy.

Amino Acids↗
PubMed1988

Purification and characterization of two soluble Cl(-)-activated arginyl aminopeptidases from human brain and their endopeptidase action on neuropeptides.

Two closely related Cl(-)-activated arginyl aminopeptidases (I and II) were purified from a soluble extract of postmortem human cerebral cortex by anion-exchange chromatography and preparative gel electrophoresis. The electrophoretic mobility of II was approximately 80% that of I; the molecular mass of both enzymes was approximately 70 kilodaltons (kDa) (gel filtration). The aminopeptidase action of I and II on aminoacyl-7-amido-4-methylcoumarin (AMC) substrates was restricted to the Arg and Lys derivatives. Both enzymes had significant endopeptidase activity, hydrolysing several biologically active peptides including neurotensin, bradykinin, angiotensin-I, substance P, luliberin, and somatostatin at internal bonds. Other peptides [Leu-enkephalin, proctolin, thyroliberin, adrenocorticotropin18-39 (ACTH18-39), ACTH11-24, and dynorphin (1-13)] were not appreciably hydrolysed. The amino- and endopeptidase activities had pH optima at 6.5 and 7, respectively, and were both inhibited by metal ion chelators and sulphydryl group blocking agents. The aminopeptidase activity was stimulated 20-fold by Cl- ions, whereas the endopeptidase activity was unaffected by the latter. Km values for neurotensin degradation were 20 microM (I) and 37 microM (II) and for Arg-AMC hydrolysis they were 167 microM (I) and 125 microM (II). The endopeptidase activity was not inhibited by the aminopeptidase inhibitors arphamenine or bestatin (IC50 = 9 nM and 0.1 microM, respectively, with Arg-AMC substrate).

Aged↗
PubMed1987

Involvement of endopeptidase 24.15 in the inactivation of bradykinin by rat brain slices.

The effect of peptidase inhibitors on the degradation of [3H]-bradykinin by rat hypothalamic slices was studied using HPLC to separate and identify the products. The degradation appears to be mainly mediated by an enzyme which cleaves the peptide at the Phe5-Ser6 bond and is inhibited by 1,10-phenanthroline, dynorphin(1-13) and carboxyphenylethyl-Ala-Ala-Phe-p-aminobenzoate. This suggest the involvement of a membrane bound variant of the soluble metalloendopeptidase (EC3.4.24.15) isolated from rat brain which degrades neurotensin, angiotensin and other neuropeptides as well as bradykinin.

4-Aminobenzoic Acid↗
PubMed1987

High affinity specific binding of the thyrotrophin releasing hormone metabolite histidylproline to rat brain membranes.

Histidylproline a metabolite of Thyrotrophin Releasing Hormone specifically binds to both high and low affinity sites in fresh rat brain membranes. Characterisation of the high affinity site under optimal conditions demonstrated an equilibrium dissociation constant (KD) of approximately 9nM and maximum binding capacity of approximately 120 fmols/mg protein. Kinetic analysis of [3H]-His-Pro binding is limited by low binding density, instability of the high affinity site and rapid degradation of the radioligand. The thiol blocking reagent pHydroxymercuriphenylsulphonic acid (HMPS) inhibited [3H]-His-Pro degradation but also reduced binding of the peptide to membranes. The results are discussed with reference to the lack of specific binding sites in brain for the proposed neuropeptide and TRH metabolite cyclo(His-Pro).

Animals↗