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M J Dunn

Publications and source records attributed to M J Dunn.

At least 73 records · Page 4Linked to original sources
PubMed1997

Activation of extracellular signal-regulated kinase in proliferative glomerulonephritis in rats.

Multiple extracellular mitogens are involved in the pathogenesis of proliferative forms of glomerulonephritis (GN). In vitro studies demonstrate the pivotal role of extracellular signal-regulated kinase (ERK) in the regulation of cellular proliferation in response to extracellular mitogens. In this study, we examined whether this kinase, as a convergence point of mitogenic stimuli, is activated in proliferative GN in vivo. Two different proliferative forms of anti-glomerular basal membrane (GBM) GN in rats were induced and whole cortical tissue as well as isolated glomeruli examined using kinase activity assays and Western blot analysis. Administration of rabbit anti-rat GBM serum to rats, preimmunized with rabbit IgG, induced an accelerated crescentic anti-GBM GN. A significant increase in cortical, and more dramatically glomerular ERK activity was detected at 1, 3, and 7 d after induction of GN. Immunization of Wistar-Kyoto rats with bovine GBM also induced a crescentic anti-GBM GN with an increase of renal cortical ERK activity after 4, 6, and 8 wk. ERK is phosphorylated and activated by the MAP kinase/ERK kinase (MEK). We detected a significant increase in the expression of glomerular MEK in the accelerated form of anti-GBM GN, providing a possible mechanism of long-term activation of ERK in this disease model. In contrast to ERK, activation of stress-activated protein kinase was only detectable at early stages of proliferative GN, indicating these related kinases to serve distinct roles in the pathogenesis of GN. Our observations point to ERK as a putative mediator of the proliferative response to immune injury in GN and suggest that upregulation of MEK is involved in the long-term regulation of ERK in vivo.

Animals↗
PubMed1997

Identification and characterization of wheat grain albumin/globulin allergens.

Bakers' asthma, an immediate-type allergic response to the inhalation of cereal flours, is an important occupational disease among workers of the baking and milling industries, and the salt-soluble proteins of wheat and rye flour dust are considered the most relevant allergens. In order to identify and characterize the major IgE-binding proteins, the polypeptide composition of the albumin/globulin protein fraction obtained from different cultivars was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and high-resolution two-dimensional polyacrylamide gel electrophoresis with immobilized pH gradients in the first dimension (IPG-Dalt), followed by immunoblotting with sera from asthmatic bakers. Relevant allergens were isolated by micropreparative IPG-Dalt and blotting onto polyvinylidenedifluoride membranes and identified by amino acid composition analysis or N-terminal amino acid sequence analysis. SDS-PAGE, IPG-Dalt, and immunoblotting demonstrated that the sera of the bakers allergic to flour contained IgE antibodies which bound to numerous albumin/globulin polypeptides in the 70, 55, 35, 26-28, and 14-18 kDa areas. More detailed investigations using IPG-Dalt revealed cultivar-specific differences in IgE-binding. It was also demonstrated that the majority of the allergens were not single polypeptide spots, but consisted of up to ten isoforms of similar molecular mass but different isoelectric points. Amino acid composition analysis and N-terminal amino acid sequence analysis, which were performed for nine allergens located in the 14-18, 26-28, and 35 kDa areas, revealed homologies to amylase/protease inhibitors, acyl-CoA oxidase and fructose-bisphosphate-aldolase from wheat, barley, maize, and rice, respectively.

Albumins↗
PubMed1997

Protein profiling in cardiac tissue in response to the chronic effects of alcohol.

An investigation was made into cardiac protein levels after chronic ethanol consumption to examine whether specific proteins are affected by alcohol. Ethanol was administered for six weeks to male Wistar rats which were fed a nutritionally complete liquid diet containing 35% of total calories as ethanol. Controls were pair-fed identical amounts of the same diet in which ethanol was replaced by isocaloric glucose; thus both groups had identical nutritional intakes, albeit differences in ethanol or carbohydrate. After six weeks' feeding, cardiac tissue was removed and analyzed by two-dimensional electrophoresis, where equal amounts of proteins were studied. Protein patterns were analyzed by computerized densitometry and characterized by comparison with a database of known cardiac proteins. Chronic alcohol feeding caused significant decreases in the relative amounts of various proteins, including several tentatively identified as heat shock protein (HSP) 60, HSP70, and desmin. The relative proportions of actin, vimentin, myosin light chain 1, myosin light chain 2, and albumin, remained unchanged. Examination of antibodies raised against HSP65 showed no overt differences in plasma levels following chronic alcohol consumption, and liver changes as assessed by histology were mild. In conclusion, chronic alcohol appears to have selective effects on particular proteins, and the effects were not directly ascribed to overt liver dysfunction or malnutrition. This may explain some of the functional and morphological characteristics observed in alcohol-induced heart muscle disease, including reduced contractility.

Alcoholism↗
PubMed1997

HSC-2DPAGE and the two-dimensional gel electrophoresis database of dog heart proteins.

A two-dimensional gel electrophoresis database of dog (Canis familiaris) proteins is presented. The database contains 1212 protein spots which have been characterised in terms of their pI and Mr. This database has been integrated into the HSC-2DPAGE database which is accessible on the Internet via the World Wide Web with the uniform resource location (URL): (http://www.harefield.nthames.nhs.uk/nhli/ protein/index.html). Identifications for 80 of the protein spots have been obtained by visual cross-matching with the human heart protein database in HSC-2DPAGE (42 spots), N-terminal microsequence analysis (25 spots) and peptide mass fingerprinting (20 spots). This database is being used in studies of alterations in protein expression in models of heart failure and heart disease.

Amino Acid Sequence↗
PubMed1997

Latex allergen database.

Two-dimensional (2-D) electrophoresis followed by immunoblotting and N-terminal protein microsequencing were used to characterize and identify the IgE-reactive proteins of Hevea latex that are the main cause of the latex type I allergy affecting especially health care workers and spina bifida children. This approach generated a comprehensive latex allergen database, which facilitated the integration of most of the latex allergen data presented in the literature. The major latex allergens Hev b 1, Hev b 3, Hev b 6 and Hev b 7 have been localized on our 2-D maps. Moreover, we were able to identify six previously undescribed IgE-binding latex proteins, namely enolase, superoxide dismutase, proteasome subunit C5, malate dehydrogenase, triosephosphate isomerase and endochitinase. The generated latex 2-D maps will provide valuable information to develop strategies for the isolation of the novel IgE binding proteins in order to study the frequency of sensitization among both risk groups. Detailed knowledge of all proteins involved in latex allergy will allow better diagnosis of latex allergy and to monitor the success of prevention strategies that are needed to reduce the high prevalence of latex allergy among both risk groups.

Allergens↗
PubMed1997

Distinct signaling pathways mediate induction of c-fos by PGE2 in glomerular mesangial cells.

Prostanoids induce expression of several immediate-early genes but the molecular mechanisms underlying these responses remain poorly characterized. We have studied induction of the proto-oncogenc c-fos by PGE2 in mesangial cells as a model of gene regulation by prostanoids. PGE2 induced marked and transient accumulation of c-fos mRNA. Induction of c-fos by PGE2 and TxA2 did not correlate with induction of phospholipase C. Addition of exogenous cAMP failed to induce c-fos mRNA, suggesting that activation of an EP2 receptor linked to adenylate cyclase did not account for induction of c-fos by PGE2. These data contrast with previous experiments in NIH 3T3 cells where PGE2 induced c-fos by a cAMP-dependent mechanism. We further showed that PGE2 induces the c-fos gene by direct activation of the serum response element. Taken together these experiments provide evidence for a pathway linking a PGE2 receptor on the plasma membrane to transcriptional activation in the nucleus.

3T3 Cells↗
PubMed1997

Characterization and identification of latex allergens by two-dimensional electrophoresis and protein microsequencing.

BACKGROUND: Proteins of natural rubber latex cause IgE-mediated sensitization in 3% to 18% of health care workers and in up to 50% of patients with spina bifida. OBJECTIVE: This study was aimed at the generation of a comprehensive latex protein database by two-dimensional electrophoresis (2-DE). METHODS: Proteins extracted from fresh Hevea brasiliensis latex were separated by 2-DE. IgE-reactive proteins were analyzed by immunoblotting with sera of health care workers with latex allergy. Protein microsequencing and monoclonal antibodies were used to identify the latex allergens. RESULTS: The latex C-serum 2-DE map was very complex and exhibited about 200 distinct polypeptides. The proteins eluted from the latex particles consisted primarily of two groups of acidic proteins located in the 8 to 14 kd and 22 to 24 kd areas of the 2-DE map. Major IgE-reactivity was detected with C-serum proteins in the 56, 45, 30, 20, 14, and <6.5 kd areas of the immunoblots. The 8 to 14 kd particle proteins exhibited distinct IgE reactivity, whereas the 22 to 24 kd proteins were not stained. Seven of the soluble IgE-reactive protein spots showed high homology with enolase, superoxide dismutase, triosephosphate isomerase, proteasome subunit, and chitinase and represent previously undescribed latex allergens; whereas nine protein spots corresponded to known latex allergens, namely prohevein, hevein, prohevein C-domain, and hevamine. As identified by monoclonal antibodies, the IgE-reactive latex particle proteins mainly represent the allergenic rubber elongation factor. CONCLUSIONS: Two-dimensional electrophoresis, followed by immunoblotting and protein microsequencing, can rapidly identify a large number of IgE-binding latex proteins. The 2-DE latex maps generated will provide valuable information for the development of strategies to isolate the relevant latex allergens. Because the novel latex allergens are common plant enzymes, they may also act as cross-reacting proteins in various foods.

Adult↗
PubMed1997

Anti-cell surface endothelial antibodies in sera from cardiac and kidney transplant recipients: association with chronic rejection.

The aetiologies of accelerated or transplant-associated coronary artery disease (TxCAD) following cardiac transplantation and chronic rejection following renal transplantation remain ill-defined. Previous studies have used Western blotting to demonstrate an association between the formation of anti-endothelial (anti-EC) antibodies and TxCAD after heart transplantation. However, Western blotting favours detection of cytosolic proteins. The objectives of this study were to determine whether flow cytometry, a method which detects antigens on the cell surface, could be used to detect anti-EC antibodies and also whether the observations would extend to renal transplant patients with chronic rejection. Flow cytometry was used to look for antibodies reactive with the surface antigens of macrovascular and microvascular endothelial cell lines in sera from 44 cardiac and 35 renal transplant recipients before and after transplantation. In addition, sera from normals (n = 20), patients with nontransplant CAD (n = 50) and patients with unrelated diseases (n = 40) were investigated. Of 23 cardiac recipients who had developed TxCAD at one or two years post-transplant, 61% had IgM and 13% had IgG anti-EC antibodies post-transplantation. In contrast, in 21 cardiac recipients who had not developed TxCAD 14% had IgM and 14% IgG anti-EC antibodies. There was little evidence for the presence of anti-EC antibodies in cardiac recipients before transplantation. Of 26 renal transplant recipients whose transplants failed due to chronic rejection, 42% had IgG and 19% IgM anti-EC antibodies post-transplantation. Of nine renal recipients whose grafts were either functioning normally or who had acutely rejected, none had IgG or IgM anti-EC antibodies either pre- or post-transplantation. The anti-EC antibodies were not found in normals and were rare (less than 4%) in the other disease groups; they do not appear to be autoantibodies. In conclusion, these results suggest the FACS assay could be an informative and rapid test to provide more information on chronic rejection following cardiac and renal transplantation.

Adolescent↗
PubMed1997

Characterization and interconversion of polymorphs of premafloxacin, a new quinolone antibiotic.

The quinolone antibiotic premafloxacin crystallizes in at least five solid modifications, including three anhydrous phases (Forms I-III), a hydrate, and a methanolate. The anhydrous phases were studied by optical microscopy, X-ray powder diffraction, HPLC, hot-stage microscopy, dynamic moisture sorption gravimetry, differential scanning calorimetry, thermal gravimetry, and solution and isothermal calorimetry. Dry samples of Form I converted to Form II and ultimately to Form III through a sequence of melts and recrystallizations. Form III was stable to its melting temperature near 200 degrees C. Humidified samples of Form I converted directly to Form III via a moisture-mediated solid-state phase transformation at temperatures as low as 40 degrees C. The calorimetric and solubility data confirmed that Form III was lower in free energy and enthalpy than Form I at room temperature. Our investigation revealed that Form I was not crystallized directly from solution. Rather, Form I was the product of facile solid-state desolvation of the methanol solvate.

Anti-Infective Agents↗
PubMed1997

Differential regulation of the dual-specificity protein-tyrosine phosphatases CL100, B23, and PAC1 in mesangial cells.

The extracellular-signal-regulated kinase (ERK), the best described MAP kinase cascade, is a major signaling system by which cells transduce extracellular cues into intracellular responses. ERK is activated by phosphorylation both on tyrosine and threonine residues. Therefore, a new clas of protein-tyrosine phosphatases (PTPases) that exhibit dual catalytic activity toward both regulatory sites on ERK is of special interest in the control of intracellular signaling. This study examined the expression and regulation of the dual-specificity PTPases CL100, B23, and PAC1. Findings included differential expression of these phosphatases in diverse cell lines and an expression of all three dual-specificity PTPases in human mesangial cells (HMC), thereby allowing investigation of their regulation in a single cell line. The MEK antagonist PD 098059 and selective extracellular agonists of ERK were used to demonstrate the induction of CL100, PAC1, and B23 in response to activation of the ERK cascade. In contrast, anisomycin, an agonist of the recently described MAP kinases stress-activated protein kinase (SAPK) and p38 MAP kinase, stimulated CL100 gene expression but had little effect on PAC1 and B23. This effect of anisomycin was partly inhibited in the presence of the p38 MAP kinase antagonist SB 203580. This study suggests a potential mechanism to regulate ERK activity through feedback inhibition by demonstrating the ERK cascade's induction of the dual-specificity PTPases CL100, PAC1, and B23. Moreover, this study suggests an ERK-independent induction of CL100 following stimulation of SAPK and p38 MAP kinase. This mode of induction of a phosphatase capable of inactivating ERK may play an important role in the cellular stress response.

Animals↗
PubMed1997

Calcium-regulated protein tyrosine phosphorylation is required for endothelin-1 to induce prostaglandin endoperoxide synthase-2 mRNA expression and protein synthesis in mesangial cells.

The role of endothelin (ET)-1-mediated cytosolic calcium ([Ca2+]i) elevation in regulating ET-1-induced prostaglandin endoperoxide synthase, prostaglandin G/H synthase (PGHS)-2 mRNA expression and protein synthesis was investigated in mesangial cells (MC). Ionomycin, a calcium ionophore, and thapsigargin, an inhibitor of calcium ATPase, mimicked the ET-1-stimulated PGHS-2 mRNA and protein induction. Inhibition of [Ca2+]i increases with (2-¿C2-bis-(carboxymethyl)-amino-5 methylphenoxy]methyl¿-6-methoxy-8-bis-(carboxymethyl)-aminoquinoline tetra-(acetoxymethyl)ester (Quin/AM), a calcium chelator, or with the combined presence of [8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, HCl] (TMB), an inhibitor of intracellular calcium stores release, and ethyleneglycol-bis-(beta-aminoethyl)- N,N,N',N'-tetra-acetic acid (EGTA) suppressed ET-1, as well as ionomycin and thapsigargin-mediated PGHS-2 mRNA and protein formation. Also, the ET-1-, ionomycin-, and thapsigargin-induced PGHS-2 mRNA expression and protein formation was inhibited in MC pretreated with inhibitors of calcium calmodulin kinase. In contrast, these conditions did not inhibit interleukin (IL)-1-induced PGHS-2 mRNA expression and protein synthesis. Pretreatment with tyrosine kinase inhibitors abolished the ET-1-, ionomycin-, thapsigargin-, and IL-1-mediated PGHS-2 mRNA and protein induction. ET-1-, ionomycin-, and thapsigargin- induced protein tyrosine phosphorylation, but not IL-1-induced protein tyrosine phosphorylation, was suppressed by inhibiting either [Ca2+]i elevation or calcium calmodulin kinase activation. It was concluded that elevation of [Ca2+]i and activation of calcium calmodulin kinases are upstream mediators of ET-1-induced PGHS-2 gene expression through activation of non-receptor-linked protein tyrosine kinase in MC.

Aminoquinolines↗
PubMed1997

Coxsackievirus B3-induced myocarditis. Characterization of stable attenuated variants that protect against infection with the cardiovirulent wild-type strain.

Coxsackievirus B3 (CVB3) is the enterovirus most frequently involved in human myocarditis or dilated cardiomyopathy. Attenuated variants were derived from a cardiovirulent CVB3 reactivated from a sequenced, full-length cDNA clone. The prophylactic potential of these variants was assessed in SWR/Ola (H-2q) mice. Animals immunized with attenuated variants of CVB3 were protected from myocarditis when challenged subsequently with the cardiovirulent wild-type virus. In contrast to nonimmunized controls, the wild-type virus was not isolated from myocardium of protected mice, nor was viral RNA detected in myocardium by reverse transcription nested polymerase chain reaction. Specific antibody to CVB3 was demonstrated by virus neutralization assay and by indirect immunofluorescence. The attenuated phenotype of one variant, p14V-1, remained stable throughout 20 consecutive passages in SWR mice and induced a markedly lower level of autoantibody against mouse cardiac myosin heavy chain than the cardiovirulent wild type. These data demonstrate that attenuated strains protect against CVB3-induced myocarditis in mice, that the attenuated phenotype is stable, and that they do not persist in myocardium nor induce a significant level of anti-heart anti-body against myosin heavy chain. These attenuants may be the basis of a live vaccine against CVB3 in the prevention of enteroviral heart muscle disease.

Animals↗
PubMed1996

Cytosolic and nuclear mitogen-activated protein kinases are regulated by distinct mechanisms.

We have investigated the regulation and localization of mitogen-activated protein kinase (MAPK) and mitogen-activated protein kinase kinase (MAPKK) in both cytosolic and nuclear fractions of glomerular mesangial cells. p42 MAPK was localized by both immunoblot and kinase activity in both cytosol and nucleus and was rapidly activated, in both fractions, by fetal bovine serum and TPA. Downregulation of protein kinase C (PKC) by TPA inhibited stimulation of cytosolic p42 MAPK, but unexpectedly had no effect on stimulated p42 MAPK in the nucleus. Next we studied the upstream kinase p45 MAPKK by indirect immunofluorescence microscopy, Western blot analysis, and kinase specific activity. Unlike MAPK, p45 MAPKK is almost exclusively cytosolic in resting cells and kinase activity stimulated by TPA is restricted to the cytosol. Interestingly, PKC downregulation for 24 h with TPA dramatically enhanced nuclear MAPKK as assessed by all three techniques. Cytosolic stimulated MAPKK was attenuated in PKC downregulation. Collectively these results show that in mesangial cells: (i) p42 MAPK and p45 MAPKK localize in both the cytosol and the nucleus, and (ii) PKC exerts a negative effect on nuclear MAPKK activity as documented by PKC downregulation, which augments p45 MAPPK nuclear mass and activity. These results indicate that the dual regulation of these two kinases is under differential control in the cytosol and the nucleus.

Animals↗
PubMed1996

Induction of mitogen-activated protein kinase phosphatase 1 by the stress-activated protein kinase signaling pathway but not by extracellular signal-regulated kinase in fibroblasts.

The intracellular mechanisms involved in the activation of extracellular signal-regulated kinase (ERK) are relatively well understood. However, the intracellular signaling pathways which regulate the termination of ERK activity remain to be elucidated. Mitogen-activated protein kinase phosphatase 1 (MKP-1) has been shown to dephosphorylate and inactivate ERK in vitro and in vivo. In the present study, we show in NIH3T3 fibroblasts that activation of the stress-activated protein kinase (SAPK) pathway by either specific extracellular stress stimuli or via induction of MEKK, an upstream kinase of SAPK, results in MKP-1 gene expression. In contrast, selective stimulation of the ERK pathway by 12-O-tetradecanoylphorbol-13-acetate or following expression of constitutively active MEK, the upstream dual specificity kinase of ERK did not induce the transcription of MKP-1. Hence, these findings demonstrate the existence of cross-talk between the ERK and SAPK signaling cascades since activation of SAPK induced the expression of MKP-1 that can inactivate ERK. This mechanism may modulate the cellular response to stimuli which employ the SAPK signal transduction pathway.

3T3 Cells↗
PubMed1996

Construction of an internal standard for semiquantitative polymerase chain reaction analysis of heat shock proteins.

We describe a simple and effective method for generating competitive cDNA fragments for use as an internal standard in semi-quantitative polymerase chain reaction (PCR). The use of a nested composite primer to a region slightly within the expected amplicon but with heat shock protein 60 kDa (hsp60) primer sequence appended to the 5' terminus of the primer produces a "mimic" DNA that will model the PCR kinetics of the target template in sequence context, and PCR primer site, and that will be similar in size. This ensures that both competitor and target template are subjected to similar PCR kinetics and so allows more meaningful quantitation.

Base Sequence↗