Biomedical subjects

M K Bennett

Publications and source records attributed to M K Bennett.

At least 37 records · Page 2Linked to original sources

PubMed1999

Differential phosphorylation of syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) isoforms.

The synaptic plasma membrane proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) are central participants in synaptic vesicle trafficking and neurotransmitter release. Together with the synaptic vesicle protein synaptobrevin/vesicle-associated membrane protein (VAMP), they serve as receptors for the general membrane trafficking factors N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein (alpha-SNAP). Consequently, syntaxin, SNAP-25, and VAMP (and their isoforms in other membrane trafficking pathways) have been termed SNAP receptors (SNAREs). Because protein phosphorylation is a common and important mechanism for regulating a variety of cellular processes, including synaptic transmission, we have investigated the ability of syntaxin and SNAP-25 isoforms to serve as substrates for a variety of serine/threonine protein kinases. Syntaxins 1 A and 4 were phosphorylated by casein kinase II, whereas syntaxin 3 and SNAP-25 were phosphorylated by Ca2+- and calmodulin-dependent protein kinase II and cyclic AMP-dependent protein kinase, respectively. The biochemical consequences of SNARE protein phosphorylation included a reduced interaction between SNAP-25 and phosphorylated syntaxin 4 and an enhanced interaction between phosphorylated syntaxin 1A and the synaptic vesicle protein synaptotagmin I, a potential Ca2+ sensor in triggering synaptic vesicle exocytosis. No other effects on the formation of SNARE complexes (comprised of syntaxin, SNAP-25, and VAMP) or interactions involving n-Sec1 or alpha-SNAP were observed. These findings suggest that although phosphorylation does not directly regulate the assembly of the synaptic SNARE complex, it may serve to modulate SNARE complex function through other proteins, including synaptotagmin I.

Animals↗

PubMed1999

Systemic and mucosal immunity is elicited after both intramuscular and intravaginal delivery of human immunodeficiency virus type 1 DNA plasmid vaccines to pregnant chimpanzees.

DNA vaccines encoding human immunodeficiency virus type 1 (HIV-1) env/rev and gag/pol were delivered intravaginally (IVAG) and intramuscularly (IM) to 2 pregnant chimpanzees. Vaccination was well tolerated and each chimpanzee developed antibodies (up to 1 year later) to both vaccines. Placental transfer of anti-Env and anti-Gag IgG was demonstrated in both maternal/infant pairs. Specific IgG was also demonstrated in saliva, vaginal, and rectal washes after IVAG immunization. Predominantly anti-HIV-1 IgA was detected in the milk of both mothers after both IM and IVAG immunization. Cellular responses included Gag-specific proliferation of lymphocytes and cytotoxic T lymphocytes against both antigens. These data suggest a strategy for induction of mucosal and systemic responses after both IM and IVAG delivery of DNA vaccines in a primate model and could ultimately be useful in lowering maternal-to-fetal transmission of HIV-1, perinatally and through breastfeeding.

AIDS Vaccines↗

PubMed1999

Syntaxin 2 splice variants exhibit differential expression patterns, biochemical properties and subcellular localizations.

The syntaxins are a large protein family implicated in the targeting and fusion of intracellular transport vesicles. A subset of proteins of this family are the four syntaxin 2 splice variants, syntaxins 2A (2), 2B (2'), 2C (2") and 2D. Each syntaxin 2 variant contains an identical, or nearly identical, amino-terminal cytoplasmic domain followed by a distinct hydrophobic (syntaxins 2A and 2B) or hydrophilic (syntaxins 2C and 2D) carboxyl-terminal domain. To investigate whether the difference among the syntaxin 2 variants is functionally important, we have examined comparatively their RNA transcript and protein expression patterns, membrane associations, protein-protein interactions and intracellular localizations. Analysis of the RNA transcript and protein expression patterns demonstrated that syntaxins 2A, 2B and 2C are broadly, but not uniformly, expressed while syntaxin 2D expression is restricted to the brain. Subcellular fractionation studies showed that syntaxins 2A and 2B behave as integral membrane proteins while syntaxin 2C is only partially associated with membranes. In vitro biochemical assays demonstrated that the syntaxin 2 variants exhibit similar yet distinct interactions with other proteins implicated in vesicular trafficking, including SNAP-25, SNAP-23, VAMP-2 and n-sec1. In a variety of nonpolarized cell types, syntaxins 2A and 2B localized to both the plasma membrane and endosomal membranes. However, in two polarized epithelial cell lines, MDCK and Caco-2, syntaxin 2A localized predominantly to the apical plasma membrane while syntaxin 2B was associated with both the apical and the basolateral membranes. These observations indicate that the distinct carboxyl-terminal domains of the syntaxin 2 variants influence their biochemical and localization properties and may therefore confer upon these variants different functional roles in the regulation of intracellular membrane trafficking.

Alternative Splicing↗

PubMed1998

Protease resistance of syntaxin.SNAP-25.VAMP complexes. Implications for assembly and structure.

A stable ternary complex formed with vesicle-associated membrane protein 2 (VAMP2) and plasma membrane proteins syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25) is proposed to function in synaptic vesicle exocytosis. To analyze the structural characteristics of this synaptic protein complex, recombinant binary (syntaxin 1A.SNAP-25), recombinant ternary, and native ternary complexes were subjected to limited trypsin proteolysis. The protected fragments, defined by amino-terminal sequencing and mass spectrometry, included a carboxyl-terminal region of syntaxin 1A, the cytoplasmic domain of VAMP2, and amino- and carboxyl-terminal regions of SNAP-25. Furthermore, separate amino- and carboxyl-terminal fragments of SNAP-25, when combined with VAMP2 and syntaxin 1A, were sufficient for stable complex assembly. Analysis of ternary complexes formed with full-length proteins revealed that the carboxyl-terminal transmembrane anchors of both syntaxin 1A and VAMP2 were protected from trypsin digestion. Moreover, the stability of ternary complexes was increased by inclusion of these transmembrane domains. These results suggest that the transmembrane domains of VAMP2 and syntaxin 1A contribute to complex assembly and stability and that amino- and carboxyl-terminal regions of SNAP-25 may function as independent domains.

Animals↗

PubMed1998

The synaptic SNARE complex is a parallel four-stranded helical bundle.

The heterotrimeric synaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, consisting of the synaptic vesicle-associated membrane protein 2 (VAMP2) and presynaptic plasma membrane proteins SNAP-25 (synaptosome-associated protein of 25,000 Mr) and syntaxin 1A, is a critical component of the exocytotic machinery. We have used spin labeling electron paramagnetic resonance spectroscopy to investigate the structural organization of this complex, particularly the two predicted helical domains contributed by SNAP-25. Our results indicate that the N- and C-terminal domains of SNAP-25 are parallel to each other and to the C-terminal domain of syntaxin 1A. Based on these findings, we propose a parallel four-stranded coiled coil model for the structure of the synaptic SNARE complex.

Animals↗

PubMed1998

Glutaraldehyde as a possible cause of diarrhoea after sigmoidoscopy.

BACKGROUND: An outbreak of proctitis at the start of a colorectal cancer screening programme utilizing flexible sigmoidoscopy prompted scrutiny of the incidence of this complication and the role of glutaraldehyde in its aetiology. METHODS: Questionnaires completed 1 day and 3 months after sigmoidoscopy were reviewed for 388 patients, and glutaraldehyde levels in the recycled rinse water of the endoscope washing machine were measured. The incidence of symptoms in the subsequent 612 patients after installation of a washer that does not recycle rinse water was examined. RESULTS: Five patients (1.3 per cent) presented to hospital with bloody diarrhoea occurring immediately after a normal flexible sigmoidoscopy. Repeat examination confirmed the presence of proctitis. Symptoms subsided rapidly with either no treatment or steroid enemas. Eight additional patients (2.1 per cent) recorded similar problems but received no treatment and the symptoms settled spontaneously. Glutaraldehyde levels rose progressively in the rinse water after each wash cycle with 2 per cent glutaraldehyde solution. Only one possible case of proctitis (0.2 per cent) was identified from the questionnaires completed by 612 patients after changing to a washer that did not recycle the rinse water. CONCLUSION: These observations should prompt the careful assessment of cleaning techniques. The use of washing machines that do not recycle rinse water may avoid this complication.

Colorectal Neoplasms↗

PubMed1998

Atrial fibrillation with neonatal pulmonary lymphangiectasia.

A female infant born at 28 weeks' gestation was found to have mild hydrops foetalis. Initial echocardiography showed a structurally normal heart. During the first week of life, episodic atrial tachycardia with 1:1 or 2:1 conduction was seen, requiring therapy with digoxin. The infant remained ventilator dependent, with a persistent, chylous pleural effusion which contained a preponderance of lymphocytes. Congenital pulmonary lymphangiectasia (CPL) was confirmed histologically. Worsening episodes of atrial tachycardia, including episodes of atrial fibrillation, were further investigated and a repeat echocardiogram revealed thickening of the entire right atrial wall. The cardiac findings of a thickened right atrial wall with the histological signs of myocarditis were thought to be the cause of paroxysms of atrial fibrillation, an extremely rare arrhythmia in the neonatal period. To the authors' knowledge there have been no previous reports of CPL in association with the cardiac abnormalities described herein.

Atrial Fibrillation↗

PubMed1998

Microscopic image analysis for quantitative measurement and feature identification of normal and cancerous colonic mucosa.

The development of an automated algorithm for the categorization of normal and cancerous colon mucosa is reported. Six features based on texture analysis were studied. They were derived using the co-occurrence matrix and were angular second moment, entropy, contrast, inverse difference moment, dissimilarity, and correlation. Optical density was also studied. Forty-four normal images and 58 cancerous images from sections of the colon were analyzed. These two groups were split equally into two subgroups: one set was used for supervised training and the other to test the classification algorithm. A stepwise selection procedure showed that correlation and entropy were the features that discriminated most strongly between normal and cancerous tissue (P < 0.0001). A parametric linear-discriminate function was used to determine the classification rule. For the training set, a sensitivity and specificity of 93.1% and 81.8%, respectively, were achieved, with an overall accuracy of 88.2%. These results were confirmed with the test set, with a sensitivity and specificity of 93.1% and 86.4%, respectively, and an overall accuracy of 90.2%.

Colon↗

PubMed1998

Nucleoside analogue therapy in fibrosing cholestatic hepatitis--a case report in an HBsAg positive renal transplant recipient.

A 45-year-old HBsAg carrier (HBeAb positive with normal liver function tests) underwent renal transplantation for mesangioproliferative glomerulonephritis. Sixteen months later he developed jaundice. Investigations showed he remained HBeAb positive, but HBV-DNA levels were 99 pg/ml, indicating active replication of a HBV pre-core mutant. He was commenced on lamivudine therapy with a subsequent rapid fall in HBV-DNA levels to 2.8 pg/ml, but liver function tests continued to deteriorate and he developed hepatorenal failure. Liver biopsy showed fibrosing cholestatic hepatitis. He underwent liver transplantation, which was complicated by lactic acidosis. Lamivudine was withdrawn and HBV prophylaxis with HB immunoglobulin was commenced. Unfortunately he died 38 days post-transplant of surgical complications with no evidence of HBV recurrence. We discuss the use of nucleoside analogues in fibrosing cholestatic hepatitis and review the literature.

Cholestasis, Intrahepatic↗

PubMed1998

Validation of an automated technique for determining the mechanical characteristics of coronary arteries during balloon angioplasty: laboratory assessment with necropsy segments.

OBJECTIVES: To develop a technique for automatic inflation of a percutaneous transluminal coronary angioplasty (PTCA) balloon, with continuous measurement of the balloon pressure and volume; to validate the technique for determining the mechanical characteristics of coronary arteries. METHODS: During necropsy examination of the hearts of nine patients, 17 coronary artery samples were obtained for histological examination. A PTCA balloon was inserted into each artery, and the balloon pressure and volume were measured continuously during four repeat automatic inflations of the balloon. RESULTS: Of the 17 arteries, eight showed elastic, six plastic, and three fracture pressure-volume deformation characteristics. For the plastic deformations, the first inflation required a higher pressure than subsequent repeat inflations of 82 (61) kPa (mean (SD), range 25 to 175 kPa). For the three in the fracture group, the pressure drop because of the fracture occurred between 210 and 540 kPa. Two of these three showed a tear on visual inspection, and the other showed disruption of the intimal plaque on blinded histological examination. Of the six with plastic deformation characteristics alone, one showed a tear, and on histological examination two others showed splitting of the internal and external elastic lamina and one showed separation of intima and media. None in the elastic group showed any of these characteristics. CONCLUSIONS: Plastic and fracture deformation characteristics could be differentiated from elastic characteristics. Visual or histological evidence of fracturing was present in all three arteries identified during angioplasty as having pressure-volume fracture characteristics.

Angioplasty, Balloon, Coronary↗

PubMed1998

Automated feature extraction and identification of colon carcinoma.

OBJECTIVE: To assess an automated algorithm, developed for the classification of normal and cancerous colonic mucosa, using geometric analysis of features and texture analysis. STUDY DESIGN: Twenty-one images were analyzed, 10 from normal and 11 from cancerous mucosa. The classification was based on a regularity index dependent on shape, object orientation for establishing parallelism and five texture features derived using the co-occurrence image analysis method. RESULTS: Geometric analysis yielded an overall classification accuracy of 80%. The corresponding sensitivity and specificity were 94% and 64%, respectively. Using texture analysis, the overall classification accuracy was 90%, with a sensitivity and specificity of 82% and 100%, respectively. CONCLUSION: This initial study demonstrated that geometric and texture analysis techniques show promise for automated analysis of colon cancer.

Algorithms↗

PubMed1997

Regulation of CFTR chloride channels by syntaxin and Munc18 isoforms.

The cystic fibrosis gene encodes a cyclic AMP-gated chloride channel (CFTR) that mediates electrolyte transport across the luminal surfaces of a variety of epithelial cells. The molecular mechanisms that modulate CFTR activity in epithelial tissues are poorly understood. Here we show that CFTR is regulated by an epithelially expressed syntaxin (syntaxin 1A), a membrane protein that also modulates neurosecretion and calcium-channel gating in brain. Syntaxin 1A physically interacts with CFTR chloride channels and regulates CFTR-mediated currents both in Xenopus oocytes and in epithelial cells that normally express these proteins. The physical and functional interactions between syntaxin 1A and CFTR are blocked by a syntaxin-binding protein of the Munc18 protein family (also called n-Secl). Our results indicate that CFTR function in epithelial cells is regulated by an interplay between syntaxin and Munc18 isoforms.

Animals↗

PubMed1997

Regulated secretion in platelets: identification of elements of the platelet exocytosis machinery.

To further characterize the molecular mechanisms of platelet function, we have sought to identify some of the proteins that mediate the secretory events of the platelet release reaction. We report that platelets contain the general elements of the membrane transport apparatus: N-ethylmaleimide sensitive fusion protein (NSF), p115/transcytosis-associated protein (p115/TAP), and the soluble NSF attachment proteins (alpha- and, gamma-SNAP). The cDNAs encoding two of these proteins, alpha- and gamma-SNAP, have been cloned from a human platelet-derived cDNA library. Platelet membrane extracts possess SNAP receptor (SNARE) activity, suggesting that the class of proteins (SNAREs) proposed to provide the specificity for vesicle docking and membrane fusion are present in platelets. To identify these proteins, we have used specific antibodies against known SNAREs to probe platelet extracts. Syntaxin 2 and 4 can be readily detected in platelet membrane preparations and are shown to participate in 20 S complex formation. Syntaxin 1, 3, and 5 could not be detected. Other known SNARE and SNARE-associated proteins such as vesicle-associated membrane protein (VAMP)/synaptobrevin 2, SNAP-25, synaptophysin, or synaptotagmin I could not be immunochemically detected in platelet membrane preparations. The presence of both the general transport proteins (NSF and SNAPs) and specific transport proteins (syntaxin 2 and 4) indicates that platelet exocytosis uses a molecular mechanism similar to other secretory cells such as neurons. However, the subcellular concentrations of these proteins suggest that, unlike neuronal secretion, granule-to plasma membrane docking may be the limiting step in platelet exocytosis.

Amino Acid Sequence↗

PubMed1997

Effect of mutations in vesicle-associated membrane protein (VAMP) on the assembly of multimeric protein complexes.

The assembly of multimeric protein complexes that include vesicle-associated membrane protein 2 (VAMP-2) and the plasma membrane proteins syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25) are thought to reflect the biochemical correlates of synaptic vesicle targeting, priming, or fusion. Using a variety of protein-protein interaction assays and a series of deletion and point mutations, we have investigated the domains of VAMP-2 required for the formation of binary complexes with either syntaxin 1A or SNAP-25 and ternary complexes with both syntaxin 1A and SNAP-25. Deletions within the central conserved domain of VAMP-2 eliminated binding to either syntaxin 1A or both syntaxin 1A and SNAP-25. Although all of the deletion mutants were able to form ternary complexes, only some of these complexes were resistant to denaturation in sodium dodecyl sulfate. These results demonstrate that cooperative interactions result in the formation of at least two biochemically distinct classes of ternary complex. Two point mutations previously shown to have effects on the intracellular trafficking of VAMP-2 (M46A, reduced endocytosis and sorting to synaptic vesicles; N49A, enhanced sorting to synaptic vesicles) lie within a domain required for both syntaxin 1A and SNAP-25 binding. Syntaxin 1A and SNAP-25 binding was reduced by the M46A mutation and enhanced by the N49A mutation, suggesting that a correlation exists between the membrane-trafficking phenotype of the two VAMP-2 point mutants and their competence to form complexes with either syntaxin 1A or SNAP-25.

Animals↗

PubMed1997

Ultrasonography in blunt abdominal trauma.

This article discusses studies of the use of ultrasound in patients with blunt abdominal trauma, both in initial assessment and ongoing evaluation. Reviews of studies of children and adults to detect the presence and extent of hemoperitoneum and organ injuries are presented. Ultrasound results are compared with diagnostic peritoneal lavage, computed tomography, clinical course, and autopsy results. The central question addressed is to what extent can ultrasonography replace or supplement other techniques, particularly diagnostic peritoneal lavage, in the assessment of patients with blunt abdominal trauma. Ultrasound equipment, technique, scoring scales, limitations, and training issues are also addressed.

Abdominal Injuries↗

PubMed1997

Ca2+ and the regulation of neurotransmitter secretion.

Ca2+ plays an important role in the regulation of multiple steps that contribute to neurotransmitter secretion. Electrophysiological approaches have defined the nature of the Ca2+ signal and its sites of action, while recent biochemical, molecular, and genetic approaches have identified and characterized candidate molecular targets for Ca2+ regulation.

Animals↗

PubMed1997

Association of syntaxin 3 and vesicle-associated membrane protein (VAMP) with H+/K(+)-ATPase-containing tubulovesicles in gastric parietal cells.

H+/K(+)-ATPase is the proton pump in the gastric parietal cell that is responsible for gastric acid secretion. Stimulation of acid secretion is associated with a reorganization of the parietal cells resulting in the incorporation of H+/K(+)-ATPase from a cytoplasmic membrane pool, the tubulovesicle compartment, into the apical canalicular membrane. To better characterize the role of membrane trafficking events in the morphological and physiological changes associated with acid secretion from parietal cells, we have characterized the expression and localization of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in these cells. Each of the six different SNARE proteins examined [syntaxins 1 through 4 of 25-kDa synaptosome-associated protein, and vesicle-associated membrane protein] were found to be expressed in parietal cells. Furthermore, two of these SNAREs, vesicle-associated membrane protein and syntaxin 3, were associated with H+/K(+)-ATPase-containing tubulovesicles while the remainder were excluded from this compartment. The expression of syntaxin 1 and synaptosome-associated protein of 25 kDa in parietal cells, two SNAREs previously thought to be restricted to neuroendocrine tissues, suggests that parietal cells may utilize membrane trafficking machinery that is similar to that utilized for regulated exocytosis in neurons. Furthermore, the localization of syntaxin 3, a putative target membrane SNARE, to the tubulovesicle compartment indicates that syntaxin 3 may have an alternative function. These observations support a role for intracellular membrane trafficking events in the regulated recruitment of H+/K(+)-ATPase to the plasma membrane after parietal cell stimulation.

Animals↗

PubMed1997

The prevalence of hereditary haemochromatosis in a diabetic population.

Hereditary haemochromatosis is an under-diagnosed and treatable cause of chronic liver disease. Its prevalence indicates that selective population screening may be worthwhile, but opinion differs as to whether diabetic patients constitute such a group. We studied 727 patients attending a teaching hospital diabetic clinic. On first testing, 7.4% had abnormally high iron indices, but only 3% remained abnormal on retesting. Of these patients, those at high risk were offered liver biopsy for histological assessment and iron assay. Only one had hereditary haemochromatosis, but all had abnormal liver histology--largely steatosis but some with fibrosis. These findings raise questions regarding the true prevalence of this disorder in North-East England, do not indicate that targeted screening of diabetic patients is worthwhile, and incidentally highlight the potential importance of diabetes as a cause of liver disease.

Adult↗