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Biomedical subjects

M J Walsh

Publications and source records attributed to M J Walsh.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of DNA-protein interactions within a distal regulatory element upstream of a mammalian housekeeping gene promoter.

We have characterized a DNA-protein interaction within a sequence element distal from the site of transcription initiation within the mouse housekeeping gene (HPRT) promoter region. This interaction occurs within a 35-base pair regulatory element which confers cell type-specific gene transcription, designated as the HPRT cis-acting regulatory element (HCRE). Competition analysis by gel mobility shift electrophoresis indicates that this DNA-protein interaction is novel and not related to many transcription factors previously reported. Cell cycle synchronization experiments and gel mobility shift assays have demonstrated that within the HCRE a specific DNA-protein complex responds to G1 activation of the cell cycle. Experiments to purify specific DNA-binding proteins that interact with the HCRE has resulted in the purification of one sequence-specific DNA-binding protein of approximately 66 kDa. To determine the putative DNA-binding sequence, footprinting analysis has mapped the protection from DNase I hydrolysis which confers a core sequence of GTCTGGGT using both affinity purified protein and crude nuclear extract. This DNA motif represents a novel protein-binding sequence. Interestingly, data base searches have identified the same or homologous sequences of this DNA motif in additional genes, potentially related to cellular growth and proliferation. This consensus was most notable within a region 5' upstream of the ornithine decarboxylase gene. The unique cell type-specific regulation of the HPRT gene in the intestinal mucosa is not completely understood at this time but because of the relationship of ornithine decarboxylase expression to cell proliferation and more specifically, to mucosal cell renewal in the intestine, the function of DNA-protein interactions within the consensus sequence may prove analogous. This may account for the cell type-specific and cell-cycle responsive gene regulation previously demonstrated with HPRT. Identification of one sequence-specific DNA-binding protein within the HCRE suggest that this protein contributes to the trans-activation of specific genes during the immediate-early response of the cell cycle.

3T3 Cells↗
PubMed1992

Lipids and fatty acids and their relationship to restenosis.

One hundred consecutive patients had fasting lipids and percutaneous fat biopsy performed at the time of percutaneous transluminal coronary angioplasty to determine if there was an association between restenosis and lipids or fatty acids. Angiographic follow-up and complete lipid and fatty acid results were available in 82 patients. Restenosis occurred in 37/82 (45%). Total cholesterol, triglyceride, high density lipoprotein, low density lipoprotein, and apolipoproteins A1 and B were not associated with restenosis. There was a significantly lower level of the monounsaturated fat palmitoleic acid (p less than 0.02), a trend towards a lower level of the monounsaturated fat oleic acid (p less than 0.09), and a trend towards a higher level of the saturated fat palmitic acid (p less than 0.08) in the restenosis group. The polyunsaturated fatty acids were not associated with restenosis. We conclude that lipids are not significantly associated with restenosis, and that lower levels of monounsaturated fatty acids may increase the risk of restenosis.

Angina Pectoris↗
PubMed1992

Effects of hypoxia and acute osmotic stress on intermediary metabolism in Leishmania promastigotes.

This study further explores the effects of hypoxia and acute osmotic stress on intermediary metabolism of Leishmania major and Leishmania donovani. Late log phase promastigotes were washed and incubated with glucose as the sole exogenous carbon source, and rates of glucose consumption and product formation were measured as a function of osmotic strength (610, 305, and 167 mOsm kg-1) and pO2 (95, 10, and 0% O2) in the presence of 5% CO2. Very mild hypoxia dramatically altered flux through the pathways of intermediary metabolism and increased the rates of production of the major metabolites, thus confirming the presence of a low-affinity O2 sensor which was active under all osmolalities tested. The data also require that as pO2 is lowered towards anoxia an endogenous carbohydrate source is mobilized. Under aerobic conditions, acute hypo-osmotic stress had little effect on product formation, whereas acute hyperosmotic stress altered metabolism in a manner similar to mild hypoxia, with the exception of decreasing the rates of acetate and succinate production. It was also shown in L. donovani promastigotes that the effects of anoxia and hyperosmolality were not additive. Thus, separate sensors with partially overlapping actions are involved in the metabolic responses to hypoxia and hyperosmolality. There was no apparent species-specificity for the responses to pO2 and osmotic stress. Uncoupling with carbonyl cyanide p-trifluoromethoxyphenylhydrazone caused changes in metabolite flux patterns which differed from the changes caused by either hypoxia or acute osmotic stress, while rotenone and calcium ionophore A23187 had no significant effects. The identity of the sensors responsive to pO2 and osmolality, and the mechanisms by which they regulate flux through the pathways of intermediary metabolism, require further study.

Animals↗
PubMed1992

The postsynaptic density: constituent and associated proteins characterized by electrophoresis, immunoblotting, and peptide sequencing.

The proteins of the postsynaptic density (PSD) fraction of cerebral cortex were resolved by two-dimensional electrophoresis (2DE) and more than 30 proteins identified by characteristic 2DE mobility, immunoblotting with specific antibodies, and N-terminal and peptide sequencing. The PSD fraction is enriched for spectrin, actin, tublin and microtubule associated protein II, myosin, enzymes of glycolysis, creatine kinase, elongation factor 1 alpha, and receptor protein. The three neurofilament proteins are detected but a 58-kDa protein is prominent and is, by peptide sequencing, the bovine homolog of the recently cloned 66-kDa neurofilament protein; in contrast to the latter, however, it is enriched in cerebrum compared with spinal cord. A 68-kDa protein is identified as a member of the hsp70/BiP family of proteins. A protein, designated dynamin, indicating its putative role as a microtubule motor, is identified as a major protein, is found, however, greatly enriched in the particulate fraction, and is significantly denaturant and detergent insoluble. A protein designated N-ethylmaleimide-sensitive factor is also detected. Thus, two proteins implicated in vesicular transport are present in the PSD fraction. Seven polyclonal antibodies were produced to 2DE separated and electroeluted proteins of the PSD and were identified by peptide sequence analysis and 2DE profile as the hsp70/BiP homologous protein, the novel neurofilament protein synapsin IIa, pyruvate kinase, dynamin, aconitase and an unknown contaminating protein, and a 115-kDa protein that by subcellular fractionation and immunoblotting is a diagnostic PSD molecule. In addition, peptide sequences are obtained for four additional higher molecular weight proteins of the PSD that are not related at the level of primary structure to any known proteins.

Actins↗
PubMed1991

Amino acid sequence of bovine muzzle epithelial desmocollin derived from cloned cDNA: a novel subtype of desmosomal cadherins.

Desmosomes are cell-type-specific intercellular junctions found in epithelium, myocardium and certain other tissues. They consist of assemblies of molecules involved in the adhesion of specific cell types and in the anchorage of cell-type-specific cytoskeletal elements, the intermediate-size filaments, to the plasma membrane. To explore the individual desmosomal components and their functions we have isolated DNA clones encoding the desmosomal glycoprotein, desmocollin, using antibodies and a cDNA expression library from bovine muzzle epithelium. The cDNA-deduced amino-acid sequence of desmocollin (presently we cannot decide to which of the two desmocollins, DC I or DC II, this clone relates) defines a polypeptide with a calculated molecular weight of 85,000, with a single candidate sequence of 24 amino acids sufficiently long for a transmembrane arrangement, and an extracellular aminoterminal portion of 561 amino acid residues, compared to a cytoplasmic part of only 176 amino acids. Amino acid sequence comparisons have revealed that desmocollin is highly homologous to members of the cadherin family of cell adhesion molecules, including the previously sequenced desmoglein, another desmosome-specific cadherin. Using riboprobes derived from cDNAs for Northern-blot analyses, we have identified an mRNA of approximately 6 kb in stratified epithelia such as muzzle epithelium and tongue mucosa but not in two epithelial cell culture lines containing desmosomes and desmoplakins. The difference may indicate drastic differences in mRNA concentration or the existence of cell-type-specific desmocollin subforms. The molecular topology of desmocollin(s) is discussed in relation to possible functions of the individual molecular domains.

Amino Acid Sequence↗
PubMed1991

Complete amino acid sequence of the epidermal desmoglein precursor polypeptide and identification of a second type of desmoglein gene.

The amino acid sequence of the precursor to desmoglein, a major desmosomal cadherin, has been determined from a cDNA clone from bovine muzzle epithelium, and the transcription start site, i.e., the beginning of the approximately 7.6 kb mRNA, identified by primer extension analysis. The precursor segment of 49 amino acids starts with a relatively hydrophobic stretch of 17 amino acids, conforming to the typical features of signal peptides, displays no sequence homology to the corresponding portion of other cadherins. The isolation of the complete cDNA has allowed the cloning of a desmoglein cDNA construct, which under the control of the human beta-actin promoter, was successfully used in cell transfection. In addition, a major N-glycosylation site has been identified by lectin affinity chromatography and amino acid sequencing at amino acid position 61, i.e., in the middle of the first extracellular domain. In the course of these studies we have identified, in colon carcinoma and other simple epithelial cells, another kind of desmoglein which by partial cDNA-derived sequence and by Southern blotting is clearly the product of a different gene. This suggests that there are multiple desmogleins which can be differentially expressed in various epithelia.

Amino Acid Sequence↗
PubMed1990

An abundant and ubiquitous homo-oligomeric ring-shaped ATPase particle related to the putative vesicle fusion proteins Sec18p and NSF.

We have discovered a ring-shaped particle of 12.5 nm diameter, 14.5S and apparent molecular weight of approximately 570,000 that displays 6-fold radial symmetry and is composed of a single kind of an acidic (pI approximately 5.5) polypeptide of Mr 97,000 (p97). Using antibodies to this protein we have detected its occurrence in a wide range of cells and tissues of diverse species from frog to man, including highly specialized cells such as mammalian erythrocytes and spermatozoa. In Xenopus laevis oocytes, the particle is found in both isolated nuclei and in manually enucleated ooplasms, which corresponds to immunofluorescence staining dispersed over both nucleoplasm and cytoplasm. The particle has a N-ethylmaleimide (NEM)-inhibitable Mg2(+)-ATPase activity, and its amino acid sequence, as deduced from cDNA clones, displays considerable homology to the mammalian NEM-sensitive fusion protein (NSF) and yeast Sec18p believed to be essential for vesicle fusion in secretory processes, indicating that these three proteins belong to the same multigene family.

Adenosine Triphosphatases↗
PubMed1990

A regulatory element is characterized by purine-mediated and cell-type-specific gene transcription.

Purines and purine nucleotides were found to affect transcription of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in whole nuclei isolated from intestinal mucosa of adult rats fed a purine- and purine nucleotide-free diet. Nuclear run-on transcription assays, performed on whole nuclei from different tissues and cell types, identified an intestine-specific decrease in the overall incorporation of [alpha-32P]UTP in HPRT transcripts from intestinal epithelial cell nuclei when exogenous purines or purine nucleotides were omitted from either the diet or culture medium. Using a 990-base-pair genomic fragment that contains the 5'-flanking region from the HPRT gene, we generated plasmid constructs with deletions, transfected the DNA into various cell types, and assayed for chloramphenicol acetyltransferase (CAT) reporter activity in vitro. We determined that an element upstream from the putative transcriptional start site is necessary to maintain the regulatory response to purine and nucleotide levels in cultured intestinal epithelial cells. These results were tissue and cell type specific and suggest that in the absence of exogenous purines, the presence of specific factors influences transcriptional initiation of HPRT. This information provides evidence for a mechanism by which the intestinal epithelium, which has been reported to lack constitutive levels of de novo purine nucleotide biosynthetic activity, could maintain and regulate the salvage of purines and nucleotides necessary for its high rate of cell and protein turnover during fluctuating nutritional and physiological conditions. Furthermore, this information may provide more insight into regulation of the broad class of genes recognized by their lack of TATA and CCAAT box consensus sequences within the region proximal to the promoter.

Actins↗
PubMed1990

Cardiac catheterisation with 5 French catheters.

From the beginning of November 1987 to the end of January 1989, 526 coronary arteriograms and left ventricular angiograms were performed with 5 French coronary catheters. In 448 (85%) patients diagnostic pictures were obtained with three standard types of 5 French catheters (No 4 Judkins): that is, left coronary, right coronary, and pigtail catheters. In 60 patients (11.4%) various other 5 French catheters were required to complete the study. In nine patients (1.7%), a 7 or 8 French catheter was used. Major complications causing cardiac arrest or requiring urgent operation developed in five patients. Sixty two patients (11.77%) had minor complications that required sublingual nitrates or a single bolus of atropine, or developed a haematoma that did not need intervention or had a mild reaction to the contrast material. Complications of moderate severity developed in 17 patients (3.2%): severe chest pain, arrhythmia requiring a temporary pacemaker, contrast reaction associated with hypotension, haematoma requiring blood transfusion, or a transient ischaemic episode. There were no deaths. 5 French catheters were used for routine coronary angiography and left ventriculography in 98.3% of patients. There were no major complications related to femoral artery puncture. The routine use of 5 French coronary catheters should increase the feasibility of safe coronary angiography in outpatients and should reduce the cost of this investigation.

Angina Pectoris↗
PubMed1990

Identification of desmoglein, a constitutive desmosomal glycoprotein, as a member of the cadherin family of cell adhesion molecules.

Monoclonal antibodies to the constitutive desmosomal glycoprotein desmoglein were characterized whose epitopes are located intracellularly, i.e., in the cytoplasmic portion of this molecule, and contribute to the structure of the desmosomal plaque. Using one of these antibodies (DG3.10), a peptide was isolated from a proteolytic digest of desmoglein purified from isolated bovine muzzle demosomes, and its amino acid sequence was determined. In comparisons of this sequence with the amino acid sequence of desmoglein as deduced from the sequence of cDNA clones from the same tissue, encompassing most of approximately 7.6 kb mRNA and the complete coding region of 959 residues (calculated molecular weight approximately 102,400), the DG3.10 epitope was identified in a region starting 163 amino acids before the carboxy terminus in the first of four consecutive repeats of a homologous element of 29 +/- 1 amino acids. This topological information, together with the identification of a single hydrophobic region of sufficient length to provide a transmembrane segment and of several extended regions showing high sequence homology to various cadherins, has allowed the construction of a model of the molecular organization of desmoglein. We conclude that desmoglein is a member of the cadherin family of cell adhesion glycoproteins which is characterized by an unusually long cytoplasmic domain which exceeds those of the cadherins by more than 275 amino acids, contains special repetitive elements and spans the desmosomal plaque at least once.

Amino Acid Sequence↗
PubMed1989

Identification of a widespread nuclear actin binding protein.

The many different cellular functions so far shown to involve actin and to be regulated by specific actin binding proteins are located primarily, if not exclusively, in the cytoplasm. Actin is also found in the nucleus of various cells, but because of the problems of cell fractionation the significance of nuclear actin has remained unclear. The large amphibian oocyte nucleus (germinal vesicle), however, can be isolated manually with little cytoplasmic contamination. This nucleus contains high concentrations (4-6 mg ml-1) of mostly soluble, although polymerization-competent beta- and gamma-actin, which exists in a nucleocytoplasmic exchange pool. The findings that drastic effects on transcription and chromosome morphology are caused by the injection of actin antibodies or actin binding proteins into germinal vesicles, and that a factor required for accurate transcription by RNA polymerase II is actin, suggest that nuclear actin is involved in specific nuclear functions. We have recently identified two main components in Xenopus laevis oocytes with actin binding activities; one of these activities is Ca2+-dependent, is located predominantly, if not exclusively, in the cytoplasm and is attributable to gelsolin. Here we report that the second component, having a Ca2+-independent activity, is a heterodimeric acting binding protein; this protein is markedly enriched in the nuclei of oocytes and somatic cells of amphibia, but also occurs in nuclei of other vertebrate cells.

Actins↗
PubMed1989

Purification and characterization of acylation stimulating protein.

We have purified to homogeneity and analyzed the amino acid composition of a small (Mr 14,000), basic (pI 9.0) protein from human plasma. This has been named acylation stimulating protein (ASP) because it markedly stimulates triacylglycerol synthesis in human adipocytes. As well, it stimulates triacylglycerol synthesis in human skin fibroblasts cultured from normal individuals. Characteristic saturation curves for the cell metabolic responses to ASP were observed in both cell types with higher stimulation of oleate incorporation into triacylglycerol being observed in adipocytes. The stimulation of triacylglycerol synthesis was much greater with ASP than with insulin. Neither fatty acid binding protein nor albumin was able to mimic the ASP effect.

Adipose Tissue↗
PubMed1989

The effect of ASP on the adipocyte of the morbidly obese.

The control of triglyceride synthesis within the adipocyte is not fully understood. Insulin is considered to be the most potent stimulant of triglyceride synthesis. In this paper, we report on the effect of a small (14000 Da), basic (pI 9.0) protein isolated from human serum. This protein has been called acylation stimulating protein (ASP). It is a potent stimulant of triglyceride synthesis in adipocytes from both normal weight and morbidly obese subjects. Its stimulatory effect on adipocytes is both rapid, occurring between 15-30 min after the start of incubation, and prolonged, lasting for up to 3 hr. Compared to insulin, it is sixfold more potent in its effect on triglyceride synthesis. As well as acting on isolated cells, ASP also has a fourfold stimulatory effect on triglyceride synthesis in human adipose microsomes at a concentration of 25 micrograms/ml. This study indicates that ASP is a potent stimulant of triglyceride synthesis and therefore may play a role in the pathogenesis of morbid obesity.

Adipose Tissue↗
PubMed1988

Extended N-terminal sequencing of proteins of archaebacterial ribosomes blotted from two-dimensional gels onto glass fiber and poly(vinylidene difluoride) membrane.

Previously uncharacterized proteins from intact ribosomes and ribosomal subunits of the extreme halophile Halobacterium marismortui (Haloarcula marismortui) were isolated and separated by high-resolution two-dimensional electrophoresis (2DE). N-Terminal amino acid sequences of 14 of these acidic large-subunit proteins were obtained by direct blotting of the separated proteins from two-dimensional electrophoresis gels to sequencer-stable supports followed by excision of the protein spots and sequencing. Furthermore, long internal sequences were obtained by in situ enzymatic cleavage of halobacterial proteins in gel pieces obtained from two-dimensional gels followed by electrophoretic separation of the fragments, blotting, and sequencing. Precautions are outlined for avoidance of N-terminal blockage of proteins, and the preparation and selection of suitable supports for obtaining extended N-terminal sequences are described. The results suggest that when prior fractionation is carried out to enrich for cell organelles, subcellular components of cells, or cell membranes, it is routinely possible to obtain numerous N-terminal sequences from one or a few 2DE gels of such fractions. Our results also indicate that, with appropriate precautions, proteins are routinely obtainable from 2DE gels in a form suitable for both N-terminal and internal sequence determination and show no detectable evidence for N-terminal blockage or destruction or modification of labile amino acid residues.

Acetates↗
PubMed1988

Purification and characterization of eight class 5 outer membrane protein variants from a clone of Neisseria meningitidis serogroup A.

Methods published for the purification of P.II proteins from Neisseria gonorrhoea have been modified to allow the purification of class 5 proteins from Neisseria meningitidis serogroup A bacteria. The five class 5 protein electrophoretic variants detected within an epidemic in the Gambia (a, b, c, d, and e) and three other variants (f, g, and h) found within other isolates of the same clone in West Africa have been purified with yields of 6-28 mg. The NH2-terminal amino acid sequence for variant c differs from those of the other class 5 proteins, whereas the latter are very similar to the sequence predicted for two class 5 proteins from DNA analyses of serogroup C meningococci and determined for 8 P.II proteins from gonococci. Numerous other regulatory, chemical, and serological differences were found between the c protein and the other class 5 proteins such that we recommend that the class 5 proteins be subdivided into two subclasses. mAbs have been isolated that distinguish between these two protein subclasses and Western blotting with these antibodies enabled us to conclude that both protein subclasses were found in bacteria isolated from different epidemics and pandemics of the last 50 yr.

Amino Acid Sequence↗
PubMed1988

A new device to aid knee surgery. A leg-positioning mechanism.

A novel leg-positioning and holding mechanism has been developed to facilitate knee surgery. The normal thigh tourniquet is used as the point of attachment of the mechanism. The knee may be placed in any of the positions desired for knee surgery by means of a single control lever. To facilitate alignment during arthroplasty a pointer has been incorporated to indicate the midline of the limb. The leg positioner has proved useful during knee arthroplasty, high tibial osteotomy, arthroscopic procedures, and ligament reconstruction. Its use has reduced fatigue, freed the assistant for other tasks, and held the limb more firmly than was possible hitherto.

Humans↗
PubMed1987

Regulation of types I, III, and IV procollagen mRNA synthesis in glucocorticoid-mediated intestinal development.

Administration of dexamethasone (0.8 mg/kg) to 9-day-old rats once daily for 3 consecutive days caused precocious induction of adult specific disaccharidase activity in the small intestine. Maturation-specific disaccharidase activity was accompanied by decreased amounts of types I and III collagen and decreased procollagen type I and III mRNA levels. Conversely, type IV procollagen, fibronectin, and laminin amounts and their respective mRNA levels were increased. In vitro transcription of nuclei isolated from small intestine and colon of suckling rats indicated a decreased rate of synthesis of procollagen types I and III mRNAs and an increased rate of synthesis of procollagen type IV mRNAs and laminin mRNAs after dexamethasone treatment. The data suggest that glucocorticoids mediate a differential regulation of interstitial and basement membrane collagen gene expression in the developing rat intestine.

Animals↗