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Biomedical subjects

M G Harrington

Publications and source records attributed to M G Harrington.

At least 19 recordsLinked to original sources
PubMed1999

Cross-linked poly(N-acetylethylenimine) as an isoelectric focusing matrix.

Agarose and polyacrylamide are the gels used for most analytical and micropreparative electrophoresis of biopolymers. In an alternative approach that offers different physico-chemical properties from these standard gels, nonionic hydrogels and amphigels composed of poly(N-acetylethylenimine) (PAEI) and a variety of cross-linkers were prepared and used as anticonvective matrices for isoelectric focusing. PAEI was prepared from the ring opening, ionic polymerization of 2-methyl-2-oxazoline. The N-acetyl side chains were hydrolyzed with aqueous sodium hydroxide to produce secondary amine sites which were used for the attachment of cross-linkers. Several cross-linkers were tested for their suitability for electrophoresis, and the cross-linker system based on the Diels-Alder reaction between a furan and maleimide tethered to PAEI gave a moldable gel that can be reversibly converted to a sol at 80 degrees C. This gel was used for isoelectric focusing under both denaturing and nondenaturing conditions. Several protein standards were resolved as well as was achieved with polyacrylamide.

Animals↗
PubMed1998

Mucosal metabolism in ulcerative colitis and Crohn's disease.

PURPOSE: Colonic mucosal metabolism of butyrate may be impaired in ulcerative colitis. In this study we sought to confirm this observation, to determine if a similar change occurs in Crohn's colitis, and to establish whether a panenteric disorder of butyrate metabolism exists in either condition. METHODS: With use of a microculture technique, mucosal metabolic fluxes of 14[C]-labeled butyrate and 14[C]-labeled glutamine were measured as 14[C] carbon dioxide production in mucosal biopsy specimens from the colon and ileum in patients with ulcerative colitis, Crohn's colitis, and healthy bowel. Results were expressed as pmol/microg biopsy DNA/hour. RESULTS: In the colon the mucosal metabolic fluxes of both butyrate and glutamine are reduced in both ulcerative colitis and Crohn's colitis compared with healthy controls. These changes were most marked in the presence of moderate to severe mucosal inflammation, there being no significant difference in mucosal metabolic flux between mildly inflamed mucosa and healthy controls. In the ileum the mucosal metabolic fluxes of butyrate and glutamine did not differ between healthy controls and those with either ulcerative colitis or Crohn's colitis. CONCLUSIONS: Changes in colonic mucosal metabolism of butyrate and glutamine in inflammatory bowel disease occur as a consequence of the inflammatory process and are not peculiar to ulcerative colitis. Ileal mucosal metabolism is unchanged in ulcerative colitis and Crohn's colitis, indicating the absence of a panenteric abnormality of mucosal metabolism in these two conditions.

Biopsy↗
PubMed1998

Metabolic substrate utilization differs in ileal faecal and urinary reservoirs.

BACKGROUND: Construction of an ileal faecal or urinary reservoir profoundly alters ileal luminal ecology and availability of mucosal metabolic substrates. The aims of this study were to measure mucosal metabolic flux of butyrate and glutamine in histologically normal (control) ileum and to determine the effect of reservoir construction on metabolic fluxes in patients with ileal pouch-anal anastomosis and ileocystoplasty. METHODS: Endoscopic biopsy samples were obtained from normal ileum (n = 10), ileum of patients with ulcerative colitis (n = 10), ileal pouch-anal anastomosis (n = 7), ileocystoplasty (n = 7) and ileal conduit (n = 7). Using a closed microculture technique, biopsy utilization of 14C-labelled butyrate and glutamine was measured as [14C]carbon dioxide production. Biopsy DNA content was measured and [14C]carbon dioxide evolution expressed as picomoles [14C]carbon dioxide per microgram DNA per hour. RESULTS: The metabolic flux of both butyrate and glutamine was reduced in ileal pouch mucosa compared with that of ileal mucosa in patients with ulcerative colitis. In contrast, the metabolic flux of buyrate alone was reduced in ileal mucosa from ileocystoplasty and ileal conduit compared with that in normal ileal mucosa, while the metabolic flux of glutamine remained unchanged. CONCLUSION: Ileal mucosal metabolic fluxes measured in vitro are altered by changing luminal ecology in vivo. These changes may affect the health and mucosal integrity of ileum used to construct these reservoirs.

Butyrates↗
PubMed1997

14-3-3 and BSE.

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14-3-3 Proteins↗
PubMed1997

SpMyb functions as an intramodular repressor to regulate spatial expression of CyIIIa in sea urchin embryos.

The CyIIIa actin gene of Strongylocentrotus purpuratus is transcribed exclusively in the embryonic aboral ectoderm, under the control of 2.3 kb cis-regulatory domain that contains a proximal module that controls expression in early embryogenesis, and a middle module that controls expression in later embryogenesis. Previous studies demonstrated that the SpRunt-1 target site within the middle module is required for the sharp increase in CyIIIa transcription which accompanies differentiation of the aboral ectoderm, and that a negative regulatory region near the SpRunt-1 target site is required to prevent ectopic transcription in the oral ectoderm and skeletogenic mesenchyme. This negative regulatory region contains a consensus binding site for the myb family of transcription factors. In vitro DNA-binding experiments reveal that a protein in blastula-stage nuclei interacts specifically with the myb target site. Gene transfer experiments utilizing CyIIIa reporter constructs containing oligonucleotide substitutions indicate that this site is both necessary and sufficient to prevent ectopic expression of CyIIIa. Synthetic oligonucleotides containing the myb target site were used to purify a protein from sea urchin embryo nuclear extracts by affinity chromatography. This protein is immunoprecipitated by antibodies specific to the evolutionarily conserved myb domain, and amino acid sequences obtained from the purified protein were found to be identical to sequences within the myb domain. Sequence information was used to obtain cDNA clones of SpMyb, the S. purpuratus member of the myb family of transcription factors. Through interactions within the middle module, SpMyb functions to repress activation of CyIIIa in the oral ectoderm and skeletogenic mesenchyme.

Actins↗
PubMed1997

Covalent variation is a general property of transcription factors in the sea urchin embryo.

Nine different transcription factors of the Strongylocentrotus purpuratus embryo, belonging to diverse structural families, were examined by high-resolution, two-dimensional gel electrophoresis. These factors had all been cloned previously, and antibodies against the recombinant proteins were available. The factors were visualized immunologically in nuclear extracts from 24-hour blastula-stage embryos. Remarkably, every one of the nine factors displayed multiple charge variants. Three factors, SpZ12-1, SpP3A2, and SpRunt-1, were studied at different stages of embryonic development. The prevalence and distribution of the variant isoforms of all three factors differed at each stage examined; and in all cases the complexity of the variants was greatest in the 24-hour blastula-stage extracts. The most complex set of variants was observed for SpP3A2, and phosphatase treatment demonstrated that some but not all, of the covalent modifications defining these variants are phosphorylations. As the transcription factors were chosen for this study merely on the basis of the availability of antibodies, we conclude that deployment of transcription factors in sea urchin embryos generally involves their covalent modification.

Animals↗
PubMed1996

The 14-3-3 brain protein in cerebrospinal fluid as a marker for transmissible spongiform encephalopathies.

BACKGROUND: There is no practical and reliable premortem test for Creutzfeldt-Jakob disease and the related transmissible spongiform encephalopathies. Two proteins, designated 130 and 131, which have been detected in low concentrations in cerebrospinal fluid from patients with Creutzfeldt-Jakob disease, appear to be sensitive and specific markers for the disease. Attempts to identify these proteins, however, have been unsuccessful. We hypothesized that they may be present in the normal brain. METHODS: We detected proteins 130 and 131 in normal human brain, partially sequenced their amino acids, and found that they matched the brain protein known as 14-3-3. We then developed a simple, rapid immunoassay for this protein and tested it in cerebrospinal fluid samples from 71 humans and 30 animals with spongiform encephalopathies and in control samples from 186 humans and 94 animals. RESULTS: The immunoassay detected the 14-3-3 protein in cerebrospinal fluid from 68 of the 71 patients with Creutzfeldt-Jakob disease (96 percent, 95 percent confidence interval, 92 to 99 percent). Among 94 patients with other dementias, the specificity was 96 percent. If one excludes the three patients with dementia who had strokes within one month before testing, the specificity was 99 percent. The test was positive in 12 of 24 patients with viral encephalitis. In animals the sensitivity of the assay was 87 percent and the specificity was 99 percent. CONCLUSION: In patients with dementia, a positive immunoassay for the 14-3-3 brain protein in cerebrospinal fluid strongly supports a diagnosis of Creutzfeldt-Jakob disease. This finding, however, does not support the use of the test in patients without clinically evident dementia.

14-3-3 Proteins↗
PubMed1996

SpRunt-1, a new member of the runt domain family of transcription factors, is a positive regulator of the aboral ectoderm-specific CyIIIA gene in sea urchin embryos.

In this paper we present a structural and functional characterization of a new sea urchin embryo transcription factor, SpRunt-1. This factor was isolated by means of its specific interaction with a cis-regulatory target site of the CyIIIa gene. Here we show that this target site, the P7I site, is required for normal embryonic activation of CyIIIa x CAT reporter gene constructs. An oligonucleotide affinity column bearing the P7I target site purifies a 21-kDa polypeptide from blastula-stage nuclear extracts, and the amino acid sequence obtained from this polypeptide was used to generate a nucleic acid probe with which the corresponding cDNA was cloned. The cDNA encodes an approximately 60-kDa protein, SpRunt-1, which includes a "runt domain" that is closely homologous to those of Drosophila and mammalian runt domain transcription factors. RNA and genomic blots show that SpRunt-1 is represented by a single embryonic transcript, encoded by one of possibly two runt-domain-containing genes. By RNA probe protection we found that transcripts of SpRunt-1 increase in concentration dramatically after the blastula stage of development, suggesting that the up-regulation of CyIIIa that occurs after blastula stage is a function of zygotically transcribed SpRunt-1. These results are discussed with reference to known features of the runt domain family of transcription factors.

Actins↗
PubMed1996

DNA-protein binding assays from a single sea urchin egg: a high-sensitivity capillary electrophoresis method.

A capillary electrophoresis method has been developed to study DNA-protein complexes by mobility-shift assay. This method is at least 100 times more sensitive than conventional gel mobility-shift procedures. Key features of the technique include the use of a neutral coated capillary, a small amount of linear polymer in the separation medium, and use of covalently dye-labeled DNA probes that can be detected with a commercially available laser-induced fluorescence monitor. The capillary method provides quantitative data in runs requiring < 20 min, from which dissociation constants are readily determined. As a test case we studied interactions of a developmentally important sea urchin embryo transcription factor, SpP3A2. As little as 2-10 x 10(6) molecules of specific SpP3A2-oligonucleotide complex were reproducibly detected, using recombinant SpP3A2, crude nuclear extract, egg lysates, and even a single sea urchin egg lysed within the capillary column.

Animals↗
PubMed1995

Stabilization of ribonuclease B activity by concentrated xylose solutions.

Ribonuclease B has become a paradigm as a simple example of an N-linked glycoprotein. We have found that certain affinity-purified preparations of this enzyme demonstrated a pronounced tendency to lose activity if stored as dilute aqueous solutions. Such inactivation is accelerated by the presence of NaCl, but can be counteracted by inclusion of high (1 mol/l) concentrations of xylose. Enzyme activity cannot be restored by addition of xylose after storage of the enzyme. In marked contrast to alpha-methyl-mannoside, xylose does not prevent ribonuclease B from binding to concanavalin A and so may be used to stabilize the enzyme during purification by lectin affinity chromatography.

Chromatography, Affinity↗
PubMed1995

SpGCF1, a sea urchin embryo DNA-binding protein, exists as five nested variants encoded by a single mRNA.

Several Strongylocentrotus purpuratus gene cis-regulatory regions contain asymmetric C4 sequences which are core elements of target sites for a specific DNA-protein interaction. Blastula stage nuclear extract contains five proteins which specifically bind to these target sites, resulting in a characteristic pattern of complexes in gel mobility shift assays. We used automated affinity chromatography to purify a protein which binds to these sites and have isolated the corresponding cDNA. This protein, SpGCF1, is a novel sea urchin DNA-binding protein with no overall homology to proteins reported in the databases currently available. The DNA-binding domain of this protein was identified by a deletion analysis. As demonstrated both for protein translated in vitro and for bacterial protein expressed from a cDNA clone, a single SpGCF1 mRNA serves as a template for the synthesis of five DNA-binding polypeptides. We show that these five polypeptides are most likely produced by differential usage of a nested set of AUG start codons in the SpGCF1 cDNA and thus contain variable amounts of a proline-rich N-terminal domain. Since proline-rich regions often serve as transcriptional activation domains, the five SpGCF1 proteins apparently possess different "activation potentials."

Amino Acid Sequence↗