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Biomedical subjects

T Gaffney

Publications and source records attributed to T Gaffney.

At least 19 recordsLinked to original sources
PubMed1996

Preproinsulin I and II mRNAs and insulin electron microscopic immunoreaction are present within the rat fetal nervous system.

Insulin-like substance has been found within the nervous system. In the rat, preproinsulin II mRNA was shown within the brain and preproinsulin I mRNA within the retina. The present study demonstrates the presence of preproinsulin mRNAs within the 15, 17 and 19 day gestational age fetal rat brain, spinal cord and dorsal root ganglia (DRG), employing RNA template-specific polymerase chain reaction (RS-PCR), semi-nested PCR and RNase protection assay. Preproinsulin I mRNA was present in the 17 and 19 day gestational age brain, spinal cord and DRG, and only in the brain of the 15 day gestational age brain. Preproinsulin II mRNA was present in all the gestational ages studied in the brain, spinal cord and DRG. The RS-PCR and the semi-nested PCR demonstrated products that co-migrated with the pancreatic control. The semi-nested products were characterized as preproinsulin I and II by restriction enzyme digestion and sequence. RNase protection assay using specific cRNA for preproinsulin I and II showed a band that co-migrated with pancreatic preproinsulin I and II mRNAs, and confirmed the PCR results. In addition, insulin receptor mRNA was detected by RS-PCR. Ultrastructural studies showed insulin immunoreaction within the endoplasmic reticulum, Golgi apparatus, cytoplasm, axon, dendrites, and in relation to the synapses. Thus, we demonstrated the presence of preproinsulin I and II mRNA, insulin receptor mRNA and insulin immunoreaction within the rat fetal central and peripheral nervous system.

Animals↗
PubMed1995

Molecular analysis of genes encoding phenazine biosynthesis in the biological control bacterium. Pseudomonas aureofaciens 30-84.

The DNA sequence of five contiguous open reading frames encoding enzymes for phenazine biosynthesis in the biological control bacterium. Pseudomonas aureofaciens 30-84 was determined. These open reading frames were named phzF, phzA, phzB, phzC and phzD. Protein PhzF is similar to 3-deoxy-D-arabino-heptulosonate-7-phosphate synthases of solanaceous plants. PhzA is similar to 2,3-dihydro-2,3-dihydroxybenzoate synthase (EntB) of Escherichia coli. PhzB shares similarity with both subunits of anthranilate synthase and the phzB open reading frame complemented an E. coli trpE mutant deficient in anthranilate synthase activity. Although phzC shares little similarity to known genes, its product is responsible for the conversion of phenazine-I-carboxylic acid to 2-hydroxy-phenazine-I-carboxylic acid. PhzD is similar to pyridoxamine phosphate oxidases. These results indicate that phenazine biosynthesis in P. aureofaciens shares similarities with the shikimic acid, enterochelin, and tryptophan biosynthetic pathways.

Amino Acid Sequence↗
PubMed1995

Characterization of tobacco plants expressing a bacterial salicylate hydroxylase gene.

Transgenic tobacco plants that express the bacterial nahG gene encoding salicylate hydroxylase have been shown to accumulate very little salicylic acid and to be defective in their ability to induce systemic acquired resistance (SAR). In recent experiments using transgenic NahG tobacco and Arabidopsis plants, we have also demonstrated that salicylic acid plays a central role in both disease susceptibility and genetic resistance. In this paper, we further characterize tobacco plants that express the salicylate hydroxylase enzyme. We show that tobacco mosaic virus (TMV) inoculation of NahG tobacco leaves induces the accumulation of the nahG mRNA in the pathogen infected leaves, presumably due to enhanced stabilization of the bacterial mRNA. SAR-associated genes are expressed in the TMV-infected leaves, but this is localized to the area surrounding necrotic lesions. Localized acquired resistance (LAR) is not induced in the TMV-inoculated NahG plants suggesting that LAR, like SAR, is dependent on SA accumulation. When SA is applied to nahG-expressing leave's SAR gene expression does not result. We have confirmed earlier reports that the salicylate hydroxylase enzyme has a narrow substrate specificity and we find that catechol, the breakdown product of salicylic acid, neither induces acquired resistance nor prevents the SA-dependent induction of the SAR genes.

Catechols↗
PubMed1991

Allele-specific activation of genetically engineered receptors.

The binding of agonists and antagonists to the beta-adrenergic receptor (beta AR) is postulated to involve an ionic interaction between the amine group of the ligand and the carboxylate side chain of Asp113 in the third hydrophobic domain of the receptor. To explore the importance of this interaction in the binding of ligands to the beta AR, a Ser residue was substituted for Asp113, and the ability of this mutant receptor to respond to compounds which could potentially interact with the hydroxyl side chain of the Ser residue was assessed. The mutant receptor was fully activated by catechol-containing esters and ketones, compounds which did not activate the wild-type beta AR. The demonstration that the molecular substitution of a single amino acid residue can alter the ligand binding specificity of the beta AR provides evidence that the chemical nature of this residue is a critical determinant in the recognition site of the receptor. Further, the ability to modify the specificity of a receptor by the replacement of amino acids at the binding site demonstrates the potential for the rational design of drugs which function specifically at genetically engineered receptors.

Adenylyl Cyclase Inhibitors↗
PubMed1990

Evidence for a third structural class of beta-1,3-glucanase in tobacco.

Glucan endo-1,3-beta-glucosidases (beta-1,3-glucanases) have been implicated in several developmental processes and they may also play a direct role in the plant's defense against fungal pathogens. In an effort to characterize the glucanase gene family, complementary DNA clones encoding an acidic form of beta-1,3-glucanase have been isolated from tobacco. The cDNA was expressed in E. coli and shown to encode a beta-1,3-glucanase activity. The protein sequence encoded by the cDNA was found to match the partial protein sequence of PR-35, a previously characterized beta-1,3-glucanase. The protein encoded by the cDNA was purified from the extracellular fluid of TMV-infected tobacco leaves and found by immunological methods to correspond to glucanase PR-Q'. From a detailed analysis of the cDNA it is clear that this glucanase represents a third structural class of enzyme which differs substantially from both the basic, vacuolar glucanase and the acidic, extracellular forms (PR-2, PR-N and PR-O). It has previously been demonstrated that the basic form of beta-1,3-glucanase is synthesized as a pre-pro-enzyme and upon maturation the 21 amino acid signal peptide and a 22 amino acid carboxy-terminal peptide are removed. This processing event has been proposed to be involved with the vacuolar localization of the enzyme. By comparing the deduced protein structure of PR-Q' to that of the basic form it is evident that this extracellular enzyme is missing the carboxy-terminal 22 amino acids. The role of a conserved phenylalanine-glycine dipeptide in the processing of glucanases and other pathogenesis-related proteins from tobacco is discussed.

Amino Acid Sequence↗
PubMed1989

Cotranscription of genes encoding indoleacetic acid production in Pseudomonas syringae subsp. savastanoi.

Indoleacetic acid (IAA) production by the plant pathogen Pseudomonas syringae subsp. savastanoi is essential for tumor formation on olive and oleander. The bacterium produces IAA from tryptophan in reactions catalyzed by tryptophan monooxygenase and indoleacetamide hydrolase. The genetic determinants are, respectively, iaaM and iaaH. In oleander isolates, the genes encoding the IAA biosynthetic enzymes are located on a plasmid; in olive isolates, the genes occur on the chromosome. The IAA genes from the oleander isolate strain EW2009 are located within a 4-kilobase (kb) segment of the 52-kb plasmid pIAA1. Escherichia coli strains harboring a recombinant plasmid, pCJP3, which contains this 4-kb fragment, excreted IAA into culture media, and crude cell extracts had both tryptophan monooxygenase and indoleacetamide hydrolase activity. In vitro coupled transcription-translation of pCJP3 demonstrated that this fragment coded for proteins of 62 and 47 kilodaltons which correspond to tryptophan monooxygenase and indoleacetamide hydrolase, respectively. Expression of these genes was dependent upon a vector promoter in pCJP3. However, in the absence of a vector promoter, E. coli containing recombinant plasmids with additional pIAA1 DNA in front of iaaM had high levels of tryptophan monooxygenase. Northern (RNA) hybridization experiments verified that iaaM and iaaH are cotranscribed as a portion of a ca. 4- to 5-kb transcript in vivo. Southern hybridization experiments with IAA plasmids from different oleander strains of P. syringae subsp. savastanoi revealed that all IAA plasmids contained a region of at least 10 kb of homology, with the IAA genes at one end. Repetitive DNA and a copy of IS51 were found at the end of this region of homology.

Amidohydrolases↗
PubMed1988

Myocardial kinetics of technetium-99m-hexakis-2-methoxy-2-methylpropyl-isonitrile.

To study the potential usefulness of technetium-99m hexakis-2-methoxy-2-methylpropyl-isonitrile (Tc-MIBI) as a cardiac perfusion imaging agent, the left circumflex coronary arteries of 12 dogs were partially occluded. Eight additional control dogs had no coronary artery stenosis. Myocardial Tc-MIBI activities in the left circumflex and left anterior descending zones were continuously monitored by miniature implantable radiation detectors for 4 hr after administration of the isotope. The dogs were then killed. Serial gamma camera images were also acquired during the study. Heart rate, arterial blood pressure, pressure distal to the stenosis, and cardiac output did not change significantly during the experiment. Microsphere-determined regional myocardial blood flow was significantly reduced in the left circumflex distribution in the 12 dogs with coronary artery stenoses. In the 12 dogs with left circumflex coronary artery stenoses, the 4 hr fractional Tc-MIBI clearances from the normal and ischemic zones were minimal and equivalent (0.15 +/- 0.05 vs 0.15 +/- 0.07). In the eight control dogs, 4 hr fractional Tc-MIBI clearances from the left anterior descending and left circumflex artery zones were minimal and equivalent (0.11 +/- 0.06 vs 0.10 +/- 0.07). Four hour fractional Tc-MIBI clearance from the blood was 0.98 +/- 0.03 for the dogs with stenosis and 0.97 +/- 0.02 for the dogs without stenosis. One additional dog had complete occlusion of the left circumflex coronary artery followed by administration of Tc-MIBI and scandium-46-labeled microspheres. This heart was immediately sectioned and counted to determine the relationship of regional blood flow to Tc-MIBI distribution (r = .92).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Treatment with desmopressin acetate to reduce blood loss after cardiac surgery. A double-blind randomized trial.

Bleeding after cardiopulmonary bypass remains a cause for concern, requiring reexploration of the chest in approximately 3 percent of patients who have had operations on the heart. We examined the possibility that this problem might be alleviated by desmopressin acetate (DDAVP), which increases the plasma level of von Willebrand factor and improves hemostasis in mild hemophilia and other conditions associated with defective platelet function. In a double-blind, prospective, randomized trial, we studied the effect of intraoperative desmopressin acetate in 70 patients undergoing various cardiac operations requiring cardiopulmonary bypass. Patients undergoing uncomplicated primary coronary-artery bypass grafting were not included. The drug significantly reduced mean operative and early postoperative blood loss (1317 +/- 486 ml in the treated group vs. 2210 +/- 1415 ml in the placebo group); of the 14 patients whose 24-hour blood loss exceeded 2000 ml, 11 had received the placebo. Plasma levels of von Willebrand factor were higher after desmopressin acetate than after placebo. Patients with the most bleeding had relatively low levels of von Willebrand factor before operation, suggesting a role for this factor in the hemorrhagic tendency induced by extracorporeal circulation. There were no untoward side effects of desmopressin acetate. We conclude that the administration of desmopressin acetate can be recommended to reduce blood loss in patients undergoing complex cardiac operations. The beneficial effect of the drug on hemostasis after cardiopulmonary bypass may be related to its effect on von Willebrand factor.

Adult↗
PubMed1981

Effect of a high energy, low carbohydrate diet on serum levels of lipids and lipoproteins.

The effect of a high energy, low carbohydrate (ketogenic) diet on serum levels of lipids and lipoproteins was studied in two normal individuals. A marked rise in serum levels of cholesterol and phospholipid occurred during the diet, but serum triglyceride levels remained within the normal range. High density lipoprotein cholesterol level did not change, and the rise in serum level of cholesterol levels with its distribution in lipoproteins, these observed changes pose a potential atherogenic risk. Serum lipid and lipoprotein levels reverted to normal on discontinuation of the diet. A modest weight loss observed during the diet was regained within 24 hours of the diet's cessation.

Adult↗