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Biomedical subjects

G Titus

Publications and source records attributed to G Titus.

14 recordsLinked to original sources
PubMed2000

Two-year mortality and its determinants following acute myocardial infarction in Trinidad and Tobago.

The purpose of this study was to determine the occurrence of coronary artery disease risk factors in patients presenting with acute myocardial infarction (AMI) to a tertiary care institution in Trinidad and to determine the factors associated with increased mortality following AMI. All patients admitted to the Eric Williams Medical Sciences Complex (EWMSC) between January 1 and December 31, 1996, with a diagnosis of AMI were identified using the hospital admissions and discharge diagnosis databases. Demographic, clinical and laboratory variables were extracted from the hospital case records of patients with confirmed AMI. Sixty-one AMI patients (38 men) were admitted during the study period. Mean age at admission was 60 +/- 11 years with an ethnic case mix of thirty-nine (62%) of East Indian descent, eight (13%) of African descent, twelve (20%) mixed ethnicity and three (5%) of Caucasian descent. Thirty patients (49%) were hypertensive. Thirty-two patients (53%) were diabetic and eighteen patients (30%) gave a history of cigarette smoking. The mean left ventricular ejection fraction was 53 +/- 14%. The mean serum cholesterol from 29 patients was 228.2 +/- 49.0 mg/dl. Increasing age, female gender, an ejection fraction less than 40%, non treatment with streptokinase and in-hospital ventricular fibrillation were associated with poor survival. Multiple regression analyses identified three independent predictors of mortality. These were gender (p = 0.04), in-hospital ventricular fibrillation (p = 0.001) and an ejection fraction less than 40% (p = 0.02). Diabetes mellitus, hypertension, hyperlipidaemia and cigarette smoking were prevalent amongst patients presenting with AMI. Ventricular function was a major determinant of two-year mortality following AMI. Aggressive risk factor modification is recommended to prevent both first and recurrent coronary events.

Adult↗
PubMed1989

Binding and autophosphorylating activity of human insulin analogs.

Insulin receptor binding and autophosphorylating activities of a number of synthetic analogs of human insulin have been examined using highly purified insulin receptor from human placenta. In general, autophosphorylation correlates well with the ability of the analogs to stimulate glucose oxidation and to inhibit lipolysis in adipocytes although their biological activities varied over a wide range. These findings support the hypothesis that autophosphorylation is an obligatory step in the pathways leading to glucose oxidation and inhibition of lipolysis. The relative biological potencies of the analogs in the autophosphorylation assay also correlated well with their receptor-binding affinities except for the peptides [endo-TyrB16a]insulin, in which an additional Tyr has been inserted between TyrB16 and LeuB17 and [ProA2]insulin. The relative receptor binding affinity of [endo-TyrB16a]insulin is significantly greater than its biological activity in the adipocyte or receptor autophosphorylation assays. The converse is true for [ProA2]insulin. These results demonstrate that the amino-acid residues involved in binding and receptor activation may not be identical.

Amino Acid Sequence↗
PubMed1987

The rat liver insulin receptor.

Using insulin affinity chromatography, we have isolated highly purified insulin receptor from rat liver. When evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, the rat liver receptor contained the Mr 125,000 alpha-subunit, the Mr 90,000 beta-subunit, and varying proportions of the Mr 45,000 beta'-subunit. The specific insulin binding of the purified receptor was 25-30 micrograms of 125I-insulin/mg of protein, and the receptor underwent insulin-dependent autophosphorylation. Rat liver and human placental receptors differ from each other in several functional aspects: (1) the adsorption-desorption behavior from four insulin affinity columns indicated that the rat liver receptor binds less firmly to immobilized ligands; (2) the 125I-insulin binding affinity of the rat liver receptor is lower than that of the placental receptor; (3) partial reduction of the rat liver receptor with dithiothreitol increases its insulin binding affinity whereas the binding affinity of the placental receptor is unchanged; (4) at optimal insulin concentration, rat liver receptor autophosphorylation is stimulated 25-50-fold whereas the placental receptor is stimulated only 4-6-fold. Conversion of the beta-subunit to beta' by proteolysis is a major problem that occurs during exposure of the receptor to the pH 5.0 buffer used to elute the insulin affinity column. The rat receptor is particularly subject to destruction. Frequently, we have obtained receptor preparations that did not contain intact beta-subunit. These preparations failed to undergo autophosphorylation, but their insulin binding capacity and binding isotherms were identical with those of receptor containing beta-subunit. Proteolytic destruction and the accompanying loss of insulin-dependent autophosphorylation can be substantially reduced by proteolysis inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1984

Ligands for insulin receptor isolation.

Biotinylated insulins are bivalent molecules having the ability to bind to insulin receptors on the one hand and to "avidins" on the other. In order to be useful as ligands for insulin receptor isolation, biotinylated insulins must be developed that have the capacity to bind simultaneously to both and insulin receptor. The present investigation addresses this problem. A series of biotinylated and dethiobiotinylated insulins has been prepared in which the distance between the biotin carboxyl group and the insulin varies from 7 to 20 atoms. These compounds form complexes with succinoylavidin. The dissociation rates (K-1) of these complexes have been determined from the [14C]biotin exchange assay. The dissociation kinetics of most of these complexes are biphasic, and the kinetic constants reported are those corresponding to the slow rate. Ligands containing dethiobiotin dissociate more rapidly than the corresponding biotin derivatives. The interposition of a spacer arm substantially decreases the rate of dissociation. The [14C]biotin exchange assay could not be used with streptavidin complexes of the above ligand since biotin dissociates more rapidly from streptavidin than from succinoylavidin. However, the relative dissociation rates of a series of ligands could be determined and were as follows: 6-(dethiobiotinylamido)-hexanoic acid greater than dethiobiotinyl-A1-insulin greater than biotinylinsulin greater than biotinyl-A1-insulin greater than biotinyl-A2-insulin. Dethiobiotin and its amide failed to form complexes with streptavidin. The affinity of the ligands for insulin receptors was determined by measuring their ability to stimulate 14CO2 formation from [1-14C]glucose in rat epididymal adipocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
PubMed1984

Avidin-biotin affinity chromatography: application to the isolation of human placental insulin receptor.

The ligand N alpha, B1-(6-biotinylamido)hexanoyl-insulin was attached noncovalently to Sepharose 4B immobilized succinoylavidin to form an insulin-affinity resin. This resin was used to isolate highly purified insulin receptor from human placental tissue by a four step process involving (i) preparation of a crude membrane fraction, (ii) solubilization with Triton X-100, (iii) wheat germ agglutinin purification, and (iv) insulin-affinity chromatography. NaDodSO4/PAGE of the purified 125I-labeled receptor under nonreducing conditions showed the presence of a major component with an approximate molecular weight of 350,000 and a minor component with a molecular weight of approximately equal to 166,000. Based on the assumption that the degree of labeling is comparable in both components, the material corresponding to the Mr 350,000 peak represents approximately equal to 94% of the receptor preparation as determined by scanning the autoradiograms. The specific insulin binding capacity of the preparation is 18 +/- 6 micrograms of 125I-labeled insulin per mg of protein as determined by the polyethylene glycol assay and analyzed by Scatchard plot. Insulin binding activity was stable at 4 degrees C and pH 7.6 for at least 12 weeks but was destroyed by freezing and thawing. The availability of highly purified receptor afforded the opportunity to explore its precipitability by polyethylene glycol under assay conditions. Whereas trichloroacetic acid precipitated 95% of the 125I-labeled receptor, polyethylene glycol precipitated only 30%. If the specific activity of the receptor is corrected for incomplete precipitability by polyethylene glycol, the apparent specific binding would be 3.5 +/- 1.2 mol of insulin per mol of receptor. These results are in disagreement with the current receptor model, which postulates that 1 mol of receptor (Mr, 350,000) binds 2 mol of insulin. Clearly, the problems associated with the method available for determining insulin binding are sufficiently serious to preclude their use in determining receptor valence.

Avidin↗
PubMed1982

Insulin receptor isolation studies.

The observation that N alpha,B1-biotinylinsulin binds firmly to resins in which succinoylavidin is covalently attached to AH Sepharose 4B and can be retrieved by exposure of the resins to 20 mM biotin provided the basis for the present investigations. Solubilized, partially purified insulin receptor from human placenta binds to affinity resins in which N alpha,B1-biotinylinsulin is noncovalently attached to AH Sepharose 4B-immobilized-succinolylavidin. Exposure of the receptor loaded resin to 20 mM biotin results in liberation of a high molecular weight material containing bound 125I-biotinylinsulin, which precipitates with polyethyleneglycol and cross reacts with human insulin receptor antibodies. The technique is biospecific and appears to be applicable to the purification of insulin receptors on a preparative scale. Crude solubilized insulin receptor from human placenta is contaminated with "insulinase" which is inhibited by N-ethylmaleimide. HPLC provides a tool to assess "insulinase" activity that is more sensitive than the TCA precipitation method.

Chromatography, High Pressure Liquid↗
PubMed1982

Recovery from thyroid-stimulating hormone-induced refractoriness in thyroid slices: effect of removal of hormone and new protein synthesis.

An initial incubation of bovine thyroid slices with TSH causes decreased responsiveness to the subsequent addition of the hormone when the adenylate cyclase -cAMP system and other metabolic parameters are measured. After the initial incubation with TSH, refractoriness persists despite incubation of thyroid slices for 24 h in the absence of added TSH. Removal of persistently bound TSH by trypsin or antibody to TSH did not reverse the refractoriness during a subsequent 2 h incubation without added TSH. However, normal TSH responsivity was restored by the removal of TSH bound during the first incubation by the addition of either trypsin or antibody to TSH at the beginning of a 24-h second incubation. Restitution of TSH responsiveness after treatment with trypsin or antibody to TSH requires new protein synthesis. While TSH-induced refractoriness does not modify stimulation of cAMP by cholera toxin, its effect on glucose oxidation is significantly diminished. Menadiol stimulation of glucose oxidation is not inhibited in thyroid slices refractory to TSH. Thus, the effect of menadiol is subsequent to the block induced by TSH, whereas that of cholera toxin is proximal to it.

Adenylyl Cyclases↗
PubMed1981

Hormonal properties of avidin-biotinylinsulin and avidin-biotinylcorticotropin complexes.

In connection with the development of affinity columns (based on the avidin-biotin interaction) for retrieval of peptide and protein hormone receptors, the hormonal properties of a number of avidin-biotinylinsulin and avidin-bioinylcorticotropin complexes were examined. Of particular interest was an evaluation of streptavidin as a ligand for the attachment of biotinylated hormones to solid supports and its possible advantage over SpHPP-avidin (S = succinoylated; pHPP = 3-(p-hydroxyphenyl)propionyl). As concerns binding kinetics using rat liver plasma membranes, streptavidin was found superior to avidin since it does not display apparently nonsaturable binding. Scatchard analyses of the binding of 125I-streptavidin, 125I-S-streptavidin and 125I-SpHPP-avidin to rat liver plasma membranes gave KD value of 6.7, 13.2, and 10.6 nM respectively. The binding was saturable and the unlabeled proteins competed with their labeled counterparts for the membrane binding sites. Biotinylinsulin, attached to either streptavidin or SpHPP-avidin was able to compete for 125I-insulin-binding sites on rat liver plasma membranes though somewhat larger concentrations of the complexes than of insulin were required to achieve comparable inhibition. The ID50 values for insulin and the biotinylinsulin complexes were 5 and 80 nM respectively. Biotinylcorticotropin was found to be a more effective activator of particulate rat adrenal adenylate cyclase when complexed with unmodified avidin than with streptavidin, S-streptavidin or SpHPP-avidin.

Adenylyl Cyclases↗
PubMed1980

Hormone-receptor studies with avidin and biotinylinsulin-avidin complexes.

Avidin can be labeled to high specific radioactivity by introducing 3-(p-hydroxyphenyl)-propionyl groups into the molecule (pHPP-avidin). 125I-pHPP-avidin binds avidly to rat liver plasma membranes and is not displaced by unlabeled pHPP-avidin. Nonspecific binding of 125I-pHPP-avidin can be substantially reduced by succinoylation of pHPP-avidin with succinic anhydride (SpHPP-avidin). Spectral changes ensuing when the dye 4-hydroxyazobenzene-2'-carboxylic acid binds to avidin cannot be used to assess the binding characteristics of the modified avidins since the absorption coefficients of the complexes are markedly different; however, the modified molecules bind theoretical amounts of [14C]biotin. Biotinylinsulin and biotinylinsulinSpHPP-avidin complexes compete with 125I-insulin for binding to receptor sites on rat liver plasma membranes. Biotinylinsulin complexes with unmodified avidin display anomalous binding behavior attributable to formation of membrane aggregates. In light of this finding, results obtained using unmodified avidin must be interpreted with caution. Biotinylinsulin125I-SpHPP-avidin binds specifically and saturably to rat liver plasma membranes. The biotinylhormoneSpHPP-avidin technique has potential for labeling peptide hormones and other compounds that cannot be iodinated by conventional procedures.

Amino Acid Sequence↗
PubMed1980

Effect of increased circulating thyroid-stimulating hormone on in vitro thyroid-stimulating hormone stimulation of thyroid and adipose tissues.

Hormones have been shown to regulate the number and/or binding properties of their own receptors. The present studies examined the effect of chronic increased endogenous TSH levels, induced by tapazole or thyroidectomy, on in vitro TSH responsiveness and binding in thyroid and adipose tissues. The results showed that TSH and prostaglandin E1 significantly increased cAMP levels in the thyroids of weight- and age-matched controls, whereas thyroids from tapazole-treated rats responded only to prostaglandin E1. Iodide organification was also measured, and the thyroids from tapazole-treated rats showed a significantly reduced effect of TSH compared to weight- and age-matched controls, although stimulation by dibutyryl cAMP was equivalent in all three groups. TSH or epinephrine stimulation of cAMP and glucose oxidation was equivalent in adipose tissue from control and hypothyroid rats. There was a significant 50% reduction in the number TSH-binding sites in thyroids from tapazole-treated rats: the affinity remained unchanged. [125I]TSH binding to adipose tissue plasma membranes was similar in control and hypothyroid groups. These studies demonstrate that elevated levels of TSH appear to regulate the number of TSH receptors in thyroid, but not adipose, tissue.

Adipose Tissue↗
PubMed1980

Effects of thyrotropin and cholera toxin on the thyroidal adenylate cyclase-adenosine 3',5'-monophosphate system.

The present experiments examined the relationship between cholera toxin and TSH stimulation of the adenylate cyclase system in bovine thyroid tissue. Preincubation of thyroid slices for 20 min at 4 C with a maximal concentration of cholera toxin (100 microgram/ml) did not impair the subsequent stimulation of cAMP by submaximal amounts of TSH (1 mU/ml) during a 5-min incubation at 37 C. Incubation of cholera toxin or TSH with mixed gangliosides, followed by the addition of thyroid slices resulted in inhibition of the cholera toxin but not the TSH stimulation of cAMP formation. Previous exposure of thyroid slices to TSH induced refractoriness to subsequent stimulation of cAMP formation by TSH, but the response to cholera toxin was unchanged. NAD is necessary for cholera toxin, but not TSH, stimulation of adenylate cyclase. In the absence of NAD, cholera toxin inhibited the effect of maximal concentrations of TSH and prostaglandin E1 on adenylate cyclase activity but had no effect on NaF stimulation. In the presence of NAD, the stimulation of adenylate cyclase activity of bovine thyroid plasma membranes by a maximal amount of TSH was not influeced by maximal amounts of cholera toxin. Cholera toxin had a biphasic action on the binding of [125I]iodo-TSH, with low concentrations enhancing and high concentrations inhibiting binding. TSH augmented the binding of [125I]iodo-cholera toxin over the range of 1-100 mU/tube. Cholera toxin at 10 microgram/ml maximally inhibited binding. In addition to the requirement for ribosylation of adenylate cyclase, the present results indicate that the mechanisms of action of TSH and cholera toxin on cAMP formation are different.

Adenylyl Cyclases↗
PubMed1980

Epidermal growth factor stimulation of human breast cancer cells in culture.

Epidermal growth factor (EGF), a polypeptide found in human and animal blood and secretions, has been found to stimulate a variety of tissues in vitro including normal and malignant rodent mammary epithelium and human breast epithelial cells and fibroadenoma. We have studied the influence of EGF on malignant human breast tissue with a model system comprising human breast carcinoma cells growing in tissue culture. EGF stimulated growth of MCF-7 cells in serum-free medium. After 7 days in culture, a 2-fold increase in cell number and DNA content and a 3-fold increase in total protein were observed in cells incubated with EGF (10 ng/ml). As little as 0.01 ng/ml of EGF stimulated growth; 10 ng/ml was maximal. EGF effects on growth were noted for cells plated at a high as well as sparse (cloning) density. EGF also stimulated the rates of thymidine, uridine, and leucine incorporation into macromolecules in a dose- and time-dependent fashion. Stimulation of uridine and leucine incorporation was evident by 3 hr, whereas EGF stimulation of thymidine incorporation was delayed until 12 to 18 hr. EGF increased the proportion of cells active in DNA synthesis by nearly 2-fold. The combination of optimal concentrations of insulin (also a growth factor for these cells) and EGF did not stimulate growth above that seen with either hormone alone, suggesting a common step in their mechanism of action. The EGF effect was not dependent on the presence of serum and was not enhanced by dexamethasone as reported for other types of cells. EGF had no effect on another human breast cancer cell line, the MDA-231. These studies suggest that growth of some human breast cancers may be influenced by EGF.

Animals↗
PubMed1979

In vitro and in vivo refractoriness to thyrotropin stimulation of iodine organification and thyroid hormone secretion.

Earlier studies indicated that initial exposure of thyroid slices to thyrotropin diminished responsiveness of the adenylate cyclase-cyclic AMP system, glucose oxidation, and (32)P(i) incorporation into phospholipids upon readdition of the hormone. The present studies demonstrate that slices from dog, beef, and human thyroid glands initially incubated with thyrotropin (TSH) were less responsive to subsequent addition of the hormone when organification of iodide was examined. Increasing the amount of TSH did not overcome the refractoriness induced by the initial exposure to the hormone. Furthermore, the stimulatory effects of dibutyryl cyclic AMP and prostagladin E(1) were abolished in slices previously incubated with TSH. Development of such refractoriness did not depend upon new protein synthesis and was not abolished by 1 mM prophylthiouracil in the first incubation. Addition of 0.1 muM thyroxine or triiodothyronine or 1.5 muM iodide during all three incubations did not modify the response to TSH, added for the first time in the third incubation. However, 1 mM iodide in the buffer during all three incubations inhibited the response to TSH during the third incubation. During the refractory period, effects of TSH on colloid droplet formation were also diminished. The in vivo effect of TSH on serum l- triiodothyronine in rats was significantly reduced when the rats had been injected with TSH 8 h earlier. These studies demonstrate that TSH-induced refractoriness also includes effects on organification of iodine and secretion of thyroid hormone. The results cannot be adequately explained by unresponsiveness of adenylate cyclase because effects of dibutyryl cyclic AMP and prostagladin E(1) were also inhibited by prior exposure to TSH.

Animals↗