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Biomedical subjects

T Hunter

Publications and source records attributed to T Hunter.

At least 235 records · Page 13Linked to original sources
PubMed1990

A one-year, open, prospective study of sulfasalazine in the treatment of rheumatoid arthritis: adverse reactions and clinical response in relation to laboratory variables, drug and metabolite serum levels, and acetylator status.

Response to sulfasalazine was studied for 1 year in 45 patients with rheumatoid arthritis. Twenty-two patients achieved a satisfactory clinical response. Adverse reactions developed in 15. Hemoglobin rose, and platelet count, erythrocyte sedimentation rate, rheumatoid factor titer, immunoglobulins, and C3 component of complement fell in relation to degree of response. At 1 year, 18 patients elected to continue the treatment. No relationship between clinical response, adverse reactions, or laboratory changes and drug disposition was observed.

Acetylation↗
PubMed1990

p34cdc2: the S and M kinase?

In the yeast cell cycle, the induction of two very different processes, DNA synthesis (S-phase) and mitosis (M-phase), requires the same serine/threonine-specific protein kinase p34cdc2, which has been highly conserved through evolution. On the basis of work conducted largely in multicellular eukaryotes, it has recently been suggested that p34cdc2 is able to perform these two mutually exclusive roles by phosphorylating different sets of substrates through a cell cycle-dependent association with other proteins that dictate the substrate specificity of the protein kinase. To recognize its mitotic substrates, p34cdc2 associates with one of the cyclins--a family of proteins of two distinct but related types (A and B) characterized by their periodic destruction at each mitosis. In interphase, the formation of a complex between p34cdc2 and another protein (or proteins) would allow the phosphorylation of a different set of proteins involved in the G1 to S transition. This review focuses on the evidence for this appealing simple model and the nature of the putative substrates proposed.

Amino Acid Sequence↗
PubMed1989

cDNA cloning and sequencing of the protein-tyrosine kinase substrate, ezrin, reveals homology to band 4.1.

Ezrin is a component of the microvilli of intestinal epithelial cells and serves as a major cytoplasmic substrate for certain protein-tyrosine kinases. We have cloned and sequenced a human ezrin cDNA and report here the entire protein sequence derived from the nucleotide sequence of the cDNA as well as from partial direct protein sequencing. The deduced protein sequence indicates that ezrin is a highly charged protein with an overall pI of 6.1 and a calculated molecular mass of 69,000. The cDNA clone was used to survey the distribution of the ezrin transcript, and the 3.2 kb ezrin mRNA was found to be expressed in the same tissues that are known to express the protein and at the same relative levels. Highest expression was found in intestine, kidney and lung. The cDNA clone hybridized to DNAs from widely divergent organisms indicating that its sequence is highly conserved throughout evolution. The amino acid sequence of ezrin revealed a high degree of similarity within its N-terminal domain to the erythrocyte cytoskeletal protein, band 4.1 and secondary structure predictions indicate that a second region of ezrin contains a long alpha-helix, a feature also common to band 4.1. The structural similarity of ezrin to band 4.1 suggests a mechanism for the observed localization to the membrane, and a role for ezrin in modulating the association of the cortical cytoskeleton with the plasma membrane.

Amino Acid Sequence↗
PubMed1989

Isolation of a human cyclin cDNA: evidence for cyclin mRNA and protein regulation in the cell cycle and for interaction with p34cdc2.

This paper reports the nucleotide and predicted amino acid sequence of a human B-type cyclin. The predicted protein sequence shows strong homology to the other known cyclins in the central third of the protein. We show that the level of cyclin mRNA is regulated during the cell cycle, increasing during G2 phase to four time that present in G1. The protein accumulates steadily during G2 to at least 20 times its level in G1 and is abruptly destroyed at mitosis. In G2/M phase, cyclin is associated with p34cdc2, the human homolog of the fission yeast gene cdc2+, and this complex has histone H1 kinase activity.

Amino Acid Sequence↗
PubMed1989

Phospholipase C-gamma is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro.

Phospholipase C-gamma (PLC-gamma) was rapidly phosphorylated on tyrosines and serines following PDGF and EGF treatment of quiescent 3T3 mouse fibroblasts and A431 human epidermoid cells, respectively, PDGF treatment increased PLC-gamma phosphorylation within 30 sec. This lasted for up to 1 hr, and occurred at high stoichiometry. Continuous receptor occupancy was required to maintain this phosphorylation. Three major sites of tyrosine phosphorylation were detected in PLC-gamma, two of which were phosphorylated in EGF-treated A431 cells. Under certain conditions PDGF receptor coimmunoprecipitated with PLC-gamma, suggesting that PDGF receptor can phosphorylate PLC-gamma directly. Indeed, purified PDGF or EGF receptor phosphorylated purified PLC-gamma on tyrosines identical to those phosphorylated in vivo. Tyrosine phosphorylation of PLC-gamma was not induced by bombesin, TPA, or insulin. Stimulation of PLC-gamma tyrosine phosphorylation and the reported ability of PDGF and EGF to induce phosphatidylinositol turnover in different cells were strongly correlated. We propose that tyrosine phosphorylation of PLC-gamma by PDGF and EGF receptors leads to its activation, and a consequent increase in phosphatidylinositol turnover.

Animals↗
PubMed1989

A novel integrin beta subunit is associated with the vitronectin receptor alpha subunit (alpha v) in a human osteosarcoma cell line and is a substrate for protein kinase C.

We demonstrate that a novel integrin beta subunit is present in association with the vitronectin receptor (VNR) alpha subunit on the surface of MG-63 human osteosarcoma cells. This beta subunit and the glycoprotein IIIa beta subunit (beta 3) were both found complexed with VNR alpha on MG-63 cells and in at least two other human cell types we examined. Tryptic peptide mapping indicated that the two beta subunits are related but distinct. The novel beta chain, referred to here as beta s, was not recognized by the monoclonal antibody AP3, which recognizes GPIIIa, nor by an antiserum raised against a peptide from the COOH-terminal cytoplasmic domain of beta 3. Both receptor complexes bound to and were specifically eluted from a column containing the cell adhesion peptide GRGDSP. The unique beta subunit became phosphorylated at high stoichiometry when MG-63 cells or AG1523 human fibroblasts were treated with the phorbol-ester tumor promoter phorbol 12-myristate 13-acetate. This phosphorylation occurred mainly on serine and probably at one major site, as determined by phosphotryptic peptide mapping. Protein kinase C phosphorylated the beta s subunit of intact receptor in vitro, at the same site phosphorylated in treated cells, indicating that protein kinase C is likely to be responsible for this phosphorylation in vivo.

Electrophoresis, Polyacrylamide Gel↗
PubMed1989

Signal transduction by epidermal growth factor occurs through the subclass of high affinity receptors.

Many cell types display two classes of epidermal growth factor receptor (EGFR) as judged from EGF binding studies; i.e., a major class of low affinity EGFR and a minor class of high affinity EGFR. We have studied their respective contribution to the cascade of events elicited by EGF in human A431 carcinoma cells, using anti-EGFR mAb 2E9. This antibody specifically blocks EGF binding to low affinity EGFR, without activating receptors in intact cells, and thus enables us to study the effects of exclusive EGF binding to high affinity EGFR. We show that blocking of low affinity EGFR by mAb 2E9 has almost no effect on the activation of the receptor protein-tyrosine kinase by EGF, suggesting that EGFR kinase activation occurs exclusively through the subclass of high affinity EGFR (5-10%). In addition, we provide evidence that high affinity EGFR exists both in monomeric and dimeric forms, and that cross-phosphorylation of low affinity EGFR by high affinity EGFR may take place in dimers of both receptor types. We demonstrate that the following early cellular response to EGF are also unimpaired in the presence of mAb 2E9: (a) inositol phosphate production, (b) release of Ca2+ from intracellular stores, (c) rise in intracellular pH, (d) phosphorylation of EGF on threonine residue 654, (e) induction of c-fos gene expression, and (f) alteration in cell morphology. As possible nonspecific side effects, we observed that the EGF induced Ca2+ influx and fluid-phase pinocytosis were inhibited in A431 cells in the presence of mAb 2E9. We conclude, therefore, that the activation of the EGFR signal transduction cascade can occur completely through exclusive binding of EGF to the subclass of high affinity EGFR.

Cell Line↗
PubMed1989

Pharmacological characterisation of a re-entry model for atrial tachycardia in conscious dogs.

We studied the effects of intravenously administered autonomic agents, the calcium channel blocker diltiazem, and Class I and III anti-arrhythmic agents on a re-entry model for atrial tachycardia in seven conscious dogs. The model was created by a Y-shaped incision in the intercaval region and front free wall of the right atrium. Acetylcholine (30 micrograms.kg-1) transiently but significantly shortened the cycle length of atrial tachycardia (CL) by 9 (SD 5)% while atropine (0.2 mg.kg-1) alone did not change or terminate the rhythm. Isoprenaline (4 micrograms.kg-1) briefly reduced CL, by 13(6)% whereas propranolol (1 mg.kg-1) alone significantly prolonged the CL and terminated the arrhythmia in 50% of the trials. Diltiazem (200 micrograms.kg-1) did not significantly change the CL. Class Ia agents procainamide (30 mg.kg-1) and quinidine (3.5 approximately 20 mg.kg-1, effective doses) increased CL by 60(30) and 43(28)% respectively and terminated the arrhythmias. Class Ib agents, phenytoin (5 and 10 mg.kg-1, cumulative doses), lignocaine (3.2 approximately 7 mg.kg-1), and mexiletine (6 approximately 20 mg.kg-1), prolonged CL by 31(14), 40(15), and 72(13)%; however, consistent conversion of the rhythm occurred only with mexiletine. Ic agents flecainide (0.5 approximately 3 mg.kg-1) and encainide (0.6 approximately 6.4 mg.kg-1) lengthened the CL by 58(17) and 75(43)% and converted the rhythm in all cases. Class III agents clofilium (0.5 mg.kg-1), d-sotolol (3.2 approximately 20 mg.kg-1), and N-acetylprocainamide (15 approximately 120 mg.kg-1) prolonged the CL by 14(4), 16(5), and 19(7)% and terminated the arrhythmia in all trials. The magnitudes of CL prolongation by the Class III agents were significantly smaller than those by the Class Ia, Ib, or Ic agents. These results show that in this re-entrant model for atrial tachycardia endogenous adrenergic but not cholinergic systems play a major role in maintaining impulse propagation and that calcium channel mediated slow inward currents do not participate in the impulse generation processes. Further, using CL as an index, the model serves as a useful tool for differentiating Class III from Class I anti-arrhythmic activities.

Acetylcholine↗
PubMed1989

A dual model for cardiac arrhythmias: coexistence of re-entry and abnormal automaticity and effects of antiarrhythmic agents.

1. We have developed a dual model for arrhythmia anaesthetized dogs. The model consists of an inducible re-entrant atrial tachycardia and spontaneous ventricular ectopies in the same heart. 2. The model for re-entrant atrial tachycardia was created by crushing the right atrium longitudinally in the intercaval region and transversely in the front free wall parallel to the atrioventricular groove. Ventricular abnormal automaticity was produced by prior (20 approximately 24 h) left anterior descending coronary artery occlusion. The ventricular arrhythmia was partially suppressed during rapid pacing-induced atrial tachycardia and resumed after atrial re-entry was terminated. 3. Mapping experiments indicate that the atrial tachycardia was due to circus movement occurring in the tissue around the tricuspid ring. This re-entrant circuit was identical to that induced in the model created by the incision method. 4. Clofilium (0.75 mg kg-1, n = 5) increased the cycle length of atrial re-entry by 14 +/- 4% from 139 +/- 12 to 159 +/- 18 ms (P less than 0.05). Flecainide (1.8 +/- 0.9 mg kg-1, n = 5) prolonged the cycle length of the tachycardia by 114 +/- 57% from 158 +/- 11 to 332 +/- 66 ms (P less than 0.05). 5. Both drugs terminated the atrial arrhythmia, but re-entry could be reinduced only in flecainide-treated dogs. Flecainide reduced ventricular ectopies by 89 +/- 19%, whereas clofilium did not change ventricular abnormal automaticity or maximum pacing cycle length that is necessary to overdrive the ventricle fully. 6. These data indicate that the dual model provides coexisting arrhythmias of different mechanisms in the same heart and that Class I and Class III anti-arrhythmic drugs may be differentiated from each other by the distinct patterns of pharmacological activities produced in this test system.

Animals↗
PubMed1989

The ets sequence is required for induction of erythroblastosis in chickens by avian retrovirus E26.

E26 is a replication-defective avian retrovirus that causes an erythroblastic leukemia in vivo and transforms hematopoietic precursor cells of both the erythroid and the myeloid lineages in vitro. The E26 genome contains two sets of cell-derived sequences, ets and myb. myb sequences are also present in avian myeloblastosis virus, which transforms myeloblasts exclusively. To determine whether the ets sequence is responsible for the erythroid specificity of E26, we analyzed the transforming activities of several viruses carrying mutations in the ets sequence constructed in vitro. The mutant viruses retained the ability to transform myeloid cells in vitro, indicating that the myb oncogene is sufficient for this viral function. However, the ets-deficient viruses did not cause an overt leukemia in chickens. The results indicate that the ets sequence is required for the induction of erythroblastosis by E26.

Alpharetrovirus↗
PubMed1989

Synthesis of p36 and p35 is increased when U-937 cells differentiate in culture but expression is not inducible by glucocorticoids.

p36 and p35 are distinct but related proteins that share many structural and biochemical features which were first identified as major substrates for protein-tyrosine kinases. Subsequently, both proteins have been shown to be Ca2+-, phospholipid-, and F-actin-binding proteins that underlie the plasma membrane and are associated with the cortical cytoskeleton. Recent reports have claimed that these proteins function as lipocortins, i.e., phospholipase A2 inhibitors that mediate the anti-inflammatory action of glucocorticoids. To investigate this possibility and to learn more about the functions of p36 and p35, we used human-specific anti-p36 and anti-p35 monoclonal antibodies to determine whether the expression or secretion of either protein was inducible by dexamethasone in the human U-937 myeloid cell line and in other human cell types. Additionally, we examined the levels of mRNA for both proteins. No effect of dexamethasone was observed on p36 or p35 expression at either the mRNA or protein level, nor were these proteins secreted under any of the culture conditions investigated. However, it was observed that in these cells the rate of synthesis and accumulation of both proteins was increased when the U-937 cells were induced to differentiate in culture to adherent macrophagelike cells. This offers a model system with which to study the control of p36 and p35 expression.

Animals↗
PubMed1989

AtT20 cells express modified forms of pp60c-src.

We have compared the properties of pp60c-src from the mouse pituitary tumor cell line, AtT20, and from mouse fibroblasts. In vitro, pp60c-src phosphotransferase activity from AtT20 cells is 2- to 3-fold that of mouse NIH 3T3 fibroblast pp60c-src. In analyzing the reason for this elevation in specific activity, we found that pp60c-src from AtT20 cells differs structurally in at least three ways from pp60c-src in fibroblasts. First, AtT20 cells and primary rat anterior pituitary cells express low levels of the neuronal form of pp60c-src. Second, pp60c-src from AtT20 cells is phosphorylated at two additional N-terminal serine residues. Last, AtT20 pp60c-src is phosphorylated to a lower overall stoichiometry.

Acid Phosphatase↗