PubMed Health⌕ Search

Biomedical subjects

E B Dowdle

Publications and source records attributed to E B Dowdle.

At least 37 records · Page 2Linked to original sources
PubMed1987

The reactive sites of proteinase inhibitors from Erythrina seeds.

Although the Kunitz-type proteinase inhibitors from the seeds of various Erythrina species have similar molecular weights (approximately 20,000), and share many other chemical characteristics, they could nevertheless be divided into three groups on the basis of their relative abilities to inhibit chymotrypsin, trypsin and tissue plasminogen activator. Group a inhibitors were relatively specific for chymotrypsin; they were poor inhibitors of trypsin and had no apparent effect upon tissue plasminogen activator. Group b proteins inhibited trypsin strongly and chymotrypsin slightly less effectively. They had no effect upon t-PA. Group c inhibitors inhibited trypsin, chymotrypsin and t-PA. Analysis of the amino acid composition of the three groups of inhibitors revealed major differences in alanine content. Minor differences in the content of most other amino acids were also noticed. Group b and group c inhibitors had, in most cases, the same reactive sites (Arg-Ser). The sequences neighbouring the reactive sites showed a significant degree of homology. Chemical modification of arginine in proteinase inhibitors from the seeds of E. latissima and soybeans using 1-2-cyclohexanedione confirmed the presence or absence of arginine in the reactive sites.

Amino Acid Sequence↗
PubMed1986

Characterization of seven human melanoma cell lines: melanogenesis and secretion of plasminogen activators.

Permanent cell lines (UCT-Mel 1 through 7) were established from biopsies of metastatic tissue taken from seven patients with malignant melanoma. Cells from these lines were all aneuploid and all grew as non-contact-inhibited, adherent monolayers. All of the lines, with the remarkable exception of UCT-Mel 6, formed tumours in nude mice, expressed the melanoma M-18 antigen and synthesized plasminogen activators exclusively of the tissue-type. UCT-Mel 6 cells were non tumourigenic, they lacked the M-18 antigen and they synthesized plasminogen activators exclusively of the urokinase type. UCT-Mel 1 and UCT-Mel 2 formed pigment in vitro and both of these lines showed an increase in pigment content and tyrosinase synthesis with increasing cell density. The rate of plasminogen activator released by UCT-Mel 1 and UCT-Mel 3 declined strikingly as the cells became confluent. Assuming that proteolytic activity is required for cell migration in vivo; that tyrosinase synthesis reflects expression of the differentiated phenotype and that melanoma cells retain some of the characteristics of neural crest cells, we suggest that the effects of confluence and close cell-cell contact provide a useful experimental counterpart for the study of normal neural crest all behaviour that is characterized by an inverse relationship between migration and a protease secretion on the one hand and pigmentation on the other.

Cell Count↗
PubMed1985

The complete amino acid sequence of trypsin inhibitor DE-3 from Erythrina latissima seeds.

Trypsin inhibitor DE-3 from Erythrina latissima seeds contains 172 amino acids, including 4 half-cystine residues, and resembles the Kunitz-type inhibitors. Limited hydrolysis of DE-3 with trypsin at pH 3 produced two fragments, F1 and F2, containing 63 and 109 amino acids, respectively. Amino-terminal sequence studies revealed that F1 was the N-terminal and that F2 was the C-terminal fragment. The complete amino acid sequence of fragments F1 and F2 was then determined on peptides produced by enzymatic digestion with trypsin and Staphylococcus aureus V8 protease. The sequence of trypsin inhibitor DE-3 from E. latissima seeds shows a high degree of homology to those of Kunitz-type trypsin inhibitors from soybeans and winged bean seeds.

Amino Acid Sequence↗
PubMed1985

Genetically determined molecular weight differences in murine complement component C6.

Plasma samples from male CBA, BALB/c and DBA/2 mice were subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Bands of C6 hemolytic activity in the washed resolving gel were identified by means of an erythrocyte/agarose overlay gel. CBA plasma was found to contain two forms of C6, one with a molecular weight (Mr) of approximately 90 000 (type A) and one with a Mr of 100 000 (type B); CBA mice were thus designated C6: A+B+. BALB/c and DBA/2 plasma on the other hand contained only the low molecular weight type A C6 and were designated C6: A+B-. Flat bed isoelectric focusing followed by functional overlays showed that CBA plasma produced two sets of hemolytic bands (pI 5.25-5.4 and 5.0-5.2) whereas BALB/c and DBA/2 plasma produced only the more cathodal band set. The additional band set in CBA plasma corresponded to the high-Mr type B C6. Results of breeding experiments demonstrated that the possession of the high Mr type B C6 was inherited in the manner of an autosomal phenotypically dominant characteristic.

Animals↗
PubMed1984

Purification of human tissue plasminogen activator with Erythrina trypsin inhibitor.

Seeds of the legume Erythrina latissima contain a 20,000-dalton, single-chain protein that has been shown to inhibit the amidolytic activity of trypsin and tissue plasminogen activator. It had no comparable effect on urokinase. IC50 values of 1.1 X 10(-7) M for tissue plasminogen activator and 6.9 X 10(-10) M for trypsin were determined by titration. When coupled to agarose, the Erythrina inhibitor provided an effective reagent for affinity purification of tissue plasminogen activator from melanoma cell-conditioned tissue culture medium. Using this as a single-step procedure, 270-fold purified enzyme was reproducibly obtained with yields of 90% or greater. Both one- and two-chain forms of tissue plasminogen activator were purified. The enzyme migrated, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as a predominant 72,000-dalton doublet with lesser amounts of immunochemically similar, 115,000- and 68,000-dalton components.

Cells, Cultured↗
PubMed1983

Diamphotoxin. The arrow poison of the !Kung Bushmen.

We have purified the arrow poison extracted from Diamphidia nigro-ornata pupae by the !Kung Bushmen of Southern Africa, and named it diamphotoxin. The toxin is a single chain polypeptide of Mr = 60,000 with an isoelectric point of pH 9.5. It blocks neuromuscular function and is cardiotoxic and hemolytic, and the minimum lethal dose for mice is 25 pg (less than 0.5 fmol). The toxin binds tightly to cells, permitting the resolution of two distinct phases. Erythrocytes exposed to toxin in the absence of divalent cations show no apparent lesion (phase I). After washing and addition of 1 mM Ca2+, there occurs a rapid efflux of K+ followed by the loss of hemoglobin (phase II). The pH optimum for phase I is pH 6.7 and for phase II pH 8.6. The action of the toxin is noncatalytic, requiring a solution concentration of approximately 65 toxin molecules/cell for hemolysis of sheep erythrocytes under standard conditions. Ca2+ ions induce a conformational change in the free, purified toxin molecule. We propose that this change also occurs in membrane-bound toxin. Hemolysis would result from the formation of channels permitting the diffusion of small cations.

Amino Acids↗
PubMed1983

Functional identification of serum complement components following electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate.

A method is described for detecting the active complement components C6 and C7 after polyacrylamide gel electrophoresis (PAGE) of whole serum in the presence of sodium dodecyl sulphate (SDS). The method involves the removal of SDS by washing with non-ionic detergent followed by the application of an erythrocyte/agarose gel to detect haemolytic activity. Two forms of human C6 with apparent molecular weights of approximately 121,000 daltons and 114,000 daltons were observed. Major activity resided in the 121,000 dalton species. The 2 forms of human C6 were not related to known genetic polymorphisms for this component. Analysis of sera from different animal species showed that not all possessed the 2 forms of C6 and that there were interspecies differences in C6 molecular weights. These are most marked in the case of human and murine C6; the major form of murine C6 had a molecular weight approximately 20,000 daltons less than the major human form. One form of human C7 with an apparent molecular weight of 104,000 daltons was seen. The molecular weights of C7 from the various animal sera tested did not differ significantly from this. Studies with reducing agents and metabolic inhibitors showed that both C6 and C7 required intact disulphide bonds and sulphydral groups for functional activity.

Animals↗
PubMed1983

The regulation of tissue plasminogen activator activity by human fibroblasts.

We have found that live and ethanol-fixed fibroblasts, when covered with conditioned medium containing tissue plasminogen activator, associate with the enzyme and remove it from the medium. Binding of tissue plasminogen activator to fixed cells showed equilibrium kinetics with maximal uptake corresponding to 2.4 units of enzyme per 10(6) fixed cells. Enzyme bound to fixed cells could activate plasminogen and produce plaques of caseinolysis in casein-plasminogen-agar overlays. Electrophoretic analysis showed it covalently attached to a fibroblast component with a molecular weight of 40,000-50,000. Sequestration of tissue plasminogen activator by live fibroblasts showed nonsaturable first order kinetics with a rate constant of 0.465/hr. We conclude that active enzyme is bound to a surface receptor, then internalized and degraded. Fibroblasts did not release the binding molecule into the medium; binding of tissue plasminogen activator from the medium was unaffected by heparin or thrombin. This phenomenon differs from that described by Baker et al. and ascribed to "proteasenexin."

Cell Count↗
PubMed1983

The effects of dexamethasone and tetradecanoyl phorbol acetate on plasminogen activator release by human acute myeloid leukemia cells.

This investigation was undertaken to examine the extent to which leukemic cell functions are susceptible to regulation in vitro and to investigate their heterogeneity in this regard. Since plasminogen activator release is known to be a modulatable cellular function that can be influenced by antiinflammatory steroids and tetradecanoyl phorbol acetate (TPA), the effect of these two compounds on the secretion of urokinase- or tissue-type enzymes by leukemic cells was studied. The release of both enzyme species could be stimulated or suppressed by these substances by mechanisms that were inhibitable by actinomycin-D and hence required transcription of new mRNA. Plasminogen activator release by cells from 41/45 patients with AML was either stimulated or inhibited by 10(-7) M dexamethasone, implying that most AML cells possess glucocorticoid receptors. In 26/45 cases, the enzyme was inhibited by this steroid to less than 25% of control values. Pronounced inhibition of this degree was not encountered with normal polymorphonuclear leukocytes. Plasminogen activator secretion by AML cells was profoundly inhibited in 20/41 cases by 1 ng/ml TPA and stimulated in 8/41 cases. Leukemic blasts varied considerably in their response to dexamethasone and TPA. Plasminogen activator release should prove a sensitive means of monitoring the responses of AML cells to biologically active compounds.

Cell Survival↗
PubMed1983

The secretion of plasminogen activators by human myeloid leukemic cells in vitro.

Peripheral blood cell preparation from 23 normal subjects and 72 patients with acute and 32 patients with chronic myeloid leukemia were cultured in vitro and released plasminogen activators were analyzed. The quantity of plasminogen activator secreted by leukemic cells varied widely and could not be correlated with the clinical severity of the disease. Immunochemical and electrophoretic techniques have been used to show that normal peripheral blood granulocytes released exclusively urokinase-like plasminogen activator, whereas leukemic cells secreted either urokinase or a tissue activator-like enzyme. The molecular species of enzyme released by acute myeloid leukemic cells may serve as a diagnostic marker of relevance to the management of this disease, since patients with acute myeloid leukemia whose cells released only tissue plasminogen activator did not respond to combination chemotherapy. Tissue plasminogen activators released by leukemic cells may display an unusual electrophoretic pattern that resembles that shown by urokinase. Immunochemical procedures are therefore essential for the correct identification of these enzymes.

Cells, Cultured↗
PubMed1982

Variable effects of retinoids on two pigmenting human melanoma cell lines.

Two melanoma cell lines, each derived from a different patient with metastatic disease, were very similar in their appearance, their growth characteristics, and their tendency to differentiate and to pigment in culture as they become confluent. These lines, UCT-Mel 1 and UCT-Mel 2, were used to study the effects of retinoic acid and other derivatives of vitamin A. When added to UCT-Mel 1 cells, retinoids had only a modest effect on plasminogen activator release and were without measurable effect on morphology, growth, or tyrosinase synthesis. In contrast, when added to UCT-Mel 2 cells, retinoids appeared to induce a more differentiated state evident as an inhibition of cell proliferation and the assumption of a dendritic morphology. Paradoxically, however, retinoids caused a striking inhibition of the density-dependent intracellular accumulation of tyrosinase and melanin that was taken to represent spontaneous in vitro differentiation. Culture of UCT-Mel 2 cells in the presence of retinoic acid resulted in initial inhibition followed by marked stimulation of cellular plasminogen activator release. The data suggest that the manner in which retinoids exert their effects on cells in vitro does not depend on the histological origin of the tumor cells being studied but on the innate responsiveness of that particular cell line to the retinoid or compound in question.

Aged↗
PubMed1981

Increased susceptibility to herpes simplex virus infections in children with acute measles.

Children with measles treated either in hospital or as outpatients were examined for clinical or laboratory evidence of oropharyngeal herpes simplex virus (HSV) infection. HSV was isolated from 43% of the hospitalized children and 37% of the outpatient children and an additional 9% of the hospitalized group had lesions without the virus being isolated. The incidence of both HSV isolation and clinical lesions in infected children was significantly greater in children hospitalized for measles than in a control group of children hospitalized for tuberculosis. Data from the outpatient group strongly suggest that malnourished children with measles are particularly susceptible to HSV infection. The immunosuppressed state of the children with acute measles was demonstrated by impaired lymphocyte transformation responses to phytohemagglutinin and the inability of their serum to support normal transformation responses by lymphocytes from normal individuals. However the extent of this immunosuppression was unrelated to the susceptibility of the children to HSV infection.

Child, Preschool↗
PubMed1980

Conractile responses of guinea-pig ileum to high-frequency electrical field stimulation.

The responses of longitudinal muscle-myenteric plexus strips prepared from guinea-pig ileum to high-frequency electrical field stimulation (10-50 Hz) were investigated. The primary high-frequency responses consisted of a cholinergic component that was less susceptible to inhibition by adenyl compounds than the 0.1 Hz twitch response, and a non-cholinergic component which included a considerable degree of direct muscle stimulation which was not affected by adenosine and adenine nucleotides at concentrations up to 100 micro M. High frequency stimulation in the presence of atropine, at concentrations that effectively blocked the contractions produced by high doses of exogenously applied acetylcholine, elicited delayed complex secondary contractions. These contractions were abolished by tetrodotoxin and were inhibited by morphine and adenyl compounds. The secondary contractions were also elicited in choline-free Krebs buffer following treatment with 100 micro M hemicholinium-3 for 2 h; and they were not enhanced by the addition of 1.5 micro M eserine sulphate. By these criteria, it is concluded that the secondary contractions were mediated by a neurogenic, non-cholinergic mechanism. The nature of the spasmogen, which was not identified, is discussed.

Acetylcholine↗
PubMed1980

The effects of dipyridamole on the guinea-pig ileal longitudinal muscle-myenteric plexus preparation.

1 The longitudinal muscle-myenteric plexus strip prepared from guinea-pig ileum has been used to study the actions of dipyridamole and its interactions with adenine derivatives. 2 Dipyridamole augmented the inhibitory effects of adenine derivatives on the twitch response induced by 0.1 Hz field stimulation of the preparation. This synergistic effect was apparent with relatively low concentrations of dipyridamole (10 to 100 nM) and after short pretreatment times (1 to 2 min) that did not inhibit the twitch response on their own. Appropriate studies suggested the dipyridamole-adenosine synergism followed a pattern of facilitative agonist competition. 3 Dipyridamole did not inhibit either uptake of [3H]-adenosine by the preparation or adenosine deaminase activity under the same conditions that it exhibited synergism with adenosine. 4 Higher concentrations of dipyridamole inhibited the twitch response, mainly by decreasing acetylcholine release but partly by a direct action on smooth muscle. The direct action of dipyridamole on muscle was not synergistic with adenosine. 5 Fluorescence microscopy showed preferential binding of dipyridamole to the myenteric plexus.

Adenosine↗
PubMed1980

The effects of calcium concentration on the inhibition of cholinergic neurotransmission in the myenteric plexus of guinea-pig ileum by adenine nucleotides.

1 Adenosine and the adenine nucleotides AMP, ADP, ATP, cyclic AMP, NAD, NADP and NADH produced a dose-related inhibition of the contractile response of guinea-pig ileum longitudinal muscle-myenteric plexus strips to low frequencies (less than 1 Hz) of electrical field stimulation. 2 These compounds inhibited hexamethonium-sensitive contractions induced by nicotine but did not alter the responses to exogenous acetylcholine, and the acetylcholine output from the myenteric plexus was inhibited by the adenyl compounds. These findings indicate that adenine derivatives act at a presynaptic site on postganglionic cholinergic neurones. 3 The degree of inhibition produced by adenine compounds was inversely related to the calcium concentration of the bath fluid over a range of calcium concentrations (1 to 5 mM) that had no effect on the responses of the muscle to exogenous acetylcholine. 4 The inhibition produced by adenine derivatives was antagonized by theophylline and augmented by dipyridamole. Both of these interactions were sensitive to, and synergistic with, alterations of the concentration of calcium in the bath fluid. 5 The results suggest that adenine compounds inhibit acetylcholine release from the myenteric plexus by diminishing the availability of intracellular calcium ions required for neurotransmitter release.

Adenine Nucleotides↗
PubMed1980

Effects of retinoids on normal and neoplastic human cells cultured in vitro.

The effects of retinoic acid on cultured human cells derived from normal and neoplastic tissues were studied. Retinoic acid consistently induced plasminogen activator synthesis by cells of mesenchymal origin (with the exception of adult skin fibroblasts) but not by cells of epithelial origin. The effect of retinoic acid was more pronounced than that of equimolar concentrations of retinol or retinyl acetate. Dexamethasone inhibited the retinoid-induced increase in plasminogen activator in lung- and foreskin-derived fibroblasts. Cells derived from normal or neoplastic tissues showed no consistent differences either in baseline rates of plasminogen activator release or in the magnitude of the retinoid effect.

Cells, Cultured↗