Eugene B. Dowdle

Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates

Abstract A new technique is described for the electrophoretic analysis of plasminogen activators in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized plasminogen and gelatin. The method depends upon the fact that the zymogen and gelatin, when incorporated into the polyacrylamide matrix at the time of casting, are retained during subsequent electrophoresis of enzyme samples, and serve as satisfactory sequential, in situ substrates for the localization of plasminogen activator bands by negative staining. The nonionic detergent, Triton X-100, is used to remove sodium dodecyl sulfate and restore enzyme activity. The method can be used to detect as little as 1 mU of urokinase and effectively distinguishes between melanoma- and urokinase-type plasminogen activators. Plasminogen-independent proteases are detected by omission of plasminogen from the gel.

The effects of sex and age on serum IgE concentrations in three ethnic groups.

Serum IgE concentrations were measured using a radio-radial immunodiffusion method in 4,440 blood donors resident in the Western Cape Province, South Africa, and the following differences were observed. Firstly, in all three ethnic groups studied, that is the Whites, the Cape Coloureds and the Africans, there was a tendency for males to have higher serum IgE concentrations than females. This differences was highly significantly in Whites. Secondly, in the Whites, serum IgE concentrations tended to be lower with advancing age. Thirdly, previous reports of elevated serum IgE concentration in indigenous African population were confirmed. No clearly defined cause for these differences emerged rom this study.

Bibrotoxin, a novel member of the endothelin/sarafotoxin peptide family, from the venom of the burrowing asp Atractaspis bibroni

A new member of the endothelin/sarafotoxin family of vasoconstrictor peptides, bibrotoxin (BTX), was isolated from the venom of the burrowing aspAtractaspis bibroni by reversed‐phase FPLC. The amino acid sequence of BTX differs from SRTX‐b in the substitution Ala4 instead of Lys4, which suggests that it represents the peptide isoform of Atractaspis bibroni corresponding to SRTX‐b. BTX competed for [125I]ET‐1 binding to human ETB‐type receptor with a Ki of 3.2 × 10−9 M compared to 4.2 × 10−9 M for SRTX‐b. In rat thorax aorta BTX induced vasoconstrictions with a threshold concentration of 3 × 10−8 M compared to 1 × 10−9 for ET‐1.

Purification and properties of a toxin from the South African sea anemone, Pseudactinia varia

A comparison was made of the hemolytic potency of aqueous extracts prepared from five species of intertidal sea anemones from the coast of South Africa. The active agent in an extract of Pseudactinia varia was purified by ammonium sulfate precipitation, gel permeation chromatography and isoelectric focusing. The hemolytic toxin, termed variolysin, is a protein having a molecular weight of 19,500 and an isoelectric pH of 9.8. It retained appreciable activity after heating to 70 degrees for 40 min. Amino acid analysis revealed that it lacked methionine and cysteine. Its hemolytic activity was inhibited by sphingomyelin. The properties of variolysin show that it is broadly similar to cytolytic toxins isolated from a number of other anthozoans.

Effects of allergy, intestinal helminthic infestation and sex on serum IgE concentrations and immediate skin hypersensitivity in three ethnic groups.

Serum IgE concentrations were measured in 232 blood donors resident in the Western Cape, South Africa. Subjects were interviewed for histories of allergy and skin tested with a variety of common allergens. Faecal specimens were collected for microscopic examination. Results confirmed that Cape Coloureds and Africans tend to have elevated serum IgE concentrations and showed that African males have a high prevalence of immediate skin hypersensitivity. Cape Coloureds and Africans had high prevalences of helminthic infestation and relatively low prevalence of allergic symptoms. Only in Whites were elevated serum IgE concentrations associated with positive allergic histories; in this group, results suggested that the sex difference in serum IgE concentrations observed previously is owing to differences in the IgE responses of allergic males and females.

Secretion of plasminogen activators by normal bone marrow cells and leukaemic myeloid cells

The secretion of tissue plasminogen activator (t-PA) and urokinase by normal human bone marrow cells is a differentiation linked property with t-PA being produced by primitive progenitor cells and urokinase being produced by more differentiated cells and by mature neutrophils and macrophages. Cells from patients with acute myeloid leukaemia also secrete both types of plasminogen activator (PA) and the type of enzyme secreted has prognostic significance. Patients whose cells secrete t-PA die rapidly and fail chemotherapy whereas 80% of those individuals whose cells secrete urokinase enter remission following chemotherapy. The generation of plasmin in the haemopoietic microenvironment would influence haemopoiesis by converting precursor cytokines to active species and would also release various haemopoietic cytokines from cell surfaces and matrix facilitating their interaction with cell surface receptors. The inappropriate secretion of PAs by leukaemic cells could result in abnormal haemopoiesis due to the aberrant plasmin-mediated activation and release of various cytokine species.

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.

The lymphocyte: Monocyte ratio: B- and T-cell ratio after radiotherapy, chemotherapy and surgery☆

Differential white blood cell counts were taken from 42 previously untreated patients with mammary carcinoma who received radiotherapy, 10 patients with Stage III mammary carcinoma who received chemotherapy, 19 patients previously treated by a simple mastectomy, and 42 untreated patients. The percentage of B- and T-cells in the peripheral blood also was measured. Severe lymphopenia was seen in patients treated with radiotherapy and chemotherapy. The percentage of B- and T-cells remained constant before and after radiotherapy, and there was no selective depression of T-cells. The monocyte count, 3 months after radiotherapy, was significantly higher than the pre-treatment mean count. The percentage of B- and T-cells in the peripheral blood remained unchanged before and after treatment with chemotherapy. The mean lymphocyte counts following surgery were slightly higher than the control values whereas the monocyte counts were slightly lower than the control values. Surgery did not appear to influence the percentages of B- and T-cells in the peripheral blood.

Neutrophil Chemotaxis: the Kinetics of Cellular Locomotion in vitro

The Boyden chamber system has been used to study the motile response of rabbit peritoneal neutrophils to partially purified casein cytotaxin. The results of these in vitro experiments indicate that neutrophils respond in two distinct ways to cytotaxin. Firstly, they increase their innate random mobility. Secondly, they respond to the information contained in a cytotaxin concentration gradient by exhibiting directional movement with a velocity vector directed towards the higher cytotaxin concentration. Since the two responses are kinetically distinct, one may infer that they are mechanistically distinct.

Contractile 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.