Reconstitution of Rh (D) antigen activity from human erythrocyte membranes solubilized by deoxycholate.

Proteins were selectively solubilized from human erythrocyte membranes with deoxycholate. After ultracentrifugation and preliminary fractionation procedures, the detergent was removed from the soluble membranes by Bio-Beads SM-2 and dialysis against 5 millimolar magnesium chloride. Reaggregation took place with the apparent formation of vesicles which showed serologically specific Rh antigen activity.

Divalent cation effects on the binding of human anti-phospholipid antibodies.

Anti-phospholipid antibodies from sera of subjects with documented syphilis were measured as a result of their individual interactions with cardiolipin, phosphatidylserine and phosphatidic acid, each impregnated in nitrocellulose paper from chloroform solution, followed by reaction with a labelled second antibody. Binding curves generated by increasing the cardiolipin concentration over a standard area of nitrocellulose paper showed saturation. The presence of Ca2+ or Mg2+ during the antibody binding step resulted in a complex pattern of binding as a function of the cation concentration. When the extent of binding was normalized to percent of maximum bound, virtually super-impossible patterns were seen with different sera. These patterns were distinctive for both the phospholipid and the cation. The speculation is presented, albeit without any direct evidence, that the extent of antibody binding is sensitive to a variety of intermediate phospholipid phase structures which may be initiated by the presence of the specific divalent cation at a particular concentration.

Cholesterol effects on the interaction of cardiolipin with anti-cardiolipin antibody.

Human antibodies to cardiolipin, phosphatidic acid and phosphatidylserine were assessed by binding to nitrocellulose paper and subsequent reaction with an enzyme-linked or radioactively labelled second antibody to human IgG. The addition of cholesterol to constant amounts of cardiolipin impregnated in the nitrocellulose paper resulted in a profound fall in antibody binding beginning at a 0.5 to 1 molar ratio of cholesterol to cardiolipin and stabilizing at about 15% of the original level. Antibody binding to phosphatidic acid and phosphatidylserine also showed extensive cholesterol-induced inhibition beginning at a slightly lower molar ratio of cholesterol to phospholipid. The structural array of neither the cardiolipin alone impregnated in nitrocellulose nor the phospholipid together with cholesterol is known. It is possible that the specific cardiolipin phase structure required for human antibody recognition was disrupted by cholesterol.

Stereospecificity of the hydroxyeicosatetraenoic and hydroxyoctadecadienoic acids produced by cultured bovine endothelial cells.

Characterization of the stereospecificity of the derivatives of arachidonic acid and linoleic acid produced by endothelial cells is needed to define the enzymatic origin of these compounds and their role in vascular physiology. In studies utilizing two bovine endothelial cell lines (CPAE and AG04762), both free 15-hydroxyeicosatetraenoic acid (15-HETE) and 11-hydroxyeicosatetraenoic acid (11-HETE) were generated during incubations with exogenous arachidonic acid and both free 9-hydroxyoctadecadienoic acid (9-HODE) and 13-hydroxyoctadecadienoic acid (13-HODE) were generated during incubations with exogenous linoleic acid. Esterification of 15-HETE, 9-HODE and 13-HODE during these incubations was demonstrated. The analyses included reversed-phase high performance liquid chromatography of the free acid and its methyl ester and chiral separation of the methyl ester on straight phase chiral columns. The ratio of 9-HODE/13-HODE averaged 2.7 in the chromatographic analyses of the extracts of the incubations with linoleic acid. The combined production of 13-HODE and 9-HODE from linoleic acid was four times greater than that of 15-HETE and 11-HETE from arachidonic acid. With regard to the products of the CPAE endothelial cell line, the S/R ratio of the stereoisomers averaged 1.5 for free 15-HETE, 5.7 for free 13-HODE and 0.2 for free 9-HODE. The 11-HETE had strict (R) stereospecificity. The products from the AG04762 endothelial cell line had similar stereochemistry. All these stereochemical findings point to the activity of a cyclooxygenase rather than that of a lipoxygenase.

Hypertonic cryohemolysis and the cytoskeletal system.

Hypertonic cryohemolysis is defined as the lysis of erythrocytes in a hypertonic environment when the temperature is lowered from above 15-18 degrees C below that temperature. This has been found to be a general phenomenon (that is, whether the solute is charged or not), to exhibit interesting temperature characteristics and to be preventable by agents such as valinomycin which tend to dissipate the concentration gradient across the cell membrane. As yet, no clear information is available to translate this phenomenon to the molecular level and to relate it to current structure/function concepts in the erythrocyte membrane. In this study, data are presented which would indicate on the basis of two entirely separate methodologies that the spectrin-actin cytoskeletal framework is involved in this phenomenon. The first of these methodologies is based on radiation-induced ablation of cryohemolysis and indicates that an intact macromolecular complex of an order of 16000 000 daltons is required for cryohemolysis with hypertonic NaCl. The second methodology is based on selective cross-linking of spectrin and actin in the agent diamide, which resulted in concentration-dependent suppression of cryohemolysis. Polyacrylamide gel electrophoresis of the erythrocyte from diamide-treated cells showed intense protein aggregation with loss of spectrin-actin and bands 4.1, 4.2. We conclude that the spectrin-actin cytoskeletal system possibly including its interaction with phospholipids is the key to the phenomenon of hypertonic cryohemolysis.

The mode of attenuation of erythrocyte membrane Rh0(D) antigen activity by 5,5'-dithiobis-(2-nitrobenzoic acid) and protection against loss of activity by bound anti-Rh0(D) antibody.

The Rh0(D) antigen activity of human erythrocyte membranes is lost on treatment with the aromatic disulfide, 5,5'-dithiobis-(2-nitrobenzoic acid), reacting by specific disulfide interchange with membrane thiols. The inactivation rate of the antigen was first order with respect to inhibitor concentration and a double reciprocal plot of the inactivation rate constants against the sulfhydryl reagent concentration was linear, giving an apparent dissociation constant of 1.25 mM and indicating that a binding step preceded covalent interaction. In a study of the binding characteristics of anti-Rh0(D) to the 2-nitro-5-thiobenzoate derivative of the membrane Rh0(D) antigen, the maximum number of binding sites fell with increasing sulfhydryl reagent concentration but the apparent association constant also diminished at the same rate. The inactivation of the Rh0(D) antigen was greatly retarded by the presence of bound anti-Rh0(D), a protective effect not seen with normal serum. Bound anti-Rh0(D) antibody protects against both the loss of sites and the change in affinity brought about by DTNB. It is concluded that a single critical thiol whose reactivity is altered by bound anti-Rh0(D) antibody is involved in the membrane Rh0(D) antigenic determinant site. These observations may permit extensive probing of the microenvironment of this essential cysteine residue.

Molecular size of the Rh0(D) antigen of the human erythrocyte in situ by radiation inactivation.

Radiation inactivation was used to determine the molecular size of the RhO(D) antigen of isolated membranes and of the intact human erythrocyte. Isolated membranes were frozen and irradiated at -50 degrees C. After thawing, the bound 14C-anti-D was measured and the log residual Rh(D) antigen activity was plotted against the radiation dose. A mol. wt of 60,000 was calculated. Intact human erythrocytes frozen in the presence of cryoprotective reagents were also studied. Rh(D) antigen inactivation occurred as a single exponential function of radiation dose which also yielded a mol. wt of approx. 56,000 upon analysis by classical target theory.

The phospholipid requirement for Rho(D) antigen activity: mode of inactivation by phospholipases and of protection by anti-Rh0(D) antibody.

Previous studies have suggested a membrane phospholipid requirement for Rho(D) antigen activity. Isolated erythrocyte membranes incubated with phospholipase A2 from both bee venom and porcine pancreas undergo loss of Rh antigen activity. The mode of attenuation of this antigen activity as indicated by double-reciprocal binding plots suggests substantial loss of sites accompanied by an apparently increased association constant. In the presence of anti-Rho(D), but not anti-A, bound to group A Rho(D)-positive membranes prior to hydrolysis, there is marked protection: almost complete preservation of sites at the expense of a decreased association constant. This pattern of protection is not seen with phospholipase C, which cleaves the polar headgroup in contrast to the A2-enzymes, which hydrolyze the fatty acid in the 2-position. Analysis of the products of digestion shows a trend to protection of bulk phospholipids of all major classes in the presence of bound specific antibody. The hydrophobic fatty acid chain may be the site with which the bound anti-Rho(D) antibody is in closest proximity.

Generation and metabolism of lipoxygenase products in normal and membrane-damaged cultured human keratinocytes.

The production and metabolism of lipoxygenase eicosanoids were studied in cultured human keratinocytes. The identity of these eicosanoid structures was established by a variety of chromatographic and analytical techniques. Normal cultured keratinocytes did not produce lipoxygenase eicosanoids either spontaneously or when given arachidonic acid in the presence of permeabilizing concentrations of ethanol or dimethyl sulfoxide. Freeze-thawing of human neonatal and adult keratinocytes resulted in a rapid release of linoleic and arachidonic acids over time. Activation of a latent 15-lipoxygenase was demonstrated by the synthesis of 15-hydroxyeicosatetraenoic acid (15-HETE) and 13-hydroxyoctadecadienoic acid, and both these products were greatly increased in amount when the corresponding fatty acid precursor was added. Eicosanoid production by cells of newborn and adult origin was indistinguishable. Rapid metabolism of exogenous 15-HETE by normal keratinocytes was observed. Measurable quantities of esterified 15-HETE were found after 1 min, but by 18-20 h all the esterified 15-HETE was degraded to the extent that 80% of the recovered radioactivity was found in water-soluble form. In contrast, when labeled or unlabeled 5-HETE was used a much larger fraction was esterified intact (30% as opposed to 10%) and at the end of 18-20 hours a substantial peak of esterified 5-HETE remained. Intact esterified [3H] HETE were recovered only in the triacylglycerol fraction. The key findings that omega-6 lipoxygenase products are generated but not esterified by membrane-damaged keratinocytes, whereas these products are esterified but not generated by normal keratinocytes, may be of importance in transcellular metabolic control.

Epidermal fatty acid oxygenases are activated in non-psoriatic dermatoses.

The extent of epidermal fatty acid oxygenase activation in non-psoriatic dermatoses and the nature of these oxygenases are not known. The monohydroxylated fatty acid derivatives produced in vivo and trapped in skin scales or produced in vitro by oxygenases preserved in scales were analyzed by high performance liquid chromatography in 10 patients with non-psoriatic dermatoses. Evidence for 15-lipoxygenase activation included the finding of 15(S)-hydroxyeicosatetraenoic acid (HETE) in scales from seven patients and the production of 15(S)-[14C]HETE and 13(S)-[14C]hydroxyoctadecadienoic acid (HODE) during scale incubations, respectively, with [14C]arachidonic and [14C]linoleic acid. Evidence for the activation of an arachidonic acid 12(R)-oxygenase included the finding of 12(R)-HETE in scales from eight patients and the production of 12(R)-[14C]HETE during scale incubations with [14C]arachidonic acid. 13-HODE was the predominant fatty acid derivative present in scale extracts; its lack of enantiopurity (mean S/R = 3.1) and the substantial formation of 9-HODE (mean S/R = 0.6; 9/13-HODE = 0.43) suggest its derivation from 15-lipoxygenase and a second oxygenase. The levels of 15(S)-HETE and 12(R)-HETE had a 125- to 144-fold range and were highest in scales from a patient with erythroderma and in three psoriatic scale samples similarly analyzed. These findings indicate that 15-lipoxygenase, most likely of keratinocyte origin, and an arachidonic acid 12(R)oxygenase of unknown type and cell origin are activated in diverse dermatoses.

Suppression of leukotriene synthesis in human leukocytes by a urea extract of Bordetella pertussis: evidence for mediation by adenylate cyclase toxin.

Incubation of human leukocytes with a urea extract of Bordetella pertussis led to inhibition of zymosan-induced leukotriene generation. The proteins in this extract are known to include an adenylate cyclase which suppresses certain defense mechanisms of leukocytes against bacterial invasion. The formation of leukotriene B4 and leukotriene C4, induced by serum-coated zymosan, was almost completely inhibited in the presence of 75 micrograms of urea-extracted proteins/ml cell suspension. This suppression by the bacterial urea extract was rapid, with the maximum effect occurring in the first min of incubation. The reduction in leukotriene generation was accompanied by a dramatic increase in intracellular cyclic AMP levels. Since leukotrienes are potent pro-inflammatory compounds, the present study indicates that Bordetella pertussis-induced suppression of leukotriene formation might be an important factor in the increased susceptibility to secondary bacterial infection, which occurs as a result of this disease.

Intracellular cold trapping of exogenous arachidonic acid and activation of the 5-lipoxygenase pathway of human neutrophils.

Exogenous arachidonic acid was trapped in human leukocytes with ethanol at 0 degrees C and 16 degrees C. Leukotriene B4 and its isomers were produced from the trapped substrate, and this production continued for at least 24 h at 0 degrees C. Synthesis from trapped substrate of these eicosanoids increased as a function of the incubation temperature. The ability to study leukotriene synthesis and intercellular transfer from exogenous but intracellularly trapped substrate without the multiple effects of cell activators may be of value in elucidating the metabolism and regulatory properties of these lipoxygenase products.

Leukotriene production associated with leukocyte membrane destruction: evidence of a terminal signal.

Four methods of severe disruption of human neutrophil membrane integrity resulted in the formation of substantial amounts of leukotriene B4 and its isomers. This synthesis took place from endogenous substrate in the absence of any cell activators. It is postulated that leukotriene formation could be a preserved cell function and that generation at this stage could constitute a terminal chemotactic signal.

Saturability of esterification pathways of major monohydroxyeicosatetraenoic acids in rat basophilic leukemia cells.

The principal monohydroxyeicosatetraenoic acids (HETEs), 5-, 12-, and 15-HETE, which can be produced by rat basophilic leukemia (RBL-1) cells, are also esterified by these cells. Exogenously added 5-, 12-, and 15-HETE were rapidly incorporated as esters in RBL cells, reaching plateau levels within 25 min. In incubations in culture medium with protein added, all three HETEs were essentially completely metabolized within 24 h. 5-HETE was esterified more rapidly and to a greater extent than 12-HETE or 15-HETE when these were incubated together with RBL cells, indicating some degree of selectivity in the esterification pathways. When arachidonic acid (AA) was incubated in increasing concentrations with constant concentrations of 15-HETE and RBL cells, the free 15-HETE concentration increased and esterified 15-HETE concentration decreased markedly at AA: 15-HETE molar ratios above 9. 15-HETE esterification in RBL cells was also markedly inhibited by the polyunsaturated fatty acids, eicosatetraynoic and eicosapentanoic acids, but not by oleic or linoleic acids. In separate experiments with unlabeled and radiolabeled substrates, the extent of incorporation of esterified HETE in RBL cells decreased at higher concentrations of 15-HETE and AA, which showed that the pathway was saturable. The shapes of the curves for these fatty acid inhibitors suggest a concentration-dependent two-compartment pathway of esterification. These data indicate that the HETEs and other 20 carbon fatty acid substrates probably compete for activity of a specific arachidonyl-CoA synthetase, which is the first and rate-limiting step for esterification of arachidonic acid by many human cells. Esterified 15-HETE was found to be predominantly in the phosphatidylethanolamine fraction of RBL cell lipids.

Dissected popliteal cyst: an unusual presentation of acute gout.

A forty-six year old man with acute gout and a dissected popliteal cyst presented with clinical features which mimicked rheumatoid arthritis and thrombophlebitis. The clinical and laboratory diagnosis of the case are presented and similar cases previously reported are briefly reviewed.

Dissected popliteal cyst: an unusual presentation of acute gout.

A 46-year-old man with acute gout and a dissected popliteal cyst presented with clinical features which mimicked rheumatoid arthritis and thrombophlebitis. The clinical and laboratory diagnosis of the case are presented and similar cases previously reported are briefly reviewed.

Transformations of 5-HETE by activated keratinocyte 15-lipoxygenase and the activation mechanism.

There is convincing evidence that normal cultured human keratinocytes possess a 15-lipoxygenase activity which, however, does not appear to manifest itself without cell membrane damage. When "activated", this enzyme transforms arachidonic acid into 15-hydroxyeicosatetraenoic acid (15-HETE), and linoleic acid into 13-hydroxyoctadecadienoic acid, presumably by peroxidase action on their respective hydroperoxy intermediates. Normal but not membrane-damaged keratinocytes metabolize exogenous 5-HETE, principally by esterifying the eicosanoid intact, primarily in the triacylglycerol fraction. In the present study, membrane-damaged keratinocytes were found to transform 5-HETE to 5,15-diHETE and also to a lipoxin-like group of tetraenes. Similar, if not identical, tetraenes were produced by action of the keratinocyte enzyme on 5(S),15(S)-diHETE, which points to the role of the latter as an intermediate between 5-HETE and the tetraenes. A direction for further study of the mechanism of the "activation" step is presented.

The last public execution in Glasgow: the case of Dr Edward Pritchard, M.D.

Dr Edward Pritchard, a Glasgow medical practitioner, was the last person to be executed in public in Glasgow. In a famous trial of the time, he was condemned to death for murdering his wife and his mother-in-law, and he was hanged on Glasgow Green in 1865. An account of Dr Pritchard's life, his trial and his death is given here, together with a reference to the medical examination which was carried out on Pritchard's remains when his grave was exhumed in 1910.