Molecular aspects of polyene- and sterol-dependent pore formation in thin lipid membranes.

Amphotericin B modifies the permeability properties of thin lipid membranes formed from solutions containing sheep red cell phospholipids and cholesterol. At 10(-6)M amphotericin B, the DC membrane resistance fell from approximately 10(8) to approximately 10(2) ohm-cm(2), and the membranes became Cl(-)-, rather than Na(+)-selective; the permeability coefficients for hydrophilic nonelectrolytes increased in inverse relationship to solute size, and the rate of water flow during osmosis increased 30-fold. These changes may be rationalized by assuming that the interaction of amphotericin B with membrane-bound sterol resulted in the formation of aqueous pores. N-acetylamphotericin B and the methyl ester of N-acetylamphotericin B, but not the smaller ring compounds, filipin, rimocidin, and PA-166, produced comparable permeability changes in identical membranes, and amphotericin B and its derivatives produced similar changes in the properties of membranes formed from phospholipid-free sterol solutions. However, amphotericin B did not affect ionic selectivity or water and nonelectrolyte permeability in membranes formed from solutions containing phospholipids and no added cholesterol, or when cholesterol was replaced by either cholesterol palmitate, dihydrotachysterol, epicholesterol, or Delta5-cholesten-3-one. Phospholipid-free sterol membranes exposed to amphotericin B or its derivatives were anion-selective, but the degree of Cl(-) selectivity varied among the compounds, and with the aqueous pH. The data are discussed with regard to, first, the nature of the polyene-sterol interactions which result in pore formation, and second, the functional groups on amphotericin B responsible for membrane anion selectivity.

Venous thrombosis in a family with defective release of vascular plasminogen activator and elevated plasma factor VIII/von Willebrand's factor.

A family is described in which venous thrombosis developed in five members as early as 14 years of age. Routine coagulation studies, plasma antithrombin III, factor V, plasminogen, beta-thromboglobulin, fibrinopeptide A, prothrombin fragment F1+2, and thrombin-antithrombin III complex were all within normal limits. However, defective release of vascular plasminogen activator was observed on several occasions in all five subjects as compared with a control population of 125 persons (0.04 Committee on Thrombolytic Agents [CTA] units/ml plasma as compared with 0.21 CTS units/ml). In addition, levels of factor VII/von Willebrand's factor were significantly elevated above the normal range in this pedigree.

Myeloma and generalized mesenchymal amyloidosis causing malnutrition and cardiac failure.

A 39-year-old Negro woman had the mesenchymal type of generalized amyloidosis and myeloma of bone. Malnutrition and heart failure dominated the clinical course and led to her death. The autopsy study is summarized in the following diagnoses.

Defective mononuclear cell support of erythropoiesis in the elderly.

As many as one third of anemic elderly patients have anemia as an isolated finding without demonstrable morphologic abnormality of the marrow, deficiency of hematinic vitamins, or minerals or disease known to cause secondary anemia. These people may have a pathologic process: anemia of senescence. Erythroid cells mature from nonlymphoid mononuclear cells; the maturation process is modulated by lymphokines. When assessed as end products of the humoral or cellular immune system, mononuclear cell function declines with host age. This study tests the hypothesis that mononuclear cell support of erythroid burst formation is defective in elderly subjects. Leukocyte-conditioned medium was prepared from each of five young and five elderly adults. Erythroid bursts from young or elderly adults were grown in culture medium containing leukocyte-conditioned medium from at least three young adults and at least three elderly adults. Erythroid bursts from either young or elderly adults were 1.4-3.0 times more numerous if the leukocyte-conditioned medium derived from a young rather than elderly adult. In addition, when culture medium contained leukocyte-conditioned medium from young adults, only half the elderly adults studied had erythroid bursts equal to those of young adults. These data support the hypothesis that mononuclear cell support of erythropoiesis in the elderly is defective.

Effect of selenium supplementation on selenium balance in the dependent elderly.

Although trace minerals are necessary constituents of enzymes, dietary requirements of these nutrients for the elderly are unknown. This study measured selenium balance in six dependent elderly men before and after five weeks daily administration of 200 micrograms organically-bound selenium; dietary selenium intake averaged 62.1 +/- 7 micrograms/day during both study periods. Selenium status was assessed not only chemically but also biologically as red cell and platelet glutathione peroxidase activities. Plasma selenium averaged 8.8 +/- 0.8 micrograms% (normal: 10 +/- 2 micrograms %) when intake derived from dietary sources alone and increased during medicinal supplementation to an average of 12.8 +/- 1.9 micrograms %. The rise in plasma selenium was not associated with an increase in red cell or platelet glutathione peroxidase activity. The effect of selenium supplementation on in vivo platelet aggregability was studied by measuring plasma levels of beta-thromboglobulin and platelet factor 4, two proteins secreted concomitant with aggregation. beta-thromboglobulin diminished 7.5 +/- 11.0 ng/ml and platelet factor 7.6 +/- 11.0 ng/ml during selenium supplementation despite no change in platelet glutathione peroxidase activity. These data support the concept that selenium nutritional status should be assessed not only by blood selenium content but also by selenium-dependent enzyme activity or selenium-dependent biologic effect.

Embryonal rhabdomyosarcoma in ascitic fluid. Immunocytochemical and DNA flow cytometric study.

We report the case of an 18-year-old woman who had a small-cell malignant neoplasm of the left paranasal sinuses and who, 18 months later, developed malignant ascites. Immunoperoxidase stains of the ascitic fluid cells and, subsequently, the original tumor, identified it as embryonal rhabdomyosarcoma. Flow cytometric DNA analysis of the ascitic fluid demonstrated an aneuploid cell population that was also present in the paraffin-embedded tissue on which the original diagnosis had been made.

The changing face of primary hyperparathyroidism.

The introduction of multiphasic screening and the development of sensitive parathormone assays have changed the demography and clinical symptomatology of patients presenting with primary hyperparathyroidism. This retrospective review includes 158 patients operated on for primary hyperparathyroidism at the Medical College of Georgia from 1973-1987. Compared to the 46 patients managed prior to 1973, the frequency of subclinical hyperparathyroidism has increased from 46% to 64%. The median patient age has increased from 50 to 59 years. Recognition of primary hyperparathyroidism in a more geriatric population modifies indications for surgical intervention in subclinical disease. Osteoporosis, myalgias, fatigue, arthralgias, memory loss, or constipation occurred in 50% of patients. These complaints are frequent in normocalcemic elderly people. They represent disease, not normal aging. Their exacerbation by hypercalcemia should not go uncorrected if neck exploration can be tolerated by the patient.

The effect of cultured endothelial cells on factor VIII procoagulant activity.

Cultured human umbilical vein endothelial cells produce a protein that has von Willebrand factor activity and forms immunoprecipitates with rabbit antibody to purified plasma factor VIII/von Willebrand factor (FVIII/vWF) protein, but it has no FVIII procoagulant activity. Of the three characteristics of plasma FVIII/vWF protein, only FVIII procoagulant activity is readily destroyed by trace proteases. A previous report from this laboratory demonstrated protease activity in culture medium under conditions that had been used by others to show that endothelial cells do not synthesize protein with FVIII procoagulant activity. However, even if cultured endothelial cells are placed in protease-free culture medium, no FVIII procoagulant activity can be detected, despite an increase in the level of protein with vWF activity from 0 to 0.57 microgram/ml by 48 hr. This observation and the lack of protease activity in medium left in contact with the cells for 48 hr led to the hypothesis that proteases exist on the surface of cultured umbilical vein endothelial cells. Protease activity was quantitated by the hydrolysis of p-nitroaniline from the substrate, N-benzoyl-phenylalanyl-valyl-arginyl-p-nitroanilide and by degradation of the procoagulant activity of added purified plasma FVIII/vWF protein. In the absence of endothelial cells, no protease activity was present in protease-free culture medium whether or not it had previously overlaid cultured cells. This medium did not cause cleavage of p-nitroaniline from the tripeptide substrate, and 83% of added FVIII procoagulant activity remained after 48 hr. When the synthetic tripeptide was incubated in contact with cultured endothelial cells, 7.3 +/- 0.8 X 10(-10) moles of p-nitroaniline/hr was released; moreover, only 47% of the added FVIII procoagulant activity remained after 48 hr. Given this rate of destruction, it can be calculated that sufficient protease activity exists on the surface of cultured endothelial cells to degrade the procoagulant activity of approximately 1.6 microgram FVIII/vWF protein/hr. This degradation rate is 45 times the rate of release of FVIII/vWF protein from cultured endothelial cells when assessed by the generation of protein with vWF activity. Hence, the detection of FVIII procoagulant activity, if in fact synthesized by cultured endothelial cells, will be most difficult.

Enhanced mobilization of iron from body stores in malnourished patients during intravenous nutritional support.

Abnormal iron metabolism, characterized by low serum iron concentration and diminished saturation of iron-binding capacity, is known to occur in patients with malignant or inflammatory diseases. In this study, serum iron concentration and iron-binding capacity were measured in 21 patients with malignant disease, trauma or inflammatory illness before and during intravenous nutritional support without iron supplementation. Prior to nutritional support, the serum iron value averaged 26.0 +/- 3.5 micrograms per deciliter and the iron-binding capacity, 160.0 +/- 14.0 micrograms per deciliter. After ten to 21 days of parenteral nutrition, the serum iron-binding value serum iron concentration rose to an average of 68 +/- 9 micrograms per deciliter and the iron-binding capacity to 200 +/- 15 micrograms per deciliter, and increased reticulocytosis was observed. The rise in serum iron value reflected a shift of iron from body stores to plasma, since the saturation of iron-binding capacity increased from 18.0 +/- 3.5 to 36.0 +/- 4.0 per cent. Fever, granulocytosis, malignant disease, pulmonary infiltrates or open wounds persisted in all patients throughout the observation, ruling out resolution of a chronic process as the cause for rising serum iron levels. Changes in serum ceruloplasmin and ascorbate values also could not account for the rise. We propose the provision of adequate nutritional substrates to establish an anabolic state during total parenteral nutrition is associated with improved mobilization of iron from body stores.

Physical conditioning augments the fibrinolytic response to venous occlusion in healthy adults.

The effects of a 10-week physical-conditioning program on fibrinolytic activity at rest and after stimulation by venous occlusion were studied in 69 healthy adults 25 to 69 years old. Physical conditioning was documented by treadmill performance, and fibrinolysis was measured with a newly developed radioenzymatic assay. Whereas fibrinolysis declined at rest from 16.2 +/- 1.3 to 11.4 +/- 0.8 units (mean +/- S.E.M.) (P = 0.0017), the increment in fibrinolysis produced by venous occlusion was increased from 21.7 +/- 2.9 to 33.8 +/- 4.7 units (P = 0.0037). This augmentation was most marked in women, persons with low initial levels of stimulated fibrinolysis, and persons with low initial physical fitness. We conclude that physical conditioning can enhance the augmentation of fibrinolytic activity that occurs in response to venous occlusion. Enhanced fibrinolysis in response to thrombotic stimuli could be an important mechanism in the beneficial effect of habitual physical activity on the risk of cardiovascular disease.