Long-term culture of human aortas

Summary Segments of human thoracic aorta were maintained in long-term explant culture for 18 weeks in serum-supplemented medium. The aortas were grossly normal in appearance, and random samples fixed for light microscopy prior to culture revealed a normal morphology. The intima contained no more than five layers of smooth muscle cells. After 7 days in culture, the intima was noticeably thicker than the uncultured segments. The increased thickness was due to proliferating smooth muscle cells and production of extracellular material. After several months in culture, extracellular material consisting of collagen and flocculent material was present in areas resembling atherosclerotic fibrous plaques. A peripheral growth, which formed around the explant, was composed of fibroblastlike cells and added to the overall thickness of the intima. However, aortic segment maintained for up to 2 months in serum-free culture medium showed no cellular proliferation. This study demonstrates that changes resembling early stages of atherosclerosis occur in human aortas maintained in explant culture using routine culture procedures. Supported in part by the Pangborn Fund and the Graduate School of the University of Maryland. This is publication 443 from the Cellular Pathobiology Laboratory.     

Report of a trisomy 8p infant with carrier father.

This report describes an infant with fatal congenital heart disease, cleft palate, brain malformations, and trisomy 8p resultant from the paternal balanced reciprocal translocation, rcp(8;15) (p11;p11). Review of six previously reported trisomy 8p patients (resultant from parental balanced translocation in each instance) revealed severe mental retardation in five, short stature in all, and a variety of brain, skeletal, and cardiac defects. The features of the seven trisomy 8p patients reviewed here are not sufficiently similar to suggest a distinct dysmorphic syndrome. In addition the features differ from those in the trisomy 8 mosaicism syndrome, in which the mental retardation and malformations are generally less severe.     

Red blood cell lysis induced by the venom of the brown recluse spider: the role of sphingomyelinase D.

Red blood cell lysis induced by the venom of Loxosceles reclusa, the brown recluse spider, may be related to the hemolytic anemia observed in several cases of spider envenomation. These investigations demonstrate that the venom of the brown recluse spider contains a calcium-dependent, heat-labile hemolysin of molecular weight approximately 19,000. The pH optimum for the hemolytic reaction was 7.1, and the optimum calcium concentration for venom-induced lysis was observed within the range of 6 to 10 mm. Sheep red blood cells were more susceptible to the spider hemolysin than human red blood cells, although both types exhibited appreciable lysis. Digestion of sheep red blood cell membranes with partially purified venom lysin resulted in degradation of the sphingomyelin component. However, reaction of the membranes with the venom lysin produced no release of water-soluble phosphate, and no free fatty acids were generated. These results indicate that the sphingomyelin-degrading activity of the venom is not a phospholipase C- or a phospholipase A₂-type activity. Sphingomyelin was employed as substrate for the venom hemolysin, and the organic and aqueous fractions of the reaction mixtures were analyzed by thin-layer chromatography. Analysis of the organic fraction revealed a phosphate-containing product with the solubility and chromatographic characteristics of N-acylsphingosine phosphate (ceramide phosphate), and analysis of the aqueous fraction demonstrated the presence of choline. The isolation and identification of these products indicate that the sphingomyelin of the red cell membrane is hydrolyzed by a sphingomyelinase D-type activity expressed by the partially purified venom hemolysin. A close correspondence between the hemolytic and sphingomyelinase D activities was observed when the partially purified hemolysin was further characterized in polyacrylamide gel electrophoresis at pH 8.3 and pH 4.9. The hemolytic and sphingomyelinase activities were coincident within the electrophoretic pattern at both pHs. The results presented demonstrate conclusively a direct lytic action of brown recluse venom upon red blood cells and report for the first time the presence of sphingomyelinase D in spider venom.     

Human lysosomal elastase. Catalytic and immunological properties.

1. The elastase of human spleen was shown to exhibit endopeptidase activity against azo-casein and elastin. 2. Activity against several synthetic substrates was detected, and benzyloxycarbonyl-L-alanine 2-naphthyl ester was found to be a good substrate for routine use. 3. The enzyme showed a broad pH optimum in the range of 8.2-9.2 against azo-casein and the synthetic substrate. 4. The effect of inhibitors on the spleen elastase showed it to be a serine proteinase with a specificity similar to that of porcine pancreatic elastase. 5. Specific antisera were raised against the enzyme, and it was shown to be immunologically identical with the lysosomal elastase of human neutrophil leucocytes.     

The effects of proteolytic enzymes on the mechanical properties of adult human articular cartilage.

The effects of the lysosomal proteinase cathepsin D on the mechanical properties of adult human articular cartilage were examined in detail in 7 joints within the age range 21 to 72 years. The results of a preliminary study on the effects of the lysosomal proteinase cathepsin B1 and clostridial collagenase on the mechanical properties of cartilage are also presented. Cartilage which had been incubated with either cathepsin D or cathepsin B1 showed increased deformation in uniaxial compression perpendicular to the articular surface. The enzyme-treated cartilage also showed decreased tensile stiffness at low values of stress. This effect was more pronounced in specimens from the deeper zone of cartilage than in specimens from the superficial zone. It was also more pronounced in specimens which were aligned perpendicular to the predominant alignment of the collagen fibres in the superficial zone than in specimens which were parallel to the collagen fibres. At higher stresses the tensile stiffness of the treated cartilage was not significantly different from that of the untreated tissue. The tensile fracture stress of the cartilage was also not significantly reduced by the action of cathepsin D. In contrast to the effects observed with the cathepsins, the preliminary results obtained by incubating cartilage for 24 h with clostridial collagenase showed that both the tensile stiffness and the fracture stress were considerably lower than the corresponding values for the untreated tissue. Biochemical analysis of the incubation media, and the specimens, revealed that a large proportion of the proteoglycans was released from the cartilage by each of the three enzymes. The proportion of the total collagen which was released from the cartilage was different for each enzyme: cathepsin D released between 0 and 1.5 per cent, cathepsin B1 released between 2.3 and 4.3 per cent and collagenase released between 5.3 and 27.8 per cent of the collagen after 24 h.     

Motoneuron dendrites: role in synaptic integration.

Dendrites constitute over 80 per cent of the receptive surface area in cat motoneurons. Calculations based on matched electrical and gemoetrical measurements in these neurons indicate that the specific resistance of dendritic membranes in resting motoneurons is at least 2,000 ohm-cm2. When the specific membrane resistance is this high, even the most distal dendritic synapses can contribute significantly to the depolarization of the soma, and hence influence the rate of action potential generation. However, dendritic membrane resistance depends strongly on the level of background synaptic activity. The conductance changes associated with excitatory synaptic activity on a dendrite can be great enough to reduce significantly both the excitatory synaptic driving potential and the effective membrane resistance on that dendrite, and thus greatly reduce the effectiveness of synapses on the dendrite. Inhibitory synaptic activity produces an even greater reduction in dendritic membrane resistance. Thus the relative effectiveness of dendritic synapses depends on the type, distribution, and intensity of background synaptic activity, as well as on dendritic geometry and resting membrane properties.     

Effect of pH alteration on growth and glucose oxidation in myoblast cultures

The growth of chick heart cells in culture declines when the cells reach confluency. The decline in growth rate is associated with both a decrease in the pH of the bicarbonate-CO₂ buffered medium and a reduced capacity for glucose oxidation by the pentose phosphate pathway. The pH of proliferating cultures supplemented with either 14 mM NaHCO₃ or with a mixture of organic buffers (pK 7.4) was increased by 0.3 pH unit over that of the controls. The rate of glucose oxidation by the pentose phosphate pathway in confluent cultures supplemented with NaHCO₃ or organic buffer increased by 60% 24 h after pH correction. This was associated with an increase in glucose uptake from the medium. We conclude that pH elevation in confluent heart cell cultures stimulates both growth and the capacity for glucose oxidation by the pentose phosphate pathway. The data also provide further evidence for a relationship between activity of the pentose phosphate pathway and cell growth.     

Xanthine dehydrogenase accumulation in developing Drosophila eyes

Xanthine dehydrogenase activity is assayed by following the oxidation of pterin to isoxanthopterin by spectrofluorometry at the reaction's wavelength peaks: excitation, 344 nm; emission, 412 nm. The method is sensitive to less than 0·1 μU of activity (0·1 pmol/min) and allows the assay of Drosophila imaginal disk homogenates.While the larval eye disk contains less than 0·1 per cent of the individual's XDH, the developing eye becomes a major store, with 30 per cent of the individual's activity by the time of eye pigmentation. The data suggest a basis for the well-known non-autonomous action of the gene rosy, the structural gene for XDH: the enzyme is synthesized in an organ of primary gene expression, and transported through the haemolymph to the eye of the pupa and pharate adult.     

Recovery of MAO-B enzyme activity after (-)deprenyl (selegiline) pretreatment, measured in vivo

The recovery of MAO-B activity after a single dose of 0.25 mg/kg s.c. (-)deprenyl was measured in rat, by following the time course of the changes in the PEA (phenyl-ethylamine)-induced hyperactivity. One hour after its administration (-)deprenyl enhanced both the PEA-induced locomotion and stereotypy, however, its effect on the latter was more marked. At 24 h and at later time-points only stereotype was enhanced. The results show that a single small dose of (-)deprenyl which selectively blocks MAO-B, causes a long-lasting inhibition of PEA metabolism, and the enzyme activity needs more than one week to restore completely.