Changes in adenosine deaminase activity in ageing cultured human cells and the role of zinc.

The level of adenosine deaminase (ADA; EC 3.5.4.4) was estimated at different passages in six confluent fibroblast cultures established from forearm skin biopsies of healthy adult normal volunteers. After determination of the zinc concentration in standard growth medium, ADA activity was estimated at different passages of subculture in media with different zinc concentrations. The results indicated that the specific activity of ADA in control confluent skin fibroblast cultures (passage 2) cultivated in standard growth medium containing 15.4 microM zinc (similar to that present in normal human plasma) was equal to 226.6+/-19.64 micromol min(-1) mg(-1) protein. The results showed that there were no significant changes in ADA specific activity in any of the control cultures as the zinc concentration of the medium was increased. To characterize the passage of subculture at which fibroblasts enter the ageing phase, three marker enzymes were assayed namely, phosphofructokinase, lactate dehydrogenase and glycogen phosphorylase. The result showed that the cells enter the ageing phase at passage 20 and beyond. Further investigation showed that ADA activity of serially subcultured confluent cultures cultivated in standard growth medium significantly dropped at passages 20, 25 and 30. ADA activity however was not significantly altered in cells at passage 2, 10 and 15 cultivated in standard growth medium and in the presence of higher zinc levels (23.1, 34.6, 53.8 and 73.1 microM). Furthermore there was significant lowering of ADA activities in cells at passages 20, 25 and 30 when cells were cultured in the presence of 15.4, 23.1 and 34.6 microM zinc. Such lowered activities of ADA were restored to normal when the cells were cultured in the presence of higher zinc concentration equal to 53.8 and 73.1 microM. From the results we concluded that it is possible to restore ADA activity in aged skin fibroblasts to normal levels by raising the zinc concentration in the culture medium to four or five times the control normal plasma zinc level.

Response of neurons and microglia/macrophages in the area postrema of adult rats following exposure to hypobaric hypoxia.

The response of neurons and microglia/macrophages in the area postrema (AP) was examined in adult rats following exposure to hypobaric hypoxia. In this connection, immunoexpression of complement type 3 (CR3) receptors, major histocompatibility complex (MHC) class I and II antigens and ED1 antigens on the macrophages/microglia was downregulated immediately after the hypoxic insult. However, it showed an upregulation at 7-14 days and was comparable to the controls thereafter. At the ultrastructural level, swollen axons showing disruption of their myelin sheaths were observed between 7 and 14 days. At this time interval microglia/macrophages in the AP were observed to phagocytose such axons. Neurons did not show any structural alteration at any time interval following hypoxic exposure and appeared comparable to the neurons in the control AP. It is suggested that alterations in CR3 receptors, ED1, and MHC I and II antigens on the macrophages/microglia in hypobaric hypoxia were in response to axonal changes. Increased permeability of blood vessels following hypoxia may also play a role in activation of these cells as they would be involved in the clearance of extravasated serum derived substances.

Separation and purification of Echis coloratus venom and some biological and biochemical effects of the proteins.

Crude venom of Echis coloratus was separated into seven protein fractions using 7% preparative native polyacrylamide gel electrophoresis. The effect of crude venom and seven venom protein fractions (F1-F7) from Echis coloratus on key metabolic activities of fibroblast cultures was investigated. Confluent cultures were incubated with the venom proteins for 3 h at 37 degrees C. The specific activity of phosphofructokinase, was significantly lowered upon incubation with the crude venom and with fractions 2, 3, 4 and 6. Citrate synthase activity was significantly lowered by the crude venom and by fractions 2 and 3. Glycogen phosphorylase activity was significantly increased by the crude venom and by fractions 2, 3, 4 and 6 leading to a significant concurrent drop in glycogen content. Creatine kinase activity was significantly increased by the crude venom and by fractions 3, 4, 5 and 6. Cellular ATP levels rose significantly upon incubation with the crude venom and with fractions 3, 4, 5 and 6. Incubation of cell sonicates with all the venom proteins did not significantly alter the activity or content of any of the studied parameters.

Ultrastructural variations of rat myocardium due to Walterinnesia aegytia snake envenomation.

This study was undertaken to investigate the toxic effect of Walterinnesia aegyptia venom on the ultrastructure of rat myocardium. Male albino rats were prepared for intraperitoneal injection of saline (control group) and saline solution of W.aegyptia venom (study group) at a dose of 0.04 mg animal-1. Biopsies from the left ventricle were prepared for electron microscopy after 1 h (D1 group), 2 h (D2 group), 18 h (D3 group) and 24 h (D4 group). Myocardial cells were in a state of partial to complete contraction. The D1 group showed some mitochondrial vacuoles; D2 group demonstrated more vacuolation and alterations in the form of disorganized cristae. Similar findings were depicted in D3 group. The D4 group demonstrated, in addition, dissolution of mitochondrial cristae. Myofilaments in D3 group experienced coalescence into ill-defined amorphous masses (foci of myolysis). These masses were characterized by the presence of multiple, parallel, Z-like dark bands with disorganization of the filamentous arrangement. In the D4 group, more myolytic foci were observed. This reaction was not limited to one myocyte but extended to the neighbouring ones. Mitochondrial vacuoles were mostly associated with electron dense deposits. Glycogen particles tended to decrease as the experiment proceeded from D1 to D4. These ultrastructural changes were time dependent. They would suggest a cardiotoxic action of W.aegyptia snake venom.

The effect of the desert cobra (Walterinnesia aegyptia) crude venom and its protein fractions on the metabolic activity of cultured human fibroblasts.

Fibroblast cultures were used to study the effect of crude venom and six venom protein fractions (F2-F7) from Walterinnesia aegyptia on their metabolic activity. This was done by incubation of six fibroblast cultures with 10 micrograms of crude venom for 3 h at 37 degrees C. The activities of phosphofructokinase, lactate dehydrogenase, and citrate synthase were significantly lowered upon incubation with all fractions except F2. Glycogen phosphorylase activity was significantly increased, leading to a significant concurrent drop of glycogen content. This effect was only seen for fractions F3 and F5. Creatine kinase activity and cellular ATP levels rose significantly upon incubation with all venom proteins except fractions F2 and F7. Increases were seen for aspartate and alanine aminotransferases by all venom proteins except fractions F2 and F4. Incubation of cell sonicates with all the venom proteins did not significantly alter activities of any of the parameters. Thus, fibroblasts in culture under such conditions appear to mobilize glycogen, phosphocreatine, and protein for ATP production to compensate for decreased glucose.