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.

GB virus C/hepatitis G virus infection in Saudi Arabian blood donors and patients with cryptogenic hepatitis.

Viral hepatitis is a common infection in the developing countries. Aside from Hepatitis A-E viruses, a novel hepatitis virus termed GBV-C, or HGV, was recently described. We have studied the prevalence of this virus among Saudi Arabian healthy blood donors (n = 200) and patients with cryptogenic (non-A-E) hepatitis (n=71). After serum extraction and RNA reverse transcription, amplification was carried out by the polymerase chain reaction (PCR), using primers for the 5' noncoding region (NCR), NS5A region and NS3 helicase region. Among the patients with cryptogenic hepatitis, PCR-positivity was 18/71 (25.4%) for the 5' NCR, 14/71 (19.7%) for the NS5A region, and 15/71 (21.1%) for the NS3 helicase region. Among the healthy blood donors, PCR-positivity was 4/200 (2%) for the 5' NCR, 0/200 (0%) for the NS5A region, and 1/200 (0.5%) for the NS3 helicase region. Since the 5' NCR is considered the most conserved segment of the virus genome, it is not unusual to find higher positivity rate when that region is used for amplification. It is noted that the positivity rate is not far different among the three amplified regions, indicating that the heterogeneity of GBV-C/HGV is not as extensive as in hepatitis C virus. Phylogenetic analysis of 5'NCR DNA sequences showed that all isolates in this study belong to genotype 2. We conclude that the prevalence of GBV-C/HGV is similar to what is reported worldwide among the general Saudi population but relatively higher among Saudi patients with cryptogenic hepatitis.

Plasmid profile and antibiotic resistance in coagulase-negative staphylococci isolated from polluted water.

Four different species of coagulase-negative staphylococci (CNS) were isolated from polluted waters in Fez, Morocco and found to be Staphylococcus simulans, Staph. lenticus, Staph. hyicus and Staph. xylosus. Eight isolates belonging to these four species were analysed for their plasmid content. Southern blot hybridizations were performed to define the resistance determinants of the plasmids harboured by these species. These determinants were found to be carried mainly by Class I staphylococcal plasmids (1-5 kb). A plasmid (4.3 kb) carrying a tetracycline resistance gene was present in five isolates from all identified species. Plasmids carrying a chloramphenicol resistance gene were more frequently encountered and found to be of different sizes. Plasmids carrying erythromycin, neomycin, and streptomycin resistance genes were less frequent and were the same size. The results indicate that the occurrence of multi-resistant CNS in polluted waters may constitute a reservoir for disseminating antibiotic-resistance into the community.

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.

Adenosine deaminase activity in serum and pleural effusions of tuberculous and non-tuberculous patients.

In a retrospective study Adenosine deaminase (ADA), was assayed in 86 serum samples and 12 pleural fluid samples of patients with pulmonary tuberculosis. Serum and pleural fluid ADA levels were also examined in a group of 38 non-tuberculous patients with pleural effusion. Highly significant increases in both serum and pleural fluid ADA levels were noted in tuberculous patients when compared to both control enzyme cut-off values and non-tuberculous pleural effusion patients. Negative Ziehl-Nielsen staining for acid fast bacilli and Purified Protein Derivative skin test results were obtained in 32.3% and 11.1% of the examined patients respectively. All these patients showed significantly elevated serum or pleural fluid ADA levels. It is hereby suggested that serum and pleural fluid ADA levels can in conjunction with other tests, serve as a marker in patients of pulmonary tuberculosis.

Enhancement of leishmanicidal activity of human macrophages against Leishmania major and Leishmania donovani infection using recombinant human granulocyte macrophage colony stimulating factor.

The in vitro effect of recombinant human Granulocyte Macrophage Colony Stimulating Factor (rh-GMCSF) on the leishmanicidal activity and superoxide anion productivity of macrophages derived from human blood monocytes (MOs) were investigated. MOs treated with 25, 125, or 250 U/mL of rh-GMCSF for 72 h prior to infection with leishmania parasites, manifested significant dose-dependent increase in its leishmanicidal activities against Leishmania major and Leishmania donovani parasites. The percentage of increase in leishmanicidal activity of L. major-infected MOs were 22.71, 64.34 and 81.34, respectively while in L. donovani-infected MOs, it reached 3.01, 32.28 and 74.38, respectively. Treatment of leishmania-infected MOs with rh-GMCSF (250 U/mL) for different periods of time up to 96 hours, induced a significant time-dependent reduction in the percentage of infected cells and the parasitic load (No. of amastigotes/100 MOs). After 96 h of treatment with rh-GMCSF, the percentages of reduction in the infection rates were 82.45 in L.major-infected MOs (p < 0.001) and 39.65 in L. donovani-infected cells (p < 0.01). The percentage of reduction in the parasitic load reached 90.82 (p < 0.001) and 36.6 (p < 0.05) in MOs infected with L. major and L. donovani, respectively. The priming effect of rh-GMCSF on superoxide anion production by human MOs stimulated with phorbol myristate acetate (PMA) was both dose-dependent and time-dependent. In 72 hour-old human MOs, the maximum superoxide anion release was generated by MOs primed for 45 min with 500 U/mL of rh-GMCSF. These cells produced 8.960 +/- 2.075 nmol/5 x 10(4) MOs/ 180 min as compared to 4.563 +/- 1.773 nmol/5 x 10(4) unprimed cell control/180 min (p < 0.001).

Prevalence of hypertension and sociodemographic characteristics of adult hypertensives in Riyadh City, Saudi Arabia.

OBJECTIVES: To estimate the prevalence of hypertension in adults residing in Riyadh city and to study the sociodemographic characteristics of adult hypertensives. DESIGN: Cross-sectional survey. SETTING: Primary Health Care Centres (PHCCs) in Riyadh city selected by stratified random sampling, the subjects resident in each PHCC catchment area were selected by systematic sampling from their records in the PHCCs. SUBJECTS AND METHODS: A total of 1394 adults aged 15 years and over were interviewed and examined during March 1993 to March 1994. The average of three measurements of blood pressure (BP) was taken to represent their current pressures. A subject is considered hypertensive if the average BP reading is 160/95 mm Hg or more, or is currently under treatment. RESULTS: The total hypertensive subjects were 214 giving an overall prevalence of hypertension of 15.4%. Of these 157 (11.3%) subjects were known hypertensives and were under some form of treatment. On the other hand 57 (4.1%) other subjects were newly detected by the study. Hypertension (BP = 160/95 mm Hg or more) was significantly related to age, marriage, education, occupation and employment status and consanguinity. Male subjects had a higher prevalence of hypertension but the differences were not significant. Nationality and income were not related to high BP. CONCLUSION: Hypertension is a problem among adults in Riyadh city. It is significantly related to some sociodemographic and family factors. About 27% of all hypertensives are not aware of their disease and more than 31% of known hypertensives are apparently not well controlled. There is a need for a programme to prevent and control hypertension in Riyadh city. Similar studies need to be done in other areas of the country to estimate the prevalence of hypertension and associated factors as prerequisites for any programme to control the disease.

Effect of rh-GMCSF and rh-GCSF on oxygen free radical production by human neutrophils and blood monocyte-derived human macrophages.

The in vitro effect of recombinant human granulocyte-macrophage colony stimulating factor (rh-GMCSF) and recombinant human granulocyte colony stimulating factor (rh-GCSF) on oxygen free radical (OFR) generation by human neutrophils and blood monocytes derived human macrophages stimulated with phorbol myristate acetate was investigated and compared. The production of OFR by neutrophils and macrophages was time dependent, and the maximum release of OFR by neutrophils and macrophages was measured 90 and 180 min after stimulation with phorbol myristate acetate, respectively. The priming effects or rh-GMCSF and rh-GCSF on OFR production by human neutrophils and macrophages was dose dependent. The maximum generation of OFR by neutrophils occurred when primed with 1,000 U/ml of rh-GMCSF and reached 2.383 +/- 0.191 nmol/10(5) neutrophils/90 min as compared with 1.072 +/- 0.113 nmol/10(5) neutrophils/90 min in the unprimed controls. This represents a 122.20% increase in OFR generation (p < 0.001). However, the percentage of maximum increase in OFR production was 57.84 when neutrophils were primed with a concentration of 5,000 U of rh-GCSF/ml. In 72-hour-old human macrophages, much higher levels of OFR production as compared with neutrophils were measured following stimulation with phorbol myristate acetate. The maximum generation of OFR was measured in macrophages primed for 45 min with 500 U/ml of rh-GMCSF. These cells produced 8.960 +/- 2.075 nmol/5 x 10(4) macrophage/180 min as compared with 4.563 +/- 1.773 nmol/5 x 10(4) unprimed macrophages/180 min (p < 0.001). In macrophages primed with rh-GCSF, however, the maximum OFR production was induced by a dose of 5,000 U/ml and reached 6.902 +/- 1.463 nmol/5 x 10(4) macrophages/180 min as compared with 4.563 +/- 1.773 nmol/5 x 10(4) macrophages/180 min in the unprimed controls (p < 0.05). In conclusion, the priming effect of rh-GMCSF on OFR generation by human macrophages and neutrophils was more potent than that of rh-GCSF, both in the extent of augmentation and in the dose required to produce maximum OFR generation.

Leishmania major: in vitro ultrastructural study of the paraxial rod of promastigotes.

The flagellum of Leishmania major promastigotes has an intraflagellar structure known as the paraxial rod (PAR) which extends from a point halfway in the flagellar pocket to the tip of the flagellum, lying opposite the axonemal microtubule doublets 4-7. An expansion of the axonemal plasma membrane envelops the PAR and may provide desmosomal attachment at the orifice of the flagellar pocket. The complex organization of the 4-6 nm thick filaments in the PAR was studied by us in cross, oblique, longitudinal and tangential sections by electron microscope. These filaments are disposed in two parallel lamellae, one alongside the axoneme (ca. 45 nm thick), and the other alongside the plasma membrane (ca. 65 nm thick), with an interlamellar gap of about 22-28 nm. In each lamella, 8-12 parallel series of longitudinal filaments at ca. 30 nm intervals interdigitate with coplanar parallel series of oblique filaments at ca. 25 nm intervals and inclined to the long axis of the flagellum at ca. 48 degrees, and ca. 55 degrees, in the inner (paraxonemal) and outer lamella, respectively. The parallel filaments in each of the longitudinal and oblique series are spaced at ca. 8 nm intervals. They are cross-striated at ca. 30 nm intervals by transverse filaments which terminate occasionally on adjacent axonemal microtubules 5 and 6 in the inner lamella, and the plasma membrane in the outer lamella. Extending across the interlamellar gap is a set of parallel rows of 7-12 nearly parallel filaments at ca. 20 nm intervals. The part of the flagellar plasma membrane enclosing the PAR has a subplasmalemmal cytoskeleton consisting of a layer of longitudinal 2 nm filaments at 8 nm intervals, obliquely striated by parallel 2 nm filament doubles at ca. (-65) degrees with the long axis of the flagellum and ca. 20 nm periodicity. Each filament doublet stria apparently gives origin to collinear short filament doublet extensions that curve into juxtaposed meshes of the outer lamella. Microtubules of the axonemal doublets 5 and 6 are connected to electron-dense (ca. 12 nm thick) strips of the inner lamella of the PAR by longitudinal series of ca. 4 nm cross-links across a ca. 12 nm cleft.

Production of superoxide anion by polymorphonuclear leukocytes from diabetic patients with or without diabetic retinopathy.

Oxygen free radicals have been implicated in the pathogenesis of diabetic microangiopathy. The production of superoxide anion (O2-.) by polymorphonuclear leukocytes (PMNs) from 45 insulin-dependent diabetes mellitus patients in the resting state and in response to a soluble stimulus (phorbol myristate acetate) was measured spectrophotometrically and compared with that of 15 age and sex matched controls. The resting superoxide anion production by PMNs from diabetic patients was significantly higher than that of controls (2.17 +/- 1.32 and 1.35 +/- 0.6 nmol/10(5) cells/60 min respectively; p = 0.037). In contrast, PMNs from diabetic patients released significantly lower levels of superoxide anion compared to controls in response to phorbol myristate acetate stimulation (2.33 +/- 2.04 and 3.55 +/- 0.98 nmol/10(5) cells/60 min respectively; p = 0.044). The stimulated superoxide anion production was significantly higher in diabetic patients with retinopathy compared to diabetic patients without retinopathy (2.7 +/- 2.08 and 1.3 +/- 1.6 nmol/10(5) cells/60 min respectively; p = 0.02). Furthermore, stimulated PMNs from diabetic patients with proliferative retinopathy generated superoxide anion at significantly higher rates than did those from diabetics with nonproliferative retinopathy or without retinopathy (3.8 +/- 1.5, 2.08 +/- 2.1 and 1.3 +/- 1.6 nmol/10(5) cells/60 min respectively; p = 0.005). These results suggest that reactive oxygen species produced by PMNs may play a role in the progression of diabetic retinopathy.

Serum factor from diabetic patients with or without retinopathy stimulates superoxide anion production by normal polymorphonuclear leukocytes.

Oxygen free radicals (OFRs) have been implicated in the pathogenesis of diabetic microangiopathy. The effects of serum from insulin-dependent diabetes mellitus patients with or without retinopathy on the production of superoxide anion by normal polymorphonuclear leukocytes (PMNs) were measured spectrophotometrically and compared with that of age matched controls. Superoxide anion production by PMNs incubated with serum from retinopathy-free patients or patients with retinopathy was significantly higher than that of controls (P=0.0002 and 0.0001, respectively). Furthermore, superoxide anion production by PMNs incubated with serum from patients with retinopathy was significantly higher than retinopathy-free patients (P=0.02). These observations suggest that a diabetic serum factor provoked a significant generation of superoxide anion in normal PMNs, a phenomenon found parallel to the presence of retinopathy, indicating that OFRs may play a role in the progression of diabetic retinopathy. The nature of this serum factor remains to be clarified.

Processing of envelope polypeptides of herpes simplex virus type 1. Demonstration of variation in different cell lines by high-performance liquid chromatography and radioimmunoprecipitation.

[35S]Methionine-labelled envelope polypeptides of herpes simplex virus type 1, strain F, propagated in mammalian cell culture of various origins, were separated by ion-exchange high-performance liquid chromatography on a TSK DEAE-3SW column. Analysis of the fractions by radioimmunoprecipitation followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis of the immunoprecipitates showed similarities as well as distinct differences in the number, migration patterns and molecular mass of the synthesized polypeptides, depending on the host cell. The results show that this method can be used to demonstrate species-specific or organ-specific differences in the processing of virus-specified polypeptides synthesized in host cells.

Establishment of a normal reference range for adenosine deaminase in plasma of healthy Saudis using the continuous spectrophotometric method.

The level of adenosine deaminase (EC. 3.5.4.4), was estimated in plasma of 389 healthy males and 493 healthy females in order to establish a normal reference range for Saudis. Using the continuous spectrophotometric method, the reference ranges were calculated in two ways using the mean +/- 2 SD and the 2.5th - 97.5th percentile value methods. In both methods of calculation, a slightly higher range was observed for children as compared to adults. The method of 2.5th - 97.5th percentile values brought almost all of our subjects within the recommended range of 11.5 - 25 U/l. In the current study, the normal range for adenosine deaminase totalled 15.0 - 23.2, 14.8 - 23.6, 15.0 - 23.0 and 16.7 - 24.6 U/l for the overall population, all males, females, and children, respectively. The ranges are discussed in the light of significantly different results obtained by the two calculation methods and recommendation of an appropriate method for healthy Saudis, namely the 2.5th - 97.5th percentile values. The choice of the Ellis and Goldberg kinetic continous monitoring method for the estimation of plasma ADA levels in the current investigation is also hereby justified.

Leishmania infecting man and wild animals in Saudi Arabia. 9. The black rat (Rattus rattus) a probable reservoir of visceral leishmaniasis in Gizan province, south-west Saudi Arabia.

Twenty-two black rats (Rattus rattus) were captured in houses where parasitologically confirmed cases of human visceral leishmaniasis had been recorded in Al-Arda Emara, Gizan province, south-west Saudi Arabia. Four of the rats were found to be infected with Leishmania; isoenzyme characterization showed that 3 were infected with L. donovani sensu lato zymodeme LON42 and the fourth with L. infantum zymodeme LON49. L. donovani s.l. LON42 has also been isolated from human visceral leishmaniasis patients living in this area, while dogs, but not humans, have been found to be infected with L. infantum LON49 in this part of Saudi Arabia.

Genomic variation among herpes simplex virus type 1 strains: virus DNA analysis of isolates from Saudi patients.

Fifty-two clinical isolates of herpes simplex virus type 1 (HSV-1) from Saudi Arabian patients were analysed by restriction endonuclease digestion of the virus DNA using the enzymes HindIII and BamHI, followed by hybridization with 32P labelled DNA of laboratory strain F. Of the isolates, 17 were resolved into four distinct cleavage patterns with HindIII restriction enzyme. The remaining 35 strains had the same cleavage pattern as the standard HSV-1-F. Further investigation of the 52 isolates with BamHI, which is a multicut enzyme and therefore capable of higher resolution, differentiated 47 of the 52 isolates and were assigned into nine cleavage groups. Comparing our findings with similar studies reported elsewhere suggest geographic clustering of HSV-1 strains. Fragments giving rise to the observed DNA polymorphism were mapped to the unique region of the long and short segments of the genome.

The effect of crude Cerastes cerastes gasperetti venom on the activity of key metabolic enzymes in cultured human fibroblasts.

Cultured human fibroblasts were used to study the effect of a crude extract of Cerastes cerastes gasperetti venom on the activity of a profile of key enzymes of metabolism. A single concentration of the crude venom was incubated with confluent fibroblasts established from six normal subjects for a period of three hours. A dramatic reduction in the specific activities of glucose and glycogen degradative enzymes was observed (23.7 +/- 3.9%, 36.3 +/- 8.7% and 71.1 +/- 5.7% of control for citrate synthase, glucose-6-phosphate and phosphofructokinase respectively). Furthermore, the specific activity of creatine kinase was doubled. No significant change in activity of three transaminases was noticed. Incubation of the same concentration of venom for the same period of time with serum did not result in any change in the activity of the enzymes studied. It is suggested that the cells mobilize stored phosphocreatine for the production of adenosine triphosphate (ATP) to compensate for the reduced rate of sugar catabolism. Furthermore, it is hereby suggested that the effects noticed on the enzyme activities are not directed at the enzyme protein itself, but are of mediated nature.

Determination of lomefloxacin in biological fluids by high-performance liquid chromatography and a microbiological method.

A high-performance liquid chromatographic method (HPLC) was developed for the determination of lomefloxacin in plasma and urine and was compared to a microbiological assay. Lomefloxacin and norfloxacin (internal standard) were extracted from plasma and urine samples using chloroform. Measurements were carried out with a fluorescence detector using an excitation wavelength of 280 nm and an emission wavelength of 430 nm with a mercury lamp. Quantification was achieved by the measurement of the peak-height ratio and the analytical recovery of the drug from plasma and urine was found to be (mean +/- SD) 99.3 +/- 3.74% and 95.7% +/- 3.82%, respectively. In the microbiological assay, E. coli ATCC 1346 was the test organism using an agar diffusion technique. The coefficients of variation for within-day analysis for both the HPLC method and microbiological assay from plasma samples were less than 7%. The minimum detectable concentration for both the HPLC and the microbiological method was 50 ng/ml and 100 ng/ml, respectively. Both methods were used to determine the lomefloxacin level in plasma following intravenous administration to mice. Excellent agreement was obtained between the results of the two methods. The HPLC method offers significant advantages in accuracy, precision, speed of analysis and turnover-time.

Influence of new quinolones on normal immune capabilities.

Ciprofloxacin, pefloxacin, norfloxacin, and ofloxacin were investigated for immunomodulatory activity on humoral and cell-mediated immune responses. Ciprofloxacin and pefloxacin altered the humoral immune responses of mice to sheep red blood cells. This effect was not exhibited by norfloxacin or ofloxacin. All four quinolones did not alter cell-mediated responses. When these antimicrobial agents were tested for their interaction with human polymorphonuclear phagocytic activity, all agents suppressed this activity. In addition, all except norfloxacin showed anti-inflammatory activity.