Selectin- and integrin-mediated T-lymphocyte rolling and arrest on TNF-alpha-activated endothelium: augmentation by erythrocytes

The adhesive and hemodynamic forces that lead to lymphocyte rolling and arrest on activated endothelium and the biophysical role of various adhesion molecules and blood elements in this process are poorly understood. By quantifying their behaviour both in vivo and in vitro, we show here that erythrocytes facilitate selectin- and integrin-mediated rolling and binding of T-lymphocytes on tumor necrosis factor alpha-activated endothelium. The relative contribution of selectins and integrins to this process can be distinguished by using a simple mathematical expression of lymphocyte capture within the range of physiological shear stress. The need for selectin participation in lymphocyte capture increases with shear stress (> 1 dyn/cm2), and both beta 1 and beta 2 integrins act in synergy to produce adhesive drag on captured cells. These findings are potentially useful in developing strategies for intervening with T-cells in a variety of normal and pathological responses as well as for the delivery of genetically modified T-cells to their targets in vivo.     

Human immunodeficiency virus (HIV) antigen testing to detect HIV infection in female sex workers in Singapore

Human immunodeficiency virus (HIV) infection is characterised by seroconversion after a ?window? period of 2 to 3 months. After this period antibodies are usually detectable by screening tests (enzyme immunoassay or particle agglutination) confirmed by Western blot analysis. We studied 1000 newly enrolled female sex workers who had not been previously tested for HIV to assess the usefulness of HIV antigen testing to improve the efficacy of HIV infection detection. Blood was taken at enrollment for HIV antigen and HIV antibody testing. The Abbott HIVAG-1 test was used to detect antigen; antibody detection was by the Abbott recombinant HIV-1/HIV-2 3rd generation enzyme immunoassay (EIA) test, the Fujirebio Serodia-HIV particle agglutination (PA) test for screening, and the Diagnostic Biotechnology HIV Blot 2.2 Western blot (WB) test for antibody confirmation. Of the 1000 samples, 26 were positive for HIV antibody testing (26/26 for EIA, 25/25 for PA, 26/26 for WB), giving a prevalence rate of 2.6%, Of these 26 seropositive samples 1 was positive on HIV antigen testing. There were no samples which were antigen-positive and antibody-negative. HIV antigen testing does not add to increased efficacy of HIV detection among female sex workers in Singapore.     

Age-dependent alterations of DNA synthesis. Terminal deoxynucleotidyl transferase and DNA polymerase activities in bone marrow subpopulations from mice

The decrease of functional capacity of cellular immunity during ageing seems to be due to cellular changes of stem cells, particularly in the growth properties and the cell density in T-cell subsets. We approached this problem at the molecular biological level by quantifying the key enzymes necessary for DNA synthesis in bone marrow cells from mice: deoxynucleotidyl transferase (TdT) and DNA polymerase alpha. The bone marrow cells were fractionated on a discontinuous bovine serum albumin density gradient and the extractable enzyme activities (expressed per 10(8) nucleated cells in the respective fraction) were determined. TdT activity was found to decrease markedly during ageing. Mature animals contain only 34% and senescent animals only 13% of the activity observed in immature mice. From the density distribution analysis it was found that a shift of TdT-containing cells to the lower density occurs. The specific DNA polymerase alpha activity also decreases in bone marrow cells with age. While the overall activity amounts in immature cells to 78 enzyme units/10(8) cells, it decreases in mature cells to 57 units/10(8) cells, and in cells from senescent animals to 36 units/10(8) cells. Density distribution analysis of the cells shows that the highest activity is observed in the low-density fraction. From these experimental data we conclude that in the fractions containing precursor T-cells, a reduced number of proliferating cells is present.     

The activation of prothrombin by the prothrombinase complex. The contribution of the substrate-membrane interaction to catalysis

The conversion of prothrombin to thrombin requires the cleavage of two peptide bonds and is catalyzed by the prothrombinase complex composed of factors Xa and Va assembled on a membrane surface. Presteady-state kinetic studies of the effects of membranes on the proteolytic reaction were undertaken using model membranes composed of phosphatidylcholine and phosphatidylserine (PCPS). The concentration of PCPS was varied to alter the concentration of free phospholipid available for substrate binding without influencing the concentration of membrane-assembled prothrombinase. In fluorescence stopped-flow measurements, increasing concentrations of PCPS resulted in an increase in the rate of product formation. Assessment of bond cleavage by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following rapid chemical quench using 125I-prothrombin revealed that the activation reaction proceeded through the ordered cleavage at Arg323-Ile324 followed by cleavage at Art274-Thr275 at all concentrations of PCPS. Increasing the PCPS concentration resulted in a large increase in the Arg323-Ile324 cleavage reaction with a much smaller effect on the subsequent cleavage at Arg274-Thr275, thereby leading to an increase in the extent of accumulation of the intermediate, meizothrombin. Fluorescence stopped-flow and rapid chemical quench measurements were also conducted using prethrombin 2 plus fragment 1.2 or meizothrombin as substrates to assess the influence of PCPS on the individual cleavage reactions. The rate of cleavage at Arg323-Ile324 by prothrombinase was increased approximately 60-fold with increasing PCPS, whereas the cleavage at Arg274-Thr275 was increased by a factor of approximately 5. These differential effects of PCPS on the two cleavage reactions adequately explain changes in the extent of accumulation of meizothrombin during prothrombin activation. Proteolytic removal of the membrane binding fragment 1 domain of the substrates, meizothrombin and prethrombin 2-fragment 1.2, had no effect on the cleavage at Arg274-Thr275 at saturating PCPS but completely eliminated the membrane-dependent rate enhancement for cleavage at Arg323-Ile324. Thus, membrane binding by the substrate is essential for the first cleavage reaction at Arg323-Ile324, which leads to the conversion of prothrombin to meizothrombin. In contrast, the substrate-membrane interaction mediated by the fragment 1 domain has no detectable effect on the second cleavage reaction at Arg274-Thr275 which is required for the conversion of meizothrombin to thrombin.     

Regulation by gangliosides and sulfatides of phospholipase A2 activity against dipalmitoyl- and dilauroylphosphatidylcholine in small unilamellar bilayer vesicles and mixed monolayers

The modulation by gangliosides GM1 and GD1a, and sulfatide (Sulf) of the activity of porcine pancreatic phospholipase A2 was studied with small unilamellar vesicles of dipalmitoylphosphatidylcholine (L-dpPC) and lipid monolayers of dilauroylphosphatidylcholine (L-dlPC). The presence of Sulf always led to an increase of the maximum rate of the enzymatic reaction, irrespective on whether the vesicles were above, in the range of, or below the bilayer transition temperature. Sulf did not modify the latency period for the reaction that is observed at the bilayer transition temperature. Gangliosides inhibited the maximum rate of enzymatic activity bilayer vesicles in the gel phase but the effect was complex. When the reaction was carried out at a temperature within the range of the bilayer phase transition, the gangliosides inhibited the maximal rate of the reaction in proportion to their content in the bilayer. However, at the same time the latency period observed with vesicles of pure phospholipid at this temperature was shortened in proportion to the mole fraction of gangliosides in the bilayer. At temperatures above the bilayer phase transition, gangliosides stimulated the activity of PLA2. Preincubation of the enzyme with Sulf or gangliosides did not affect the activity against bilayer vesicles of pure substrate. These glycosphingolipids did not modify the rate or extent of desorption of the enzyme from the interface, nor the pre-catalytic steps for the interfacial activation of PLA2, or the enzyme affinity for the phospholipid substrate. Also, the activity of the enzyme was not altered irreversibly by glycosphingolipids. Our results indicate that Sulf and gangliosides modulate the catalytic activity of PLA2 at the interface itself, beyond the initial steps of enzyme adsorption and activation, probably through modifications of the intermolecular organization and surface electrostatics of the phospholipid substrate.     

Dye leakage of four root end filling materials: effects of blood contamination

OBJECTIVES: A measurement of cell DNA content would be highly useful in determining the malignant nature of thyroid tumours in cases without distinctive features such as metastases, capsule invasion or emboli. Abnormal cell ploidy can be recognized with flow cytometry, but it is not known whether such results have diagnostic value. We therefore compared--in a double blind prospective study--the results of flow cytometry and pathologic diagnosis in fresh tumoural and non-tumoural thyroid cells. METHODS: Fifty unselected cold thyroid nodules were obtained from 50 consecutive patients (40 women, 10 men; age 18-80 years; mean 46) who underwent surgery within a 6 month period. Surrounding non-tumoural tissue was also obtained in 46 of them. Cell ploidy and the percentage of cells in each cell phase was determined with flow cytometry for both tumoural and nontumoural tissues. Two pathologists, unaware of the flow cytometric results, independently established the histologic diagnosis according to the WHO classification. RESULTS: The pathologic diagnosis was carcinoma in 7 cases (papillary carcinoma 6, vesicular carcinoma 1) and benign adenomas in 43 (29 macrovesicular, 11 microvesicular, 3 oncocytal). All the non-tumoural tissue samples were diploid. All 7 carcinomas were diploid and 10 of the 43 benign adenomas were aneuploid (4 near-diploid, 3 hyperploid, 1 near-tetraploid, 2 multiploid). The mean proliferation index was increased in 5 diploid tumours. CONCLUSION: These findings confirm that cell ploidy measured by flow cytometry is of no diagnostic value in the thyroid gland. It was also revealed that aneuploidy in adenomas may be related to tissue rearrangements of undetermined prognostic significance.     

Long-term effect of N and P fertilization on a pearlmillet—wheat cropping system

Results of an eight-year study on long-term effect of N and P application in a pearlmillet—wheat sequence is reported. There was little or no residual effect of N on any of the crops. Pearlmillet needed 70 to 80 kg N and wheat required more than 120 kg N ha^–1 every year for optimum grain yield. There was no soluble P build up in soil by continuous P application. Fertilizing wheat every year with 19 kg P and pearlmillet with 13 kg P ha^–1 is considered optimum.Continuous cropping leading to a production of 216 tonnes of biomass ha^–1 in 17 crops and use of high analysis N (urea) and P (triple superphosphate) fertilizers had not impaired the K and Zn supplying capacity of these alluvial soils containing illite clay minerals. The experiment is being continued to monitor the productivity of the soil as affected by continuous cropping.