Analysis of Con A-binding glycoproteins in synaptosomal membranes

Concanavalin A (Con A)-binding proteins obtained from solubilized synaptosomal membranes of bovine brain were analyzed by two-dimensional electrophoresis (2DE), and were identified by peroxidase conjugated Con A (Con A-peroxidase staining), after transfer from 2DE gel to nitrocellulose paper. The Con A-binding proteins were resolved up to 40 spots, ranging in isoelectric points (pI) from 4.5 to 8.0 and molecular weight (MW) from 10 kDa to 120 kDa. Most of the Con A-binding proteins were streaked across a pH gradient and/or exhibited as multiple spots, indicating broad charge and molecular weight heterogeneity. The presence of protein groups that showed high affinities for Con A were revealed. Most interesting group (named GP51), which consisted of seven spots separated horizontally in charge heterogeneity (pI5.85-7.5) with MW 51kDa, was characterized by its binding to an immobilized protein A gel. This implies that GP51 is related to immunoglobulins and/or GP51 may be a new member of the immunoglobulin supergene family.     

Prostacyclin stimulation of adenylate cyclase activity in human thyroid membranes

Effect of prostacyclin (PGI2) on adenylate cyclase activity in human thyroid membranes was examined. PGI2 caused a dose- and time-dependent production of cyclic AMP (cAMP) with high potency. When GTP was added in concentrations up to 100 uM, the activation of adenylate cyclase by PGI2 was increased. In the assay medium containing 3 mM ATP, 10 uM GTP and nucleotide regenerating system, the replacement of Mg2+ by increasing concentrations of Mn2+ caused a progressive loss of PGI2 as well as TSH-stimulated adenylate cyclase activities, while high concentrations of Mg2+ (12 or 18 mM) slightly suppressed the activity stimulated by either PGI2 or TSH. Both agents had an additive effect on the stimulation of adenylate cyclase activity in the presence of either 6 mM Mg2+ or 6 mM Mn2+. Gamma-globulin fraction containing non-stimulatory TSH receptor antibody which was prepared from a patient with chronic thyroiditis, suppressed only TSH- but not PGI2-stimulation of the adenylate cyclase activity. These results suggest that PGI2 can stimulate the adenylate cyclase activity in human thyroid tissue, and that PGI2-stimulation may be mediated by the different system from TSH-dependent one.     

Hepatic lesions in experimental Campylobacter jejuni infection of mice

Mice orally infected with Campylobacter jejuni developed focal infiltrative necrotic lesions in the liver, as determined by both histology and liver function tests. The initial histopathological feature was a focal infiltrative lesion in the parenchyma and portal triads. Foci of infiltrative lesions became necrotic between days 30 and 60 post-inoculation (p.i.). During this period, portal infiltrates increased in severity. From month 4 p.i., focal areas of infiltrative necrosis in the liver parenchyma became extensive. Study of liver function demonstrated mild elevations of transaminases, alkaline phosphatase and lactic dehydrogenase, and also the presence of hypoalbuminaemia. Although histopathological changes of the liver became gradually more marked after day 30 p.i., liver functions of infected mice were most affected at 2 months p.i. The capacity of C. jejuni to induce hepatic lesions seemed to be related to that of organisms to persist in the gall bladder; there was no correlation between biliary carriage in infected mice and positive faecal culture.     

O3 Tolerance and the Ascorbate-Dependent H2O2 Decomposing System in Chloroplasts

The relationship between O₃ tolerance and the chloroplast H₂O₂scavenging system (PS I     

Tumor cytotoxicity of human monocyte membrane-bound interleukin-1α induced by synergistic actions of interferon-γ and synthetic acyltripeptide,FK-565

Summary Human blood monocytes were isolated by counter-flow centrifugal elutriation from healthy donors and these noncytotoxic monocytes were rendered tumoricidal to allogeneic melanoma (A375) cells by activation with a synthetic acyltripeptide (FK-565), as assessed by measuring release of [¹²⁵I]iododeoxyuridine in 72 h. When monocytes were treated with FK-565 for 16 h, and then fixed with paraformaldehyde, they showed cytotoxicity to A375 melanoma cells. The fixed-monocyte-mediated cytotoxicity to A375 cells was induced by the synergistic actions of FK-565 and recombinant interferon- (rIFN-), but not other cytokines [rIFN-A, rIFN-, tumor necrosis factor (TNF), interleukin (IL)-2, -3 and -6]. For synergistic activation of monocytes with induction of a membrane-associated antitumor monokine, the monocytes had to be incubated first with rIFN- and then with FK-565. FK-565 also acted synergistically with rIFN- to stimulate monocytes to produce membrane-associated IL-1 activity, which induced C3H/HeJ thymocyte blastogenesis in response to phytohemagglutinin P. The tumoricidal and thymocytestimulating activities of the fixed monocytes were almost completely inhibited by a specific anti-(IL-1) antiserum, but not by a specific anti-(IL-1) antiserum or monoclonal anti-TNF antibody. These results suggest that membrane-associated IL-1 of human blood monocytes can be induced by two activation signals (rIFN- then FK-565) at their suboptimal concentrations.Abbreviations IL interleukin - IFN interferon - TNF tumor necrosis factor     

Immunoreactive endothelin concentrations in maternal and fetal blood

Immunoreactive-endothelin (ir-ET) concentrations were determined in peripheral maternal blood and in umbilical cord blood just after delivery. The concentrations in both the umbilical artery (2.83 +/- 1.36 pmol/l plasma, Mean +/- SD) and vein (3.37 +/- 1.53 pmol/l) were significantly higher than those found in maternal venous blood (1.43 +/- 1.02 pmol/l). On the other hand, ir-ET levels in maternal blood were not significantly different when compared with those found in non-pregnant women (1.50 +/- 0.83 pmol/l). No significant difference of ir-ET levels between the umbilical artery and vein was observed. A highly significant correlation (r = 0.60, p less than 0.01) of ir-ET levels between the umbilical artery and vein was observed. Also, a significant correlation (r = 0.48, p less than 0.01) between umbilical vein and maternal vein ir-ET levels with a weaker correlation (r = 0.36, p less than 0.05) between umbilical artery and maternal vein ir-ET levels was demonstrated. The present study indicates that ir-ET may be actively secreted in fetal circulation and the plasma levels in maternal and fetal circulation may have a possible relation.     

Regulation of thyroid peroxidase activity by thyrotropin, epidermal growth factor and phorbol ester in porcine thyroid follicles cultured in suspension

The activity of thyroid peroxidase (TPO) in porcine follicles cultured for 96 h in suspension with five hormones (5H) still attained over 50% of that in the freshly isolated follicles. On the other hand, the activity in those cultured with 5H + TSH (6H) was several times higher than that cultured with 5H after 96 h, although an initial decrease of TPO activity during the first 24 h of culture was observed in both conditions. The ability of follicles to metabolize iodide (uptake and organification) when cultured with 6H for 96 h was also several times higher than that of those cultured with 5H. The half-maximal dose of TSH for stimulation of TPO activity and iodide metabolism was 0.03-0.04 mU/ml and the effect was mediated by cAMP. These results indicate that in porcine thyroid follicles in primary suspension culture, TPO activity as well as the ability of iodide metabolism is induced by chronic TSH stimulation. In addition, epidermal growth factor (EGF, 10(-9)M) and phorbol 12-myristate 13-acetate (PMA, 10(-8) M) completely inhibited TSH stimulation on both activities and also basal (5H) activity of iodide metabolism.     

NMR Studies of Pi-Containing Extracellular and Cytoplasmic Compartments in Brain

Abstract: The inorganic phosphate (P_i) NMR peak in brain has an irregular shape, which suggests that it represents more than a single homogeneous pool of P_i. To test the ability of the Marquardt-Levenberg (M-L) nonlinear curve fit algorithm software (Peak-Fit) to separate multiple peaks, locate peak centers, and estimate peak heights, we studied simulated P_i spectra with defined peak centers, areas, and signal-to-noise (S/N) ratios ranging from ∞ to 5.8. As the S/N ratio decreased below 15, the M-L algorithm located peak centers accurately when they were detected; however, small peaks tended to grow smaller and disappear, whereas the amplitudes of larger peaks increased. We developed an in vitro three-compartment model containing a mixture of P_i buffer, phosphocreatine, phosphate diester, and phosphate monoester (PME), portions of which were adjusted to three different pHs before addition of agar. Weighed samples of each buffered gel together with phospholipid extract and bone chips were placed in an NMR tube and covered with mineral oil. Following baseline correction, it was possible to separate the P_i peaks arising from the three compartments with different pH values if each peak made up 10–35% of total P_i area. In vivo, we identified the plasma compartment by intraarterial infusion of P_i. It was assumed that intracellular compartments contained high-energy phosphates and took up glucose. Based on these assumptions we subjected the brains to complete ischemia and observed that P_i compartments at pH 6.82, 6.92, 7.03, and 7.13 increased markedly in amplitude. If the brain cells took up and phosphorylated 2-deoxyglucose (2-DG), 2-DG-6-phosphate (2-DG-6-P) would appear in the PME portion of the spectrum ionized according to pH_i. Four 2-DG-6-P peaks with calculated pH values of 6.86, 6.94, 7.04, and 7.15 did appear in the spectrum, thereby confirming that the four larger P_i peaks represented intracellular spaces.     

Induction and Repair of Damage to DNA in Cucumber Cotyledons Irradiated with UV-B

Photoinduced lesions in DNA, namely, cyclobutane pyrimidinedimers (CPDs) and pyrimidine-(6-4)-pyrimidone photoproducts[(6-4)photoproducts], in cucumber cotyledons that had been irradiatedwith naturally occurring levels of UV-B (290–320 nm) werequantitated by enzyme-linked immunosorbent assays with monoclonalantibodies specific to each type of photolesion. Induction ofthese photolesions was dependent on temperature and their extentwas reduced by simultaneous irradiation with white light. Thedark repair of both types of photolesion was undetectable. Light-dependentremoval of (6-4)photoproducts was very slow, with 50% removalin 4 h. By contrast, 50% of initial CPDs were removed within15 min. Both photorepair processes were dependent on the intensityof white light and were sensitive to temperature. These resultsindicate that high photolyase activity is present in cucumbercotyledons and that repair activities in cucumber cotyledonsare different from those reported in Arabidopsis, in which (6-4)photoproductsare photorepaired more rapidly than CPDs. (Received October 13, 1995; Accepted December 28, 1995)