Endogenous phosphorylation of soluble enzymes in human red cells. Cyclic 3',5'-AMP-dependent phosphorylation of phosphofructokinase without detectable regulatory effect

ATP-depleted human red cells have been incubated in a glucose-containing medium with [32P]orthophosphate in the presence and in the absence of cyclic 3',5'-AMP and dibutyril cyclic 3',5'-AMP. Spectrin, pyruvate kinase, phosphofructokinase, glucose-6-phosphate dehydrogenase and hemoglobin A1 have been purified and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein-bound radioactivity has been measured from the sodium dodecyl sulfate polyacrylamide gels and the trichloroacetic acid-precipitated proteins. In the cytosol, the most intense phosphorylation was found for pyruvate kinase whose, in the presence of cyclic AMP, specific radioactivity was comparable to that of the membrane protein and spectrin. In the absence of cyclic nucleotides it was five times less phosphorylated. Phosphofructokinase was only phosphorylated when the red cells were incubated with cyclic nucleotides; the extent of phosphorylation was four times less than for pyruvate kinase. Hemoglobin, glucose-6-phosphate dehydrogenase and a contaminant protein copurified with phosphofructokinase were not phosphorylated: the 'background' of the radioactivity found for these proteins was 100 times less than for pyruvate kinase and spectrin, and 20 times less than for phosphofructokinase (+cyclic AMP).     

Modification by simetryn sulphoxide of a specific thiol group in rat haemoglobin.

Native rat haemoglobins were found to bind simetryn sulphoxide to an extent 40-fold greater than human haemoglobin. This specific behaviour was studied by using only high-pressure ('performance') liquid chromatography for the preparative separation of globin chains and the isolation of peptides resulting from chemical and enzymic degradation. High recoveries (greater than 80%) of peptides throughout the procedures in combination with microsequence techniques, allow a definitive assignment of the residue undergoing modification. The haemoglobin beta-chain cystine-125 residue, with a stoichiometry of one per tetramer of rat haemoglobin, was found to be modified. Stereochemical implications of this finding are discussed. Simetryn sulphoxide would appear to be useful as a specific reagent for the mapping of exposed thiol residues in proteins.     

Toxicological evaluation of agromet (Metalaxyl) preparation.

A complex toxicological study was carried out in relation to the hygienic standardization of the fungicide preparation Ridomil, whose forthcoming production in this country will be under the name Metalaxyl. The study was performed on sexually mature white rats of both sexes, at oral, dermal and inhalation exposure, acute, subacute and chronic experiments, according to the Bulgarian State Standard. Besides the routine toxicological studies were carried also studies for establishing the long-term effects of the preparation (gonadotropic, embryotoxic and teratogenic, cardiovascular). The results from the study permitted the Agromet preparation to be put in III class--moderately toxic according to limiting index LD50 oral. On the basis of data from the authors study on the general toxicity and long-term effects of Metalaxyl a temporary MAC 4 mg/m3 was proposed.     

Cyclosporin A regulates the expression of HLA-DR on human monocytes by two different mechanisms.

Cyclosporin A (CsA), but not its nonimmunosuppressive analog cyclosporin H (CsH), inhibited the expression of HLA-DR in human monocytes. Induction of HLA-DR by interferon (IFN)-gamma in fresh monocytes was also inhibited by CsA and not by CsH. However, when monocytes were pretreated with either CsA or CsH for 16 hr prior to the addition of IFN-gamma, HLA-DR expression was increased, probably because of a cyclosporin-induced increase in the number of IFN-gamma receptors. Down-regulation of the HLA-DR mRNA by CsA was found to be dependent on continuous protein synthesis. IFN-alpha also inhibited the IFN-gamma-induced HLA-DR mRNA expression and showed synergy with CsA at low concentrations but not at high concentrations of the drugs. A common mechanistic element in the pathways of CsA and IFN-alpha is proposed.     

Plant-induced changes in the redox potentials of rice rhizospheres

Redox potentials in microsites of the rhizosphere of flooded rice were continuously measured for several days. Close to the root tips redox potential markedly increased. The highest increase was measured in the rhizosphere of the tips of short lateral roots. Aerobic redox conditions were reached there, except in a very strongly reduced soil. Both the extension of the oxidation zone around the root tips and the maximum redox potential reached were influenced by the reducing capacity of the soil. The radius of the redox rhizosphere varied from less than 1 mm in a strongly reduced soil up to 4 mm in a weakly reduced one. The root-induced oxidation processes in the rhizosphere depended on the atmospheric oxygen supply to the roots.     

Morphologic, phenotypic, and cytotoxic analyses of C57BL/6 murine non-parenchymal liver cells: new evidence associating murine liver large granular lymphocytes with monocyte precursors and implications for tumor immunotherapy.

Non-parenchymal liver cells (NPCs) have been implicated in murine host resistance to hepatic metastases. We have examined the relative cell number, morphology, phenotype, and cytotoxic potential of Percoll fractionated C57BL/6 murine liver NPCs. Low density (Percoll fractions 2 and 3) cells showed a large granular lymphocyte morphology and made up 76% of all NPCs recoverable, while high density (fractions 5 and 6) showed a small lymphocyte morphology and made up 10% of all NPCs. Low density cells demonstrated the following phenotype: 14% of the cells demonstrated the Thy 1.2 marker; 12%, the Lyt-2 marker; 67%, the L3T4 marker; 74%, the asialo GM1 marker; 30%, the 49H.8 marker; and 65%, the F4/80 marker. The high density cells expressed the same markers on 71%, 21%, 33%, 68%, 37%, and 19% of their cell surface, respectively. There were no differences phenotypically between high density NPCs and splenocytes except for the F4/80 expression (fractions 5 and 6 NPCs, F4/80 expression 19%, fresh splenocytes 60%). Dual color analysis of L3T4+ NPCs documented that fractions 2 and 3 cells also expressed the F4/80 marker on 85% of their cell surface and the Thy 1.2 marker on 11% of their cell surface. The high density fractions 5 and 6 L3T4+ cells expressed the F4/80 marker on 16% of their cell surface, and the Thy 1.2 marker on 89% of their cell surface. Cytotoxicity against YAC-1 [a natural killer (NK) sensitive target], MCA-102 (a NK resistant target), and WEHI-164 (a natural cytotoxicity target) were similar for fractions 2 and 3, and 5 and 6 cells. Based upon the expression of the F4/80 marker on L3T4+ cells that are Thy 1.2 negative and appear to be similar to LGLs morphologically (fractions 2 and 3 NPCs), we propose that these cells are monocyte precursors while fractions 5 and 6 cells are small lymphocytes. These findings with liver LGLs support the need for the evaluation of monocyte directed biological response modifiers in therapeutic models of murine hepatic metastases.