Evidence for negative regulation of T cell growth by low affinity interleukin 2 receptors

Two experimental situations have been studied, and the results provide evidence for a negative regulatory role for the low affinity interleukin 2 receptor (LA-IL 2R). The IL 2-dependent T helper cell line L-14, deprived of IL 2, becomes quiescent and expresses comparable numbers of high affinity IL 2R (HA-IL 2R) and LA-IL 2R. After activation by recombinant IL 2, this cell line preferentially expresses LA-IL 2R. The IL 2 responsiveness of the L-14 cell line was found to vary according to the ratio of LA-IL 2R to HA-IL 2R: the relative predominance of the LA-IL 2R coincides with a hyporeactivity of cells to IL 2. In contrast, a predominance of HA-IL 2R is accompanied by an increase in cellular IL 2 reactivity. Treatment of three IL 2-dependent T cell lines (L-14, HT-2, and C30.1) with limited amounts of recombinant IL 2 and moderate concentrations of anti-IL 2R monoclonal antibodies stimulates T cell growth. This treatment was shown to selectively diminish the expression of membrane LA-IL 2R. The stimulation was attributed to the decrease of expression of LA-IL 2R.     

Complementarity of particles and pits in freeze-fractured hepatic and cardiac gap junctions

Summary Particles and pits of freeze-fractured gap junctions are considered as complementary structures despite the frequent observations of more regular and closer spacings of pits, ascribed to plastic deformation of particle arrays. Recently, however, the noncomplementarity of pits and particles in Purkinje fibers has been reported. To ascertain the relationship between both structures, gap junctions from fixed, cryoprotected liver and myocardium were investigated using spacing and density measurements and complementary replicas.In hepatocyte gap junctions, the center-to-center distances (mean±sd) among pits, 9.57±1.49 nm, and particles, 9.70±1.77 nm, are not significantly different. Density determinations yielded a slightly higher value for the pits, (11,510±830)/m², than for the particles, (11,230±950)/m². In the myocardium, the spacing of the regularly arrayed pits, 9.55±1.33 nm barely exceeds the value of 9.44±1.62 nm for the particles, which show some clustering. However, the packing density for the pits, (10,090±740)/m², appears a little higher than that of the particles (9,890±920)/m². As density and spacing measurements provided no decisive answers, the positions of individual pits and particles of complementary junctional faces were recorded on transparent sheets and compared. In this fashion, a one-to-one correspondence between particles and pits could be established, while small discrepancies may be attributed to plastic deformation. Moreover, the collinearity of pits and particles may be suggested by the observation of a platinum grain in the center of many pits.     

Circumferential analysis of digitalized gamma angiocardiography by assessment of regional left ventricular contraction

After acquisition of a digital equilibrium gamma-angiocardiographie, circumferential analysis of end-diastolic and end-systolic frames gives 120 points diastolic and systolic curves. Their difference represents systolic volume and leads to regional left ventricular ejection fraction assessment at the considered radius level. The circumferential analysis evolute gives the regional left ventricular ejection fraction representative curves which allows especially differential diagnosis between left ventricular akinesia and dyskinesia.     

Binding of anti-fibronectin to early amphibian ectoderm does not result in inhibition of neural induction under in vitro conditions

Summary Antibodies directed to fibronectin (anti-FN) were injected into the blastocoel of late blastulae of Xenopus laevis. Two animal caps (ectoderm) were isolated, when control embryos reached the early gastrula stage, and were combined with untreated upper blastopore lip in the sandwich method. In two control series fibronectin or Holtfreter solution was injected into the blastocoel. The results of the experiments suggest that neural induction cannot be prevented by binding anti-FN to fibronectin, which covers the blastocoelic side of the ectoderm. The data support the view that extracellular matrix proteins are not themselves responsible for neural induction. However, in comparison with the control series a slight shift of the differentiation pattern in the spinocaudal direction could be observed in the anti-FN series. The possible role of extracellular proteins in the formation of a close juxtaposition of mesodermal and ectodermal target cells as a prerequisite for shortdistance transmission of neural inducers is discussed.     

Dibutyryl-cyclic AMP inhibits cholesterol esterification in J 774 monocyte-like cells

The effect of dibutyryl-cyclic AMP (dbcAMP) and theophylline was investigated on oleic acid incorporation into cholesteryl esters and triacylglycerols in the mouse monocyte-macrophage cell line J 774. 24h pretreatment of macrophages with dbcAMP decreased cholesteryl ester formation in a dose-dependent manner (about 4 fold reduction for dbcAMP 10(-4)M + theophylline 10(-3)M), while oleic acid incorporation into triacylglycerols was markedly (2 to 3 fold) enhanced. The catabolism of acetylated LDL was only slightly affected (about 15-20% reduction with dbcAMP 5 X 10(-4)M + theophylline 10(-3)M). Acyl Coenzyme A: cholesterol-O-acyl-transferase activity, measured in vitro on cell homogenates, was reduced in dbcAMP-treated cells, whereas diacylglycerol acyltransferase activity was increased. These results suggest that cyclic AMP can modulate cholesteryl ester and triacylglycerol formation in macrophages, and that these metabolisms are inversely regulated. Agents which increase cyclic AMP intracellular level could be of interest for reducing cholesteryl ester accumulation in macrophages.     

Microheterogeneity of the malate dehydrogenase from several sources

Different homogeneously purified cytosolic malate dehydrogenases gave, on isoelectric focusing, several active bands. The phenomenon could not be assigned to differences in their molecular weights or to alterations in the enzyme preparations during the purification procedure. Resolution of the multiple malate dehydrogenase active bands was achieved by chromatofocusing. The aged isolated subforms always yielded the original electrofocusing pattern. This fact suggests that conformational isomerism is a likely explanation for the charge heterogeneity of the enzymes studied.     

5'-modified agonist and antagonist (2'-5')(A)n analogues: synthesis and biological activity

Two 5'-modified (2'-5')(A)4 oligomers with an increased resistance to phosphatase degradation were synthesized and evaluated for their ability to develop an antiviral response when introduced into intact cells by microinjection or by chemical conjugation to poly(L-lysine). The enzymatic synthesis of 5'-gamma-phosphorothioate and beta,gamma-difluoromethylene (2'-5')(A)4 from adenosine 5'-O-(3-thiotriphosphate) and adenosine beta,gamma-difluoromethylenetriphosphate by (2'-5')-oligoadenylate synthetase is described. The isolation and characterization of these (2'-5')(A)4 analogues were achieved by high-performance liquid chromatography. The structures of 5'-modified tetramers were corroborated by enzyme digestion. These two 5'-modified tetramers compete as efficiently as natural (2'-5')(A)4 for the binding of a radiolabeled (2'-5')(A)4 probe to ribonuclease (RNase) L. Nevertheless, at the opposite to 5'-gamma-phosphorothioate (2'-5')(A)4, beta,gamma-difluoromethylene (2'-5')(A)4 failed to induce an antiviral response after microinjection in HeLa cells. In addition, it behaves as an antagonist of RNase L as demonstrated by its ability to inhibit the antiviral properties of 5'-gamma-phosphorothioate (2'-5')(A)4 when both are microinjected in HeLa cells. The increased metabolic stability of 5'-gamma-phosphorothioate (2'-5')(A)4 as compared to that of (2'-5')(A)4 was first demonstrated in cell-free extracts and then confirmed in intact cells after introduction in the form of a conjugate to poly(L-lysine). Indeed, 5'-gamma-phosphorothioate (2'-5')(A)4-poly(L-lysine) conjugate induces protein synthesis inhibition and characteristic ribosomal RNA cleavages for longer times than unmodified (2'-5')(A)4-poly(L-lysine) in the same cell system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production and characterization of four monoclonal antibodies against porcine pancreatic colipase

Four monoclonal antibodies directed against porcine colipase have been generated by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. Antibodies were screened by binding to immobilized colipase in a solid-phase assay. Monoclonal antibodies were purified by affinity chromatography on colipase coupled to Sepharose. All monoclonal antibodies are of the IgG1 class with high affinity for the antigen. The dissociation constant of the complex formed in solution between porcine colipase and antibody varied from 1.1 X 10(-10) M to 1.8 X 10(-8) M. Epitope specificity was studied for each antibody and in pairs with an enzyme-linked immunosorbent assay (ELISA). Results indicate that the four monoclonal antibodies react with at least three different antigenic regions of colipase. Finally, three monoclonal antibodies were found to be potent inhibitors of colipase activity. Antiporcine monoclonal antibodies appear to be suitable probes for studying the lipid affinity site of the protein cofactor of pancreatic lipase.     

Regional assignment of the gene for diphtheria toxin sensitivity using subchromosomal fragments in microcell hybrids

Human x mouse microcell hybrids resistant to G418 were constructed between mouse hepatoma cells and human x mouse whole cell hybrids containing only intact human chromosome 5 and 22 with an integrated neo ^r-gene. Among these, microcell hybrid BG15 produced four subclones, BG15-4, BG15-6, BG15-7 and BG15-9, which contained variously sized complements of human chromosome 5. BG15-6 contained an intact human chromosome 5, BG15-7 a deleted human chromosome 5 (5pter-q22) and BG15-4 and BG15-9 a translocation between parts of human chromosome 5 (pter-qter? and pter-q23, respectively) and a mouse chromosome. Southern DNA blot analysis showed that the human dihydrofolate reductase (DHFR) gene was present in all four subclones, whereas the human homolog of the v-fms gene was present in BG15-4 and 15-6, but absent from BG15-7 and 15-9. BG15-4, 15-6 and 15-9 were sensitive to diphtheria toxin, and only BG15-7 was resistant to the toxin. We used these microcell hybrids to restrict further the regional location of the gene for diphtheria toxin sensitivity to the q23 region of human chromosome 5.     

In vitro and in vivo synthesis of dolichol and other main mevalonate products in various organs of the rat

The relative rate of biosynthesis of dolichol from [3H]mevalonate in nine rat organs was studied in slices and in the whole animal. This biosynthesis was also compared to that of cholesterol and ubiquinone. All tissues examined are able to synthesize dolichol, as well as ubiquinone and cholesterol. Comparison of the data from slices in vitro with the in vivo studies demonstrated relatively good agreement for dolichol and ubiquinone synthesis. Although dolichol of high specific radioactivity was recovered in the blood, redistribution between organs, such as occurs with cholesterol, appears to be insignificant. The highest rates of dolichol biosynthesis were found in kidney, spleen and liver. On the other hand, muscle makes the largest contribution to total body dolichol synthesis. Newly synthesized dolichol also appears in the bile, but excretion by this route is far from sufficient to account for dolichol turnover. Incorporation of mevalonate into the final products is mainly dependent on biosynthetic activity. For comparison of the biosynthetic rates in different organs, possible sources of errors (such as variations in the size of the precursor pool, limitation by the rate of precursor uptake or non-linear incorporation) were investigated the size of the mevalonate pool in various organs. Equilibration of this pool with exogenous mevalonate is a rapid and passive process. The size of the mevalonate pool does not determine the rates of cholesterol and dolichol biosynthesis, indicating the presence of regulatory steps in the terminal portion of these biosynthetic pathways.