János Somogyi

Numerical estimation of γ-aminobutyric acid (GABA)-containing neurons in three thalamic nuclei of the cat: Direct GABA immunocytochemistry

The percentage of neurons that are immunoreactive for the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA) was determined within: (1) the lateral geniculate nucleus (LGN), (2) the ventrobasal complex (VB) and (3) the antero-ventral nucleus (AV) of the thalamus in the cat. An antiserum to GABA was used to stain GABA-containing perikarya in 1.0 micrometer thick Araldite-embedded sections. Immunostained somata in all three nuclei were invariably smaller than the immuno-negative nerve cells. 27% of all neurons in the LGN, 33% in the VB and 25% in the AV were immunoreactive for GABA.

The Hsp90-specific inhibitor geldanamycin selectively disrupts kinase-mediated signaling events of T-lymphocyte activation

Abstract The 90-kDa heat shock protein (Hsp90) is the most abundant molecular chaperone of eukaryotic cells. Its chaperone function in folding nascent proteins seems to be restricted to a subset of proteins including major components of signal transduction pathways (eg, nuclear hormone receptors, transcription factors, and protein kinases). Improper function of these proteins can be induced by selective disruption of their complexes with Hsp90 using the benzoquinonoid ansamycin geldanamycin. In this study, we demonstrate that geldanamycin treatment blocks interleukin (IL)-2 secretion, IL-2 receptor expression, and proliferation of stimulated T-lymphocytes. Moreover, geldanamycin decreases the amount and phosphorylation of Lck and Raf-1 kinases and prevents activation of the extracellular signal regulated kinase (ERK)-2 kinase. Geldanamycin also disrupts the T-cell receptor–mediated activation of nuclear factor of activated T-cells (NF-AT). Treatment with geldanamycin, however, does not affect the activation of lysophosphatide acyltransferase, which is a plasma membrane enzyme coupled to the T-cell receptor after T-cell stimulation. Through demonstrating the selective inhibition of kinase-related T-lymphocyte responses by geldanamycin, our results emphasize the substantial role of Hsp90–kinase complexes in T-cell activation.

Alterations in the properties and isoform ratios of brain Na+/K+-ATPase in streptozotocin diabetic rats

In this study we analysed the changes in the properties of rat cerebral cortex Na+K(+)-ATPase in streptozotocin induced diabetes (STZ-diabetes). Special attempt was made to determine whether insulin treatment of diabetic animals could restore the altered parameters of this enzyme. Na+/K(+)-ATPase activity was found to be decreased by 15% after 2 weeks, and by 37% after 4 weeks in diabetic rat brains with a parallel decrease in maximal capacity of low affinity ouabain binding sites. There was no significant change in the high affinity ouabain binding sites. The Kd values did not change significantly. Western blot analysis of brain Na+/K(+)-ATPase isoforms indicated a 61 +/- 5.8% and 20 +/- 2.8% decrease of the alpha 1 and alpha 3 isoforms, respectively in 4 weeks diabetic animals. Change in the amount of the alpha 2 isoform proved to be less characteristic. Both types of beta subunit isoform showed a significant decrease in four weeks diabetic rats. Our data indicate a good correlation in diabetic rats between changes in Na-/K(+)-ATPase activity, low affinity ouabain binding capacity and the level of alpha 1 isoform. While insulin treatment of diabetic animals restored the blood glucose level to normal, a complete reversal of diabetes induced changes in Na+/K(+)-ATPase activity, ouabain binding capacity and Na+/K(+)-ATPase isoform composition could not be achieved.

Zinc increases the affinity of phorbol ester receptor in T lymphocytes

In the primary structure of the major phorbol ester receptor, protein kinase C the presence of putative metal (zinc) binding sites has been suggested. We have demonstrated earlier that zinc activates protein kinase C and contributes to its binding to plasma membranes in T lymphocytes. Here we report that zinc increases the phorbol ester binding affinity of cytosolic protein kinase C. The effect of zinc on the membrane-bound enzyme is much less pronounced. Our results raise the possibility that cytosolic protein kinase C is a mixture of isoenzymes with different sensitivity towards zinc ions.

Changes in the expression of Na+/K+-ATPase isoenzymes in the left ventricle of diabetic rat hearts: effect of insulin treatment

SummaryNa+/K+-ATPase related strophanthidin sensitive 3-O-methylfluorescein-phosphatase activity, [3H]ouabain binding and expression of Na+/K+-ATPase subunit isoforms were measured in the left ventricle of the heart of normal and streptozotocin-diabetic rats with and without insulin treatment. Compared to control animals, the enzyme activity was 0.75 ± 0.09 and 0.62 ± 0.06 times lower in rats diabetic for 2 and for 4 weeks, respectively. This was associated with a proportional decrease of the [3H]ouabain binding sites. Immunoblots indicated a 0.76 ± 0.08 and 0.61 ± 0.08-fold decrease of alpha1, a 0.68 ±0.09 and 0.41 ± 0.04-fold decrease of alpha2 subunit in 2- and 4-week diabetic rats, respectively relative to controls. Beta1 subunit decreased proportionally 0.71 ± 0.07 and 0.38 ± 0.06-fold, and beta2 decreased 0.75 ± 0.08 and 0.31 ± 0.06-fold, respectively. Northern blot analysis revealed a significant reduction in mRNA level of Na+/K+-ATPase subunit isoforms after 2 and 4 weeks of diabetes (for alpha1 66.2 ± 8.2 and 55.9 ± 7.8% of controls for alpha2 91.7 ± 12.1 and 41.1 ± 7.1 % of controls and for beta subunit 93.4 ±11.1 and 49.8 ±6.8% of controls, respectively). Although, mRNA levels of isoform reverted to even higher levels than the control values after insulin treatment, insulin caused only a partial recovery of enzyme activity, [3H]ouabain binding capacity and protein expression. We have obtained evidence that in cardiac left ventricle there are more than one type of Na+/K+-ATPase alpha and beta subunit isoforms which are affected in diabetes and by insulin treatment. The time course of diabetes induced changes and the degree of involvement suggest that the Na+/K+-ATPase isoforms are altered individually.

The effect of proteases on the (Na+ + K+)-activated adenosine triphosphatase system of rat brain

Abstract A 10-min incubation of the (Na + + K + )-activated ATPase preparation with 20 μg trypsin per mg protein at 25° decreased ATPase activity by approx. 50%, whereas only 10–15-fold greater concentrations of subtilisin A and of chymotrypsin decreased the activity to the same extent. Yet the simultaneously measured proteolysis was 5–6-fold more with subtilisin A or with chymotrypsin than with trypsin. The loss in ATPase activity with proteases was partly counteracted by Mg 2+ , Na + , and K + . The protective effect of cations against trypsin could be modified by ATP: the action of Mg 2+ was completely abolished; the protection by K + , in the presence of Mg 2+ , was considerably lessened by ATP, whereas, under similar conditions, the effect of Na + was not influenced at all by ATP. The experiments suggest that trypsin, in contrast to chymotrypsin and subtilisin A, acts at the region of the active centre of ATPase. The effect of cations and ATP are manifested by a change in the conformation of the ATPase molecule; the conformation is different in the presence of Mg 2+ + ATP + Na + and in the presence of Mg 2+ + ATP + K + . These changes in conformation of the enzyme system seem to constitute the basis of the active ion transport across the cell membrane. It is likely that arginine or lysine has an important functional role in the active centre of (Na + + K + )-activated ATPase.

The Hsp90-specific inhibitor, geldanamycin, blocks CD28-mediated activation of human T lymphocytes

The 90 kDa heat shock protein (Hsp90) is a molecular chaperone aiding the folding of nuclear hormone receptors and protein kinases. Hsp90-mediated folding can be disrupted by the Hsp90-specific drug, geldanamycin. Here we provide evidence for the inhibition of the CD28-specific BW 828 antibody-mediated activation of human T lymphocyte proliferation, IL-2 secretion and IL-2 receptor expression by geldanamycin. Our results suggest that the major cytoplasmic chaperone, Hsp90, plays an important role in CD28-mediated T lymphocyte activation.

Formation of γ-glutamyl-taurine in the rat brain

Abstract After intraventricular administration of 14C- or 3H-labelled taurine, γ-glutamyl-taurine was detected in the protein free extract of rat brain using a combination of ion exchange chromatography, electrophoretic separation and thin-layer chromatography. Under in vitro conditions γ-glutamyl-transpeptidase (EC 2.3.2.2.) prepared from rat brain catalyzes the formation of γ-glutamyl-taurine from taurine and glutathione or γ-glutamyl-p-nitroanilide. The enzymes of the γ-glutamyl cycle are presumed to be responsible for the in vivo metabolism of this dipeptide.

The nonapeptide leucinostatin A acts as a weak ionophore and as an immunosuppressant on T lymphocytes

Earlier studies have disclosed that leucinostatin A, a hydrophobic nonapeptide antibiotic, assumes an alpha-helical secondary structure in nonpolar environments. The present report demonstrates that the peptide acts as a weak ionophore facilitating the transport of mono-and divalent cations through the plasma membrane of T lymphocytes and through artificial membranes. Leucinostatin A does not change the thymidine uptake of both resting mouse thymocytes and peripheral blood lymphocytes but dose-dependently prevents the activation of T lymphocytes by tetradecanoyl-phorbol-acetate and by anti-T cell receptor antibody.

The tumor promoter tetradecanoyl-phorbol-acetate (TPA) elicits the redistribution of zinc in subcellular fractions of rabbit thymocytes measured by X-ray fluorescence

Prolonged (90 minutes) incubation with tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) elicits the redistribution of zinc from the nuclear fraction and mitochondria to the cytosol and microsomes in rabbit thymocytes. The zinc redistribution might play a role in the TPA-caused desensitization of T lymphocytes towards calcium and in the TPA-induced inhibition of Gl----S phase transition and cytotoxicity of T lymphocytes.