Effects of phenylhydrazine hydrochloride on energy metabolism in rabbit erythrocytes and reticulocytes.

The effects of 1, 5 and 20 mmol/l phenylhydrazine hydrochloride on energy metabolism of rabbit erythrocyte and reticulocyte were studied. Significant depression of glycolysis, accompanied by loss of adenine nucleotides, mainly due to an extensive decline of ATP, was found in erythrocytes. Energy metabolism of reticulocytes appears to be more sensitive to deleterious effects of this drug. The declines of ATP and sum of all adenine nucleotides, as well as the accumulation of hypoxanthine were twofold in reticulocyte-rich red cell suspensions compared with suspensions of mature erythrocytes. It can be concluded that these changes are the consequence of lower energy production due to phenylhydrazine hydrochloride-induced inhibition of oxidative phosphorylation.

Degenerative neurological disorders associated with deficiency of glutamate dehydrogenase.

The activity of glutamate dehydrogenase, the enzyme of glutamate degradation, was measured in platelets of 27 healthy controls and 85 patients with different degenerative cerebellar and/or basal ganglia disorders. A group of 7 patients was selected with slowly progressive multiple-system atrophy, in whom a clinical diagnosis of olivopontocerebellar atrophy appeared tenable, with decreased activity of glutamate dehydrogenase (38% of the mean control value). In 4 patients data on inheritance were compatible with the genetic pattern of autosomal recessive inheritance, while 3 patients were sporadic cases. In an effort to define this group of patients more precisely, it is suggested that decreased activity of glutamate dehydrogenase induces an increase in extracellular glutamate levels in the central nervous system with subsequent development of excitotoxicity.

Phorbol ester inhibition of insulin-stimulated deoxyribonucleic acid synthesis in BC3H-1 myocytes.

Insulin and 12-O-tetradecanoyl phorbol 13-acetate (TPA) each acutely stimulates hexose transport, amino acid uptake, and pyruvate dehydrogenase activity in the BC3H-1 myocyte in a nonadditive fashion, suggesting that the acute effects of insulin and TPA are mediated through a common mechanism of action. Here we have demonstrated that while chronic incubation with insulin stimulated DNA synthesis by 3- to 6-fold, TPA, in contrast, did not stimulate DNA synthesis and, indeed, caused a 70% inhibition of insulin-stimulated DNA synthesis in a dose-dependent fashion. In differentiated myocytes, insulin maximally stimulated hydroxyurea-sensitive [3H]thymidine incorporation into DNA at 200-400 nM with an ED50 of 5-8 nM, suggesting that insulin stimulates DNA synthesis via the insulin receptor rather than through growth factor receptors. Phorbol ester inhibition of insulin-stimulated DNA synthesis was specific for the active tumor-promoting phorbols and the synthetic diacylglycerol 1-oleoyl-2-acetyl-sn-glycerol. Maximal TPA inhibition of insulin-stimulated DNA synthesis was observed at 100 nM with an ID50 of 30 nM TPA, values analogous to those required for TPA stimulation of hexose transport in the myocyte. Chronic incubation with TPA did not inhibit insulin-stimulated protein synthesis, acute K+ flux, K+ accumulation, cytosolic thymidine levels, or insulin binding, indicating that TPA inhibits a specific intracellular event mediating DNA synthesis and suggesting that the acute and chronic effects of insulin in BC3H-1 myocytes are regulated by distinct pathways.

Epidermolysis bullosa acquisita. Direct immunofluorescence and ultrastructural studies.

A case of epidermolysis bullosa acquisita (EBA), associated with inflammatory bowel disease in which cicatricial alopecia was present, was studied by electron microscopy and direct immunofluorescence microscopy. Direct immunofluorescence studies were performed on both clinically normal and perilesional skin, with and without previous separation of the epidermis from the dermis by incubation with 1 M sodium chloride. We propose the use of this separation technique to identify the level of antibody deposition in patients with EBA in whom circulating antibodies are lacking. This technique may be particularly beneficial in delineating between EBA and the clinically similar scarring localized forms of bullous pemphigoid in which circulating antibodies are often absent.

Insulin but not phorbol ester treatment increases phosphorylation of vinculin by protein kinase C in BC3H-1 myocytes.

Insulin was found to increase protein kinase C activity in BC3H-1 myocytes as determined by in vitro phosphorylation of both a lysine-rich histone fraction (histone III-S) and vinculin. TPA treatment for 20 min or 18 h provoked an apparent loss of histone-directed but not vinculin-directed phosphorylation by cytosolic C-kinase. Thus, chronic TPA-induced 'desensitization' or 'depletion' of cellular protein kinase C is more apparent than real, and is not a valid means for evaluating the role of C-kinase in hormone action.

Epidermolysis bullosa herpetiformis with mottled pigmentation and an unusual punctate keratoderma.

We report the clinical and pathological features of an epidermolytic form of epidermolysis bullosa (EB) present in a family with six affected members that was transmitted in an autosomal dominant manner in four generations. The essential clinical features included generalized herpetiform blistering of the skin, mottled pigmentation and palmo-plantar hyperkeratosis, both punctate and diffuse. Biopsy material obtained from fresh blisters, clinically intact preblistering skin, hyperkeratotic areas, and skin with mottled pigmentation was examined by light and/or electron microscopy. In addition to reporting a heretofore undescribed association of EB herpetiformis with mottled pigmentation and punctate keratoderma, we report previously undescribed histologic changes in the areas of punctate hyperkeratosis. Specifically, the unique histologic findings consisted of the presence of dyskeratotic cells with clear cytoplasm at the cellular periphery, parakeratosis, and involvement of the intradermal portion of the sweat duct. The possibility that these findings represent a new type of epidermolytic EB, rather than a variant of other types of epidermolytic EB, particularly EB herpetiformis or EB with mottled pigmentation, is discussed.

[The role of cAMP in the energy metabolism of human erythrocytes]. / Uloga cAMP u energetskom metabolizmu eritrocita coveka.

In human red blood cell there is complete adenyl cyclase--cAMP system. However, the role of this system and its connection with energy metabolism in red cell is not known. Hence, we studied the effects of cAMP and DB-cAMP on energy metabolism in human red cell. Blood was taken out from haematologically healthy volunteers. Red cells were isolated by differential centrifugation and washed three times in Tris-Ringer buffer (pH 7.4). The red cells suspensions (in the above buffer) were simultaneously incubated without additions (control) and with cAMP or DB-cAMP (5 mmol/L). Incubation temperature was 37 degrees C. The samples of suspensions for the extraction of metabolites and cofactors were taken at 0 and 180 minutes of incubation. The lactate, glucose, G6P, F6P, TP, ATP, ADP and AMP were determined in neutralized perchloric acid extracts by specific enzymatic methods. Results of our experiments show that cAMP and DB-cAMP significantly increase red cell glycolysis. Under the same conditions these nucleotides induce positive "cros-over" point at the phosphofructokinase (PFK) step of the glycolysis. The levels of adenine nucleotides (ATP, ADP, AMP) were unchanged through a whole period of incubation. We concluded that cAMP and DB-cAMP stimulates glycolytic process in red cells probably by allosteric activation of PFK.

Effects of dibutyryl-cANP on glycolysis in washed human erythrocytes.

The experiments were carried out with washed human erythrocytes in order to study the effects of dibutyryl-cAMP (DB-cAMP) on glycolysis. 5 mM DB-cAMP significantly increases glucose consumption and lactate production in incubated erythrocytes. The cross-over plot of glycolytic intermediates shows that increased glycolysis is probably the result of activation of phosphofructokinase by DB-cAMP. Under the same condition DB-cAMP significantly protects the 2,3-diphosphoglycerate level in incubated erythrocytes.