Modulation of inhibitory efficiency of rat skeletal muscle calpastatin by phosphorylation.

Rat skeletal muscle calpastatin form is markedly modified in its inhibitory properties by means of a reverse reaction which involves both phosphorylation and dephosphorylation. Dephospho-calpastatin shows greater inhibitory efficiency versus mu-calpain, whereas phospho-calpastatin shows maximal inhibition versus m-calpain. Both forms are present in fresh rat muscle.

Echocardiographic parameters and indices in the normal beagle dog.

M-mode and two-dimensional echocardiographic measurements were made from the right sternal border of 50 healthy Beagles (25 males and 25 females) approximately 7 months old. The dogs were conscious and standing during the investigation. The following parameters, in systole and diastole, were measured on the echocardiographic images: left ventricular posterior wall thickness (LVWT); intraventricular septum thickness (IST); left ventricular internal dimension (LVID); and circumference (LVC).

The calpain-calpastatin system in mammalian cells: properties and possible functions.

All mammalian cells contain a calcium-dependent proteolytic system, composed by a proteinase, calpain, and an inhibitor, calpastatin. In some cell types an activator protein has also been identified. Moreover, two calpain isoforms, distinguishable on the basis of a different calcium requirement, can be present in a single cell.

Mechanism of action of the calpain activator protein in rat skeletal muscle.

Rat skeletal muscle contains a calpain activator protein characterized by a high specificity for calpain II, the high Ca(2+)-requiring isoform of this class of proteinases. The activator protein increases the rate of intramolecular conversion of the native 80-kDa catalytic subunit of calpain into the autolysed 75-kDa forms with maximal rate at concentrations of calcium approximately 25 times lower than those required by the native proteinase. The activator protein interacts with native calpain II forming a 1:1 complex; interaction does not occur with the fully activated form, produced by autoproteolysis.

Respiratory burst in activated neutrophils is directly correlated to the intracellular level of protein kinase C.

The production of superoxide anion in human and rat neutrophils is directly correlated to the level of protein kinase C. Such correlation has been established on a comparative basis by analysis of neutrophils from normal and hypertensive subjects, characterized by an increased amount of protein kinase C, and of neutrophils from normal and genetically hypertensive rats characterized by low amounts of the kinase. Protein kinase C activity in all these different populations of neutrophils is modulated by specific inhibitors in an identical dose-dependent fashion which results in a linearly correlated decrease in O2- production.

Differentiation of murine erythroleukemia cells by hexamethylenebisacetamide involves secretion and binding to membranes of a differentiation enhancing factor.

A protein factor previously shown to enhance terminal differentiation of transformed erythroid cells is synthesized by murine erythroleukemia cells and secreted in the early stages of differentiation induced by hexamethylenebisacetamide (HMBA). Secretion also occurs, constitutively, in the absence of inducer, from a murine erythroleukemia cell variant characterized by an accelerated response to HMBA. The protein factor binds to intact cells following addition of HMBA and enhances translocation of protein kinase C to the nuclear fraction.

Identification of two calpastatin forms in rat skeletal muscle and their susceptibility to digestion by homologous calpains.

Two forms of calpastatin, differing in their specificity for the homologous calpain isozymes I and II, have been separated from rat skeletal muscle extracts and purified to homogeneity. Calpastatin I, the first form to elute in chromatography on DE32, is more effective against calpain I, while calpastatin II is more effective as an inhibitor of calpain II. Based on their molecular mass (approximately 105 kDa) both calpastatin forms belong to the high molecular mass class found in muscles of other animal species (Murachi, T.

The calpastatin defect in hypertension is possibly due to a specific degradation by calpain.

Calpastatin activity, significantly reduced in erythrocytes of patients affected by essential hypertension, is restored to normal values by appropriate therapeutical treatments in a time-dependent fashion and in parallel with the decline in blood pressure. Evidence is also presented indicating that red cell calpastatin is degraded in human and rat red cells by homologous calpain, and that the rate of degradation is approx. 5-times higher in rat erythrocytes.

A vincristine-resistant murine erythroleukemia cell line secretes a differentiation enhancing factor.

A clone of vincristine resistant murine erythroleukemia cells V3.17[44], characterized by high sensitivity to terminal erythroid differentiation induced by hexamethylene bisacetamide, secretes into the extracellular medium a protein factor which partially reduces the latent period before commitment and accelerates the expression of the terminal differentiated phenotype in a slow responding murine erythroleukemia N23 cell variant. This differentiation enhancing factor increases the rate of protein kinase C down-regulation which occurs at slower rate during cell differentiation.

Identification of a protein kinase C activating factor from murine erythroleukemia cells: characterization of the activation kinetics.

A protein kinase C (PKC) activating factor (AF) has been identified in the extracellular medium of V3.17 vincristine resistant murine erythroleukemia (MEL) cells clone. The factor is a protein that stimulates the activity of PKC alpha and beta isozymes isolated from MEL cells, rat and mouse brain approximately 2 to 2.

Inactivation of hepatocyte protein kinase C by carbon tetrachloride: involvement of drug's metabolic activation and prooxidant effect.

The involvement of CCl4 biotransformation mechanism in decreasing the Protein Kinase C activity has been analyzed in hepatocytes isolated from phenobarbital-pretreated rats. A significant inhibition (55%) and an almost total disappearance (87%) of the enzyme activity were observed at 15 min and at 30 min incubation with CCl4, respectively. Cell preincubation with Trolox or desferrioxamine allowed a marked whilst not complete protection of both cytosolic and particulate Protein Kinase C activity.

Identification of an endogenous activator of calpain in rat skeletal muscle.

An additional component of the regulatory system of rat skeletal muscle calpain has been identified. It exerts a potent activating effect on calpain activity and is a heat stable small molecular weight protein. Of the two calpain isozymes present in muscle, the activator is specific for calpain II, being uneffective with calpain I.

Photodynamic therapy by topical meso-tetraphenylporphinesulfonate tetrasodium salt administration in superficial basal cell carcinomas.

The efficacy of an originally developed photodynamic approach, using topical administration of tetraphenylporphinesulfonate as the photosensitizer, was evaluated in a series of 292 basal cell carcinoma lesions (less than 2-mm thick) in 50 treated patients. The lack of indication for conventional therapies was the main selection criterion. The photosensitizing agent (2% solution) was topically applied at 0.

Introduction of the beta isozyme of protein kinase C accelerates induced differentiation of murine erythroleukemia cells.

Induction of differentiation in murine erythroleukemia cells (MELCs) involves a protein kinase C (PKC)-mediated step. Vincristine-resistant cells respond more rapidly to hybrid polar/apolar inducers than the parental cells. These vincristine-resistant MELCs contain elevated levels of the beta isozyme of PKC (PKC-beta).

Identification of the proteolytically activated form of protein kinase C in stimulated human neutrophils.

The proteolytically activated form of protein kinase C has been identified in human neutrophils by using a monoclonal antibody that recognizes both the native kinase and the catalytically active proteolytic fragment (protein kinase M). Stimulation with fMet-Leu-Phe results in the conversion of approximately 30% of native protein kinase C to protein kinase M, with little evidence of further degradation. Stimulation with phorbol 12-myristate 13-acetate, on the other hand, causes only a transient formation of protein kinase M, with complete loss of total kinase activity.

Epidemiological study of urinary tract stones in a northern Italian city.

An epidemiological study of stone disease in a Northern Italian city was carried out by means of a postal questionnaire mailed to 6000 individuals (2.5% of the entire population). It was found that the incidence of stone disease was comparable to that of industrialised Western Europe.

Isovalerylcarnitine is a specific activator of the high calcium requiring calpain forms.

Isovalerylcarnitine, a product of the catabolism of L-leucine, is a potent activator of rat calpains isolated from erythrocytes, kidney, liver, skeletal and heart muscle. Only calpains II, but not calpains I, are activated by IVC, with the only exception of rat erythrocyte calpain I, the only species present in these cells which has a Ca2+ requirement higher than that of most calpain I isoenzymes. Activation by IVC involves a dual effect: 1) a ten fold increase in the affinity of calpain for Ca2+, and 2) an increase in the Vmax 1.

Isozymes of protein kinase C in human neutrophils and their modification by two endogenous proteinases.

Two major protein kinase C (PKC) isozymes, accounting for approximately 95% of the total activity in human neutrophils, were separated by hydroxyapatite chromatography and were identified as beta-PKC (60% of the total) and alpha-PKC (35% of the total). No gamma-PKC was detected. A minor Ca2+/phospholipid requiring kinase that eluted from hydroxyapatite after alpha-PKC did not react significantly with any of the specific antisera employed for identification.

Differential expression of protein kinase C isozymes and erythroleukemia cell differentiation.

Hexamethylene bisacetamide (HMBA) and other polar/apolar chemical agents are potent inducers of erythroid differentiation in murine erythroleukemia cells (MELC), as well as other transformed cell lines. Although the mechanism of action of HMBA is not yet known, evidence has been obtained that protein kinase C (PKC) plays a role in this process. In this study we provide further evidence that establishes this relationship.

The calpains.

In recent years interest has increased concerning the characterization of the structural-functional properties and the identification of the physiological role of non-lysosomal intracellular proteinases. Among these, calpain, a calcium-dependent cysteine proteinase ubiquitously present in a variety of tissues and cells, has been most extensively investigated in terms of activation, regulatory mechanisms, specificity and biological function. This review discusses each of these points on the basis of the most recent results concerning the general characteristics of calpain activity, and its preferential site of action within the cell as related to the specific functions of the proteinase in different cell types.

Extinction and absorption coefficients and scattering phase functions of human tissues in vitro.

Optical properties of different human tissues in vitro have been evaluated by measuring extinction and absorption coefficients at 635- and 515-nm wavelengths and a scattering angular dependence at 635 nm. Extinction was determined by the on-axis attenuation of light transmitted through sliced specimens of various thicknesses. The absorption coefficient was determined by placing samples into an integrating sphere.

Activation of neutrophil calpain following its translocation to the plasma membrane induced by phorbol ester or fMet-Leu-Phe.

Stimulation of human neutrophils with phorbol myristate acetate or fMet-Leu-Phe results in translocation to the plasma membrane of approximately 25-40% of the cellular calpain activity. In the membrane-bound form the Ca2+-requirement for proteolytic activity is substantially reduced. An anti-calpain monoclonal antibody that is internalized by stimulated neutrophils is recovered in the same subcellular fraction that contains the membrane-bound calpain, apparently in the form of pinocytotic vesicles.

Enhanced activation of the respiratory burst oxidase in neutrophils from hypertensive patients.

In neutrophils of patients with essential hypertension the NADPH-dependent O2- production elicited by stimulation with f-Met-Leu-Phe is three to four fold higher in comparison with neutrophils of normotensive control subjects. Neutrophils from hypertensive patients are less responsive to priming, by non-stimulating doses of the agonist, as compared to control cells, which following this pretreatment augment superoxide anion production up to levels close to those expressed by neutrophils from hypertensive patients. No difference in NADPH oxidase activity, between neutrophils from the two groups of subjects, was observed when the rate of O2- production was evaluated in a reconstructed cell-free system containing the membrane fraction and the cytosolic cofactors.

Effect of low-energy laser irradiation on colony formation capability in different human tumor cells in vitro.

Fibroblasts and lymphocytes are the most widely used cells for studying the so-called biostimulative effect of low-power laser in vitro. In contrast, stimulation of cancer cells by laser light has not been investigated extensively. The present study attempted to evaluate whether or not human tumor cells could exhibit an increase in colony-forming capability following low-watt laser irradiation.

The role of intracellular proteinases in human neutrophil activation.

In addition to other proteinases human neutrophils contain two non granular neutral endopeptidases: a serine proteinase and a cysteine Ca2+ dependent proteinase named calpain. Serine proteinase localized in association with the cytoskeleton-membrane proteins, apparently exerts a dual role: it is partially released into the medium during neutrophil stimulation by phorbol myristate acetate (PMA), presumably acting as one of the cytotoxic factors; and in its intracellular localization is presumably involved in the process of down regulation of native protein kinase C (PKC). Calpain, predominantly present in resting conditions in an inactive form, becomes activated in the course of neutrophil stimulation and appears to be involved both in the down regulation of native PKC as well as in the formation of a proteinase-activated kinase form, presumably derived from PKC and defined as PKC-M.