Safinamide Modulates Striatal Glutamatergic Signaling in a Rat Model of Levodopa-Induced Dyskinesia.

Safinamide (Xadago) is a novel dual-mechanism drug that has been approved in the European Union and United States as add-on treatment to levodopa in Parkinson's disease therapy. In addition to its selective and reversible monoamine oxidase B inhibition, safinamide through use-dependent sodium channel blockade reduces overactive glutamatergic transmission in basal ganglia, which is believed to contribute to motor symptoms and complications including levodopa-induced dyskinesia (LID). The present study investigated the effects of safinamide on the development of LID in 6-hydroxydopamine (6-OHDA)-lesioned rats, evaluating behavioral, molecular, and neurochemical parameters associated with LID appearance. 6-OHDA-lesioned rats were treated with saline, levodopa (6 mg/kg), or levodopa plus safinamide (15 mg/kg) for 21 days. Abnormal involuntary movements, motor performance, molecular composition of the striatal glutamatergic synapse, glutamate, and GABA release were analyzed. In the striatum, safinamide prevented the rearrangement of the subunit composition of N-methyl-d-aspartate receptors and the levodopa-induced increase of glutamate release associated with dyskinesia without affecting the levodopa-stimulated motor performance and dyskinesia. Overall, these findings suggest that the striatal glutamate-modulating component of safinamide's activity may contribute to its clinical effects, where its long-term use as levodopa add-on therapy significantly improves motor function and "on" time without troublesome dyskinesia.

Role of calpain-1 in the early phase of experimental ALS.

Elevation in [Ca(2+)]i and activation of calpain-1 occur in central nervous system of SOD1(G93A) transgenic mice model of amyotrophic lateral sclerosis (ALS), but few data are available about the early stage of ALS. We here investigated the level of activation of the Ca(2+)-dependent protease calpain-1 in spinal cord of SOD1(G93A) mice to ascertain a possible role of the protease in the aetiology of ALS. Comparing the events occurring in the 120 day old mice, we found that [Ca(2+)]i and activation of calpain-1 were also increased in the spinal cord of 30 day old mice, as indicated by the digestion of some substrates of the protease such as nNOS, αII-spectrin, and the NR2B subunit of NMDA-R. However, the digestion pattern of these proteins suggests that calpain-1 may play different roles depending on the phase of ALS. In fact, in spinal cord of 30 day old mice, activation of calpain-1 produces high amounts of nNOS active species, while in 120 day old mice enhanced-prolonged activation of calpain-1 inactivates nNOS and down-regulates NR2B. Our data reveal a critical role of calpain-1 in the early phase and during progression of ALS, suggesting new therapeutic approaches to counteract its onset and fatal course.

Human collecting lymphatic glycocalyx identification by electron microscopy and immunohistochemistry.

Blood flow is translated into biochemical inflammatory or anti-inflammatory signals based onshear stress type, by means of sensitive endothelial receptors. Recognition of the phenomenon is of paramount importance for enhanced insights into the pathophysiological processes of vascular remodeling. The endothelial glycocalyx is a pericellular matrix, identified in both arteries and veins, acting collectively as a sensor responsive to blood flow changes. Venous and lymphatic physiology is interconnected; however, to our knowledge, a lymphatic glycocalyx structure has never been identified in humans. The objective of this investigation is to identify glycocalyx structures from ex vivo lymphatic human samples. Lower limb vein and lymphatic vessels were harvested. The samples were analyzed by transmission electron microscopy. The specimens were also examined by immunohistochemistry. Transmission electron microscopy identified a glycocalyx structure in human venous and lymphatic samples. Immunohistochemistry for podoplanin, glypican-1, mucin-2, agrin and brevican characterized lymphatic and venous glycocalyx-like structures. To our knowledge, the present work reports the first identification of a glycocalyx-like structure in human lymphatic tissue. The vasculoprotective action of the glycocalyx could become an investigational target in the lymphatic system as well, with clinical implications for the many patients affected by lymphatic disorders.

Role of full-length osteoprotegerin in tumor cell biology.

Osteoprotegerin (OPG) is a soluble tumor necrosis factor receptor family member, which potently inhibits RANKL-mediated osteoclastogenesis. Numerous constructs have been created for therapeutic purposes in which the heparin-binding and death homology domains of OPG were removed and the remaining peptide (amino acids 22-194) was fused to the Fc domain of human IgG1 (OPG-Fc). The administration of OPG-Fc efficiently counteracted bone loss in a variety of preclinical models of cancers. However, several in vitro studies have shown that native or recombinant full-length OPG not only neuralizes RANKL, but also the death-inducing ligand TRAIL, suggesting that OPG might potentially counteract the anti-tumor activity of TRAIL. Additional evidence suggests that full-length OPG possesses RANKL- and TRAIL-independent biological properties, mainly related to the promotion of endothelial cell survival and angiogenesis. Finally, breast tumor cells overexpressing OPG have shown increased bone metastatic potential in vivo. The relevance of these apparently conflicting findings in tumor cell biology is highlighted.

IGF-I induced rapid recruitment of integrin beta1 to lipid rafts is Caveolin-1 dependent.

Caveolin-1 (Cav-1) regulates both insulin like growth factor receptor (IGF-IR) and integrin beta1 function. However, the role of Cav-1 in IGF-IR/integrin beta1 cross talk remains to be established. In this study, we observed that IGF-I did not induce integrin beta1 internalization but its plasma membrane reorganization. In particular, we found a rapid and transient association between integrin beta1 and Cav-1 followed by the enrichment of integrin beta1 in lipid rafts. To determine the role of Cav-1 in this process, we transfected Hacat cells with small interfering RNA specific for Cav-1 (siRNA-Cav-1) and with a scrambled siRNA as control (siRNA-Ctr). Cav-1 down regulated Hacat cells were then stimulated with IGF-I and analyzed by immunofluorescence and flow cytometry. We found that Cav-1 silencing abolished the recruitment of integrin beta1 to lipid rafts in the presence of IGF-I. These data demonstrate that IGF-IR/integrin beta1 cross talk is followed by integrin beta1 lipid raft compartmentalization and that Cav-1 is required for this process.

Effects of calpain inhibition on dopaminergic markers and motor function following intrastriatal 6-hydroxydopamine administration in rats.

The neurotoxin 6-hydroxydopamine has been widely used to model aspects of Parkinson's disease in rodents, but the mechanisms underlying toxin-induced dopaminergic degeneration and functional impairment have not been fully elucidated. The main aim of the present study was to assess a possible role for calpains in neurochemical and behavioral deficits following unilateral infusion of intrastriatal 6-hydroxydopamine in adult rats. Toxin administration produced a profound dopaminergic denervation, as indicated by a 90-95% reduction in dopamine transporter radiolabeling measured in the caudate-putamen at 2 weeks post-lesion. Treatment with 6-hydroxydopamine also resulted in calpain activation in both caudate-putamen and substantia nigra, as measured by the appearance of calpain-specific spectrin breakdown products. Calpain activation peaked at 24 h after 6-hydroxydopamine infusion and remained elevated at later time points. In contrast, caspase-3-mediated spectrin cleavage subsided within 48 h in both brain areas. In a subsequent experiment, calpain inhibition was achieved by intrastriatal infusion of an adenovirus expressing the endogenous calpain inhibitor, calpastatin. Calpastatin delivery abolished the lesion-induced calpain-mediated spectrin cleavage and alleviated forelimb asymmetries resulting from unilateral intrastriatal 6-hydroxydopamine. Unexpectedly, dopamine transporter and tyrosine hydroxylase labeling revealed significant neuroprotection, not in the nigrostriatal pathway but rather in the ventral tegmental area. These findings support a role for calpain activation in 6-hydroxydopamine-induced degeneration of dopaminergic neurons. However, after near-total dopaminergic depletion, the primary benefit of calpain inhibition may not occur within the nigrostriatal dopaminergic pathway itself.

Selection and characterization of a novel agonistic human recombinant anti-TRAIL-R2 minibody with anti-leukemic activity.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising natural anticancer therapeutic agent because through its death receptors, TRAIL-R1 and TRAIL-R2, it induces apoptosis in many transformed tumor cells, but not in the majority of normal cells. Hence, agonistic compounds directed against TRAIL death receptors have the potential of being excellent cancer therapeutic agents, with minimal cytotoxicity in normal tissues. Here, we report the selection and characterization of a new single-chain fragment variable (scFv) to TRAIL-R2 receptor isolated from a human phage-display library, produced as minibody (MB), and characterized for the in vitro anti-leukemic tumoricidal activity. The anti-TRAIL-R2 MB2.23 efficiently and specifically bound to membrane-associated TRAIL-R2 on different leukemic cell lines and could act as a direct agonist in vitro, initiating apoptotic signaling as well as complement-dependent cytotoxicity and antibody-dependent cell cytotoxicity, providing a rationale for further investigations of MB2.23 in anticancer therapy.

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. As with other proteinases, calpain has to be under a continuous spatial and temporal control, and the structural and functional properties of the natural calpain inhibitor, calpastatin, must also be considered. The calpain-calpastatin system is the functional proteolytic unit that governs the activity of this intracellular proteolytic system, which is tightly correlated to the control of calcium homeostasis and thereby to the biological process of transmembrane signalling.

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 0.1 ml/cm2, followed by light irradiation with a dye laser emitting at 645 nm (120 or 150 J/cm2). After initial treatment, all lesions responded, with 273 (93.5%) complete responses. Recurrences were observed in 29 (10.6%). A second application of photoradiation was performed in 15 persistent lesions and 11 relapsed lesions, producing 19/26 complete responses. Our results suggest that this technique can be considered a promising alternative treatment modality in selected cases of superficial basal cell carcinomas.

Identification of a novel neutrophil membrane protein involved in modulation of oxidative burst.

On the basis of selective recognition by antibodies directed against neutrophil membrane determinants, a new neutrophil protein (molecular mass 82 kDa) has been identified, and shown to be functionally correlated with the oxidative response evoked in these cells by agonist stimulation. The protein is present in neutrophil membrane fraction but only upon activation it becomes accessible to recognition by a specific monoclonal antibody. In these conditions a complete and selective inhibition of O2- production occurs. The presence of a new protein antigen in neutrophil membranes linked to the activation of the O2- producing multienzyme complex that becomes external to the cell surface in primed or activated cells, might be important for future approaches aiming at the control of neutrophil response and at the identification of the activated forms of these cells.

Characterization of the single peptide generated from the amino-terminus end of alpha- and beta-hemoglobin chains by the Ca2+-dependent neutral proteinase.

Human erythrocyte Ca2+-dependent neutral proteinase catalyzes a limited proteolysis of isolated globin chains. The rate of hydrolysis is very rapid using heme-deprived alpha- or beta-globin chains and is reduced to one-fifth with their corresponding native forms. In both cases, the proteinase specifically cleaves a single peptide bond, this resulting in the removal from the amino-terminus end of an octapeptide in beta-globin and of an undecapeptide in alpha-globin. Both peptides have been isolated, their amino acid composition has been characterized and the susceptible site of cleavage has been identified. Hemoglobin variants show a different rate of digestion as compared to that of normal chains. The alpha-Hasharon [alpha 47(CE5) Asp----His] undergoes rapid digestion, while the beta-G San Josè chain [beta 7(A4) Glu----Gly], which carries the mutation near the site of cleavage, reveals a high degree of resistance to proteolytic degradation by the neutral proteinase.

Following association to the membrane, human erythrocyte procalpain is converted and released as fully active calpain.

When exposed to inside-out human erythrocyte vesicles, in the presence of micromolar Ca2+, the 80 kDa catalytic subunit of procalpain is processed through three successive and sequential steps. These include binding to the cytosolic surface of the membrane, followed by a very rapid conversion into the 75 kDa active subunit, and ultimately by spontaneous and complete release of this active proteinase form. Binding to the membranes is competitively inhibited by the endogenous natural inhibitor through the formation of the proteinase-inhibitor complex, in which form the 80 kDa subunit can no longer be associated to the membranes. Calcium ions and the natural endogenous inhibitor appear to be crucially involved in the modulation of this novel membrane-bound mediated activation of human red cell procalpain.

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. Thus, increased proteolytic degradation catalyzed by calpain could explain both the decrease in the amount of calpastatin activity and the profound difference between the intracellular level of the calpain inhibitor observed in erythrocytes from patients with essential hypertension and the genetically hypertensive rats.

Differences and similarities among three acidic endopeptidases associated with human erythrocyte membranes. Molecular and functional studies.

The three acidic proteinases (designated I, II and III, respectively) associated with human erythrocyte membranes were solubilized and purified to an electrophoretically homogeneous state by conventional procedures. Comparative analysis of chemical properties, including amino acid composition and fragmentation by cyanogen bromide cleavage, revealed significant differences among proteinases I, II and III. On the other hand, complete identity among the three proteolytic enzymes was observed on the basis of the peptide bonds specifically hydrolyzed in both glucagon- and phenylalanine-deprived oxidized B chain of insulin. In fact, each of the three proteinases produced splitting of the glucagon molecule between phenylalanine-22 and valine-23, while the susceptible bonds in the oxidized B chain of insulin proved to be those between leucine-15 and tyrosine-16 and between phenylalanine-25 and tyrosine-26, respectively.

Decay of proteinase and peptidase activities of human and rabbit erythrocytes during cellular aging.

Variations in activity of the membrane-bound and cytosolic proteinases and peptidases were analyzed in human and rabbit erythrocytes at various stages of their life-span. The patterns observed with human erythrocytes were the following. (a) The acidic endopeptidase activity associated with the membranes undergoes a substantial decline during cellular aging, with an estimated half-life of 65 days. Concomitantly it appears to become progressively more latent. (b) All cytosolic proteinase and peptidase activities described previously (Pontremoli, S., Melloni, E., Salamino, F., Sapartore, B., Michetti, M., Benatti, U., Morelli, A. and De Flora, A. (1980) Eur. J. Biochem. 110, 421-430) decline exponentially throughout the erythrocyte life-span, with the exception of dipeptidyl aminopeptidase III. The calculated half-lives were: 60 days for the neutral endopeptidase; 87 days for the total acidic endopeptidase activity which is accounted for by three distinct enzymes; 49 days for aminopeptidase B and 133 days for a second aminopeptidase with broad substrate specificity; 84 days for dipeptidyl aminopeptidase II. The results obtained with the rabbit erythrocytes were: (a) no significant decline of leucine aminopeptidase, dipeptidyl aminopeptidase II and III activities in the transition from reticulocytes to mature erythrocytes; (b) very limited decline of aminopeptidase B activity; (c) a pronounced age-dependent decay, in increasing order, of neutral endopeptidase, aminopeptidase A, carboxypeptidase and acidic endopeptidase activities.

The plasma membrane calcium pump is the preferred calpain substrate within the erythrocyte.

The activation of calpain in normal human erythrocytes incubated in the presence of Ca2+ and the Ca2+ ionophore A23187 led to the decline of the Ca(2+)-dependent ATPase activity of the cells. Preloading of the erythrocyte with an anticalpain antibody prevented the decline. The pump was also inactivated by applied to isolated erythrocyte plasma membranes. The decline of the pump activity corresponded to the degradation of the pump protein and was inversely correlated to the amount of the natural inhibitor of calpain, calpastatin, present in the cells. In erythrocytes containing only 50% of the normal level the degradation started at a concentration of Ca2+ significantly lower than in normal cells. A comparison of the concentrations of Ca2+ required for the degradation of a number of erythrocyte membrane proteins showed that the Ca2+ pump and band 3 were the most sensitive. All other membrane proteins tested were attacked at higher levels of intracellular Ca2+. Thus, the degradation of the Ca2+ pump protein may be a simple and sensitive means to monitor calpain activation in vivo. Furthermore, the results have shown that the calpastatin level correlated directly with the amount of activable calpain and with the concentration of Ca2+ required to trigger the activation process.

Mechanisms of perturbation of erythrocyte calcium homeostasis in favism.

Favism is an acute hemolytic anemia triggered by ingestion of fava beans in genetically susceptible subjects with severe deficiency of glucose-6-phosphate dehydrogenase (G6PD) activity. Erythrocytes from 10 favic patients had constantly and markedly increased calcium levels, as compared with values detected in 4 asymptomatic G6PD-deficient controls. Correspondingly, the calcium permeability of erythrocytes, estimated as the fraction of intracellular calcium exchangeable with externally added 45Ca2+, was invariably enhanced in favism and returned to normal patterns after several months from the acute hemolytic crisis. In favic patients, the levels of erythrocyte calcium ATPase activities showed wide variability, ranging from 2.0-12.9 mumol Pi/ml RBC/h, while control values in asymptomatic G6PD-deficient subjects were 10.62 +/- 2.03 mumol Pi/ml RBC/h. Analysis of the calcium ATPase in situ in erythrocyte membranes from favic patients showed the same molecular mass of 134 kD as observed in the control subjects. Exposure of G6PD-deficient erythrocytes in vitro to autoxidizing divicine, a pyrimidine aglycone strongly implicated in the pathogenesis of favism which leads to late accumulation of intracellular calcium, caused: (i) a marked inactivation of calcium ATPase, without changes in the molecular mass of 134 kD; and (ii) the concomitant loss of spectrin, band 3 and band 4.1, all known substrates of the calcium activated procalpain-calpain proteolytic system. Thus, the increased intraerythrocytic calcium apparently results in the degradation of calcium ATPase observed in some favic patients. It is proposed that both enhanced calcium permeability and a calcium-stimulated degradation of the calcium pump are the mechanisms responsible for the perturbation of erythrocyte calcium homeostasis in favism.

Erythrocyte deficiency in calpain inhibitor activity in essential hypertension.

The calpain-calpain inhibitor system was evaluated in erythrocytes of patients with essential hypertension and normotensive controls, either with or without a family history of hypertension. Calpain levels were similar in the controls and hypertensive patients, whereas the inhibitor activity level was significantly reduced in the latter (301.8 +/- 26.4 vs 220 +/- 14 U/mg hemoglobin, p less than 0.001). Borderline hypertensive patients and a few controls with a history of hypertension showed low inhibitor activity. Similar results have recently been reported in genetically hypertensive rats of the Milan strain. A significant inverse correlation (r = -0.43, p less than 0.001) was found between mean arterial pressure and calpain inhibitor. Although the pathophysiological significance of these observations is not yet clear, they suggest a new area of investigation into the molecular mechanisms underlying essential hypertension and its complications.

A 6 kDa protein homologous to the N-terminus of the HMG1 protein promoting stimulation of murine erythroleukemia cell differentiation.

Murine erythroleukemia (MEL) cells, in addition to an mRNA coding for a 30 kDa high mobility group (HMG)-1 protein, contain an mRNA coding for a 6 kDa HMG1 protein having the following structural properties: (1) its primary structure has 90% homology with the N-terminal sequence of the 30 kDa HMG1 protein; (2) it contains a consensus region of the HMG1 protein family; (3) it is deprived of the cluster of acidic amino acids that characterizes the C-terminal region of the 30 kDa HMG1 protein. This novel small Mr HMG1 protein has been expressed in prokaryotic cells and tested to establish similarities and differences in activity compared to the homologous higher Mr HMG1 protein. It has been found that the low Mr HMG1 form is not released from MEL cells following induction to erythroid differentiation, but is still effective, although with much less efficiency, when added to the external medium, in promoting acceleration in the rate of MEL cell differentiation as well as in activation of alpha-protein kinase C. Altogether these results provide evidence for the presence in MEL cells of a multigene family that encodes at least two different HMG1-type sequences most presumably involved, at distinct cellular sites, in different functions although commonly related to the promotion of cell differentiation. Additional information can be considered concerning the relationship between the characteristic N-terminal sequence of HMG1 protein and the extracellular activity on MEL cell differentiation.