Safinamide: from molecular targets to a new anti-Parkinson drug.

Ideal treatment in Parkinson's disease (PD) aims at relieving symptoms and slowing disease progression. Of all remedies, levodopa remains the most effective for symptomatic relief, but the medical need for neuroprotectant drugs is still unfulfilled. Safinamide, currently in phase III clinical trials for the treatment of PD, is a unique molecule with multiple mechanisms of action and a very high therapeutic index. It combines potent, selective, and reversible inhibition of MAO-B with blockade of voltage-dependent Na+ and Ca2+ channels and inhibition of glutamate release. Safinamide has neuroprotective and neurorescuing effects in MPTP-treated mice, in the rat kainic acid, and in the gerbil ischemia model. Safinamide potentiates levodopa-mediated increase of DA levels in DA-depleted mice and reverses the waning motor response after prolonged levodopa treatment in 6-OHDA-lesioned rats. Safinamide has excellent bioavailability, linear kinetics, and is suitable for once-a-day administration. Therefore, safinamide may be used in PD to reduce l-dopa dosage and also represents a valuable therapeutic drug to test disease-modifying potential.

[Aortic debris in cerebrovascular ischemic disease]. / Debris aórtico en la enfermedad cerebrovascular isquémica.

BACKGROUND: The aortic atherosclerotic debris is considered a high risk embolic source, being an independent predictor for cerebrovascular ischemia. The incidence is higher in the elderly and in patients with coronary artery disease. Transesophageal echocardiogram (TEE) is an important diagnostic tool that allows its detection. OBJECTIVE: To describe characteristics of patients with ischemic stroke and echocardiographic diagnosis of aortic debris. PATIENTS AND METHODS: We analyzed the group of patients with debris diagnosis in 209 TEE performed between 01/01/99 and 31/05/02, in 835 consecutive ischemic events. The information was collected from the Stroke Database of the Neurology Department of Policlinica Bancaria. RESULTS: TEE was accomplished in 25% of all assisted events. The mean age was 66.56 years (SD 11.22). In 30 studies (14%) aortic debris was detected. In this group of patients, 26 men and 4 women, was also found: plaques grade IV 60%, left atrial dilatation 40% and spontaneous echo contrast 20%. The most frequent risk factors were hypertension, dislipemia and smoking, with no significative difference compared to the group without debris. 40% had a prior cerebrovascular event. They presented with clinical subtype LACI 53%, PACI 27%, POCI 17%. 63% of patients had lacunar infarct (53% anterior and 10% posterior). CONCLUSION: The contribution of TTE for detection of embolic sources is relevant. A high percentage of the population with echocardiographic diagnosis of aortic debris, had a lacunar infarct, defined radiologically and by clinical features.

[Ischemic stroke: transesophageal echocardiographic findings]. / Accidente cerebrovascular isquémico: hallazgos en ecocardiograma transesofágico.

INTRODUCTION: Since its initial application in 1976, the transesophageal echocardiogram (TEE) has improved the detection of cardiovascular emboligenic sources. Even though its indication in patients with stroke is still controversial, its use has contributed to the identification of potential embolic stroke sources. OBJECTIVE: To describe the transesophageal echocardiographic findings in ischemic stroke patients. PATIENTS AND METHODS: We analyzed case series of 162 TEE performed on a total of 576 ischemic events dated between 01/01/99 to 01/05/01. The required information was collected prospectively in the Stroke Data Bank of the Neurology Department at Policl nico Bancario in Buenos Aires. RESULTS: TEE was carried out in 162 (28.1%) cases. Of theses cases 13% belonged to the clinical subtype TACI, 37% to PACI, 17% to POCI, and 37% to LACI subtype. Pathologic findings corresponded to cardiac level: spontaneous contrast in 29% of the cases, and to aortic level: plaques grade IV in 34% and debris in 13% of the cases. According to the etiology of ischemic stroke, 67 patients had been registered under the diagnosis of lacunar infarct (60 in the anterior region and 7 in the posterior region), 93 had been diagnosed medium and grand artery infarct (73 in the anterior region and 29 in the posterior region), and 2 had remained unclassified. Emboligenic sources were found in 69.5% of TACI, 65% of PACI, 52% of POCI, and 53% of LACI. CONCLUSIONS: A high percentage of aortic artheroembolic pathology was detected in the population under study. However, spontaneous contrast was the echocardiographic phenomenon more frequently reported. It is to be pointed out the presence of potential cardiac and/or aortic emboligenic sources in 48% of the population with lacunar infarct

Neuroprotective effects of Gly-Pro-Glu, the N-terminal tripeptide of IGF-1, in the hippocampus in vitro.

Insulin-like growth factor 1 (IGF-1) plays a critical role in CNS development. IGF-1 can block neuronal apoptosis in vitro and in vivo. IGF-1 is thought to be cleaved into des-N-(1-3)-IGF-1 and an amino terminal glycine-proline-glutamate (GPE tripeptide). Here we report a neuroprotective role for GPE tripeptide, with enhanced survival of the CA1-2 hippocampal neurons following an excitotoxic insult in vitro. Binding and displacement studies suggest uniquely distributed sites of action within the rat including the hippocampal CA1-2, pyriform cortex, amygdala, choroid plexus, blood vessels and to a lesser extent in the cortical regions. A similar pattern of binding was seen in the human. This finding could lead to new strategies to reduce neuronal death after injury and in disease.

Recombinant human IL-2 is cytotoxic to oligodendrocytes after in vitro self aggregation.

Interleukin 2 (IL-2) is a cytokine produced by activated T cells that plays a crucial role in the immune response and exerts multiple functions in different tissues including the nervous system. The present study was carried out to investigate the effect of recombinant human IL-2 (rhIL-2) on the survival of differnet glial cells type and of cortical neurons in culture. The results demonstrate that rhIL-2 is selectively cytotoxic to oligodendrocytes only if preincubated in aqueous solution (1200 U/ml) for at least two days before being added to the culture. Other glial and neuronal cells were unaffected. The cytotoxic effect was temperature- and concentration-dependent occurring only when rhIL-2 was reincubated at 37 degrees C and was dose-dependently neutralized by antibodies raised against IL-2 indicating that an immunoreactive IL-2 like compound is the active principle. Polyacrilamide gel electrophoresis under native (PAGE) and denaturing (SDS-PAGE) conditions and gel filtration analysis of the rhIL-2 incubated solution suggest that rhIL-2 is cytotoxic to oligodendrocytes only after association into soluble high weight molecular aggregates.

Big endothelin-converting enzyme activities in subcellular fractions of bovine aortic endothelial cells.

A combination of HPLC elution patterns and peptide sequencing has been used to characterize two distinct activities present in subcellular fractions of bovine aortic endothelial cells (BAECs) capable of converting human big endothelin-1 (big ET-1) to mature (ET-1). A pepstatin-inhibitable activity with an acidic pH optimum present in a lysosome-enriched fraction cleaved big ET-1 at positions 18 and 21 at similar rates. A neutral pH activity present in a postlysosomal organelles subfraction was also able to convert big ET-1, and was inhibited by EDTA, but not by 1-chloro-3-tosylamido-4-phenyl-2-butanone (TPCK), an inhibitor of chymotrypsin-like serine proteases.

Procoagulant activity of mouse transformed cells: different expression in freshly isolated or cultured cells.

This study was originally designed to investigate whether there is any correlation between the type of procoagulant activity (PCA) and the tumorigenicity of transformed cells. The data obtained are relevant to this question and to defining the differences in the expression of cellular activities depending on the in vitro system used. PCA was measured and characterized in normal, immortalized, and tumorigenic mouse fibroblasts. In all the cell lines studied the activity was of tissue factor type, as established with functional, enzymatic, and immunochemical criteria. However, the PCA of cells freshly isolated from the tumors induced by tumorigenic cell lines was of cancer procoagulant type, i.e. a cysteine protease with direct factor X activator activity. The same cells, when cultured in vitro, expressed again PCA of tissue-factor type. These results suggest that either a tumor-host interaction is required for the expression of cancer procoagulant or the latter activity, produced by tumor cells under in vitro conditions, is destroyed or inactivated during the culture period. Our findings caution against defining the procoagulant activity of tumors based on experiments on cultured cells.

Characterization of transformed cell lines evolving from a normal C3H line.

The development of transformed cell lines evolving from an embryo fibroblastic C3H primary culture was followed before and after the ageing crisis using different techniques. By flow cytometry, alteration of subpopulations having different DNA content and altered metabolic activity was observed after the crisis, with the trend to assume a near tetraploid DNA index at higher passages. The fibrin clot retractile activity was lost in all cases during the ageing crisis, but the outcome did not present uniform values of growth characteristics or chromosome number and tumorigenicity appeared to be a nonstable property of the transformed cell lines.

Procoagulant activity of mouse and human cultured cells following various types of transformation.

The presence of fibrin deposits in the microenvironment of tumor cells has been reported repeatedly and considered to play an important role in tumor biology. Among the mechanisms by which fibrin may be deposited in tumors, procoagulant activities (PCA) of different types have been described in cancer cells. The present study was aimed at establishing whether the nature of cellular PCA was a characteristic associated with malignant transformation. PCA of normal and transformed cells was investigated on pairs of murine and human origin. The transformed counterparts were obtained after treatment with low-dose radiation, chemical carcinogen, viral infection or after in vitro spontaneous immortalization. Both before and after any type of transformation cell PCA was of the tissue thromboplastin type, identified on the basis of biological criteria: requirement of factor VII for its expression and lack of inhibition by the serine protease inhibitor diisopropylfluorophosphate (DFP). Transformed cells of murine origin showed significantly lower activity than their normal counterparts, whereas all the transformed human cell lines expressed significantly higher activity than normal. An inverse correlation between the levels of PCA and the cell density in culture was observed in all but one of the lines tested. These findings suggest that the factor X activating property described in some tumors or in transformed cells cannot be considered as a general marker of transformation.

Culture conditions induce the appearance of immortalized C3H mouse cell lines.

Mouse fibroblasts were cultured by three different procedures: (a) changing the 0.2 ml/cm2 of growth medium every 2nd d and seeding 1 X 10(5) cells/cm2 after confluency; (b) changing the 0.4 ml/cm2 of growth medium only at subculture performed at confluency by a 1:2 split and keeping the bottles incubated on a rocking platform; (c) the same as Method b but keeping the bottles stationary throughout culture. By Method a no lines were immortalized over 36 experiments whereas Method b gave 1/4 immortalized lines and Method c gave 10:12 immortalized lines. Cells always went into crisis at the 9th to 11th doubling. Immortalized lines had a tetraploid DNA content.

Fibrin clot retractile activity in normal, established, and tumorigenic human epithelial cells in culture.

Normal and established human epithelial cell lines obtained from the same organs were compared for their capacity to retract a fibrin clot. Fibrin clot retraction was maximal in normal epithelial cells, reduced in established nontumorigenic lines, and lost in tumorigenic cancer cell lines. Fibrin clot retraction efficiency seemed to be related to the degree of cellular spreading within the clot at the end of the test. Previous works and the present study suggest that fibrin clot retraction is correlated with some steps of cell transformation in vitro.

Follitropin binding to receptors in testis: studies on the reversibility and thermodynamics of the reaction.

Studies were designed to assess the extent to which binding of follitropin (FSH) to its receptor is a reversible process. [125I]hFSH was allowed to interact with membrane receptors from calf testis for 2 h at various temperatures by which time significant specific binding of [125I]hFSH had taken place in all instances. Unlabeled FSH was then added (delayed addition) and the amount of [125I]hFSH remaining bound was monitored as a function of time. In order to assess reversibility, [125I]hFSH binding in the latter samples was compared to that occurring when unlabeled FSH had not been added or when unlabeled FSH was present from the start of the incubation. Binding was essentially fully reversible at 4 degrees C, but reversibility decreased with increasing temperature. Reversibility of FSH binding decreased markedly at temperatures greater than 26 degrees C and was considered irreversible at temperatures above 30 degrees C. At 4 degrees C essentially full reversibility (greater than 90%) was observed when the unlabeled hormone was added after 7 h of incubation, but decreased when added after 12 h. At warmer temperatures (22 or 30 degrees C) 'there was a progressive decrease in reversibility as the time of delay before addition of unlabeled hormone was lengthened. By determining the affinity constant (in separate experiments) at various temperatures, a thermodynamic analysis was possible. This analysis was restricted to the temperature range 4-26 degrees C in order to minimize complications arising from irreversible binding. The reaction was endothermal at low temperatures (T less than 12.5 degrees C) and exothermal at higher temperatures (T greater than 12.5 degrees C) and was associated with a decrease in heat capacity of 1800 cal [mol deg]-1 at 25 degrees C. The results are consistent with the concept that the hydrophobic effect plays an important role in FSH binding, but that the reaction is complex and may be composed of more than one step.

Impaired spreading of transformed cells on fibrin substrate.

Fibroblast-like cells derived from C3H mice at the first passage in culture, and a line from the same origin, which had undergone spontaneous transformation at the eleventh passage, were seeded on fibrin plates and tested for their attachment and spreading. After 4-24 hours normal cells had a spider-like morphology, while transformed cells remained in round clusters, flattened on the substrate. This data represent the morphological counterpart of the abnormal fibroblast-fibrin interaction shown in a tridimensional model, where transformed cells were found unable to induce the retraction of a fibrin clot (FCR).

Comparison of fibrin clot retraction with other transformation parameters after hydridization of normal and established cell lines.

The expression of transformation parameters (inhibition of cell division during cell crowding, anchorage dependence, loss of fibrin clot retractile activity and secretion of plasminogen activator) was studied in a heterospecific cellular hybrid, made between established L(TK-) cells and the normal human MRC-5 cells. The hybrid nature of the cross was confirmed by the ability to incorporate [3H]-thymidine, by growth in selective HAT medium, by the identification of human chromosomes and by the expression on the surface of 100% of hybrid cells of a human glycoprotein, which is recognized by the 4F2 monoclonal antibody. The hybrid cultures showed cell cycle inhibition which became less stringent with increasing population doublings and the loss of human chromosomes. Fibrin clot retraction and anchorage dependence were absent in spite of the presence of many human chromosomes. The two properties were present or lost simultaneously in the normal parent cells and in the transformed parent or hybrid cells respectively. The human type of plasminogen activator was secreted even with very little human genetic material left, and a complete dissociation between fibrin clot retraction and production of plasminogen activator was observed. The data strengthen the hypothesis that transformation is a multistep process that involves complex genetic control and where cells progressively express different phenotypes and escape growth control.

Actin organization and fibrin-clot retractile activity of cultured mouse fibroblasts.

It is known that human and animal fibroblasts are able to induce the retraction of a fibrin clot. In the present study the correlation between (i) fibrin-fibroblasts during growth, (ii) the number of actin stress-lines in mouse fibroblasts during growth in culture, and (iii) the sensitivity of actin stress-lines to a powerful actin-depolymerizing factor (ADF), present in plasma and serum of humans and laboratory animals was investigated. Fibroblasts at early passages (2-4) were tested for these parameters at various intervals after seeding (24, 96, and 168 h). The number of actin stress-lines was progressively higher, while the sensitivity to ADF action was progressively lower in cells cultured from 24 to 168 h; the FCR capacity was significantly decreased at 168 h. These data suggest that cells containing weakly polymerized and/or stabilized actin are more active than those containing highly polymerized and/or stabilized actin in triggering fibroblast contraction.

Fibrin clot retractile activity of mouse fibroblasts during growth and aging.

This study aimed at evaluating by a quantitative assay the fibrin clot retractile activity (FCR) of C3H embryo fibroblasts during their growth and aging in culture. Cell from primary and subsequent subcultures were tested at defined times from seeding, in a specially devised micromethod. Results indicate that cell-induced FCR has a kinetic similar to platelet-induced FCR; it depends on the number of cells and time of incubation in the system. It is absent or low in cells harvested from primary culture, then increases and remains high in the following doublings decreasing sharply at the end of the replicative life span in culture.