Clinicopathologic significance of S100A4 expression in osteosarcoma.

OBJECTIVE: Osteosarcoma is the most common primary malignancy, mainly arising from the metaphysis of the long bones of adolescents and young adults. Its poor prognosis is strongly associated with invasion and distant metastasis. The calcium-binding protein S100A4 promotes metastasis in several experimental animal models, including osteosarcoma (OS), and S100A4 protein expression is associated with patient outcome in a number of tumor types. In the present study, we investigated the expression of S100A4 and its clinicopathologic significance in OSs. PATIENTS AND METHODS: S100A4 were examined immunohistochemically in resected OSs from 120 patients with OS to clarify their clinicopathologic significance. Multivariate survival analyses were carried out on all investigated parameters. RESULTS: The immunohistochemical assays revealed that S1004A expression in osteosarcoma tissues was significantly higher than that in corresponding noncancerous bone tissues (p < 0.001). In addition, positive S100A4 expression more frequently occurred in osteosarcoma tissues with advanced clinical stage (p = 0.003), positive distant metastasis (p = 0.001) and poor response to chemotherapy (p = 0.04). In Kaplan-Meier analysis, only S100A4 positively stained cases showed a significantly decreased overall survival time and disease-free survival compared with negatively stained cases (both p < 0.001). On Cox multivariate analysis, positive S100A4 expression was an independent and significant prognostic factor to predict poor overall survival and disease-free survival (both p = 0.001). CONCLUSIONS: Expression of S100A4 protein in OS may be related to the prediction of metastasis potency, response to chemotherapy and poor prognosis for osteosarcoma patients, suggesting that S100A4 may serve as a prognostic marker for the optimization of clinical treatments.

The mitochondrial calcium uniporter participates in ischemia/reperfusion injury and in cardioprotection by ischemic preconditioning.

The objective of the present study was to determine whether the mitochondrial calcium uniporter plays a role in cardioprotection by ischemic preconditioning (IPC). Isolated rat hearts were subjected to 30 min regional ischemia by ligation of the left anterior descending artery followed by 120 min reperfusion. We found that both IPC and inhibition of the mitochondrial calcium uniporter during reperfusion improved recovery of left ventricular developed pressure, maximal rise velocity and end-diastolic pressure, and reduced infarct size and lactate dehydrogenase release. These protective effects were attenuated by activating the mitochondrial calcium uniporter. We conclude that the mitochondrial calcium uniporter is involved in the cardioprotection of ischemic preconditioning.

Interleukin-2 confers cardioprotection by inhibiting mitochondrial permeability transition pore.

In the present study, we determined whether interleukin-2 (IL-2) confers cardioprotection by inhibiting mitochondria permeability transition pore (MPTP) opening. In isolated rat hearts subject to 30 min ischemia and 120 min reperfusion (IR), IL-2 (50 U/ml) decreased the infarct size and LDH release, effects blocked by a selective kappa-opioid receptor antagonist, Nor-BNI (5 microM) or an opener of MPTP, atractyloside (Atr, 20 microM). In isolated ventricular myocytes subjected to anoxia and reoxygenation (AR), which reduced both the amplitude of the electrically induced [Ca2+]i transient and diastolic [Ca2+]i, IL-2 attenuated the AR-induced alterations and their effects were abolished by Atr. In addition, IL-2 attenuated the reduction in calcein fluorescence in myocytes subject to AR and reduced calcium-induced swelling in mitochondria of rat hearts subjected to IR, which were similar to effect of inhibitor of MPTP. The observations indicated that IL-2 confers cardioprotection by inhibiting the MPTP opening.

Effects of interleukin-2 on the mechanical restitution and post-rest contraction in rat ventricular papillary muscle.

To determine whether application of interleukin-2 (IL-2) alters function of sarcoplasmic reticulum (SR), we measured mechanical restitution and post-rest potentiation (PRP) in isolated rat papillary muscles. Mechanical restitution curves were constructed by interpolating extrasystoles at different test intervals following a train of steady state beats. In control group, the maximal PRP was reached after 60-120s of rest and the maximal potentiation factor was 2.36 +/- 0.23. IL-2 at 200 U/ml decreased the steady-state force of contraction to 56.4 +/- 7.2% of pre-drug control. But the time constant of recovery of steady-state force was not altered after IL-2. IL-2 decreased PRP at all intervals, shifted the potentiation curve parallel to lower values. But the potentiation was enhanced when compared with pre-rest control value in the presence of IL-2. In papillary muscle treated with IL-2, the onset of maximal PRP was delayed and the potentiation factor after 300s was 4.72 +/- 0.58 times that at the steady-state. Recirculation fraction of calcium calculated from the decay of PRP was 0.78 +/- 0.09 in control and 0.59 +/- 0.08 after IL-2 treatment. We conclude that IL-2 decreases the function of SR, which suggests that an impaired function of SR may contribute to the negative inotropic effect of IL-2.

Role of the mitochondrial permeability transition pore in TNF-alpha-induced recovery of ventricular contraction and reduction of infarct size in isolated rat hearts subjected to ischemia/reperfusion.

Pretreatment with tumor necrosis factor-alpha (TNF-alpha) is known to trigger cardioprotection. TNF-alpha can activate multiple downstream signaling cascades. However, it is not known whether the mitochondrial permeability transition pore (MitoPTP) is involved in TNF-alpha-induced cardioprotection. In the present study, we examined whether TNF-alpha inhibits MitoPTP opening. In isolated rat hearts subjected to 30 min regional ischemia and 120 min reperfusion, pretreatment with 10 U/ml TNF-alpha for 7 min followed by 10 min washout improved the recovery of left ventricular developed pressure (LVDP) and rate-pressure product (RPP = LVDP x heart rate) during reperfusion and reduced the infarct size. Administration of 20 micromol/L atractyloside, a MitoPTP opener, for 20 min (last 5 min of ischemia and first 15 min of reperfusion) and pretreatment with 1 mu inhibitor of the Ca2+-activated K+ mol/L paxilline, an channel, for 5 min before ischemia, attenuated the recovery of LVDP and RPP and the reduction of infarct size induced by TNF-alpha. The findings indicate that, in the isolated heart model, TNF-alpha protects myocardium against ischemia/reperfusion injury via inhibiting MitoPTP opening as well as by activating the Ca2+-activated K+ channel.

Negative inotropic effect of interleukin-2 in the isolated ventricular myocytes: role of NO/sGC pathway.

The present study is to investigate the effect and possible mechanism of interleukin-2 (IL-2) on the cell contractility in isolated rat cardiomyocytes. Ventricular myocytes were isolated from adult male Sprague-Dawley rats. Contractile responses were evaluated by use of the video tracking system. Contractile properties analyzed in cells electrically stimulated at 0.2Hz included peak velocity of cell shortening (+dL/dtmax), peak velocity of cell relengthening (-dL/dtmax), contraction amplitude (dL) and end-diastolic cell length. Calcium transients of ventricular myocytes were determined by the spectrofluorometric techniques. IL-2 (2.0, 10, 50, 200 and 1000 U/ml) exhibited a dose-dependent inhibition in +dL/dtmax, -dL/dtmax, dL and end-diastolic cell length. Pretreatment with the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME, 100 microM) and 1H-[1,2,4]oxadiazolo[4,3a]quinoxalin-1-one (ODQ, soluble guanylyl cyclase inhibitor) blocked IL-2-induced inhibition of the contractility. IL-2 at 200 U/ml decreased the amplitude of the [Ca2+]i transient. Pretreatment with the L-NAME or ODQ abolished IL-2-induced inhibition of amplitude of the calcium transient. We conclude that the depressant effect of IL-2 on the contraction and calcium transient of isolated ventricular myocytes is mediated by nitric oxide/soluble guanylyl cyclase pathway.

Influence of interleukin-2 on Ca2+ handling in rat ventricular myocytes.

In the present study, we examined the effect of interleukin-2 (IL-2) on cardiomyocyte Ca(2+) handling. The effects of steady-state and transient changes in stimulation frequency on the intracellular Ca(2+) transient were investigated in isolated ventricular myocytes by spectrofluorometry. In the steady state (0.2 Hz) IL-2 (200 U/ml) decreased the amplitude of Ca(2+) transients induced by electrical stimulation and caffeine. At 1.25 mM extracellular Ca(2+) concentration ([Ca(2+)](o)), when the stimulation frequency increased from 0.2 to 1.0 Hz, diastolic Ca(2+) level and peak intracellular Ca(2+) concentration ([Ca(2+)](i)), as well as the amplitude of the transient, increased. The positive frequency relationships of the peak and amplitude of [Ca(2+)](i) transients were blunted in the IL-2-treated myocytes. The effect of IL-2 on the electrically induced [Ca(2+)](i) transient was not normalized by increasing [Ca(2+)](o) to 2.5 mM. IL-2 inhibited the frequency relationship of caffeine-induced Ca(2+) release. Blockade of sarcoplasmic reticulum (SR) Ca(2+)-ATPase with thapsigargin resulted in a significant reduction of the amplitude-frequency relationship of the transient similar to that induced by IL-2. The restitutions were not different between control and IL-2 groups at 1.25 mM [Ca(2+)](o), which was slowed in IL-2-treated myocytes when [Ca(2+)](o) was increased to 2.5 mM. There was no difference in the recirculation fraction (RF) between control and IL-2-treated myocytes at both 1.25 and 2.5 mM [Ca(2+)](o). The effects of IL-2 on frequency relationship, restitution, and RF may be due to depressed SR functions and an increased Na(+)-Ca(2+) exchange activity, but not to any change in L-type Ca(2+) channels.

[Effect of interleukin-2 on intracellular calcium transients in rat ventricular myocytes].

In the present study, we investigated the effect of interleukin-2 (IL-2) on the intracellular calcium in enzymatically isolated ventricular myocytes with the use of the spectrofluorometric techniques. It was shown that IL-2 (2.5 200 U/ml) depressed electrically induced Ca(2+) (i) transients of ventricular myocytes in a dose dependent manner. IL-2 (200 U/ml) did not alter the caffeine releasable pool of Ca(2+). Pretreatment with the non selective opioid antagonist naloxone (10(-8)mol/L) or a specific kappa opioid antagonist nor binaltorphimine (nor-BNI, 10(-8) mol/L) abolished the inhibitory effect of IL-2 (200 U/ml) on the Ca(2+) (i) transients of cardiomyocytes, whereas the specific delta opioid antagonist naltrindole (10(-6) mol/L) did not abolish the inhibitory effect. The effect of IL-2 (200 U/ml) was also abolished after pretreatment with pertussis toxin (PTX, 5 mg/L) as well as phospholipase C (PLC) inhibitor U73122 (5 10(-6) mol/L), but not by tyrosine kinase inhibitor genistein (10(-4) mol/L). It is concluded that the depressant effect of IL-2 on the Ca(2+) (i) transients of isolated ventricular myocytes is mainly mediated by cardiac kappa opioid receptor pathway including a PTX sensitive Gi-protein and PLC, but not by tyrosine kinase.

[Hydrogen peroxide augments the injury effect of iron on the isolated rat heart and cardiomyocytes].

By using Langendorff perfused rat heart and enzymatically isolated cardiomyocytes, we investigated the augmented injury effect of iron on the myocardium by hydrogen peroxide and the underlying mechanisms. Cell-permeable iron (Fe-HQ) decreased the contractile amplitude, velocity and end-diastolic cell length of the cardiomyocyte but increased the contents of lactate dehydrogenase (LDH) and creatine kinase (CK) in the coronary effluent and the myocardial malondialdehyde (MDA) while the left ventricular developed pressure (LVDP), +/-dp/dt(max), heart rate and coronary flow showed biphasic alterations. Hydrogen peroxide augmented the injury effect of iron accompanied by increases of coronary LDH, CK release and myocardial MDA content and decreases of LVDP, +/-dp/dt(max), and heart rate. Reduced glutathione could antagonize the injury effect of iron and hydrogen peroxide on the myocardium while dimethyl sulfoxide had no injury effect on the isolated heart. It is suggested that the functional injury of sulfhydryl group containing proteins may be involved in the augmentation of myocardial injury due to the increase of intracellular iron by hydrogen peroxide, but hydroxyl radicals may not.

[Prostaglandin E2 obtained by semisynthetic procedures from Gorgonia plexaura homomalla cubesis. Study of its bronchodilator effect]. / Prostaglandina E-2, obtenida por procedimientos semisintéticos de la gorgonia plexaura homomalla cubesis. Estudio de su efecto broncodilatador.

The bronchodilator effects of PGE-2, isolated by semisynthetic procedures from the plexaura homomalla of the insular platform of the Cuban archipelago, was studied. Fifty bronchial asthma patients were evaluated by spirometry, before and after inhalation of an aerosol of PGE-2, in alcohol solution. The patients were divided into 4 groups, each group being tested spirometically at different moments before and after aerosol administration. The results obtained within each group were compared. Systemic blood pressure (T.A.) and heart rate (F.C.C.) were recorded simultaneously in order to detect any secondary effects on the cardiovascular system. In parallel with this study, asthmatic patients were divided into 2 control group with whom the same study was done using a different aerosol composition. In one group, a placebo of ethyl alcohol was administered, in the same proportion as the PGE-2 would have been. In the other, the aerosol contained isoproterenol and ethyl alcohol. These tests were planned to determine the significance of the initiating effect of the alcohol. The significance of the difference in the results obtained was determined statistically in all cases. Finally, the PGE-2 aerosol was used by asthmatics during acute crises, with similar methodology. Results indicated that the product tested has initiating effects on the respiratory tract mucosa, lacks a significant bronchodilating capacity and has no secondary effects on the cardiovascular system.

[Determination of cholinesterase activity in Fasciola hepatica homogenates]. / Determinación de actividad colinesterásica en homogeneizados de Fasciola hepatica.

The cholinesterase activity in Fasciola hepatica homogenates was studied through biological techniques. Results depict that the contractile stimulating action of a constant acetylcholine dose on the isolated rat duodenum is withdrawn when the agent is previously incubated at 37 degrees C during 30 minutes with different dilutions (20%, 40% and 80%) of Fasciola hepatica homogenates. The action is recovered when an anticholinesterase, as neostigmine is previously added to the homogenate. Since these effects are similar to those obtained when different dilutions of human blood serum with a high content of cholinesterase are led to act upon the acetylcholine dose, it is concluded that the acetylcholine inactivation induced by Fasciola hepatica homogenates results from the existence of such enzyme within this parasite.