Signal transduction induced in Trypanosoma cruzi metacyclic trypomastigotes during the invasion of mammalian cells

Penetration of Trypanosoma cruzi into mammalian cells depends on the activation of the parasite's protein tyrosine kinase and on the increase in cytosolic Ca2+ concentration. We used metacyclic trypomastigotes, the T. cruzi developmental forms that initiate infection in mammalian hosts, to investigate the association of these two events and to identify the various components of the parasite signal transduction pathway involved in host cell invasion. We have found that i) both the protein tyrosine kinase activation, as measured by phosphorylation of a 175-kDa protein (p175), and Ca2+ mobilization were induced in the metacyclic forms by the HeLa cell extract but not by the extract of T. cruzi-resistant K562 cells; ii) treatment of parasites with the tyrosine kinase inhibitor genistein blocked both p175 phosphorylation and the increase in cytosolic Ca2+ concentration; iii) the recombinant protein J18, which contains the full-length sequence of gp82, a metacyclic stage surface glycoprotein involved in target cell invasion, interfered with tyrosine kinase and Ca2+ responses, whereas the monoclonal antibody 3F6 directed at gp82 induced parasite p175 phosphorylation and Ca2+ mobilization; iv) treatment of metacyclic forms with phospholipase C inhibitor U73122 blocked Ca2+ signaling and impaired the ability of the parasites to enter HeLa cells, and v) drugs such as heparin, a competitive IP3-receptor blocker, caffeine, which affects Ca2+ release from IP3-sensitive stores, in addition to thapsigargin, which depletes intracellular Ca2+ compartments and lithium ion, reduced the parasite infectivity. Taken together, these data suggest that protein tyrosine kinase, phospholipase C and IP3 are involved in the signaling cascade that is initiated on the parasite cell surface by gp82 and leads to Ca2+ mobilization required for target cell invasion.

Linear competitive inhibition of human tissue kallikrein by 4-aminobenzamidine and benzamidine and linear mixed inhibition by 4-nitroaniline and aniline

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide (7.5-90.0 µM) by human tissue kallikrein (hK1) (4.58-5.27 nM) at pH 9.0 and 37ºC was studied in the absence and in the presence of increasing concentrations of 4-aminobenzamidine (96-576 µM), benzamidine (1.27-7.62 mM), 4-nitroaniline (16.5-66 µM) and aniline (20-50 mM). The kinetic parameters determined in the absence of inhibitors were: Km = 12.0 ± 0.8 µM and k cat = 48.4 ± 1.0 min-1. The data indicate that the inhibition of hK1 by 4-aminobenzamidine and benzamidine is linear competitive, while the inhibition by 4-nitroaniline and aniline is linear mixed, with the inhibitor being able to bind both to the free enzyme with a dissociation constant Ki yielding an EI complex, and to the ES complex with a dissociation constant Ki', yielding an ESI complex. The calculated Ki values for 4-aminobenzamidine, benzamidine, 4-nitroaniline and aniline were 146 ± 10, 1,098 ± 91, 38.6 ± 5.2 and 37,340 ± 5,400 µM, respectively. The calculated Ki' values for 4-nitroaniline and aniline were 289.3 ± 92.8 and 310,500 ± 38,600 µM, respectively. The fact that Ki'>Ki indicates that 4-nitroaniline and aniline bind to a second binding site in the enzyme with lower affinity than they bind to the active site. The data about the inhibition of hK1 by 4-aminobenzamidine and benzamidine help to explain previous observations that esters, anilides or chloromethyl ketone derivatives of Nalpha-substituted arginine are more sensitive substrates or inhibitors of hK1 than the corresponding lysine compounds.

Cyclic AMP increases the survival of ganglion cells in mixed retinal cell cultures in the absence of exogenous neurotrophic molecules, an effect that involves cholinergic activity

Natural cell death is a well-known degenerative phenomenon occurring during development of the nervous system. The role of trophic molecules produced by target and afferent cells as well as by glial cells has been extensively demonstrated. Literature data demonstrate that cAMP can modulate the survival of neuronal cells. Cultures of mixed retinal cells were treated with forskolin (an activator of the enzyme adenylyl cyclase) for 48 h. The results show that 50 µM forskolin induced a two-fold increase in the survival of retinal ganglion cells (RGCs) in the absence of exogenous trophic factors. This effect was dose dependent and abolished by 1 µM H89 (an inhibitor of protein kinase A), 1.25 µM chelerythrine chloride (an inhibitor of protein kinase C), 50 µM PD 98059 (an inhibitor of MEK), 25 µM Ly 294002 (an inhibitor of phosphatidylinositol-3 kinase), 30 nM brefeldin A (an inhibitor of polypeptide release), and 10 µM genistein or 1 ng/ml herbimycin (inhibitors of tyrosine kinase enzymes). The inhibition of muscarinic receptors by 10 µM atropine or 1 µM telenzepine also blocked the effect of forskolin. When we used 25 µM BAPTA, an intracellular calcium chelator, as well as 20 µM 5-fluoro-2'-deoxyuridine, an inhibitor of cell proliferation, we also abolished the effect. Our results indicate that cAMP plays an important role controlling the survival of RGCs. This effect is directly dependent on M1 receptor activation indicating that cholinergic activity mediates the increase in RGC survival. We propose a model which involves cholinergic amacrine cells and glial cells in the increase of RGC survival elicited by forskolin treatment.

Correlation of mixed lymphocyte culture with chronic graft-versus-host disease following allogeneic stem cell transplantation

The purpose of the present study was to evaluate the mixed lymphocyte culture as a predictive assay of acute and chronic graft-versus-host disease (GVHD). We studied 153 patients who received a first bone marrow transplantation from human leukocyte antigen-identical siblings. Acute GVHD was observed in 26 of 128 (20.3%) patients evaluated and chronic GVHD occurred in 60 of 114 (52.6%). One-way mixed lymphocyte culture (MLC) assays were performed by the standard method. MLC results are reported as the relative response (RR) from donor against patient cells. The responses ranged from -47.0 to 40.7%, with a median of 0.5%. The Kaplan-Meier probability of developing GVHD was determined for patients with positive and negative MLC. There was no significant difference in incidence of acute GVHD between the groups studied. However, the incidence of chronic GVHD was higher in recipients with RR >4.5% than in those with RR <=4.5%. The Cox Proportional Hazards model was used to examine the effect of MLC levels on incidence of chronic GVHD, while adjusting for the potential confounding effect of others suspected or observed risk factors. The relative risk of chronic GVHD was 2.5 for patients with positive MLC (RR >4.5%), 2.9 for those who received peripheral blood progenitor cells as a graft, and 2.2 for patients who developed previous acute GVHD. MLC was not useful for predicting acute GVHD, but MLC with RR >4.5% associated with other risk factors could predict the development of chronic GVHD, being of help for the prevention and/or treatment of this late complication.

Abnormalities of hippocampal signal intensity in patients with familial mesial temporal lobe epilepsy

Mesial temporal lobe epilepsy (MTLE) is associated with hippocampal atrophy and hippocampal signal abnormalities. In our series of familial MTLE (FMTLE), we found a high proportion of hippocampal abnormalities. To quantify signal abnormalities in patients with FMTLE we studied 152 individuals (46 of them asymptomatic) with FMTLE. We used NIH-Image® for volumetry and signal quantification in coronal T1 inversion recovery and T2 for all cross-sections of the hippocampus. Values diverging by 2 or more SD from the control mean were considered abnormal. T2 hippocampal signal abnormalities were found in 52% of all individuals: 54% of affected subjects and 48% of asymptomatic subjects. T1 hippocampal signal changes were found in 34% of all individuals: 42.5% of affected subjects and 15% of asymptomatic subjects. Analysis of the hippocampal head (first three slices) revealed T2 abnormalities in 73% of all individuals (74% of affected subjects and 72% of asymptomatic subjects) and T1 abnormalities in 59% (67% of affected subjects and 41% of asymptomatic subjects). Affected individuals had smaller volumes than controls (P < 0.0001). There was no difference in hippocampal volumes between asymptomatic subjects and controls, although 39% of asymptomatic patients had hippocampal atrophy. Patients with an abnormal hippocampal signal (133 individuals) had smaller ipsilateral volume, but no linear correlation could be determined. Hippocampal signal abnormalities in FMTLE were more frequently found in the hippocampal head in both affected and asymptomatic family members, including those with normal volumes. These results indicate that subtle abnormalities leading to an abnormal hippocampal signal in FMTLE are not necessarily related to seizures and may be determined by genetic factors.

The control of deliberate waiting strategies in a stop-signal task

To inhibit an ongoing flow of thoughts or actions has been largely considered to be a crucial executive function, and the stop-signal paradigm makes inhibitory control measurable. Stop-signal tasks usually combine two concurrent tasks, i.e., manual responses to a primary task (go-task) are occasionally countermanded by a stimulus which signals participants to inhibit their response in that trial (stop-task). Participants are always instructed not to wait for the stop-signal, since waiting strategies cause the response times to be unstable, invalidating the data. The aim of the present study was to experimentally control the strategies of waiting deliberately for the stop-signal in a stop-task by means of an algorithm that measured the variation in the reaction times to go-stimuli on-line, and displayed a warning legend urging participants to be faster when their reaction times were more than two standard deviations of the mean. Thirty-four university students performed a stop-task with go- and stop-stimuli, both of which were delivered in the visual modality and were lateralized within the visual field. The participants were divided into two groups (group A, without the algorithm, vs group B, with the algorithm). Group B exhibited lower variability of reaction times to go-stimuli, whereas no significant between-group differences were found in any of the measures of inhibitory control, showing that the algorithm succeeded in controlling the deliberate waiting strategies. Differences between deliberate and unintentional waiting strategies, and anxiety as a probable factor responsible for individual differences in deliberate waiting behavior, are discussed.

Experimental context modulates warning signal effects

Previous studies have shown that saccadic eye responses but not manual responses were sensitive to the kind of warning signal used, with visual onsets producing longer saccadic latencies compared to visual offsets. The aim of the present study was to determine the effects of distinct warning signals on manual latencies and to test the premise that the onset interference, in fact, does not occur for manual responses. A second objective was to determine if the magnitude of the warning effects could be modulated by contextual procedures. Three experimental conditions based on the kind of warning signal used (visual onset, visual offset and auditory warning) were run in two different contexts (blocked and non-blocked). Eighteen participants were asked to respond to the imperative stimulus that would occur some milliseconds (0, 250, 500 or 750 ms) after the warning signal. The experiment consisted in three experimental sessions of 240 trials, where all the variables were counterbalanced. The data showed that visual onsets produced longer manual latencies than visual offsets in the non-blocked context (275 vs 261 ms; P < 0.001). This interference was obtained, however, only for short intervals between the warning and the stimulus, and was abolished when the blocked context was used (256 vs 255 ms; P = 0.789). These results are discussed in terms of bottom-up and top-down interactions, mainly those related to the role of attentional processing in canceling out competitive interactions and suppressive influences of a distractor on the relevant stimulus.

The effect of an aerobic training program on the electrical remodeling of the heart: high-frequency components of the signal-averaged electrocardiogram are predictors of the maximal aerobic power

Increased heart rate variability (HRV) and high-frequency content of the terminal region of the ventricular activation of signal-averaged ECG (SAECG) have been reported in athletes. The present study investigates HRV and SAECG parameters as predictors of maximal aerobic power (VO2max) in athletes. HRV, SAECG and VO2max were determined in 18 high-performance long-distance (25 ± 6 years; 17 males) runners 24 h after a training session. Clinical visits, ECG and VO2max determination were scheduled for all athletes during thew training period. A group of 18 untrained healthy volunteers matched for age, gender, and body surface area was included as controls. SAECG was acquired in the resting supine position for 15 min and processed to extract average RR interval (Mean-RR) and root mean squared standard deviation (RMSSD) of the difference of two consecutive normal RR intervals. SAECG variables analyzed in the vector magnitude with 40-250 Hz band-pass bi-directional filtering were: total and 40-µV terminal (LAS40) duration of ventricular activation, RMS voltage of total (RMST) and of the 40-ms terminal region of ventricular activation. Linear and multivariate stepwise logistic regressions oriented by inter-group comparisons were adjusted in significant variables in order to predict VO2max, with a P < 0.05 considered to be significant. VO2max correlated significantly (P < 0.05) with RMST (r = 0.77), Mean-RR (r = 0.62), RMSSD (r = 0.47), and LAS40 (r = -0.39). RMST was the independent predictor of VO2max. In athletes, HRV and high-frequency components of the SAECG correlate with VO2max and the high-frequency content of SAECG is an independent predictor of VO2max.

Tracheal cryopreservation: caspase-3 immunoreactivity in tracheal epithelium and in mixed glands

Cryopreservation has an immunomodulating effect on tracheal tissue as a result of class II antigen depletion due to epithelium exfoliation. However, not all epithelium is detached. We evaluated the role of apoptosis in the remaining epithelium of 30 cryopreserved tracheal grafts. Caspase-3 immunoreactivity of tracheal epithelium was studied in canine tracheal segments cryopreserved with F12K medium, with or without subsequent storage in liquid nitrogen at -196°C for 15 days. Loss of structural integrity of tracheal mixed glands was observed in all cryopreserved tracheal segments. Caspase-3 immunoreactivity in tracheal mucosa and in mixed glands was significantly decreased, in contrast to the control group and to cryopreserved tracheal segments in which it remained high, due to the effect of storage in liquid nitrogen (P < 0.05, ANOVA and Tukey test). We conclude that apoptosis can be triggered in epithelial cells during tracheal graft harvesting even prior to cryopreservation, and although the epithelial caspase-3 immunoreactivity is reduced in tracheal cryopreservation, this could be explained by increased cell death. Apoptosis cannot be stopped during tracheal cryopreservation.

MMP-1/PAR-1 signal transduction axis and its prognostic impact in esophageal squamous cell carcinoma

The matrix metalloprotease-1 (MMP-1)/protease-activated receptor-1 (PAR-1) signal transduction axis plays an important role in tumorigenesis. To explore the expression and prognostic value of MMP-1 and PAR-1 in esophageal squamous cell carcinoma (ESCC), we evaluated the expression of two proteins in resected specimens from 85 patients with ESCC by immunohistochemistry. Sixty-two (72.9%) and 58 (68.2%) tumors were MMP-1- and PAR-1-positive, respectively, while no significant staining was observed in normal esophageal squamous epithelium. MMP-1 and PAR-1 overexpression was significantly associated with tumor node metastasis (TNM) stage and regional lymph node involvement. Patients with MMP-1- and PAR-1-positive tumors, respectively, had poorer disease-free survival (DFS) than those with negative ESCC (P = 0.002 and 0.003, respectively). Univariate analysis showed a significant relationship between TNM stage [hazard ratio (HR) = 2.836, 95% confidence interval (CI) = 1.866-4.308], regional lymph node involvement (HR = 2.955, 95%CI = 1.713-5.068), MMP-1 expression (HR = 2.669, 95%CI = 1.229-6.127), and PAR-1 expression (HR = 1.762, 95%CI = 1.156-2.883) and DFS. Multivariate analysis including the above four parameters identified TNM stage (HR = 2.035, 95%CI = 1.167-3.681), MMP-1 expression (HR = 2.109, 95%CI = 1.293-3.279), and PAR-1 expression (HR = 1.967, 95%CI = 1.256-2.881) as independent and significant prognostic factors for DFS. Our data suggest for the first time that MMP-1 and PAR-1 were both overexpressed in ESCC and are novel predictors of poor patient prognosis after curative resection. The MMP-1/PAR-1 signal transduction axis might be a new therapeutic target for future therapies tailored against ESCC.

Propofol inhibits burn injury-induced hyperpermeability through an apoptotic signal pathway in microvascular endothelial cells

Recent studies have revealed that an intrinsic apoptotic signaling cascade is involved in vascular hyperpermeability and endothelial barrier dysfunction. Propofol (2,6-diisopropylphenol) has also been reported to inhibit apoptotic signaling by regulating mitochondrial permeability transition pore (mPTP) opening and caspase-3 activation. Here, we investigated whether propofol could alleviate burn serum-induced endothelial hyperpermeability through the inhibition of the intrinsic apoptotic signaling cascade. Rat lung microvascular endothelial cells (RLMVECs) were pretreated with propofol at various concentrations, followed by stimulation with burn serum, obtained from burn-injury rats. Monolayer permeability was determined by transendothelial electrical resistance. Mitochondrial release of cytochrome C was measured by ELISA. Bax and Bcl-2 expression and mitochondrial release of second mitochondrial-derived activator of caspases (smac) were detected by Western blotting. Caspase-3 activity was assessed by fluorometric assay; mitochondrial membrane potential (Δψm) was determined with JC-1 (a potential-sensitive fluorescent dye). Intracellular ATP content was assayed using a commercial kit, and reactive oxygen species (ROS) were measured by dichlorodihydrofluorescein diacetate (DCFH-DA). Burn serum significantly increased monolayer permeability (P<0.05), and this effect could be inhibited by propofol (P<0.05). Compared with a sham treatment group, intrinsic apoptotic signaling activation - indicated by Bax overexpression, Bcl-2 downregulation, Δψm reduction, decreased intracellular ATP level, increased cytosolic cytochrome C and smac, and caspase-3 activation - was observed in the vehicle group. Propofol not only attenuated these alterations (P<0.05 for all), but also significantly decreased burn-induced ROS production (P<0.05). Propofol attenuated burn-induced RLMVEC monolayer hyperpermeability by regulating the intrinsic apoptotic signaling pathway.