Cell homeostasis in a Leishmania major mutant overexpressing the spliced leader RNA is maintained by an increased proteolytic activity

Although several stage-specific genes have been identified in Leishmania, the molecular mechanisms governing developmental gene regulation in this organism are still not well understood. We have previously reported an attenuation of virulence in Leishmania major and L braziliensis carrying extra-copies of the spliced leader RNA gene. Here, we surveyed the major differences in proteome and transcript expression profiles between the spliced leader RNA overexpressor and control lines using two-dimensional gel electrophoresis and differential display reverse transcription PCR, respectively. Thirty-nine genes related to stress response, cytoskeleton, proteolysis, cell cycle control and proliferation, energy generation, gene transcription, RNA processing and post-transcriptional regulation have abnormal patterns of expression in the spliced leader RNA overexpressor line. The evaluation of proteolytic pathways in the mutant revealed a selective increase of cysteine protease activity and an exacerbated ubiquitin-labeled protein population. Polysome profile analysis and measurement of cellular protein aggregates showed that protein translation in the spliced leader RNA overexpressor line is increased when compared to the control line. We found that L major promastigotes maintain homeostasis in culture when challenged with a metabolic imbalance generated by spliced leader RNA surplus through modulation of intracellular proteolysis. However, this might interfere with a fine-tuned gene expression control necessary for the amastigote multiplication in the mammalian host. (c) 2010 Elsevier Ltd. All rights reserved.

Tyrosines in the Carboxyl Terminus Regulate Syk Kinase Activity and Function

The Syk tyrosine kinase family plays an essential role in immunoreceptor tyrosine-based activation motif (ITAM) signaling. The binding of Syk to tyrosine-phosphorylated ITAM subunits of immunoreceptors, such as Fc epsilon RI on mast cells, results in a conformational change, with an increase of enzymatic activity of Syk. This conformational change exposes the COOH-terminal tail of Syk, which has three conserved Tyr residues (Tyr-623, Tyr-624, and Tyr-625 of rat Syk). To understand the role of these residues in signaling, wild-type and mutant Syk with these three Tyr mutated to Phe was expressed in Syk-deficient mast cells. There was decreased Fc epsilon RI-induced degranulation, nuclear factor for T cell activation and NF kappa B activation with the mutated Syk together with reduced phosphorylation of MAP kinases p38 and p42/44 ERK. In non-stimulated cells, the mutated Syk was more tyrosine phosphorylated predominantly as a result of autophosphorylation. In vitro, there was reduced binding of mutated Syk to phosphorylated ITAM due to this increased phosphorylation. This mutated Syk from non-stimulated cells had significantly reduced kinase activity toward an exogenous substrate, whereas its autophosphorylation capacity was not affected. However, the kinase activity and the autophosphorylation capacity of this mutated Syk were dramatically decreased when the protein was dephosphorylated before the in vitro kinase reaction. Furthermore, mutation of these tyrosines in the COOH-terminal region of Syk transforms it to an enzyme, similar to its homolog ZAP-70, which depends on other tyrosine kinases for optimal activation. In testing Syk mutated singly at each one of the tyrosines, Tyr-624 but especially Tyr-625 had the major role in these reactions. Therefore, these results indicate that these tyrosines in the tail region play a critical role in regulating the kinase activity and function of Syk.

Pediatric epilepsy surgery and sudden unexpected death epilepsy: the contribution of a Brazilian epilepsy surgery program

Individuals with epilepsy are at higher risk of death than those from the general population, and sudden unexpected death in epilepsy (SUDEP) is the most important direct epilepsy-related cause of death. Epilepsies in the pediatric group are more frequently associated with known potentially risk factors for SUDEP, and a treatment resulting in an improved seizure control may also decrease mortality. The aim of this study is to identify the incidence of SUDEP in a group of operated-on children and adolescents. We analyzed 267 patients up to 18 years old, with medically intractable epilepsy submitted to surgery. We considered the age at surgery, the seizure type, the pathological findings, and the seizure outcome. Data were prospectively collected, according to the protocols of our institution`s ethics committee. The percentage of boys was 58.05. A good outcome was achieved in 72.6% of the cases and a bad outcome in 27.4%. Nine patients died during follow-up, six from clinical complications, and one from SUDEP. All patients who died during the long-term follow-up had persisted with refractory postoperative seizures. The patient who died from SUDEP died during a generalized tonic-clonic seizure. Of the patients, 72.6% had excellent postoperative outcome, and one patient died of SUDEP. All patients who died had had disabling seizures` persistence. The surgical treatment of epilepsy in children and adolescents is an efficient therapy for the medically intractable symptomatic epilepsies and also for the reduction of mortality and SUDEP risks.

Impact of a program for the prevention of traffic accidents in a Southern Brazilian city: a model for implementation in a developing country

Background: Traffic accidents constitute the main cause of death in the first decades of life. Traumatic brain injury is the event most responsible for the severity of these accidents. The SBN started an educational program for the prevention of traffic accidents, adapted from the American model ""Think First"" to the Brazilian environment, since 1995, with special effort devoted to the prevention of TBI by using seat belts and motorcycle helmets. The objective of the present study was to set up a traffic accident prevention program based on the adapted Think First and to evaluate its impact by comparing epidemiological variables before and after the beginning of the program. Methods: The program was executed in Maringa city, from September 2004 to August 2005, with educational actions targeting the entire population, especially teenagers and young adults. The program was implemented by building a network of information facilitators and multipliers inside the organized civil society, with widespread population dissemination. To measure the impact of the program, a specific software was developed for the storage and processing of the epidemiological variables. Results: The results showed a reduction of trauma severity due to traffic accidents after the execution of the program, mainly TBI. Conclusions: The adapted Think First was systematically implemented and its impact measured for the first time in Brazil, revealing the usefulness of the program for reducing trauma and TBI severity in traffic accidents through public education and representing a standardized model of implementation in a developing country. (C) 2009 Elsevier Inc. All rights reserved.

Heart failure disease management program experience in 4,545 heart failure admissions to a community hospital

Background Disease management programs (DMPs) are developed to address the high morbi-mortality and costs of congestive heart failure (CHF). Most studies have focused on intensive programs in academic centers. Washington County Hospital (WCH) in Hagerstown, MD, the primary reference to a semirural county, established a CHF DMP in 2001 with standardized documentation of screening and participation. Linkage to electronic records and state vital statistics enabled examination of the CHF population including individuals participating and those ineligible for the program. Methods All WCH inpatients with CHF International Classification of Diseases, Ninth Revision code in any position of the hospital list discharged alive. Results Of 4,545 consecutive CHF admissions, only 10% enrolled and of those only 52.2% made a call. Enrollment in the program was related to: age (OR 0.64 per decade older, 95% CI 0.58-0.70), CHF as the main reason for admission (OR 3.58, 95% CI 2.4-4.8), previous admission for CHF (OR 1.14, 95% CI 1.09-1.2), and shorter hospital stay (OR 0.94 per day longer, 95% CI 0.87-0.99). Among DMP participants mortality rates were lowest in the first month (80/1000 person-years) and increased subsequently. The opposite mortality trend occurred in nonenrolled groups with mortality in the first month of 814 per 1000 person-years in refusers and even higher in ineligible (1569/1000 person-years). This difference remained significant after adjustment. Re-admission rates were lower among participants who called consistently (adjusted incidence rate ratio 0.62, 95% CI 0.52-0.77). Conclusion Only a small and highly select group participated in a low-intensity DMP for CHF in a community-based hospital. Design of DMPs should incorporate these strong selective factors to maximize program impact. (Am Heart J 2009; 15 8:459-66.)

Constitutional hypomorphic telomerase mutations in patients with acute myeloid leukemia

Loss-of-function mutations in telomerase complex genes can cause bone marrow failure, dyskeratosis congenita, and acquired aplastic anemia, both diseases that predispose to acute myeloid leukemia. Loss of telomerase function produces short telomeres, potentially resulting in chromosome recombination, end-to-end fusion, and recognition as damaged DNA. We investigated whether mutations in telomerase genes also occur in acute myeloid leukemia. We screened bone marrow samples from 133 consecutive patients with acute myeloid leukemia and 198 controls for variations in TERT and TERC genes. An additional 89 patients from a second cohort, selected based on cytogenetic status, and 528 controls were further examined for mutations. A third cohort of 372 patients and 384 controls were specifically tested for one TERT gene variant. In the first cohort, 11 patients carried missense TERT gene variants that were not present in controls (P<0.0001); in the second cohort, TERT mutations were associated with trisomy 8 and inversion 16. Mutation germ-line origin was demonstrated in 5 patients from whom other tissues were available. Analysis of all 3 cohorts (n = 594) for the most common gene variant (A1062T) indicated a prevalence 3 times higher in patients than in controls (n = 1,110; P = 0.0009). Introduction of TERT mutants into telomerase-deficient cells resulted in loss of enzymatic activity by haploinsufficiency. Inherited mutations in TERT that reduce telomerase activity are risk factors for acute myeloid leukemia. We propose that short and dysfunctional telomeres limit normal stem cell proliferation and predispose for leukemia by selection of stem cells with defective DNA damage responses that are prone to genome instability.

Shared and Unique Gene Expression in Systemic Lupus Erythematosus Depending on Disease Activity

Patients presenting with active Systemic lupus erythematosus (SLE) manifestations may exhibit distinct pathogenetic features in relation to inactive SLE. Also, cDNA microarrays may potentially discriminate the gene expression profile of a disease or disease variant. Therefore, we evaluated the expression profile of 4500 genes in peripheral blood lymphocytes (PBL) of SLE patients. We studied 11 patients with SLE (seven with active SLE and four with inactive SLE) and eight healthy controls. Total RNA was isolated from PBL, reverse transcribed into cDNA, and postlabeled with Cy3 fluorochrome. These probes were then hybridized to a glass slide cDNA microarray containing 4500 human IMAGE cDNA target sequences. An equimolar amount of total RNA from human cell lines served as reference. The microarray images were quantified, normalized, and analyzed using the R environment (ANOVA, significant analysis of microarrays, and cluster-tree view algorithms). Disease activity was assessed by the SLE disease activity index. Compared to the healthy controls, 104 genes in active SLE patients (80 repressed and 24 induced) and 52 genes in nonactive SLE patients (31 induced and 21 repressed) were differentially expressed. The modulation of 12 genes, either induced or repressed, was found in both disease variants; however, each disease variant had differential expression of different genes. Taken together, these results indicate that the two lupus variants studied have common and unique differentially expressed genes. Although the biological significance of the differentially expressed genes discussed above has not been completely understood, they may serve as a platform to further explore the molecular basis of immune deregulation in SLE.

Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency

The Myc oncogene regulates the expression of several components of the protein synthetic machinery, including ribosomal proteins, initiation factors of translation, RNA polymerase III and ribosomal DNA(1,2). Whether and how increasing the cellular protein synthesis capacity affects the multistep process leading to cancer remains to be addressed. Here we use ribosomal protein heterozygote mice as a genetic tool to restore increased protein synthesis in E mu-Myc/+ transgenic mice to normal levels, and show that the oncogenic potential of Myc in this context is suppressed. Our findings demonstrate that the ability of Myc to increase protein synthesis directly augments cell size and is sufficient to accelerate cell cycle progression independently of known cell cycle targets transcriptionally regulated by Myc. In addition, when protein synthesis is restored to normal levels, Myc- overexpressing precancerous cells are more efficiently eliminated by programmed cell death. Our findings reveal a new mechanism that links increases in general protein synthesis rates downstream of an oncogenic signal to a specific molecular impairment in the modality of translation initiation used to regulate the expression of selective messenger RNAs. We show that an aberrant increase in cap- dependent translation downstream of Myc hyperactivation specifically impairs the translational switch to internal ribosomal entry site ( IRES)- dependent translation that is required for accurate mitotic progression. Failure of this translational switch results in reduced mitotic- specific expression of the endogenous IRES- dependent form of Cdk11 ( also known as Cdc21 and PITSLRE)(3-5), which leads to cytokinesis defects and is associated with increased centrosome numbers and genome instability in E mu-Myc/+ mice. When accurate translational control is re- established in E mu-Myc/+ mice, genome instability is suppressed. Our findings demonstrate how perturbations in translational control provide a highly specific outcome for gene expression, genome stability and cancer initiation that have important implications for understanding the molecular mechanism of cancer formation at the post- genomic level.

Investigation of the in vivo activity of CYP3A in Brazilian volunteers: comparison of midazolam and omeprazole as drug markers

Objective This study compares midazolam with omeprazole as marker drugs for the evaluation of CYP3A activity in nine healthy self-reported white Brazilian volunteers. Methods Omeprazole was also used to evaluate the CYP2C19 phenotype. The volunteers received p.o. 20 mg omeprazole, and blood samples were collected 3.5 h after drug administration. After a washout period of 10 days, the volunteers received p.o. 15 mg midazolam maleate, and serial blood samples were collected up to 6 h after administration of the drug. CYP2C19 was genotyped for the allelic variants CYP2C19*1, CYP2C19*2, CYP2C19*3, and CYP2C19*17. Analysis of omeprazole, hydroxyomeprazole, omeprazole sulfone, and midazolam in plasma was carried out by LC-MS/MS. Results The volunteers genotyped as CYP2C19*1*17, CYP2C19*17*17, CYP2C19*1*1 (n=8), or CYP2C19*17*2 (n=1) presented a median hydroxylation index (omeprazole/hydroxyomeprazole) of 1.35, indicating that all of them were extensive metabolizers of CYP2C19. The volunteers (n=9) presented a 0.12 log of the omeprazole/sulfone ratio and a median oral clearance of midazolam of 17.89 ml min(-1) kg(-1), suggesting normal CYP3A activity. Conclusions Orthogonal regression analysis between midazolam clearance and log of the plasma concentrations of the omeprazole/omeprazole sulfone ratio (R=-0.7544, P < 0.05) suggests that both midazolam and omeprazole can be used as markers of CYP3A activity in the population investigated.

Discordant Nadir GH After Oral Glucose and IGF-I Levels on Treated Acromegaly: Refining the Biochemical Markers of Mild Disease Activity

Biochemical markers for remission on acromegaly activity are controversial. We studied a subset of treated acromegalic patients with discordant nadir GH levels after oral glucose tolerance test (oGTT) and IGF-I values to refine the current consensus on acromegaly remission. We also compared GH results by two GH immunoassays. From a cohort of 75 treated acromegalic patients, we studied 13 patients who presented an elevated IGF-I despite post-oGTT nadir GH of <= 1 mu g/l. The 12-h daytime GH profile (GH-12 h), nadir GH after oGTT, and basal IGF-I levels were studied in patients and controls. Bland-Altman method showed high concordance between GH assays. Acromegalic patients showed higher mean GH-12 h values (0.71+/-0.36 vs. 0.31+/-0.28 mu g/l; p<0.05) and nadir GH after oGTT (0.48+/-0.32 vs. 0.097+/-0.002 mu g/l; p<0.05) as compared to controls. Nadir GH correlated with mean GH-12 h (r=0.92, p<0.05). The mean GH-12 h value from upper 95% CI of controls (0.54 mu g/l) would correspond to a theoretical normal nadir GH of <= 0.27 mu g/l. Patients with GH nadir <= 0.3 mu g/l had IGF-I between 100-130% ULNR (percentage of upper limit of normal range) and mean GH-12 h of 0.35+/-0.15, and patients with GH nadir >0.3 and <= 1 mu g/l had IGF-I >130% ULNR and mean GH-12 h of 0.93+/-0.24 mu g/l. Our data integrate daytime GH secretion, nadir GH after oGTT, and plasma IGF-I concentrations showing a continuum of mild residual activity in a subgroup of treated acromegaly with nadir GH values <= 1 mu g/l. The degree of increased IGF-I levels and nadir GH after oGTT are correlated with the subtle abnormalities of daytime GH secretion.

INHIBITION OF GUANYLYL CYCLASE RESTORES NEUTROPHIL MIGRATION AND MAINTAINS BACTERICIDAL ACTIVITY INCREASING SURVIVAL IN SEPSIS

Sepsis results from an overwhelming response to infection and is a major contributor to death in intensive care units worldwide. In recent years, we and others have shown that neutrophil functionality is impaired in sepsis. This correlates with sepsis severity and contributes to aggravation of sepsis by precluding bacterial clearance. Nitric oxide (NO) is a major contributor to the impairment of neutrophil function in sepsis. However, attempts to inhibit NO synthesis in sepsis resulted in increased death despite restoring neutrophil migration. This could be in part attributed to a reduction of the NO-dependent microbicidal activity of neutrophils. In sepsis, the beneficial effects resulting from the inhibition of soluble guanylyl cyclase (sGC), a downstream target of NO, have long been appreciated but poorly understood. However, the effects of sGC inhibition on neutrophil function in sepsis have never been addressed. In the present study, we show that TLR activation in human neutrophils leads to decreased chemotaxis, which correlated with chemotactic receptor internalization and increased G protein-coupled receptor kinase 2 expression, in a process involving the NO-sGC-protein kinase G axis. We also demonstrate that inhibition of sGC activity increased survival in a murine model of sepsis, which was paralleled by restored neutrophil migratory function and increased bacterial clearance. Finally, the beneficial effect of sGC inhibition could also be demonstrated in mice treated after the onset of sepsis. Our results suggest that the beneficial effects of sGC inhibition in sepsis could be at least in part attributed to a recovery of neutrophil functionality.

Phosphoinositide-3 Kinase gamma Activity Contributes to Sepsis and Organ Damage by Altering Neutrophil Recruitment

Rationale Sepsis is a leading cause of death in the intensive care unit, characterized by a systemic inflammatory response (SIRS) and bacterial infection, which can often induce multiorgan damage and failure. Leukocyte recruitment, required to limit bacterial spread, depends on phosphoinositide-3 kinase gamma (PI3K gamma) signaling in vitro; however, the role of this enzyme in polymicrobial sepsis has remained unclear. Objectives: This study aimed to determine the specific role of the kinase activity of PI3K gamma in the pathogenesis of sepsis and multiorgan damage. Methods. PI3K gamma wild-type, knockout, and kinase-dead mice were exposed to cecal ligation and perforation induced sepsis and assessed for survival; pulmonary, hepatic, and cardiovascular damage; coagulation derangements; systemic inflammation; bacterial spread; and neutrophil recruitment. Additionally, wild-type mice were treated either before or after the onset of sepsis with a PI3K gamma inhibitor and assessed for survival, neutrophil recruitment, and bacterial spread. Measurements and Main Results: Both genetic and pharmaceutical PI3K gamma kinase inhibition significantly improved survival, reduced multiorgan damage, and limited bacterial decompartmentalization, while modestly affecting SIRS. Protection resulted from both neutrophil-independent mechanisms, involving improved cardiovascular function, and neutrophil-dependent mechanisms, through reduced susceptibility to neutrophil migration failure during severe sepsis by maintaining neutrophil surface expression of the chemokine receptor, CXCR2. Furthermore, PI3K gamma pharmacological inhibition significantly decreased mortality and improved neutrophil migration and bacterial control, even when administered during established septic shock. Conclusions: This study establishes PI3K gamma as a key molecule in the pathogenesis of septic infection and the transition from SIRS to organ damage and identifies it as a novel possible therapeutic target.

Cannabidiol injected into the bed nucleus of the stria terminalis modulates baroreflex activity through 5-HT(1A) receptors

Cannabidiol (CBD) is a non-psychotomimetic constituent of the Cannabis sativa plant that inhibits behavioral and cardiovascular responses to aversive situations. facilitating 5-HT(1A)-mediated neurotransmission. Previous results from our group suggest that the bed nucleus of the stria terminalis (BNST) may be involved in CBD`s anti-aversive effects. To investigate whether the cardiovascular effects of the CBD could involve a direct drug effect on the BNST, we evaluated the effects of CBD microinjection into this structure on baroreflex activity. We also verified whether these effects were mediated by the activation of 5-HT(1A) receptors. Bilateral microinjection of CBD (60 nmol/100 nL) into the BNST increased the bradycardiac response to arterial pressure increases. However, no changes were observed in tachycardiac responses evoked by arterial pressure decreases. Pretreatment of the BNST with the selective 5-HT(1A) receptor antagonist WAY100635 (0.37 nmol/100 nL) prevented CBD effects on the baroreflex activity. Moreover, microinjection of the 5-HT(1A) receptor agonist 8-OH-DPAT (4 nmol/100 nL) caused effects that were similar to those observed after the microinjection of CBD, which were also blocked by pretreatment with WAY100635. In conclusion, the present studies show that the microinjection of CBD into the BNST has a facilitatory influence on the baroreflex response to blood pressure increases, acting through the activation of 5-HT(1A) receptors. (C) 2010 Elsevier Ltd. All rights reserved.

Antileishmanial activity of ruthenium(II)tetraammine nitrosyl complexes

The complexes trans-[Ru(NO)(NH(3))(4)L](X)(3) (X = BF(4)(-), PF(6)(-) or Cl(-) and L = N-heterocyclic ligands, P (OEt)(3), SO(3)(-2)), and [Ru(NO)Hedta)] were shown to exhibit IC(50pro) in the range of 36 (L = imN) to 5000 mu M (L = imC). The inhibitory effects of trans-[Ru(NO)(NH(3))(4)imN](BF(4))(3) and of the Angeli`s salt on the growth of the intramacrophage amastigote form studied were found to be similar while the trans-[Ru(NH(3))(4)imN(H(2)O)](2+) complex was found not to exhibit any substantial antiamastigote effect. The trans-[Ru(NO)(NH(3))(4)imN](BF(4))(3) compound, administered (500 nmol kg(-1) day(-1)) in BALB/c mice infected with Leishmania major, was found to exhibit a 98% inhibition on the parasite growth. Furthermore, this complex proved to be at least 66 times more efficient than glucantime in in vivo experiments. (C) 2010 Elsevier Masson SAS. All rights reserved.

Experimental Chemotherapy in Paracoccidioidomycosis Using Ruthenium NO Donor

Paracoccidioidomycosis (PCM) is a granulomatous disease caused by a dimorphic fungus, Paracoccidioides brasiliensis (Pb). To determine the influence of nitric oxide (NO) on this disease, we tested cis-[Ru(bpy)2(NO)SO3](PF6), ruthenium nitrosyl, which releases NO when activated by biological reducing agents, in BALB/c mice infected intravenously with Pb 18 isolate. In a previous study by our group, the fungicidal activity of ruthenium nitrosyl was evaluated in a mouse model of acute PCM, by measuring the immune cellular response (DTH), histopathological characteristics of the granulomatous lesions (and numbers), cytokines, and NO production. We found that cis-[Ru(bpy)2(NO)SO3](PF6)-treated mice were more resistant to infection, since they exhibited higher survival when compared with the control group. Furthermore, we observed a decreased influx of inflammatory cells in the lung and liver tissue of treated mice, possibly because of a minor reduction in fungal cell numbers. Moreover, an increased production of IL-10 and a decrease in TNF-alpha levels were detected in lung tissues of infected mice treated with cis-[Ru(bpy)2(NO)SO3](PF6). Immunohistochemistry showed that there was no difference in the number of VEGF- expressing cells. The animals treated with cis-[Ru(bpy)2(NO)SO3](PF6) showed high NO levels at 40 days after infection. These results show that NO is effectively involved in the mechanism that regulates the immune response in lung of Pb-infected mice. These data suggest that NO is a resistance factor during paracoccidioidomycosis by controlling fungal proliferation, influencing cytokine production, and consequently moderating the development of a strong inflammatory response.