(-)-CGP 12177 increases contractile force and hastens relaxation of human myocardial preparations through a propranolol-resistant state of the beta1-adrenoceptor

Two forms of the activated beta(1)-adrenoceptor exist, one that is stabilized by (-)-noradrenaline and is sensitive to blockade by (-)-propranolol and another which is stabilized by partial agonists such as (-)-pindolol and (-)-CGP 12177 but is relatively insensitive to (-)-propranolol. We investigated the effects of stimulation of the propranolol-resistant PI-adrenoceptor in the human heart. Myocardium from non-failing and failing human hearts were set up to contract at 1 Hz. In right atrium from non-ailing hearts in the presence of 200 nM (-)-propranolol, (-)-CGP 12177 caused concentration-dependent increases in contractile force (-logEC(50)[M] 7.3+/-0.1, E-max 23+/-1% relative to maximal (-)-isoprenaline stimulation of beta(1)- and beta(2)-adrenoceptors, n=86 patients), shortening of the time to reach peak force (-logEC(50)[M] 7.4+/-0.1, E-max 37+/-5%, n=61 patients) and shortening of the time to reach 50% relaxation (t(50%), -logEC(50)[M] 7.3+/-0.1, E-max 33+/-2%, n=61 patients). The potency and maxima of the positive inotropic effects were independent of Ser49Gly- and Gly389Arg-beta(1)-adrenoceptor polymorphisms but were potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (-logEC(50)[M] 7.7+/-0.1, E-max 68+/-6%, n=6 patients, P

Cardiac involvement in human rabies: case report

A case of human rabies with cardiac involvement and viral inclusion bodies in the heart is presented. The Negri bodies were found in the Schwann cells of the right epicardial atrium, with secondary mononuclear cells inflammation. In the myocardium, an interstitial edema, proliferation of Anitschkov and rare mononuclear inflammatory cells were seen. There was no relevant cardiovascular signs or symptoms. The rarity of histological descriptions of Negri bodies in the heart is stressed, as well as the importance of cardiac involvement as a potential complication for cases with life prolonged by intensive care units, or in the end-stages of the disease.

Sarcomeric M-band alterations as a marker for human dilated cardiomyopathy

Purpose: The M-band is an important cytoskeletal structure in the centre of the sarcomere, believed to cross-link the thick filament lattice. Its main components are three closely related modular proteins from the myomesin gene family: Myomesin, M-protein and myomesin-3. Each muscle is characterized by its unique M-band protein composition, depending on the contractile parameters of a particular fiber. To investigate the role of the M-band in one of the most relevant and clinically increasing cardiac diseases, we analyzed the expression of myomesin proteins in dilated cardiomyopathy (DCM).Methods: In a previous study we analyzed mouse models suffering from DCM, demonstrating that the embryonic heart specific EH-myomesin splicing isoform was up-regulated directly corresponding to the degree of cardiac dysfunction and ventricular dilation. Based on this study, human ventricular and atrial samples (n=32) were obtained during heart surgery after informed consent and approval by an institutional review board. Patients were aged 30-70 years and suffered from dilated cardiomyopathy (DCM;n=13), Hypertrophic Cardiomyopathy (HCM;n=10) or served as controls (n=9). Patients suffering from DCM or HCM were in endstage heart-failure (NYHA III-IV) and either underwent heart transplantation or Left Ventricular Assist Device (LVAD) implantation. Heart samples from patients who underwent valve surgery or congenital heart surgery served as controls. Heart Samples were analyzed using RT-PCR, Western blot, and immunofluorescence.Results: By investigating the expression pattern of myomesins, we found that DCM is accompanied by specific M-band alterations, which were more pronounced in ventricular samples compared to the atrium. Changes in the amounts of different myomesins during DCM occurred in a cell-specific manner, leading to a higher heterogeneity of the cytoskeleton in cardiomyocytes through the myocardial wall with some cells switching completely to an embryonic phenotype.Conclusions: Here we present that the embryonic heart specific EH-myomesin isoform is up-regulated in human DCM. The alterations of the M-band protein composition might be part of a general adaptation of the sarcomeric cytoskeleton to unfavorable working conditions in the failing heart and may modify the mechanical properties of the cardiomyocytes. We suggest that the upregulation of EH-myomesin might play a pivotal role in DCM and might support classical imagingas a novel sarcomeric marker for this disease.

Artificial muscle: the human chimera is the future.

Severe heart failure and cerebral stroke are broadly associated with the impairment of muscular function that conventional treatments struggle to restore. New technologies enable the construction of "smart" materials that could be of great help in treating diseases where the main problem is muscle weakness. These materials "behave" similarly to biological systems, because the material directly converts energy, for example electrical energy into movement. The extension and contraction occur silently like in natural muscles. The real challenge is to transfer this amazing technology into devices that restore or replace the mechanical function of failing muscle. Cardiac assist devices based on artificial muscle technology could envelope a weak heart and temporarily improve its systolic function, or, if placed on top of the atrium, restore the atrial kick in chronic atrial fibrillation. Artificial sphincters could be used to treat urinary incontinence after prostatectomy or faecal incontinence associated with stomas. Artificial muscles can restore the ability of patients with facial paralysis due to stroke or nerve injury to blink. Smart materials could be used to construct an artificial oesophagus including peristaltic movement and lower oesophageal sphincter function to replace the diseased oesophagus thereby avoiding the need for laparotomy to mobilise stomach or intestine. In conclusion, in the near future, smart devices will integrate with the human body to fill functional gaps due to organ failure, and so create a human chimera.

Characterization of a voltage-gated K+ channel beta subunit expressed in human heart.

Voltage-gated K+ channels are important modulators of the cardiac action potential. However, the correlation of endogenous myocyte currents with K+ channels cloned from human heart is complicated by the possibility that heterotetrameric alpha-subunit combinations and function-altering beta subunits exist in native tissue. Therefore, a variety of subunit interactions may generate cardiac K+ channel diversity. We report here the cloning of a voltage-gated K+ channel beta subunit, hKv beta 3, from adult human left ventricle that shows 84% and 74% amino acid sequence identity with the previously cloned rat Kv beta 1 and Kv beta 2 subunits, respectively. Together these three Kv beta subunits share > 82% identity in the carboxyl-terminal 329 aa and show low identity in the amino-terminal 79 aa. RNA analysis indicated that hKv beta 3 message is 2-fold more abundant in human ventricle than in atrium and is expressed in both healthy and diseased human hearts. Coinjection of hKv beta 3 with a human cardiac delayed rectifier, hKv1.5, in Xenopus oocytes increased inactivation, induced an 18-mV hyperpolarizing shift in the activation curve, and slowed deactivation (tau = 8.0 msec vs. 35.4 msec at -50 mV). hKv beta 3 was localized to human chromosome 3 by using a human/rodent cell hybrid mapping panel. These data confirm the presence of functionally important K+ channel beta subunits in human heart and indicate that beta-subunit composition must be accounted for when comparing cloned channels with endogenous cardiac currents.

Carvedilol blocks beta(2)- more than beta(1)-adrenoceptors in human heart

Objective: To understand the basis of the effectiveness of carvedilol in heart failure by determining its specific properties at human heart and beta(2)-adrenoceptors. Methods: The positive inotropic effects of noradrenaline (in the presence of the beta(2)-selective antagonist ICI118551) and adrenaline (in the presence of the beta(1)-selective antagonist CGP20712), mediated through beta(1)- and beta(2)-adrenoceptors, respectively, were investigated in atrial and ventricular trabeculae. The patch-clamp technique was used to investigate effects of noradrenaline and adrenaline on L-type Ca2+ current in human atrial myocytes. Results: Carvedilol was a 13-fold more potent competitive antagonist of the effects of adrenaline at 1 2-adrenoceptors (-logK(B) = 10.13 +/- 0.08) than of noradrenaline at beta(1)-adrenoceptors (-logK(B) = 9.02 +/- 0.07) in human right atrium. Chronic carvedilol treatment of patients with non-terminal heart failure reduced the inotropic sensitivity of atrial trabeculae to noradrenaline and adrenaline 5.6-fold and 91.2-fold, respectively, compared to beta(1)-blocker-treated patients, consistent with persistent preferential blockade of beta(2)-adrenoceptors. In terminal heart failure carvedilol treatment reduced 1.8-fold and 25.1-fold the sensitivity of right ventricular trabeculae to noradrenaline and adrenaline, respectively, but metoprolol treatment did not reduce the sensitivity to the catecholamines. Increases of current (I-Ca,I-L) produced by noradrenaline and adrenaline were not different in atrial myocytes obtained from non-terminal heart failure patients treated with metoprolol or carvedilol, consistent with dissociation of both beta-blockers from the receptors. Conclusions: Carvedilol blocks human cardiac beta(2)-adrenoceptors more than beta(1)-adrenoceptors, thereby conceivably contributing to the beneficial effects in heart failure. The persistent blockade of beta-adrenoceptors is attributed to accumulation of carvedilol in cardiac tissue. (c) 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Prognostic Value Of Dna And Mrna E6/e7 Of Human Papillomavirus In The Evolution Of Cervical Intraepithelial Neoplasia Grade 2.

This study aimed at evaluating whether human papillomavirus (HPV) groups and E6/E7 mRNA of HPV 16, 18, 31, 33, and 45 are prognostic of cervical intraepithelial neoplasia (CIN) 2 outcome in women with a cervical smear showing a low-grade squamous intraepithelial lesion (LSIL). This cohort study included women with biopsy-confirmed CIN 2 who were followed up for 12 months, with cervical smear and colposcopy performed every three months. Women with a negative or low-risk HPV status showed 100% CIN 2 regression. The CIN 2 regression rates at the 12-month follow-up were 69.4% for women with alpha-9 HPV versus 91.7% for other HPV species or HPV-negative status (P < 0.05). For women with HPV 16, the CIN 2 regression rate at the 12-month follow-up was 61.4% versus 89.5% for other HPV types or HPV-negative status (P < 0.05). The CIN 2 regression rate was 68.3% for women who tested positive for HPV E6/E7 mRNA versus 82.0% for the negative results, but this difference was not statistically significant. The expectant management for women with biopsy-confirmed CIN 2 and previous cytological tests showing LSIL exhibited a very high rate of spontaneous regression. HPV 16 is associated with a higher CIN 2 progression rate than other HPV infections. HPV E6/E7 mRNA is not a prognostic marker of the CIN 2 clinical outcome, although this analysis cannot be considered conclusive. Given the small sample size, this study could be considered a pilot for future larger studies on the role of predictive markers of CIN 2 evolution.

Integrated Proteomics Identified Up-regulated Focal Adhesion-mediated Proteins In Human Squamous Cell Carcinoma In An Orthotopic Murine Model.

Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by combining an orthotopic murine model of oral squamous cell carcinoma (OSCC), mass spectrometry-based proteomics and biological network analysis. Our results indicated the up-regulation of proteins involved in actin cytoskeleton organization and cell-cell junction assembly events and their expression was validated in human OSCC tissues. In addition, the functional relevance of talin-1 in OSCC adhesion, migration and invasion was demonstrated. Taken together, this study identified specific processes deregulated in oral cancer and provided novel refined OSCC-targeting molecules.

Hypertrophic Adenoid Is A Major Infection Site Of Human Bocavirus 1.

Human bocavirus 1 (HBoV1) is associated with respiratory infections worldwide, mainly in children. Similar to other parvoviruses, it is believed that HBoV1 can persist for long periods of time in humans, probably through maintaining concatemers of the virus single-stranded DNA genome in the nuclei of infected cells. Recently, HBoV-1 was detected in high rates in adenoid and palatine tonsils samples from patients with chronic adenotonsillar diseases, but nothing is known about the virus replication levels in those tissues. A 3-year prospective hospital-based study was conducted to detect and quantify HBoV1 DNA and mRNAs in samples of the adenoids (AD), palatine tonsils (PT), nasopharyngeal secretions (NPS), and peripheral blood (PB) from patients undergoing tonsillectomy for tonsillar hypertrophy or recurrent tonsillitis. HBoV1 was detected in 25.3% of the AD samples, while the rates of detection in the PT, NPS, and PB samples were 7.2%, 10.5%, and 1.7%, respectively. The viral loads were higher in AD samples, and 27.3% of the patients with HBoV had mRNA detectable in this tissue. High viral loads and detectable mRNA in the AD were associated with HBoV1 detection in the other sample sites. The adenoids are an important site of HBoV1 replication and persistence in children with tonsillar hypertrophy. The adenoids contain high HBoV1 loads and are frequently positive for HBoV mRNA, and this is associated with the detection of HBoV1 in secretions.

The Effect Of Celastrol, A Triterpene With Antitumorigenic Activity, On Conformational And Functional Aspects Of The Human 90kda Heat Shock Protein Hsp90α, A Chaperone Implicated In The Stabilization Of The Tumor Phenotype.

Hsp90 is a molecular chaperone essential for cell viability in eukaryotes that is associated with the maturation of proteins involved in important cell functions and implicated in the stabilization of the tumor phenotype of various cancers, making this chaperone a notably interesting therapeutic target. Celastrol is a plant-derived pentacyclic triterpenoid compound with potent antioxidant, anti-inflammatory and anticancer activities; however, celastrol's action mode is still elusive. In this work, we investigated the effect of celastrol on the conformational and functional aspects of Hsp90α. Interestingly, celastrol appeared to target Hsp90α directly as the compound induced the oligomerization of the chaperone via the C-terminal domain as demonstrated by experiments using a deletion mutant. The nature of the oligomers was investigated by biophysical tools demonstrating that a two-fold excess of celastrol induced the formation of a decameric Hsp90α bound throughout the C-terminal domain. When bound, celastrol destabilized the C-terminal domain. Surprisingly, standard chaperone functional investigations demonstrated that neither the in vitro chaperone activity of protecting against aggregation nor the ability to bind a TPR co-chaperone, which binds to the C-terminus of Hsp90α, were affected by celastrol. Celastrol interferes with specific biological functions of Hsp90α. Our results suggest a model in which celastrol binds directly to the C-terminal domain of Hsp90α causing oligomerization. However, the ability to protect against protein aggregation (supported by our results) and to bind to TPR co-chaperones are not affected by celastrol. Therefore celastrol may act primarily by inducing specific oligomerization that affects some, but not all, of the functions of Hsp90α. To the best of our knowledge, this study is the first work to use multiple probes to investigate the effect that celastrol has on the stability and oligomerization of Hsp90α and on the binding of this chaperone to Tom70. This work provides a novel mechanism by which celastrol binds Hsp90α.

Lawsonia Inermis-mediated Synthesis Of Silver Nanoparticles: Activity Against Human Pathogenic Fungi And Bacteria With Special Reference To Formulation Of An Antimicrobial Nanogel.

Lawsonia inermis mediated synthesis of silver nanoparticles (Ag-NPs) and its efficacy against Candida albicans, Microsporum canis, Propioniabacterium acne and Trichophyton mentagrophytes is reported. A two-step mechanism has been proposed for bioreduction and formation of an intermediate complex leading to the synthesis of capped nanoparticles was developed. In addition, antimicrobial gel for M. canis and T. mentagrophytes was also formulated. Ag-NPs were synthesized by challenging the leaft extract of L. inermis with 1 mM AgNO₃. The Ag-NPs were characterized by Ultraviolet-Visible (UV-Vis) spectrophotometer and Fourier transform infrared spectroscopy (FTIR). Transmission electron microscopy (TEM), nanoparticle tracking and analysis sytem (NTA) and zeta potential was measured to detect the size of Ag-NPs. The antimicrobial activity of Ag-NPs was evaluated by disc diffusion method against the test organisms. Thus these Ag-NPs may prove as a better candidate drug due to their biogenic nature. Moreover, Ag-NPs may be an answer to the drug-resistant microorganisms.

Overlapped Sequence Types (sts) And Serogroups Of Avian Pathogenic (apec) And Human Extra-intestinal Pathogenic (expec) Escherichia Coli Isolated In Brazil.

Avian pathogenic Escherichia coli (APEC) strains belong to a category that is associated with colibacillosis, a serious illness in the poultry industry worldwide. Additionally, some APEC groups have recently been described as potential zoonotic agents. In this work, we compared APEC strains with extraintestinal pathogenic E. coli (ExPEC) strains isolated from clinical cases of humans with extra-intestinal diseases such as urinary tract infections (UTI) and bacteremia. PCR results showed that genes usually found in the ColV plasmid (tsh, iucA, iss, and hlyF) were associated with APEC strains while fyuA, irp-2, fepC sitDchrom, fimH, crl, csgA, afa, iha, sat, hlyA, hra, cnf1, kpsMTII, clpVSakai and malX were associated with human ExPEC. Both categories shared nine serogroups (O2, O6, O7, O8, O11, O19, O25, O73 and O153) and seven sequence types (ST10, ST88, ST93, ST117, ST131, ST155, ST359, ST648 and ST1011). Interestingly, ST95, which is associated with the zoonotic potential of APEC and is spread in avian E. coli of North America and Europe, was not detected among 76 APEC strains. When the strains were clustered based on the presence of virulence genes, most ExPEC strains (71.7%) were contained in one cluster while most APEC strains (63.2%) segregated to another. In general, the strains showed distinct genetic and fingerprint patterns, but avian and human strains of ST359, or ST23 clonal complex (CC), presented more than 70% of similarity by PFGE. The results demonstrate that some zoonotic-related STs (ST117, ST131, ST10CC, ST23CC) are present in Brazil. Also, the presence of moderate fingerprint similarities between ST359 E. coli of avian and human origin indicates that strains of this ST are candidates for having zoonotic potential.

Impact Of Pharmacist Interventions On Drug-related Problems And Laboratory Markers In Outpatients With Human Immunodeficiency Virus Infection.

Substantial complexity has been introduced into treatment regimens for patients with human immunodeficiency virus (HIV) infection. Many drug-related problems (DRPs) are detected in these patients, such as low adherence, therapeutic inefficacy, and safety issues. We evaluated the impact of pharmacist interventions on CD4+ T-lymphocyte count, HIV viral load, and DRPs in patients with HIV infection. In this 18-month prospective controlled study, 90 outpatients were selected by convenience sampling from the Hospital Dia-University of Campinas Teaching Hospital (Brazil). Forty-five patients comprised the pharmacist intervention group and 45 the control group; all patients had HIV infection with or without acquired immunodeficiency syndrome. Pharmaceutical appointments were conducted based on the Pharmacotherapy Workup method, although DRPs and pharmacist intervention classifications were modified for applicability to institutional service limitations and research requirements. Pharmacist interventions were performed immediately after detection of DRPs. The main outcome measures were DRPs, CD4+ T-lymphocyte count, and HIV viral load. After pharmacist intervention, DRPs decreased from 5.2 (95% confidence interval [CI] =4.1-6.2) to 4.2 (95% CI =3.3-5.1) per patient (P=0.043). A total of 122 pharmacist interventions were proposed, with an average of 2.7 interventions per patient. All the pharmacist interventions were accepted by physicians, and among patients, the interventions were well accepted during the appointments, but compliance with the interventions was not measured. A statistically significant increase in CD4+ T-lymphocyte count in the intervention group was found (260.7 cells/mm(3) [95% CI =175.8-345.6] to 312.0 cells/mm(3) [95% CI =23.5-40.6], P=0.015), which was not observed in the control group. There was no statistical difference between the groups regarding HIV viral load. This study suggests that pharmacist interventions in patients with HIV infection can cause an increase in CD4+ T-lymphocyte counts and a decrease in DRPs, demonstrating the importance of an optimal pharmaceutical care plan.

Carotenoids Are Effective Inhibitors Of In Vitro Hemolysis Of Human Erythrocytes, As Determined By A Practical And Optimized Cellular Antioxidant Assay.

β-Carotene, zeaxanthin, lutein, β-cryptoxanthin, and lycopene are liposoluble pigments widely distributed in vegetables and fruits and, after ingestion, these compounds are usually detected in human blood plasma. In this study, we evaluated their potential to inhibit hemolysis of human erythrocytes, as mediated by the toxicity of peroxyl radicals (ROO•). Thus, 2,2'-azobis (2-methylpropionamidine) dihydrochloride (AAPH) was used as ROO• generator and the hemolysis assay was carried out in experimental conditions optimized by response surface methodology, and successfully adapted to microplate assay. The optimized conditions were verified at 30 × 10(6) cells/mL, 17 mM of AAPH for 3 h, at which 48 ± 5% of hemolysis was achieved in freshly isolated erythrocytes. Among the tested carotenoids, lycopene (IC(50) = 0.24 ± 0.05 μM) was the most efficient to prevent the hemolysis, followed by β-carotene (0.32 ± 0.02 μM), lutein (0.38 ± 0.02 μM), and zeaxanthin (0.43 ± 0.02 μM). These carotenoids were at least 5 times more effective than quercetin, trolox, and ascorbic acid (positive controls). β-Cryptoxanthin did not present any erythroprotective effect, but rather induced a hemolytic effect at the highest tested concentration (3 μM). These results suggest that selected carotenoids may have potential to act as important erythroprotective agents by preventing ROO•-induced toxicity in human erythrocytes.

Propylthiouracil Quantification In Human Plasma By High-performance Liquid Chromatography Coupled With Electrospray Tandem Mass Spectrometry: Application In A Bioequivalence Study.

A rapid, sensitive and specific method for quantifying propylthiouracil in human plasma using methylthiouracil as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid-liquid extraction using an organic solvent (ethyl acetate). The extracts were analyzed by high performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-MS/MS) in negative mode (ES-). Chromatography was performed using a Phenomenex Gemini C18 5μm analytical column (4.6mm×150mm i.d.) and a mobile phase consisting of methanol/water/acetonitrile (40/40/20, v/v/v)+0.1% of formic acid. For propylthiouracil and I.S., the optimized parameters of the declustering potential, collision energy and collision exit potential were -60 (V), -26 (eV) and -5 (V), respectively. The method had a chromatographic run time of 2.5min and a linear calibration curve over the range 20-5000ng/mL. The limit of quantification was 20ng/mL. The stability tests indicated no significant degradation. This HPLC-MS/MS procedure was used to assess the bioequivalence of two propylthiouracil 100mg tablet formulations in healthy volunteers of both sexes in fasted and fed state. The geometric mean and 90% confidence interval CI of Test/Reference percent ratios were, without and with food, respectively: 109.28% (103.63-115.25%) and 115.60% (109.03-122.58%) for Cmax, 103.31% (100.74-105.96%) and 103.40% (101.03-105.84) for AUClast. This method offers advantages over those previously reported, in terms of both a simple liquid-liquid extraction without clean-up procedures, as well as a faster run time (2.5min). The LOQ of 20ng/mL is well suited for pharmacokinetic studies. The assay performance results indicate that the method is precise and accurate enough for the routine determination of the propylthiouracil in human plasma. The test formulation with and without food was bioequivalent to reference formulation. Food administration increased the Tmax and decreased the bioavailability (Cmax and AUC).