Elba Carrillo

Diagnostic Problems Caused by HBsAg Mutants – A Consensus Report of an Expert Meeting

A panel of 16 experts from 9 countries convened on April 14 at Schloss Reinhartshausen near Wiesbaden in Germany, to discuss the diagnostic significance of mutants, variants and genotypes of hepatitis B virus (HBV). Since the description of Australia antigen in 1965 and the subsequent observation that it was the envelope of the HBV and now designated hepatitis B surface antigen (HBsAg), this lipoprotein has been a mainstay in the diagnosis of HBV infections. HBsAg tests are used routinely in the diagnosis of acute and chronic liver disease, the screening of blood or organ donors and the surveillance of persons at risk to acquire or to transmit HBV. Current immunoassays for HBsAg are very specific and sensitive (both 199%) and are usually able to detect !0.5 ng HBsAg/ml serum. Their performance is validated in extensive trials before licensing and their detection limit is assayed with an International Standard for HBsAg. An immanent problem of virology is the variability of viruses. Due to the low fidelity of the viral nucleic acid polymerases and the high replication rates, virtually all nucleotide positions of a viral genome can be mutated within a relatively short time. However, the viability of the virus and its adaptation to the host allow the selection and outgrowth of only a limited number of mutants. The survival strategy of HBV in the population is mainly based on induction of immune tolerance and persistence of high viremia. In this state of infection the existing viral genome is favored whereas mutants are less fit and selected against or may be subject to an immune reaction and preferably eliminated. In fact, most of the HBsAg carriers in Germany have a very similar S gene sequence. However, under selective pressure the virus can express many different viable HBsAg mutants. Summary of the Presentations

Posttranscriptional regulation of the β2-subunit of cardiac L-type Ca2+ channels by MicroRNAs during long-term exposure to isoproterenol in rats.

Introduction and Methods The effects of long-term &bgr;-adrenergic administration on the expression levels of the cardiac L-type Ca2+ channel &bgr;2 subunit, which regulates channel trafficking and function, were characterized in adult rats. Results Systemic administration of isoproterenol (150 mg·kg·h−1) for 2 d led to a 50% increase in the ventricular wet weight-to-body weight ratio (mg/g) and of more than two-fold in the expression of actin protein. In contrast, &bgr;2 subunit protein levels decreased (down to 49%), while mRNA levels remained unchanged. Furthermore, levels of microRNAs (miRs), including miR-21 and miR-132, were upregulated (7.2 and 7.9 fold, respectively). Transfection of these miRs into HEK293 cells attenuated expression of a luciferase reporter gene controlled by a conserved 3′-untranslated region (UTR) of the &bgr;2 subunit (down to 67% and 56%, respectively). Systemic administration of isoproterenol also led to briefer intracellular Ca2+ transients during action potentials measured in isolated cardiomyocytes (down to 65%). Conclusion These results suggest that cardiac L-type Ca2+ channel &bgr;2 subunit protein expression may be downregulated by miRs in response to long-term activation of &bgr;-adrenergic signaling, possibly as an adaptive response in cardiac hypertrophy and sustained &bgr;-adrenergic states.

The β1a subunit regulates the functional properties of adult frog and mouse L-type Ca2+ channels of skeletal muscle

The β1a subunit, one of the auxiliary subunits of CaV1.1 channels, was expressed in COS‐1 cells, purified by electroelution and electrodialysis techniques and identified by Western blot using monoclonal antibodies. The purified β1a subunit strongly interacted in vitro with the alpha interaction domain (AID) of CaV1.1 channels. The actions of the purified β1a subunit on CaV1.1 channel currents were assessed in whole cell voltage clamp experiments performed in vesicles derived from frog and mouse adult skeletal muscle plasma membranes. L‐type inward currents were recorded in solutions containing Ba2+ (IBa). Values of peak IBa were doubled by the β1a subunit in frog and mouse muscle vesicles and the amplitude of the slow component of tail currents was greatly increased. The actions of the β1a subunit on CaV1.1 channel currents reached a steady state within 20 min. The β1a subunit had no effect on the time courses of activation or inactivation of IBa or shifted the current‐voltage relation. Non‐linear capacitive currents were recorded in solutions that contained mostly impermeant ions. Charge movement depended on voltage with average Boltzmann parameters: Qmax+ 28.0 ± 6.6 nC μF−1, V+−58.0 ± 2.0 mV and k+ 15.3± 1.1 mV (n= 24). In the presence of the β1a subunit, these parameters remained unchanged: Qmax+ 29.8 ± 3.5 nC μF−1, V+−54.5 ± 2.2 mV and k+ 16.4± 1.3 mV (n= 21). Overall, the work describes a novel preparation to explore in situ the role of the β1a subunit on the function of adult CaV1.1 channels.

Pharmacological preconditioning by diazoxide downregulates cardiac L-type Ca2+ channels

BACKGROUND AND PURPOSE Pharmacological preconditioning (PPC) with mitochondrial ATP‐sensitive K+ (mitoKATP) channel openers such as diazoxide, leads to cardioprotection against ischaemia. However, effects on Ca2+ homeostasis during PPC, particularly changes in Ca2+ channel activity, are poorly understood. We investigated the effects of PPC on cardiac L‐type Ca2+ channels.

Specific in vitro interaction between papillomavirus E2 proteins and TBP-associated factors.

The bovine and human papillomavirus (BPV/HPV) E2 proteins bind specifically to palindromic sequences ACCGN4CGGT that are concentrated within the viral long control region, where they regulate viral oncogene transcription. E2 can activate viral promoters over relatively large distances within the viral genome and was shown to cooperate with a number of cellular transcription factors. Transcriptional activator proteins, such as E2, are thought to act, at least in part, by influencing the assembly and/or stability of preinitiation complexes and it has been suggested that the transcription factor IID, composed by the TATA-binding protein (TBP) and numerous TBP-associated factors (TAFs), is a possible target of this important viral protein. In this paper, we demonstrate that E2 proteins associate in vitro with several TAFs, in particular with TAFII250 and TAFII80. In addition, we observed that the association of TAFII250 with BPV1 E2 is stronger than with HPV18 E2 and that the carboxy terminal domain of both viral proteins is involved in this interaction. On the other hand, TAFII80 binds with similar strength to both E2 proteins through their amino terminal region. These observations may help to explain the different behavior of bovine and human E2 proteins, since BPV E2 is a stronger transcriptional activator than HPV18 E2.

Regulation of Muscle Cav1.1 Channels by Long-term Depolarization Involves Proteolysis of the α1s Subunit

The effects of long-term depolarization on frog skeletal muscle Cav1.1 channels were assessed. Voltage-clamp and Western-blot experiments revealed that long-term depolarization brings about a drastic reduction in the amplitude of currents flowing through Cav1.1 channels and in the levels of the α1s subunit, the main subunit of muscle L-type channels. The decline of both phenomena was prevented by the action of the protease inhibitors E64 (50 μM) and leupeptin (50 μM). In contrast, long-term depolarization had no effect on β1, the auxiliary subunit of α1s. The levels of mRNAs coding the α1s and the β1 subunits were measured by RNase protection assays. Neither the content of the α1s nor the β1 subunit mRNAs were affected by long-term depolarization, indicating that the synthesis of Cav1.1 channels remained unaffected. Taken together, our experiments suggest that the reduction in the amplitude of membrane currents and in the α1s subunit levels is caused by increased degradation of this subunit by a Ca2+-dependent protease.

MiR-132 Regulates Rem Expression in Cardiomyocytes During Long-Term β-Adrenoceptor Agonism

Aims: To characterize the effects of long-term β-adrenergic receptor stimulation on Rem protein and mRNA expression in rat heart and possible involvement of miR-132. Methods: Adult rats were treated with isoproterenol (ISO, 150 µg.kg.h-1) for 2 d and Rem, miR-132, and α1c (the principal subunit of Cav1.2 channels) were measured at protein and mRNA levels with western blot and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) experiments, respectively. Ca2+ currents and intracellular Ca2+ signals were evaluated in isolated cardiomyocytes. Results: Systemic administration of ISO led to decreases in Rem protein and mRNA levels (down to 49%). Furthermore, levels of the microRNAs (miRs) miR-132 and miR-214 were upregulated 5- and 9-fold, respectively. Transfection of miR-132, but not miR-214, into HEK293 cells reduced the expression of a luciferase reporter gene controlled by a conserved 3´-untranslated region (UTR) of Rem by half. Chronic ISO administration also led to a 25% decrease in the amplitude of peak L-type Ca2+ currents, a 40% decrease in α1c subunit protein abundance at the membrane level, and a 60% decrease in expression of α1c channel subunit mRNA. Conclusions: These results suggest that Rem expression is down-regulated posttranscriptionally by miR-132 in response to long-term activation of β-adrenergic signaling, but this down-regulation does not produce a larger Ca2+ influx through Cav1.2 channels.

An embryonic heart cell line is susceptible to dengue virus infection.

Dengue virus (DENV) is the causative agent of dengue fever. In recent years, patients with more severe form of the disease with acute heart failure or progression to cardiogenic shock and death have been reported. However, the pathogenesis of myocardial lesions and susceptibility of cardiomyocytes to DENV infection have not been evaluated. Under this perspective, the susceptibility of the myoblast cell line H9c2, obtained from embryonic rat heart, to DENV infection was analyzed. Our findings indicate that H9c2 cells are susceptible to the infection with the four DENV serotypes. Moreover, virus translation/replication and viral production in this cell line is as efficient as in other susceptible cell lines, supporting the idea that DENV may target heart cells as evidenced by infection of H9c2 cells. This cell line may thus represent an excellent model for the study and characterization of cardiac physiopathology in DENV infection.

Parvalbumin is overexpressed in the late phase of pharmacological preconditioning in skeletal muscle

Pharmacological preconditioning (PPC) with mitochondrial ATP-sensitive K(+) channel openers such as diazoxide, provides protection against ischemia in cardiac muscle, skeletal muscle, and other tissues. Effects on Ca(2+) homeostasis during the late phase of PPC have been described in cardiomyocytes, but no information is available regarding intracellular Ca(2+) changes in skeletal muscle fibers during late PPC. Intracellular Ca(2+) signals were measured in single fibers of adult mouse skeletal muscle, with fluorescent probes, 48 h after the administration of diazoxide. Parvalbumin levels in the myofibers were quantitated by Western blot. Diazoxide induction of late PPC was confirmed by partial protection of muscles from peroxide-induced damage. Late PPC was associated with a significant decrease in the duration of Ca(2+) signals during single twitches and tetanus with no changes in peak values. This effect was prevented by the reactive oxygen species (ROS) scavenger tiron. Late PPC was accompanied by a 30% increase in parvalbumin levels, and this effect was also blocked by tiron. Our data show, for the first time, a role of parvalbumin in late PPC in skeletal muscle.