Patterns of expression of TSC-22 protein in astrocytic gliomas.

AIM: To evaluate expression patterns of protein product of putative tumor suppressor gene TSC-22 in human astrocytic tumors by immunohistochemical approach. METHODS: Plasmid pET-23d-TSC22 was constructed for the expression of human TSC-22 protein in bacterial system, and polyclonal rabbit antibodies against recombinant TSC-22 were produced. Immunohistochemical analysis of TSC-22 and GFAP expression with the use of anti-human-TSC-22- and anti-human-GFAP-antibodies was performed on histological slides of astrocytic tumors. RESULTS: Immunohistochemical analysis has shown that the number of cells expressing TSC-22 was significantly lower in glioblastoma tissues than that in diffuse astrocytoma. Double immunohistochemical staining of astrocytic tumors using anti-human-TSC-2- and anti-human-GFAP-antibodies showed that both TSC-22 and GFAP expression is co-localized in astrocytes. CONCLUSION: TSC-22 protein is expressed in astrocytes, but not in macrophage/microglial cells. In more aggressive forms of astrocytic tumors decreased expression of TSC-22 mRNA correlates with its lowered expression on protein level.

Testosterone-mediated modulation of HERG blockade by proarrhythmic agents.

Diverse drugs from many therapeutic classes exert cardiotoxic side effects by inducing torsades de pointes (TdP), a life threatening cardiac arrhythmia, which often results from drug interaction with HERG (human ether-a-go-go related gene) encoded K(+) channels, that generate an I(Kr) component of the delayed rectifier cardiac K(+) current. Men are known to be at a lower risk for drug-induced TdP than women suggesting a role of sex steroid hormones, androgens and estrogens, in modulation of drug sensitivity of cardiac K(+) channels, particularly those encoded by HERG. Here by using neuroleptic agents haloperidol, pimozide, and fluspirilene, all of which can induce TdP, and a steroid hormone-sensitive system Xenopus oocytes for HERG channels expression we show that testosterone is able to reduce HERG-blocking potency of neuroleptics. Haloperidol, pimozide, and fluspirilene inhibited HERG current with IC(50) of 1.36, 1.74, and 2.34 microM, and maximal block of 73%, 76% and 65%, respectively. The action of these neuroleptics was voltage-dependent, most consistent with an open-channel blocking mechanism. Pretreatment of HERG-expressing oocytes with 1 microM testosterone increased the IC(50) values to 2.73, 2.08, and 5.04 microM, reduced the maximal block to 65%, 59%, and 64%, and strongly diminished voltage-dependence of the blockade. Testosterone treatment per se produced about a 35% reduction of HERG current compared with untreated oocytes. Our data suggest that androgens may protect against the arrhythmogenic actions of some cardiotoxic drugs.

A novel molecular determinant for cAMP-dependent regulation of the frog heart Na+-Ca2+ exchanger.

Na+-Ca2+ exchanger is one of the major sarcolemmal Ca2+ transporters of cardiac myocytes. In frog ventricular myocytes the exchanger is regulated by isoproterenol via a beta-adrenoreceptor/adenylate-cyclase/cAMPdependent signaling pathway providing a molecular mechanism for the relaxant effect of the hormone. Here, we report on the presence of a novel exon of 27-base pair insertion, which generates a nucleotide binding motif (P-loop) in the frog cardiac Na+-Ca2+ exchanger. To examine the functional role of this motif, we constructed a full-length frog heart Na+-Ca2+ exchanger cDNA (fNCX1a) containing this exon. The functional expression of fNCX1a in oocytes showed characteristic voltage dependence, divalent (Ni2+, Cd2+) inhibition, and sensitivity to cAMP in a manner similar to that of native exchanger in frog myocytes. In oocytes expressing the dog heart NCX1 or the frog mutant (DeltafNCX1a) lacking the 9-amino acid exon, cAMP failed to regulate Na+-dependent Ca2+ uptake. We suggest that this motif is responsible for the observed cAMP-dependent functional differences between the frog and the mammalian hearts.

Glass-funnel technique for the recording of membrane currents and intracellular perfusion of Xenopus oocytes.

In this report we present a description of a modified version of the "glass-funnel" technique for the recording of membrane currents and intracellular perfusion of Xenopus laevis oocytes. The technique is based on the ability of the devitellinated oocyte to form a high-resistance seal with the glass, permitting separation of the oocyte into two, i.e., extra- and intracellular, compartments. The technique is fairly simple to use, provides a much higher clamp speed compared to the double-microelectrode voltage-clamp technique, and allows effective control of the composition of the intracellular milieu. To elucidate the performance of the technique with respect to various membrane currents we present data relating to the recording of Ca-channel currents expressed in X. laevis oocytes by means of mRNA extracted from the rat cerebellum and heart, as well as currents induced by cRNA for the skeletal muscle micro1 Na+ channel and the dog heart NCX1 Na+-Ca2+ exchanger. Due to effective elimination of intra- and extracellular Cl- it became possible to measure not only Ba2+ but also Ca2+ current through the expressed Ca channels, and to record the activity of the Na+-Ca2+ exchanger following dialysis of the oocyte with high-Ca2+ intracellular solutions. Corresponding currents showed properties identical to those obtained with other techniques, suggesting the adequacy of the glass-funnel technique for critical analysis of membrane ionic currents in Xenopus oocytes.

Characterization of hypothalamic low-voltage-activated Ca channels based on their functional expression in Xenopus oocytes.

Ca-channel currents expressed in Xenopus oocytes by means of messenger RNA extracted from rat thalamohypothalamic complex were studied using the double microelectrode technique. Currents were recorded in Cl(-)-free extracellular solutions with 40 mM Ba2+ as a charge carrier. In response to depolarizations from a very negative holding potential (Vh = -120 mV), inward Ba2+ current activated at around -80 mV, peaked at -30 to -20 mV and reversed at +50 mV indicating that it may be transferred through the low voltage-activated calcium channels. The time-dependent inactivation of the current during prolonged depolarization to -20 mV was quite slow and followed a single exponential decay with a time-constant of 1550 ms and a maintained component constituting 30% of the maximal amplitude. The current could not be completely inactivated at any holding potential. As expected for low voltage-activated current, steady-state inactivation curve shifted towards negative potentials. It could be described by the Boltzmann equation with half inactivation potential -78 mV, slope factor 15 mV and maintained level 0.3. Expressed Ba2+ current could be blocked by flunarizine with Kd = 0.42 microM, nifedipine, Kd = 10 microM, and amiloride at 500 microM concentration. Among inorganic Ca-channel blockers the most potent was La3+ (Kd = 0.48 microM) while Cd2+ and Ni2+ were not very discriminative and almost 1000-fold less effective than La3+ (Kd = 0.52 mM and Kd = 0.62 mM, respectively). Our data show that messenger RNA purified from thalamohypothalamic complex induces expression in the oocytes of almost exclusively low voltage-activated calcium channels with voltage-dependent and pharmacological properties very similar to those observed for T-type calcium current in native hypothalamic neurons, though kinetic properties of the expressed and natural currents are somewhat different.

[Transmembrane currents in Xenopus oocytes after an injection of brain mRNA]. / Transmembrannye toki v ootsitakh Xenopus posle in''ektsii mozgovoi mRNK.

The possibility of the translation of mRNA isolated from bovine brain and injected into Xenopus oocytes has been shown. It is found that mRNA injection induced the appearance of voltage-operated TTX-sensitive sodium and calcium currents, GABA receptors in oocytes membrane.