Molecular correlates of the calcium-independent, depolarization-activated K+ currents in rat atrial myocytes.
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1. In adult rat atrial myocytes, three kinetically distinct Ca2+-independent depolarization-activated outward K+ currents, IK, fast, IK,slow and Iss, have been separated and characterized. 2. To test directly the hypothesis that different voltage-dependent K+ channel (Kv channel) alpha subunits underlie rat atrial IK,fast, IK, slow and Iss, the effects of antisense oligodeoxynucleotides (AsODNs) targeted against the translation start sites of the Kv alpha subunits Kv1.2, Kv1.5, Kv4.2, Kv4.3, Kv2.1 and KvLQT1 were examined. 3. Control experiments on heterologously expressed Kv alpha subunits revealed that each AsODN is selective for the subunit against which it was targeted. 4. Peak outward K+ currents were attenuated significantly in rat atrial myocytes exposed to AsODNs targeted against Kv4.2, Kv1.2 and Kv1.5, whereas AsODNs targeted against Kv2.1, Kv4.3 and KvLQT1 were without effects. 5. No measurable effects on inwardly rectifying K+ currents (IK1) were observed in atrial cells exposed to any of the Kv alpha subunit AsODNs. 6. Kinetic analysis of the currents evoked during long (10 s) depolarizing voltage steps revealed that AsODNs targeted against Kv4.2, Kv1.2 and Kv1.5 selectively attenuate rat atrial IK,fast, IK, slow and Iss, respectively, thus demonstrating that the molecular correlates of rat atrial IK,fast, IK,slow and Iss are distinct. 7. The lack of effect of the Kv4.3 AsODNs on peak outward K+ currents reveals that Kv4.2 and Kv4.3 do not heteromultimerize in rat atria in vivo. In addition, the finding that Kv1.2 and Kv1.5 contribute to distinct K+ currents in rat atrial myocytes demonstrates that Kv1.2 and Kv1.5 also do not associate in rat atria in vivo. (+info)
Quantitation of clobazam in human plasma using high-performance liquid chromatography.
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A rapid and simple reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of clobazam concentrations in human blood samples is developed and validated. Solid-phase column extraction is performed to clean up blood samples before running the analytical HPLC system. The chromatography is isocratic with a mobile phase consisting of acetonitrile (20%, v/v), methanol (23%, v/v), and 0.1 M potassium hydrogen phosphate buffer (pH 3.6; 57%, v/v) at a constant flow rate of 2 mL/min. Clobazam is detected at 226 nm. Chromatography is completed within less than 25 min. The recovery rate is greater than 95% and linear over a wide range of drug concentrations. The intra-assay coefficient of variation percentage varies between 4.3 and 12. This method is used for therapeutic drug monitoring in patients undergoing antiepileptic therapy with clobazam. Plasma levels of clobazam ranged from 21 to 663 ng/mL. Other antiepileptic compounds, such as clonazepam and phenobarbital, did not interfere with the detection of clobazam. (+info)
Chemistry for the analysis of protein-protein interactions: rapid and efficient cross-linking triggered by long wavelength light.
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Chemical cross-linking is a potentially useful technique for probing the architecture of multiprotein complexes. However, analyses using typical bifunctional cross-linkers often suffer from poor yields, and large-scale modification of nucleophilic side chains can result in artifactual results attributable to structural destabilization. We report here the de novo design and development of a type of protein cross-linking reaction that uses a photogenerated oxidant to mediate rapid and efficient cross-linking of associated proteins. The process involves brief photolysis of tris-bipyridylruthenium(II) dication with visible light in the presence of the electron acceptor ammonium persulfate and the proteins of interest. Very high yields of cross-linked products can be obtained with irradiation times of <1 second. This chemistry obviates many of the problems associated with standard cross-linking reagents. (+info)
Synthesis and in vivo murine evaluation of Na4[1-(1'-B10H9)-6-SHB10H8] as a potential agent for boron neutron capture therapy.
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Reaction of the normal isomer of [B20H18]2- and the protected thiol anion, [SC(O)OC(CH3)3]-, produces an unexpected isomer of [B20H17SC(O)OC(CH3)3]4- directly and in good yield. The isomer produced under mild conditions is characterized by an apical-apical boron atom intercage connection as well as the location of the thiol substituent on an equatorial belt adjacent to the terminal boron apex. Although the formation of this isomer from nucleophilic attack of the normal isomer of [B20H18]2- has not been reported previously, the isomeric assignment has been unambiguously confirmed by one-dimensional and two-dimensional 11B NMR spectroscopy. Deprotection of the thiol substituent under acidic conditions produces a protonated intermediate, [B20H18SH]3-, which can be deprotonated with a suitable base to yield the desired product, [B20H17SH]4-. The sodium salt of the resulting [B20H17SH]4- ion has been encapsulated in small, unilamellar liposomes, which are capable of delivering their contents selectively to tumors in vivo, and investigated as a potential agent for boron neutron capture therapy. The biodistribution of boron was determined after intravenous injection of the liposomal suspension into BALB/c mice bearing EMT6 mammary adenocarcinoma. At low injected doses, the tumor boron concentration increased throughout the time-course experiment, resulting in a maximum observed boron concentration of 46.7 micrograms of B per g of tumor at 48 h and a tumor to blood boron ratio of 7.7. The boron concentration obtained in the tumor corresponds to 22.2% injected dose (i.d.) per g of tissue, a value analogous to the most promising polyhedral borane anions investigated for liposomal delivery and subsequent application in boron neutron capture therapy. (+info)
Transcription, biochemistry and localization of nematode annexins.
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The transcription of three annexin genes in the nematode, Caenorhabditis elegans, was detected by reverse transcriptase/polymerase chain reaction amplification of messenger RNAs. The highest level of expression was from the nex-1 gene, with lower levels detected for the nex-2 and nex-3 genes. The expression of nex-1 was reduced in the Dauer larval stage relative to the other annexins, correlating with the absence of the spermathecal valves, a major site of nex-1 protein localization. Recombinant nex-1 protein was expressed in yeast, isolated by calcium-dependent binding to acidic phospholipids, and its membrane binding and aggregating activities characterized using bovine chromaffin granules as a representative intracellular substrate. Binding to granule membranes was promoted by calcium with half-maximal binding seen at 630 microM calcium. Chromaffin granule aggregation was similarly promoted by the nex-1 protein at 630 microM calcium. This low sensitivity to calcium suggests the annexin can only be activated in vivo near the plasma membrane or other sources of calcium. Sequences including the nex-1 promoter were fused to the gene for green fluorescent protein and this construct was introduced into nematodes by microinjection. Examination of transgenic offspring revealed specific nex-1 promoter activity in the pharynx, the hypodermal cells, the vulva, and the spermathecal valve, locations in which the annexin may function in collagen secretion/deposition and membrane-membrane interactions. A sensitive anti-nex-1 antibody labelled with rhodamine was injected into body cavities of the nematode but did not detect extracellular nex-1 protein. Therefore, this annexin is apparently cytosolic and may function on the cytoplasmic side of the plasma membrane of the spermathecal valve to chaperon the folding of this membrane during the opening and closing of the valve. (+info)
EGF-and NGF-stimulated translocation of cytohesin-1 to the plasma membrane of PC12 cells requires PI 3-kinase activation and a functional cytohesin-1 PH domain.
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ADP-ribosylation factors (ARFs) are small GTP-binding proteins that function as regulators of eukaryotic vesicle trafficking. Cytohesin-1 is a member of a family of ARF guanine nucleotide-exchange factors that contain a C-terminal pleckstrin homology (PH) domain which has been proposed to bind the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3). Here we demonstrate that in vitro, recombinant cytohesin-1 binds, via its PH domain, the inositol head group of PIP3, inositol 1,3,4, 5-tetrakisphosphate (IP4), with an affinity greater than 200-fold higher than the inositol head group of either phosphatidylinositol 4, 5-bisphosphate or phosphatidylinositol 3,4-bisphosphate. Moreover, addition of glycerol or diacetylglycerol to the 1-phosphate of IP4 does not alter the ability to interact with cytohesin-1, data which is entirely consistent with cytohesin-1 functioning as a putative PIP3 receptor. To address whether cytohesin-1 binds PIP3 in vivo, we have expressed a chimera of green fluorescent protein (GFP) fused to the N terminus of cytohesin-1 in PC12 cells. Using laser scanning confocal microscopy we demonstrate that either EGF- or NGF-stimulation of transiently transfected PC12 cells results in a rapid translocation of GFP-cytohesin-1 from the cytosol to the plasma membrane. This translocation is dependent on the cytohesin-1 PH domain and occurs with a time course that parallels the rate of plasma membrane PIP3 production. Furthermore, the translocation requires the ability of either agonist to activate PI 3-kinase, since it is inhibited by wortmannin (100 nM), LY294002 (50 microM) and by coexpression with a dominant negative p85. This data therefore suggests that in vivo cytohesin-1 can interact with PIP3 via its PH domain. (+info)
A nuclear localization signal can enhance both the nuclear transport and expression of 1 kb DNA.
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Although the entry of DNA into the nucleus is a crucial step of non-viral gene delivery, fundamental features of this transport process have remained unexplored. This study analyzed the effect of linear double stranded DNA size on its passive diffusion, its active transport and its NLS-assisted transport. The size limit for passive diffusion was found to be between 200 and 310 bp. DNA of 310-1500 bp entered the nuclei of digitonin treated cells in the absence of cytosolic extract by an active transport process. Both the size limit and the intensity of DNA nuclear transport could be increased by the attachment of strong nuclear localization signals. Conjugation of a 900 bp expression cassette to nuclear localization signals increased both its nuclear entry and expression in microinjected, living cells. (+info)
Analysis of urine samples containing cardiovascular drugs by micellar liquid chromatography with fluorimetric detection.
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A simple direct injection chromatographic procedure with fluorimetric detection is successfully applied to the determination of mixtures of 4 diuretics (amiloride, bendroflumethiazide, piretanide, and triamterene) and 6 beta-blockers (acebutolol, atenolol, labetalol, metoprolol, nadolol, and propranolol), which are usually administered in combinations for the treatment of hypertension, in urine samples. The procedure makes use of C18 columns and micellar mobile phases of sodium dodecyl sulphate (SDS), propanol, and phosphate buffer at pH 3. The adequate resolution of most drugs is obtained with a chemometrics approach where the retention is modeled as a first step using the retention factors in only 5 mobile phases. Afterward, an optimization criterion that takes into account the position and shape of the chromatographic peaks is applied. A mobile phase of 0.11M SDS--8% propanol could resolve mixtures of 8 drugs and was adequate for the analysis of the combinations of diuretic and beta-blocker usually prescribed. However, a mobile phase of larger elution strength, such as 0.15M SDS--15% propanol, is preferred for the analysis of mixtures of amiloride-metoprolol, amiloride-labetalol, and triameterene-propranolol. The method is sensitive enough for the routine analysis of diuretics and beta-blockers at therapeutic urine levels with limits of detection in the 0.5-28-ng/mL range. Urinary excretion studies show that the detection of most drugs is possible up to 24-72 h after their ingestion. (+info)