Bile acids induce a cationic current, depolarizing pancreatic acinar cells and increasing the intracellular Na+ concentration

Biliary disease is a major cause of acute pancreatitis. In this study we investigated the electrophysiological effects of bile acids on pancreatic acinar cells. In perforated patch clamp experiments we found that taurolithocholic acid 3-sulfate depolarized pancreatic acinar cells. At low bile acid concentrations this occurred without rise in the cytosolic calcium concentration. Measurements of the intracellular Na(+) concentration with the fluorescent probe Sodium Green revealed a substantial increase upon application of the bile acid. We found that bile acids induce Ca(2+)-dependent and Ca(2+)-independent components of the Na(+) concentration increase. The Ca(2+)-independent component was resolved in conditions when the cytosolic Ca(2+) level was buffered with a high concentration of the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). The Ca(2+)-dependent component of intracellular Na(+) increase was clearly seen during stimulation with the calcium-releasing agonist acetylcholine. During acetylcholine-induced Ca(2+) oscillations the recovery of cytosolic Na(+) was much slower than the recovery of Ca(2+), creating a possibility for the summation of Na(+) transients. The bile-induced Ca(2+)-independent current was found to be carried primarily by Na(+) and K(+), with only small Ca(2+) and Cl(-) contributions. Measurable activation of such a cationic current could be produced by a very low concentration of taurolithocholic acid 3-sulfate (10 microm). This bile acid induced a cationic current even when applied in sodium- and bicarbonate-free solution. Other bile acids, taurochenodeoxycholic acid, taurocholic acid, and bile itself also induced cationic currents. Bile-induced depolarization of acinar cells should have a profound effect on acinar fluid secretion and, consequently, on transport of secreted zymogens.     

Glutaraldehyde and glyoxal fixation of ribonucleoproteins for their analysis by cesium chloride equilibrium gradient centrifugation]

Conditions for fixation of different RNP (ribosomes, poliribosomes, informosomes) by glutaraldehyde and glyoxal for their subsequent analysis in CsCl density-gradient has been developed. Higher dialdehyde concentration and longer incubation time should be used for fixation of ribosomes and polyribosomes than for that of informosomes. For the fixation of all RNP studied their incubation with 0.01 M (0.1%) glutaraldehyde for several minutes is sufficient. Much higher concentration of the fixating agent (about 0.2-0.5 M i. e. 1-3%) and more prolonged time of incubation (in order of several 10 hours) are needed for the fixation of the RNP in the case of glyoxal. Conditions for selective aldehyde fixation of informosomes in the presence of ribosomes and polyribosomes has been developed.     

Electrographic correlates of the antiamnestic action of nootropic agents following deprivation of the paradoxical sleep phase

During experiments conducted on albino rats the deficiency of passive avoidance retention was shown to correlate not only with the reduction in REM sleep and SWS, but also with disappearance of phasic component of theta-rhythm. Drugs with nootropic mode of action (cleregyl, centrophenoxin, antioxidant 3-xypyridine) recovered the deficiency of passive avoidance retention and increased phasic component of theta-Rhythm, while phenazepam enhanced tonic component of theta-rhythm, and failed to act upon learning deficits. It seems likely from these results that the electrophysiological correlates of antiamnestic effect is the maintenance of proper two-component theta-rhythm and the increase in its phasic component, whereas the destructuring of sleep, including REM sleep reduction is not considered to be key determinant in the action upon memory and learning procedure.     

Genetic control for resistance to leaf rust in wheat-Agropyron lines: Agro 139 and Agro 58

Leaf rust resistance lines of Triticum aestivum carry highly effective Lr genes from Agropyron intermedium (Host) Beauv. This Agro 58 and Agro 139 resistance segregated independently of Agropyron leaf-rust resistance genes Lr-19, Lr-24 and Lr-9 from Ae. umbellulata. Monosomic analysis showed that the Lr gene in Agro 139 was incorporated into wheat chromosome 6D. C-banding analysis could not determine the C-banding pattern of A. intermedium in wheat -Agropyron lines Agro 58 and Agro 139. It is assumed that the transfers occurred from the euchromatin regions of the Agropyron chromosomes to the euchromatin regions of the wheat chromosomes. It is suggested that the Lr gene from Agro 139 be designated LrAg ⁱ-1 and the Lr gene from Agro 58 designated LrAg ⁱ-2.     

RNA isolation from the ribonucleoproteins fixed with formaldehyde

A method for isolating RNA from the polyribosomes and informosomes fixed with formaldehyde was developed. The ribonucleoproteins were obtained by centrifugation in CsCl density gradient. It has been demonstrated that this method makes it possible to obtain full-sized rRNA and mRNA appropriate for molecular hybridization. We succeeded in amplifying 150-nucleotide sequences of individual mRNA and demonstrating the applicability of these RNA preparations for synthesis of labeled probes for RNA arrays. The method proposed is recommended for the search for untranslated mRNA and the study of the changes in translation of individual proteins during early ontogenesis and various pathologies.     

Change in the absorptive capacity of macrophages from different organs after administration of hydrocortisone

Two, twenty-four and 48 h after hydrocortisone treatment in a dose of 125 mg/kg bw the blood clearance rate for colloidal carbon particles in rats turned to be 2, 2.1. and 1.6 times less whereas that for 51Cr-SRBC in CBA mice 2.1, 2.2 and 1.7 times less as compared to untreated controls. Within 24 and 72 h after hormone injection the efficacy of red blood cell uptake by Kupffer cells decreased 1.35 and 1.8 times whereas the similar uptake by lung or spleen macrophages changed but insignificantly and that by bone marrow cells was even greater than in controls. Toward the 5th day after zymosan treatment the uptake capacity of Kupffer cells was the greatest whereas the plasma 11-OHCS content was 1.3-fold less versus the control values.     

The role of GABA-benzodiazepine receptor complex in affecting the anxiolytic effect of 3-hydroxypyridines--new non-benzodiazepine tranquilizers

The experiments on rats, using conflict situation method, have established that anxiolytic effect of 3-hydroxypyridine derivative is removed by bicucullin and picrotoxin, but not by Ro 5-3663, specific antagonist of benzodiazepine receptors Ro 15-1788 and inversive antagonist ethyl beta-carboline-3-carboxylate. The data obtained suggest that GABA-chloriontophor complex is involved into the realization of 3-hydroxypyridine anxiolytic effect.     

<Emphasis Type="Italic">In vivo</Emphasis> expression of copper-transporting proteins in rat brain regions

Expression of two copper-transporting P1-type ATPases (ATP7A and ATP7B), the CTR1 protein, a high-affinity copper transporter, and ceruloplasmin (Cp), a copper-containing ferroxidase was studied. The level of mRNA of these proteins was determined by RT-PCR analysis, the distribution of polypeptides encoded by these genes was determined by immunoblotting, and the type of cells expressing these genes was identified immunohistochemically. It was found that the major product of Cp gene in the brain is the cell membrane-bound Cp. Secretory Cp, whose molecule contains the greatest number of weakly associated copper atoms, is synthesized in the choroid plexus. CTR1 mRNA is evenly distributed in the brain; however, its content is twice higher in the vascular plexus. The Atp7a gene is active in all brain regions, whereas the Atp7b gene is active only in the hypothalamus. The membrane-bound Cp is expressed in glial cells of all types and in ependyma cells. ATP7B and ATP7A are expressed predominantly in ependymyocytes and neurons, respectively. The organization of copper transport in mammalian brain is discussed.Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 2, 2005, pp. 141–154.Original Russian Text Copyright © 2005 by Platonova, Barabanova, Povalikhin, Tsymbalenko, Danilovskii, Voronina, Dorokhova, Puchkova.     

Targeted Intracellular Site-Specific Drug Delivery: Photosensitizer Targeting to Melanoma Cell Nuclei

A number of drugs are regarded as possessing local activity because their effects take place at an extremely short distance from their location site in the cell. The response of different cellular compartments to these effects is different. Such substances as photosensitizers (PSs), which are used in photodynamic cancer therapy, should be targeted to the cell compartments where their effect is the most pronounced. This study describes the construction and properties of the chimeric modular recombinant transporters (MRTs) expressed in Escherichia coli and used for PS targeting. These constructs include (1) the -melanocyte-stimulating hormone as a ligand module, which is internalized by the target cells (mouse melanoma); (2) the optimized SV40 large T-antigen nuclear localization signal; (3) the hemoglobin-like protein from E. coli as a carrier module; (4) the endosomolytic module, the translocation domain of the diphtheria toxin. These MRTs were used for PS targeting to the mouse melanoma cell nuclei, the most PS-damaged intracellular compartment, which resulted in a PS photocytotoxic effect increase of several orders of magnitude. In our opinion, MRTs, which target locally active drugs into the desired cell compartment and thereby enhance the drug response, represent a new generation of the pharmacological agents.