Na+/K+-ATPase Inhibition Partially Mimics the Ethanol-Induced Increase of the Golgi Cell-Dependent Component of the Tonic GABAergic Current in Rat Cerebellar Granule Cells

Cerebellar granule cells (CGNs) are one of many neurons that express phasic and tonic GABAergic conductances. Although it is well established that Golgi cells (GoCs) mediate phasic GABAergic currents in CGNs, their role in mediating tonic currents in CGNs (CGN-I_tonic) is controversial. Earlier studies suggested that GoCs mediate a component of CGN-I_tonic that is present only in preparations from immature rodents. However, more recent studies have detected a GoC-dependent component of CGN-I_tonic in preparations of mature rodents. In addition, acute exposure to ethanol was shown to potentiate the GoC component of CGN-I_tonic and to induce a parallel increase in spontaneous inhibitory postsynaptic current frequency at CGNs. Here, we tested the hypothesis that these effects of ethanol on GABAergic transmission in CGNs are mediated by inhibition of the Na⁺/K⁺-ATPase. We used whole-cell patch-clamp electrophysiology techniques in cerebellar slices of male rats (postnatal day 23–30). Under these conditions, we reliably detected a GoC-dependent component of CGN-I_tonic that could be blocked with tetrodotoxin. Further analysis revealed a positive correlation between basal sIPSC frequency and the magnitude of the GoC-dependent component of CGN-I_tonic. Inhibition of the Na⁺/K⁺-ATPase with a submaximal concentration of ouabain partially mimicked the ethanol-induced potentiation of both phasic and tonic GABAergic currents in CGNs. Modeling studies suggest that selective inhibition of the Na⁺/K⁺-ATPase in GoCs can, in part, explain these effects of ethanol. These findings establish a novel mechanism of action of ethanol on GABAergic transmission in the central nervous system.     

Design,synthesis, characterization,antibacterial and antifungal activities of a novel class of 5,7-diaryl-4,4-dimethyl-4,5,6,7-tetrahydropyridino[3,4-d]-1,2,3-selenadiazoles

Compound 26 is more potent against Escherichia coli. and 24 is more active against Staphylococcus aureus, β-Heamolytic streptococcus, Vibreo cholerae, Salmonella typhii, and Shigella flexneri than the standard drug ciprofloxacin. Moreover, of all the compounds tested, 26 is more effective against Aspergillus flavus and Mucor, than the standard drug fluconazole.     

Hepatocellular Carcinoma in Hepatitis B Surface Antigen Carriers in Eight Institutions

We reviewed records of all persons dying between 1979 and 1986 in eight California institutions for the mentally retarded. Autopsies had been done in 71% of the 1,181 deaths. Nine deaths were due to hepatocellular carcinoma, which invariably developed in carriers of hepatitis B surface antigen (HBsAg) and was fatal within four months of diagnosis. The mean age at death was 32.7 years. The incidence of hepatocellular carcinoma in HBsAg carriers was 140 times greater than in the US population. Persistent hepatitis B infection was probably etiologically related to hepatocellular carcinoma in this population, which is relatively free of exposure to other hepatocarcinogens.     

Detection of Two Membrane Polypeptides Induced by Abscisic Acid and Cold Acclimation: Possible Role in Freezing Tolerance

Membrane proteins labeled in vivo from cold-acclimated and ABA-treatedalfalfa seedlings of two cultivars differing in cold-tolerancehave been compared by SDS polyacrylamide gel electrophoresisand fluorography. Results thus obtained indicate that severalqualitative changes occur in the membrane protein-profile specificallyin response to cold acclimation or ABA treatment. While somepolypeptides disappear from the non-acclimated protein patterns,others specifically appear in response to acclimation. Separationby two-dimensional gel electrophoresis and fluorography hasconfirmed the above and has enabled us to detect two proteinsof Mr 42 kDa and 120 kDa that are induced by both acclimationand ABA treatment in the freezing tolerant cultivar. (Received November 30, 1987; Accepted February 22, 1988)     

Carcinogen-nucleic acid interactions: equilibrium binding studies of aflatoxins B1 and B2 with DNA and the oligodeoxynucleotide d(ATGCAT)2

Equilibrium binding is believed to play an important role in directing the subsequent covalent attachment of many carcinogens to DNA. We have utilized UV spectroscopy to examine the non-covalent interactions of aflatoxin B1 and B2 with calf thymus DNA, poly(dAdT):poly(dAdT), and poly(dGdC):poly(dGdC), and have utilized NMR spectroscopy to examine non-covalent interactions of aflatoxin B2 with the oligodeoxynucleotide d(ATGCAT)2. UV-VIS binding isotherms suggest a greater binding affinity for calf thymus DNA and poly(dAdT):poly(dAdT) than for poly(dGdC):poly(dGdC). Scatchard analysis of aflatoxin B1 binding to calf thymus DNA in 0.1 M NaCl buffer indicates that binding of the carcinogen at levels of bound aflatoxin less than 1 carcinogen per 200 base pairs occurs with positive cooperativity. The cooperative binding effect is dependent on the ionic strength of the medium; when the NaCl concentration is reduced to 0.01 M, positive cooperativity is observed at carcinogen levels less than 1 carcinogen per 500 base pairs. The Scatchard data may be fit using a "two-site" binding model [L.S. Rosenberg, M.J. Carvlin, and T.R. Krugh, Biochemistry 25, 1002-1008 (1986)]. This model assumes two independent sets of binding sites on the DNA lattice, one a high affinity site which binds the carcinogen with positive cooperativity, the second consisting of lower affinity binding sites to which non-specific binding occurs. NMR analysis of aflatoxin B2 binding to d(ATGCAT)2 indicates that the aflatoxin B2/oligodeoxynucleotide complex is in fast exchange on the NMR time scale. Upfield chemical shifts of 0.1-0.5 ppm are observed for the aflatoxin B2 4-OCH3, H5, and H6a protons. Much smaller chemical shift changes (less than or equal to 0.06 ppm) are observed for the oligodeoxynucleotide protons. The greatest effect for the oligodeoxynucleotide protons is observed for the adenine H2 protons, located in the minor groove. Nonselective T1 experiments demonstrate a 15-25% decrease in the relaxation time for the adenine H2 protons when aflatoxin B2 is added to the solution. This result suggests that aflatoxin B2 protons in the bound state may be in close proximity to these protons, providing a source of dipolar relaxation. Further experiments are in progress to probe the nature of the aflatoxin B1 and B2 complexes with polymeric DNA and oligodeoxynucleotides, and to establish the relationship between the non-covalent DNA-carcinogen complexes observed in these experiments, and covalent aflatoxin B1-guanine N7 DNA adducts.     

Construction of a new universal vector for insertional mutagenesis by homologous recombination

We describe here the construction of a vector (pSSC-9) which can be used for the insertional mutagenesis of any gene for which genomic sequences have been cloned. This vector contains a neomycin-resistance-encoding gene (neo^R) which is driven by a modified thymidine kinase (tk) promoter for positive selection. Flanking neo^R are two tk genes driven by their own promoters for negative selection of nonhomologous insertions. The neo^R and tk cassettes are separated by four unique cloning sites on the right-hand side of the neo^R cassette and three unique sites on the left-hand side. The vector also includes two SfiI sites, one on each side of the tk cassettes, for the excision of the cloned genomic DNA fragments along with the selectable markers. Electroporation of pSSC-9 into mouse embryonic stem (ES) cells and cultured diploid mouse adrenal Y-1 cells conferred resistance to G418 and sensitivity to ganciclovir in both cell lines. These results illustrate the expression of the positive and negative selectable markers in two different cell lines and thus suggest that the vector could be used in ES cells, as well as in cultured somatic cells.     

Barley aleurone layer cell protoplasts as a transient expression system

Protoplasts were prepared from barley aleurone layers using Onozuka cellulase digestion and purification through a Percoll gradient. Protoplasts prepared by this procedure had a viability ranging from 60% to 80% during the first two days of culture. They were responsive to gibberellic acid (GA) as measured by the stimulation of -amylase synthesis. The GA stimulation was counteracted by abscisic acid (ABA). In the presence of polyethylene glycol (PEG), the protoplasts took up exogenously added plasmid DNA containing the reporter gene coding for chloramphenicol acetyl transferase (CAT) linked to a 35S promoter from cauliflower mosaic virus (CaMV) or to barley -amylase gene promoters and expressed CAT activity. Therefore, barley aleurone layer protoplasts are suitable for analysis of hormoneresponsive elements in hydrolase genes.