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.     

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)     

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.     

Genomic mapping within the albino-deletion complex using individual early postimplantation mouse embryos

Sensitive methods for analysis of DNA from limited amounts of tissue are often difficult, error prone, and time consuming. Here, we describe a procedure for molecular analysis of individual early post-implantation mouse embryos by Southern analysis. The procedure involves embedding single embryos in agarose before lysing and deproteinizing in situ. The embedded DNA can be digested with restriction enzymes and analyzed by standard Southern-blotting procedures. The procedure is sensitive enough to detect single-copy sequences in embryos as early as day 6.5 of development. We have used the technique to genotype embryos homozygous for an embryonic lethal deletion. Normally, the lethal phenotype associated with such mutations is identified by a retrospective statistical analysis of abnormal embryos produced from a heterozygous cross as compared to those produced from a control cross. Now, if associated with a detectable DNA abnormality, the mutant embryo can be genotyped directly. We also report the use of this method for mapping cloned markers relative to deletion breakpoints. This approach can save considerable time since mapping would conventionally be done using restriction fragment length polymorphisms (RFLPs) detected in Mus musculus/Mus spretus interspecies hybrids. Using this procedure, we have been able to redefine the distal limits of the region of Chromosome (Chr) 7 containing a gene (eed) needed for development of the embryonic ectoderm.     

Intergeneric protoplast fusion between Brassica carinata and Camelina sativa

Camelina sativa is a wild crucifer that is reported to be resistant to Alternaria blight. Polyethylene glycol mediated fusion was attempted between protoplasts from etiolated hypocotyls of Brassica carinata and mesophyll protoplasts of Camelina sativa. The mean frequency of heterokaryons was 6.8%. Three hybrid shoots were regenerated, each from a single fusionderived callus. These shoots failed to produce roots capable of withstanding transplantation. Confirmation of hybridity was obtained from the morphology of in vitro produced leaves, somatic chromosome number in leaf tips, and restriction fragment length polymorphism for a nuclear rDNA probe. Analysis for organelle constitution using RFLPs indicated that the hybrid contained chrloroplasts derived from the wild species and mitochondria from the cultivated Brassica species.Abbreviations 2,4-D 2,4-dichlorophenoxy-acetic acid - IAA Indole-3-acetic acid - NAA -Naphthaleneacetic acid - IBA Indole-3-butyric acid - GA₃ gibberellic acid - BAP 6-Benzylaminopurine - MS Murashige and Skoog (1962) basal medium     

The electron impact, chemical ionization and fast atom bombardment positive ion mass spectra of 1,2-bis(sulfonyl)methylhydrazines.

A series of bis(sulfonyl)-1-methylhydrazines were analyzed by positive ion electron impact (EI), chemical ionization (CI) and fast atom bombardment (FAB) mass spectrometry. Since these compounds showed activity against the L1210 leukemia, an understanding of their mass spectral behavior is important should the structural characterization of metabolites be required. FAB proved to be the most useful technique, generally providing abundant protonated molecule ion peaks, in contrast to the weak peaks observed with CI (ammonia or isobutane) and the total absence of molecular ion peaks in the EI mass spectra. In addition, utilizing FAB eliminated the problem of thermal decomposition, which was very difficult to control under EI and CI experimental conditions. Fragments observed in FAB and CI mass spectra were consistent with protonation at the methyl-bearing nitrogen. One can locate the R1 and R2 moieties relative to the methyl-bearing nitrogen in FAB and CI by assigning that nitrogen as the site of protonation, with subsequent elimination of R2SO2H.