The role of protein kinases and protein phosphatases in the regulation of cardiac sarcoplasmic reticulum function

Summary Canine cardiac sarcoplasmic reticulum is phosphorylated by adenosine 3,5-monophosphate (cAMP)-dependent and by calcium · calmodulin-dependent protein kinases on a 27 000 proteolipid, called phospholamban. Both types of phosphorylation are associated with an increase in the initial rates of Ca²⁺ transport by SR vesicles which reflects an increased turnover of elementary steps of the calcium ATPase reaction sequence. The stimulatory effects of the protein kinases on the calcium pump may be reversed by an endogenous protein phosphatase, which can dephosphorylate both the CAMP-dependent and the calcium · calmodulin-dependent sites on phospholamban. Thus, the calcium pump in cardiac sarcoplasmic reticulum appears to be under reversible regulation mediated by protein kinases and protein phosphatases.     

Lack of a relationship between immune function and chemically induced hepatocarcinogenesis in B6C3F1 mice

Summary The relationship between immune function and chemically induced hepatocarcinogenesis was studied employing an in vivo murine model. Neonatal B6C3F₁ mice were given a single carcinogenic dose of diethylnitrosamine (DEN) and the time-response kinetics for the early (foci of alteration) and late (adenomas/carcinomas) phases of hepatocellular carcinogenesis were compared to changes in hematopoiesis and immune functions associated with immune surveillance and natural resistance. Increases in hematopoiesis occurred just prior to or concurrent with the appearance of hepatocellular carcinomas, while increased macrophage and natural killer cell cytotoxicity and suppression of cell-mediated immunity occurred following tumor appearance and progressed with increasing tumor burden. Neither immunological nor hematopoietic changes were associated with early phases of hepatocarcinogenesis, as monitored by the appearance of altered hepatocellular foci. Although changes in hematopoiesis may represent an early indicator for hepatocarcinogenesis in the mouse tumor model, the data suggest that altered immune surveillance and natural resistance are not factors in the development of chemically induced hepatocellular tumors, and the changes in immune function are probably secondary to tumor development.     

Mutations for the autosomal recessive and autosomal dominant forms of polycystic kidney disease are not allelic

Summary The autosomal dominant form of polycystic kidney disease (ADPKD) has been linked to the -globin gene locus on 16p. Linkage studies between the autosomal recessive type (ARPKD) and the 3 HVR of the -globin gene cluster showed that the ARPKD and ADPKD are not allelic.     

XY sex reversal syndrome in the mare: clinical and behavioral studies,H-Y phenotype

Summary An inherited genetic disorder causes XY embryos of the horse to develop as mares. On the basis of our study of 38 such mares, we have identified four grades or classes of XY sex reversal according to this scheme: class I, nearly normal female, of which some are fertile; class II, female with gonadal dysgenesis, normal mullerian development; calss III, intersex mare with gonadal dysgenesis, abnormal mullerian development, enlarged clitoris; class IV, virilized intersex characterized by high levels of testosterone. In general, class I and calss II mares were typed H-Y antigen-negative whereas class III and class IV mares were typed H-Y antigen-positive.     

Spontaneous release of endogenous aspartate and glutamate from rat striatal slices is increased following destruction of local neurons by ibotenic acid

We sought to determine in rat striatum whether the release of neurotransmitter amino acids aspartate (Asp), glutamate (Glu) and gamma-aminobutyric acid (GABA) were affected by local neurons. To do so, unilateral microinjections of ibotenic acid, an excitotoxin that destroys local neurons without affecting fibers of passage, were made into the striatum. Release of endogenous amino acids from lesioned and intact striatal slices were measured by HPLC one week later. The effectiveness and specificity of the lesion were confirmed by measuring the enzyme activity associated with extrinsic dopamine neurons (tyrosine hydroxylase; 111±14%), intrinsic GABA neurons (glutamic acid decarboxylase; 19±7%) and intrinsic acetylcholine neurons (choline acetyltransferase; 37±10%). Destruction of local striatal neurons markedly attenuated the release of GABA (41±12% of control) elicited by depolarization with K⁺ (35 mM), but did not significantly reduce the K⁺-evoked release of Asp (80±17%) and Glu (92±8%). However, spontaneous release of Asp and Glu was significantly greater than that observed in unlesioned tissue (159±18% and 209±27%, respectively), while the spontaneous release of GABA was not significantly reduced (75±43%). Although release of the neurotransmitter amino acids Asp, Glu and GABA were affected by the lesion, the release of the non-neurotransmitter amino acid tyrosine was unaffected. These data are consistent with the hypotheses that: 1) the predominant source of releasable stores of endogenous Asp and Glu in the striatum arises from extinsic neurons, and 2) that the spontaneous release of Asp and Glu from axon terminals in the striatum may be regulated, at least in part, by local inhibitory neurons.     

Isolation of single taste cells from lingual epithelium

A method is described for obtaining large numbers of isolatedtaste cells with identified polarity from lingual epithelium.The procedure involves incubating lingual epithelium in collagenase,staining the apical surface with fluorescein-conjugated wheatgerm agglutinin (FTTC-WGA), peeling non-gustatory surface epitheliumfrom the underlying taste buds and connective tissue, and dissociatingisolated taste buds with Ca²⁺-free saline. Isolated taste cellsretain their characteristic morphology for at least 30 min afterdissociation, and the apical specialization can be identifiedas a single patch of fluorescence usually located at the tipof an elongate process. Isolated taste cells are amenable tostudy with the patch-clamp technique, and whole-cell patch-clamprecordings show that isolated taste cells have membrane propertiessimilar to taste cells of intact lingual epithelium. Evidenceis presented that FITC-WGA staining does not alter the voltage-dependentionic currents of the taste cell membrane.     

Isolation and Characterization of a Thermophilic Bacterium Which Oxidizes Acetate in Syntrophic Association with a Methanogen and Which Grows Acetogenically on H(2)-CO(2)

We previously described a thermophilic (60 degrees C), syntrophic, two-membered culture which converted acetate to methane via a two-step mechanism in which acetate was oxidized to H(2) and CO(2). While the hydrogenotrophic methanogen Methanobacterium sp. strain THF in the biculture was readily isolated, we were unable to find a substrate that was suitable for isolation of the acetate-oxidizing member of the biculture. In this study, we found that the biculture grew on ethylene glycol, and an acetate-oxidizing, rod-shaped bacterium (AOR) was isolated from the biculture by dilution into medium containing ethylene glycol as the growth substrate. When the axenic culture of the AOR was recombined with a pure culture of Methanobacterium sp. strain THF, the reconstituted biculture grew on acetate and converted it to CH(4). The AOR used ethylene glycol, 1,2-propanediol, formate, pyruvate, glycine-betaine, and H(2)-CO(2) as growth substrates. Acetate was the major fermentation product detected from these substrates, except for 1,2-propanediol, which was converted to 1-propanol and propionate. N,N-Dimethylglycine was also formed from glycine-betaine. Acetate was formed in stoichiometric amounts during growth on H(2)-CO(2), demonstrating that the AOR is an acetogen. This reaction, which was carried out by the pure culture of the AOR in the presence of high partial pressures of H(2), was the reverse of the acetate oxidation reaction carried out by the AOR when hydrogen partial pressures were kept low by coculturing it with Methanobacterium sp. strain THF. The DNA base composition of the AOR was 47 mol% guanine plus cytosine, and no cytochromes were detected.     

Calmodulin colocalization with cold-stable and nocodazole-stable microtubules in living PtK1 cells

To investigate the association of calmodulin (CaM) with microtubules (MTs) in the mitotic apparatus (MA), the distributions of both CaM and tubulin were examined in mitotic PtK1 cells in which MT subclasses had been selectively removed or altered by treatment with cold or with the MT inhibitor, nocodazole. A fluorescent CaM conjugate with tetramethylrhodamine isothiocyanate (CaM-TRITC) was microinjected into living cells, and the CaM distribution in the living cell was compared to the distribution of MTs indicated by tubulin immunofluorescence. In cells which had been treated for 2 h at 0 to 4 degrees C or with a low (0.03 micrograms/ml) dose of nocodazole, the only MTs remaining appeared to be kinetochore MTs (kMTs). The distribution of microinjected CaM-TRITC in these cells was indistinguishable from that found in untreated cells and appeared to be colocalized with the kMTs. In cells which were treated with a high (3.0 micrograms/ml) dose of nocodazole, only short MTs remained. When CaM-TRITC was injected into these cells, it formed a somewhat punctate distribution near the chromosomes and, after tubulin immunofluorescence processing, colocalized with what appeared to be remnants of kMTs. We believe that these observations support the hypothesis that CaM exists in the MA in a structural association with kMTs.     

Irreversible Binding and Recovery of the Norepinephrine Uptake System Using an Alkylating Derivative of Norepinephrine

The effects of bromoacetylaminomenthylnorepinephrine (BAAN) on the sodium-dependent, high-affinity norepinephrine (NE) uptake system in rat brain synaptosomes and CNS neuronal cultures were investigated. BAAN inhibited [3H]NE uptake into synaptosomes in a dose- and time-dependent manner (IC50, 6.5 microM). Pretreatment of cortical synaptosomes or neuronal cells with BAAN alone, followed by washing to remove free drug, reduced the Vmax but did not alter the Km value for [3H]NE uptake. The BAAN-induced reduction in Vmax was attenuated by concurrent pretreatment with desipramine and blocked by the reaction of BAAN with dithiothreitol or cysteine. In contrast, BAAN was 19-fold less potent at inhibiting [3H]dopamine uptake in striatal synaptosomes, and no change in the Vmax or Km value for [3H]dopamine uptake was observed after a pretreatment with BAAN followed by washing. Furthermore, the irreversible beta-antagonist, bromoacetylalprenololmentane, was equipotent to BAAN for inhibiting [3H]NE uptake into cortical synaptosomes, but did not alter the Vmax or Km for [3H]NE after pretreatment. In neuronal cultures, BAAN inhibited sodium-dependent uptake of [3H]NE (IC50, 5.6 microM) with no effect on sodium-independent uptake. After pretreatment of cultures with 30 microM BAAN followed by washing, there was a 74% decrease in the Vmax for [3H]NE uptake. Following a 24-h lag period, uptake recovered to the control level within 48 h; however, recovery was completely blocked by cycloheximide. The data indicate that BAAN irreversibly binds to the [3H]NE uptake system in both CNS synaptosomes and neuronal cultures and may be a useful probe for studying the turnover of the [3H]NE uptake system.     

Transformation of plant mitochondria with mitochondrial DNA plasmids via protoplast fusion

Summary Brassica napus cybrid plants which contain novel nucleus-mitochondria-chloroplast combinations have been constructed, via protoplast fusion. Such fusions resulted in mitochondrial DNA plasmids being lost (at a frequency of 12.5%) or, more surprisingly, being transferred from mitochondria of one protoplast population to mitochondria of the other population (at a frequency of 6.1%). Mitochondria containing their new DNA complement became the dominant organelle population in regenerated plants and were faithfully maternally inherited through successive sexnal generations. No concomitant alterations in mitochondrial chromosome organization or nuclear chromosome number occurred. Protoplast fusion can, therefore, cure plant mitochondria of extrachromosomal DNA and, more importantly, be used to transform plant mitochondria with naturally occurring mitochondrial plasmids. The potential for mitochondrial transformation with recombinant vectors is discussed.