Generation of heteroplastidic Nicotiana cybrids by protoplast fusion: analysis for plastid recombinant types

Summary Protoplasts of a mutant line of Nicotiana tabacum having a maternally-transmitted chlorophyll deficiency were fused with protoplasts of two alloplasmic-male-sterile Nicotiana lines by the donor-recipient technique. In both fusion experiments variegated plantlets were regenerated which were shown to contain cytoplasms of mixed chloroplast nature. This confirms that with the donor-recipient method one can obtain mixed cytoplasms of genetically different chloroplasts. We present a convenient system to assay for genetic recombination between chloroplasts by combining use of several cytoplasmic markers: vis. chlorophyll pigmentation, chloroplast DNA restriction patterns, tentoxin resistance and male sterility. Within the limits of the experiment no recombinant types were recovered.     

Leu-enkephalin-stimulated growth hormone and prolactin release in the rat: comparison with the effect of morphine

The effect of Leu⁵-enkephalin on growth hormone (GH) and prolactin (PRL) release was studied $\text{in vivo}$ in the infant rat and compared to that of morphine. In 10 day-old pups, intracerebroventricular injection of Leu⁵-enkephalin (50, 75 and 100 μg) resulted in a dose-related increase in plasma GH; morphine was active as GH releaser at the dose of 5 and 10 μg, but not at 2.5 μg. Pretreatment with naloxone (2 mg/kg ip) suppressed the GH-releasing effect of either Leu⁵-enkephalin (100 μg) or morphine (10 μg). Leu⁵-enkephalin (75 and 100 μg) induced a rise in plasma PRL which was neither dose-related nor antagonized by naloxone; morphine (5 and 10 μg) was active as PRL releaser and its effect was antagonized by naloxone. These results indicate that: 1) Leu⁵-enkephalin stimulates both GH and PRL release; 2) the release of GH by Leu⁵-enkephalin but likely not that of PRL involves specific opiate receptors; 3) morphine releases GH and PRL through specific opiate receptors.     

The role of sodium-channel density in the natriferic response of the toad urinary bladder to an antidiuretic hormone

Summary Urinary bladders ofBufo marinus were depolarized, by raising the serosal K concentration, to facilitate voltage-clamping of the apical membrane. Passive Na transport across the apical membrane was then studied with near-instantaneous current-voltage curves obtained before and after eliciting a natriferic response with oxytocin. Fitting with the constant-field equation showed that the natriferic effect is accounted for by an increase in the apical Na permeability. It is accompanied by a small increase in cellular Na activity. Furthermore, fluctuation analysis of the amiloride-induced shot-noise component of the short-circuit current indicated that the permeability increase is not due to increased Na translocation through those Na channels which were already conducting prior to hormonal stimulation. Rather, the natriferic effects is found to be based on an increase in the population of transporting channels. It appears that, in response to the hormone, Na channels are rapidly recruited from a pool of electrically silent channels.     

Brain hypoxia and control of breathing: neuromechanical control

The effects of graded brain hypoxia on respiratory cycle timing, the lung inflation reflex, and respiratory compensation for an inspiratory flow-resistive load were studied in unanesthetized goats. Two models, inhalation and CO and acute reduction of brain blood flow (BBF) were used to produce comparable levels of brain hypoxia. The lung inflation reflex was assessed as the ratio of inspiratory time of an occluded breath to that of the preceding spontaneous breath (TIoccl/TIspont). Compensation for flow-resistive loading was assessed as the effect of the load upon the airway occlusion pressure response to rebreathing CO2 (delta P 0.1/delta PCO2). Major findings were 1) severe brain hypoxia (HbCO of 60% or BBF of 42%) caused tachypnea due to a 50% or more reduction of expiratory time but only a 20% or less reduction of inspiratory time; 2) moderate carboxyhemoglobinemia (HbCO of 25-30%) enhanced TIoccl/TIspont from 1.5 +/- 0.1 at control to 2.1 +/- 0.1, while severe brain hypoxia (HbCO of 60% and BBF of 42%) reduced the ratio to 1.0 +/- 0.2; and 3) compensation for a flow-resistive load, manifested by increases of delta P 0.1/delta PCO2 of 75-300% in the control state, was abolished at HbCO of 45-50% and BBF of 60%. The data suggest that in unanesthetized animals brain hypoxia elicits tachypnea largely by an effect on the expiratory phase of the bulbopontine timing mechanism. The observed enhancement of the lung inflation reflex and abolition of flow-resistive load compensation are best explained by hypoxic depression of higher than brain stem neural function.     

Isolation of radio-iodinated apical and basal-lateral plasma membranes of toad bladder epithelium

Summary The apical and basal-lateral plasma membranes of toad bladder epithelium were radio-iodinated with the glucose-glucose oxidase-lactoperoxidase system. The covalently bound radio-iodine was used as a marker during subcellular fractionation and membrane isolation. Homogenization conditions that ensured rupture of more than 80% of the cells without substantial nuclear damage were defined by Nomarski optics. The nuclei were separated by differential centrifugation and the apical and basal-lateral components were resolved by differential and sucrose density gradient centrifugation. The apical components yielded two radioactive bands that were identified as glycocalyx and plasma membrane labeled with¹²⁵I. The basal-lateral components yielded a heterodisperse pattern made up of at least 3 radioactive bands, but the bulk of the activity of ouabain-sensitive ATPase comigrated with only one of these bands. The mitochondia, identified by assays for cytochrome oxidase and NADH cytochromec reductase activities, were separated from the radio-iodine labeled components by centrifugation in sucrose density gradients under isokinetic conditions. The labeled glycocalyx and the slowly migrating components of basal-lateral labeling were separated from the radio-iodinated membranes by centrifugation at 100,000 × g × 1 hr after removal of the mitochondria by the isokinetic method. The labeled membranes were then subjected to ultracentrifugation in sucrose density gradients under isopycnic conditions; the basal-lateral membranes containing ouabain-sensitive ATP-ase were well resolved from the apical membranes by this method. These results provide a relatively rapid method of attaining partial purification of the apical and basal-lateral plasma membranes of toad bladder epithelium.     

Effect of aldosterone on incorporation of amino acids into renal medullary proteins

Summary Studies on the effects of pretreatment with aldosterone on the incorporation of³H leucine or³H methionine into proteins in renal slices were carried out in Joklik-modified minimal essential medium. Administration of aldosterone (2 g/100 g body wt) to adrenalectomized rats increased³H leucine incorporation into trichloroacetic acid insoluble fractions of crude homogenates of cortical slices by 15.5±0.4% and of medullary slices by 53.5±1.3%. No increase in isotope incorporation was observed in slices of renal papilla or spleen prepared from the same rats. Aldosterone had no effect on the³H-leucine content of the trichloroacetic acid-soluble fractions of all three renal zones and the spleen. The dose of aldosterone that elicited a half-maximal increase in³H-methionine incorporation into proteins of renal medullary slices (0.45 g of aldosterone/100 g body wt) was indistinguishable from that needed to elicit a halfmaximal increase in the urinary K⁺/Na⁺ ratio (0.35 g of aldosterone/100 g body wt). Dexamethasone, a potent glucocorticoid, at a dose of 0.8 g/100 g body wt did not augment³H-leucine incorporation into renal medullary proteins but was effective at 8 g/100 g body wt. Spirolactone (SC-26304), a potent anti-mineralocorticoid, abolished the effect of aldosterone on amino acid incorporation into medullary proteins when administered at a 100-fold higher dosage [i.e., 80 gvs. 0.8 g (per 100 g body wt)]. These results imply that the action of aldosterone on amino acid incorporation is mediated by the mineralocorticoid rather than the glucocorticoid pathway, presumably the mineralocorticoid receptors. Moreover, pretreatment of the rats with actinomycin D (70–80 g/100 g body wt) erased the effect of aldosterone (0.8 g/100 g body wt) on amino acid incorporation into medullary proteins.In paired experiments with³H and³⁵S methionine, aldosterone (0.8 g/100 g body wt) increased methionine incorporation into trichloroacetic acid precipitable proteins of subcellular fractions of the renal medulla. The effect of aldosterone on incorporation of methionine into medullary cytosol proteins was analyzed further by polyacrylamide gel electrophoresis at pH 8.3 in tris-glycine buffer. The gel profiles indicate that aldosterone significantly increased methionine incorporation into at least one protein (independent of the isotope) with a molecular weight of 31,000. This increase was inhibited by either pretreatment of the rat with actinomycin D (70–80 g/100 g body wt or SC-26304 (80 g/100 g body wt). Dexamethasone (0.8 g/100 g body wt) did not increase incorporation of methionine into the medullary cytosol proteins resolved by polyacrylamide gel electrophoresis.     

Induction of citrate synthase by aldosterone in the rat kidney

Summary The possible induction of renal citrate synthase (E.C. 4.1.3.7), by aldosterone was evaluated in the adrenalectomized rat. Three hours after administration of aldosterone (0.8 g/100 g body wt), renal cortical and medullary citrate synthase activity was significantly increased as reported previously by Kinne and Kirsten (Kinne, R., Kirsten, R. 1968.Pfleugers Arch. 300:244). In contrast, no change in this activity was detected in the renal papilla or the liver, under the same conditions. Kinetic analysis revealed that injection of aldosterone had no effect on theK _m s for acetyl-CoA and oxalacetate but augmentedV _max of renal medullary citrate synthase activity by 40%. The aldosterone-dependent increase in medullary citrate synthase activity was proportionate to the associated increase in the quantity of antiserum (specific for citrate synthase) required for half-maximal immuno-precipitation.The possibility that aldosterone induced the synthesis of citrate synthase was evaluated in two sets of experiments. In the first set, adrenalectomized rats were injected intraperitoneally with either aldosterone (0.8 g/100 g body wt) or the diluent, and simultaneously with³H or³⁵S methionine (500 Ci/rat). The isotopes were reversed in about half of the experiments. Three hours after the injection, renal citrate synthase was isolated by ATP-sepharose column chromatography and immuno-precipitation with the specific antiserum. Aldosterone augmented methionine incorporation into renal citrate synthase by 55% but had no effect on incorporation into the hepatic enzyme. In the second set, adrenalectomized rats were injected with either aldosterone (0.8 g/100 g body wt) or the diluent, the kidneys were removed 1 hr later and medullary slices were incubated in either³H-or³⁵S-methionine at 20° for 2 hr. Mitochondrial citrate synthase was isolated either by ATP-sepharose column chromatography and immuno-precipitation, or by polyacrylamide gel electrophoresis. Aldosterone increased methionine incorporation into the immuno-precipitates by 30% and into the enzyme peak resolved by polyacrylamide gel electrophoresis by 43%. The latter increase was eliminated by prior administration of either actinomycin D (70–80 g/100 g body wt) or spirolactone (SC-26304) (80 g/100 g body wt). An equimolar dose of dexamethasone (0.8 g/100 g body wt) had no effect on the isotope ratio associated with citrate synthase activity in the polyacrylamide gels.     

Structure of the chicken neuron-glia cell adhesion molecule, Ng-CAM: origin of the polypeptides and relation to the Ig superfamily

The neuron-glia cell adhesion molecule (Ng-CAM) mediates both neuron-neuron and neuron-glia adhesion; it is detected on SDS-PAGE as a predominant 135-kD glycoprotein, with minor components of 80, 190, and 210 kD. We have isolated cDNA clones encoding the entire sequence of chicken Ng-CAM. The predicted extracellular region includes six immunoglobulin-like domains followed by five fibronectin-type III repeats, structural features that are characteristic of several neural CAMs of the N-CAM superfamily. The amino acid sequence of chicken Ng-CAM is most similar to that of mouse L1 but the overall identity is only 40% and Ng-CAM contains a short fibronectin-like segment with an RGD sequence that has no counterpart in L1. These findings suggest that Ng-CAM and L1 may not be equivalent molecules in chicken and mouse. The amino-terminal sequences of the 210-, 190-, and 135-kD components of Ng-CAM are all the same as the predicted amino terminus of the molecule, whereas the 80-kD component begins within the third fibronectin repeat. The cDNA sequence is continuous across the junction between the 135- and 80-kD components, and a single 170-kD Ng-CAM polypeptide was isolated from tunicamycin-treated cells. In addition, all cDNA probes hybridized on Northern blots to a 6-kb RNA, and most hybridized to single bands on Southern blots. These results indicate that the Ng-CAM components are derived from a single polypeptide encoded by a single gene, and that the 135- and 80-kD components are generated from the 210/190-kD species by proteolytic cleavage. The 135-kD component contains most of the extracellular region including all of the immunoglobulin-like domains. It has no transmembrane segment, but it is tightly associated with the membrane. The 80-kD component contains two and a half type III repeats plus the RGD-containing segment, as well as the single transmembrane and cytoplasmic domains. These structural features of Ng-CAM provide a framework for understanding its multiple functions in neuron-neuron interactions, neurite fasciculation, and neuron-glia interactions.     

Anomalous mobilities of Na,K-ATPase alpha subunit isoforms in SDS-PAGE: identification by N-terminal sequencing.

Three isoforms of the alpha subunit of Na,K-ATPase, alpha 1, alpha 2, and alpha 3 have been characterized at the DNA, mRNA and protein levels. In admixtures, isoforms migrate as doublets (i.e. alpha 1 and another band originally designated alpha +, comprising alpha 2 + alpha 3) when analyzed by SDS-PAGE. As deduced from cDNA sequences their masses range from 111.7 to 112.6 kDa. With conventional protein standards, however, SDS-PAGE yields nominal masses of 85-105 kDa. In this system, the presence of a doublet that reacted with a polyclonal anti-Na,K-ATPase antibody in the kidney was interpreted as indicating two molecular or conformational species of the kidney alpha sub-unit (Siegel, G.J. and Desmond, T.J. (1989) J. Biol. Chem. 264, 4751-4754). We report that Na,K-ATPase purified from dog, guinea pig and rat kidney medulla or from rat brain, can yield two distinct bands when analyzed by SDS-PAGE or STS-PAGE, migrating between 85 and 105 kDa. An additional band migrating at 117 and 120 kDa appears often in enzyme purified from rat and guinea pig kidney medulla. The apparent molecular weights and relative intensities of these bands vary with temperature and duration of incubation during sample preparation. N-terminal sequencing and monospecific antibody probes revealed that the two distinct bands obtained from the kidney enzyme consist only of the alpha 1 isoform. The band appearing at 117-120 kDa also contains only the alpha 1 N-terminal sequence. In contrast, as reported earlier (Sweadner, K.J. (1979) J. Biol. Chem. 254, 6060-6067), the doublet seen in brain preparations consists of alpha 1 and alpha 2 or (alpha 2 + alpha 3). We conclude that monospecific antibody probes or N-terminal sequencing must be used to identify Na,K-ATPase isoforms by SDS- or STS-PAGE. In addition, gel conditions that may affect the mobilities of the isoforms are discussed.