Internode length in Pisum. Action of gene lw

Jolly, C. J., Reid, J. B. and Ross, J. J. 1987. Internode length in Pisum. Action of gene lw.
Mutant K29 of Pisum sativum L. is shown to possess a recessive gene at a new locus, lw , which results in reduced internode length, delayed flowering and increased symptoms of water congestion compared with the parental cv. Torsdag. The interaction of gene lw with the internode length genes na, le, la and cry ⁵ is examined. Extracts from the shoots of Iw plants are shown to contain similar levels of gibberellin (GA)-like substances to comparable Lw plants, but Iw plants do not elongate to the same extent as Lw plants when treated with GA₁₉ GA₁₉, or GA₂₀. The effect of gene Iw is not graft-transmissible. Unlike essentially isogenic dwarf lines possessing the GA-synthesis genes le, Ih or Is, lw plants show a relative increase in elongation similar to Torsdag in response to photoperiod extensions from sources rich in far-red light. These results suggest that gene lw probably does not reduce elongation by influencing GA-synthesis and that the response to photoperiod extensions with far-red light may depend on the level of GA.     

Human lymphokine-activated killer cells: further isolation and characterization of the precursor and effector cell

In a series of experiments we have demonstrated the progressive enrichment (5- to 40-fold) in lymphokine-activated killer (LAK) precursor activity by adherence depletion, sheep red cell rosetting, and depletion of CD3- and DR-positive lymphocytes. The LAK precursor cell thus appears to fall within the 'null' cell population. CD16 and CD11 are cell surface antigens expressed on the surface of the LAK precursor as demonstrated in sorting experiments. A 6- to 100-fold enrichment compared to unseparated peripheral blood was noted when sorted cells positive for CD16 and CD11 were tested. The LAK effector has been identified as being primarily CD3- and CD2+. Similar sorting equipment demonstrated a 7- to 500-fold difference in lytic activity for fresh tumor when comparing CD2+/CD3- and CD2+/CD3+ cells. The CD16+/CD11+ lymphocyte can proliferate in response to interleukin-2 (IL-2) alone in the absence of accessory cells and can be expanded in IL-2 alone with maintenance of lytic activity.     

HCO3- transport in basolateral membrane vesicles isolated from rat renal cortex

The mechanism of HCO3- translocation across the proximal tubule basolateral membrane was investigated by testing for Na+-HCO3- cotransport using isolated membrane vesicles purified from rat renal cortex. As indicated by 22Na+ uptake, imposing an inwardly directed HCO3- concentration gradient induced the transient concentrative accumulation of intravesicular Na+. The stimulation of basolateral membrane vesicle Na+ uptake was specifically HCO3(-)-dependent as only basolateral membrane-independent Na+ uptake was stimulated by an imposed hydroxyl gradient in the absence of HCO3-. No evidence for Na+-HCO3- cotransport was detected in brush border membrane vesicles. Charging the vesicle interior positive stimulated net intravesicular Na+ accumulation in the absence of other driving forces via a HCO3(-)-dependent pathway indicating the flow of negative charge accompanies the Na+-HCO3- cotransport event. Among the anion transport inhibitors tested, 4-4'-diisothiocyanostilbene-2,2'-disulfonic acid demonstrated the strongest inhibitor potency at 1 mM. The Na+-coupled transport inhibitor harmaline also markedly inhibited HCO3- gradient-driven Na+ influx. A role for carbonic anhydrase in the mechanism of Na+-HCO3- cotransport is suggested by the modest inhibition of HCO3- gradient driven Na+ influx caused by acetazolamide. The imposition of Cl- concentration gradients had a marked effect on HCO3- gradient-driven Na+ influx which was furosemide-sensitive and consistent with the operation of a Na+-HCO3- for Cl- exchange mechanism. The results of this study provide evidence for an electrogenic Na+-HCO3- cotransporter in basolateral but not microvillar membrane vesicles isolated from rat kidney cortex. The possible existence of an additional basolateral membrane HCO3(-)-translocating pathway mediating Na+-HCO3- for Cl- exchange is suggested.     

Multiple carbohydrate receptors on lymphocytes revealed by adhesion to immobilized polysaccharides

Phosphomannan polysaccharides and fucoidan, a polymer of fucose 4-sulfate, have been demonstrated to inhibit adhesion of lymphocytes to tissue sections that contain high endothelial venules (Stoolman, L. M., T. S. Tenforde, and S. D. Rosen, 1984, J. Cell Biol., 99:1535-1540). We have investigated the potential cell surface carbohydrate receptors involved by quantitating adhesion of rat cervical lymph node lymphocytes to purified polysaccharides immobilized on otherwise inert polyacrylamide gels. One-sixth of the lymphocytes adhered specifically to surfaces derivatized with PPME (a phosphomannan polysaccharide prepared from Hansenula holstii yeast), whereas up to half of the cells adhered to surfaces derivatized with fucoidan. Several lines of evidence demonstrated that two distinct receptors were involved. Adhesion to PPME-derivatized gels was labile at 37 degrees C (decreasing to background levels within 120 min) whereas adhesion to fucoidan-derivatized gels was stable. Soluble PPME and other phosphomannans blocked adhesion only to PPME-derivatized gels; fucoidan and a structurally related fucan blocked adhesion to fucoidan-derivatized gels. Other highly charged anionic polysaccharides, such as heparin, did not block adhesion to either polysaccharide-derivatized gel. Adhesion to PPME-derivatized gels was dependent on divalent cations, whereas that to fucoidan-derivatized gels was not. The PPME-adherent lymphocytes were shown to be a subpopulation of the fucoidan-adhesive lymphocytes which contained both saccharide receptors. These data reveal that at least two distinct carbohydrate receptors can be found on peripheral lymphocytes.     

Distribution of activities of aspartate aminotransferase isoenzymes and malate dehydrogenase in guinea pig retinal layers

Distributions of activity of the cytosolic (cAAT) and mitochondrial (mAAT) isoenzymes of aspartate aminotransferase and of malate dehydrogenase (MDH) were determined in guinea pig retinal layers. The distribution of total AAT activity (tAAT = cAAT + mAAT) and of mAAT activity correlated well (r = 0.88-0.91) with the distribution of MDH activity. mAAT activity was highest in the inner segments of the photoreceptors; there was a greater than twelve-fold difference between activity in that layer and in the inner retinal layers. cAAT activity was also highest in the inner segments, but the difference between the activity in the inner segments and the other layers was not nearly as great as with mAAT. cAAT activity was also relatively high in the outer nuclear layer, outer plexiform layer, and part of the inner plexiform layer. The high activity of cAAT, mAAT, and MDH in the inner segments indicates that all of these enzymes are involved in metabolic reactions related to energy production and/or to photoreceptive processes in the outer segments and, therefore, that the enzymes are probably involved in energy-related metabolism at synapses. However, other functions, including those related to neurotransmission, are not excluded.     

Morphine and Enkephalins Potently Inhibit [3H]Noradrenaline Release from Rat Brain Cortex Synaptosomes: Further Evidence for a Presynaptic Localization of μ-Opioid Receptors

Synaptosomes prepared from rat cerebral cortex and labeled with [3H]noradrenaline (NA) were superfused with calcium-free Krebs-Ringer-bicarbonate medium and exposed to 10 mM K+ plus 0.1 mM Ca2+ so that [3H]NA release was induced. 6,7-Dihydroxy-N,N-dimethyl-2-aminotetralin (TL-99) strongly inhibited synaptosomal K+-induced [3H]NA release (EC50 = 5-10 nM) by activating alpha 2-adrenoceptors. Release was also inhibited (maximally by 40-50%) by morphine (EC50 = 5-10 nM), [Leu5]enkephalin (EC50 = approximately 300 nM), [D-Ala2,D-Leu5]enkephalin (DADLE), and Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAGO) (EC50 values = approximately 30 nM). In contrast to the mu-selective opioid receptor agonists morphine and DAGO, the highly delta-selective agonist [D-Pen2,D-Pen5]enkephalin (1 microM) did not affect [3H]-NA release. Furthermore, the inhibitory effect of DADLE, an agonist with affinity for both delta- and mu-opioid receptors, was antagonized by low concentrations of naloxone. The findings strongly support the view that, like alpha 2-adrenoceptors, mu-opioid receptors mediating inhibition of NA release in the rat cerebral cortex are localized on noradrenergic nerve terminals.     

Changes in responsiveness to ethylene and gibberellin during corolla expansion ofIpomoea nil

Corolla expansion inIpomoea nil appears to be triggered by changes in gibberellin concentration and ethylene production during development. We investigated the role of responsiveness to GA and ethylene in corolla expansion. The effects of growth regulators applied in vitro were measured as a change in area of corolla segments from younger (15–17 mm) and older (18–20 mm) whole corollas. Applied gibberellic acid (GA₃) significantly (p < 0.05) promoted growth in the younger segments but was less effective in the older segments. Moreover, applications of the GA biosynthesis inhibitors, PP333 (paclobutrazol) AMO₁₆₁₈ (2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride), chlorocholine chloride, and tetcyclasis had little effect on younger segments but inhibited growth of older segments. The older corollas have apparently synthesized and accumulated enough GA-like substances to become less responsive to additional applied GA₃. The amount of growth induced by applied or endogenous GA depended on the amount of ethylene simultaneously produced in the tissue. The younger corollas rapidly produced ethylene from endogenous 1-aminocyclopropane-1-carboxylic acid (ACC) and did not respond to applied ACC whereas the older corollas naturally produced much less ethylene and were significantly (p < 0.05) inhibited by applied ACC. When ethylene production was inhibited by applying aminoethoxyvinylglycine (AVG), growth was promoted in all segments. However, only the growth of the younger segments was further stimulated by simultaneously applied AVG and GA₃ over the GA₃ control. Thus the differential responses of segments from 15- to 20-mm long corollas to applied growth regulators reflect developmental changes in responsiveness of the developing corolla. The change in responsiveness is attributed in part to the changes in production of endogenous growth regulators and to the effect of one endogenous plant growth regulator (PGR) on the responsiveness of the corolla to another PGR.     

Binding of an antagonistic monoclonal antibody to an intact and fragmented EGF-receptor polypeptide

A murine monoclonal antibody (No. 425) raised against human A431 carcinoma cells specifically immunoprecipitates the 170,000 molecular weight epidermal growth factor (EGF)-receptor from extracts of A431 cells as well as from extracts of human placenta and cultured fibroblasts, but does not recognize the murine receptor. Binding to the external domain of the human EGF-receptor was indicated by indirect immunofluorescent staining of fixed nonpermeable cells. The antibody binds to both glyco- and aglycoreceptor forms, indicating that the epitope is a part of the polypeptide chain. Binding of the antibody to the receptor is conformation dependent; i.e., denatured receptors lacking EGF-binding activity are not recognized by the antibody. The results of antibody binding studies indicate that the epitope is closely linked to the EGF binding active site, and is common to both high- and low-affinity EGF-receptors. Interaction of this epitope with the antibody inhibits EGF binding and bioactivity, and triggers receptor down-regulation, but does not generate EGFlike kinase-stimulatory or mitogenic responses either in vitro or in vivo. The antibody was tested for its ability to bind to domain-sized fragments of the 170-kDa EGF-receptor. It can recognize both the proteolytically generated 110-kDa EGF binding peptide, and a soluble 100-kDa EGF-receptor secreted by A431 cells. This indicates that the epitope recognized this antibody retains its conformation after proteolytic separation of the EGF binding domain from the rest of the receptor molecule.     

Cytotoxicity of the redox cycling compound diquat in isolated hepatocytes: involvement of hydrogen peroxide and transition metals

Diquat is a hepatotoxin whose toxicity in vivo and in vitro is mediated by redox cycling and greatly enhanced by pretreatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. The mechanism by which redox cycling mediates diquat cytotoxicity is unclear, however. Here, we have attempted to examine the roles of three potential products of redox cycling, namely superoxide anion radical (O2-.), hydrogen peroxide (H2O2), and hydroxyl radical (.OH), in the toxicity of diquat to BCNU-treated isolated hepatocytes. Addition of high concentrations of catalase, but not superoxide dismutase, to the incubations provided some protection against the toxic effect of diquat, but much better protection was observed when catalase was added in combination with the iron chelator desferrioxamine. Addition of desferrioxamine alone also provided considerable protection, whereas the addition of copper ions enhanced diquat cytotoxicity. Taken together, these results indicate that both H2O2 and the transition metals iron and copper could play major roles in the cytotoxicity of diquat. The role of O2-. remains less clear, however, but studies with diethylenetriaminepentaacetic acid indicate that O2-. is unlikely to significantly contribute to the reduction of Fe3+ to Fe2+. The hydroxyl radical or a related species seems the most likely ultimate toxic product of the H2O2/Fe2+ interaction, but hydroxyl radical scavengers afforded only minimal protection.     

Skeletal muscle sarcolemma in malignant hyperthermia: evidence for a defect in calcium regulation

Sarcolemmal properties implicated in the skeletal muscle disorder, malignant hyperthermia (MH), were examined using sarcolemma-membrane vesicles isolated from normal and MH-susceptible (MHS) porcine skeletal muscle. MHS and normal sarcolemma did not differ in the distribution of the major proteins, cholesterol or phospholipid content, vesicle size and sidedness, (Na+ + K+)-ATPase activity, ouabain binding, or adenylate cyclase activity (total and isoproterenol sensitivity). The regulation of the initial rates of MHS and normal sarcolemmal ATP-dependent calcium transport (calcium uptake after 1 min) by Ca2+ (K1/2 = 0.64-0.81 microM), calmodulin, and cAMP-dependent protein kinase were similar. However, when sarcolemmal calcium content was measured at either 2 or 20 min after the initiation of active calcium transport, a significant difference between MHS and normal sarcolemmal calcium uptake became apparent, with MHS sarcolemma accumulating approximately 25% less calcium than normal sarcolemma. Calcium transport by MHS and normal sarcolemma, at 2 or 20 min, had a similar calmodulin dependence (C1/2 = 150 nM), and was stimulated to a similar extent by cAMP-dependent protein kinase or calmodulin. Halothane inhibited MHS and normal sarcolemmal active calcium uptake in a similar fashion (half-maximal inhibition at 10 mM halothane), while dantrolene (30 microM) and nitrendipine (1 microM) had little effect on either MHS or normal sarcolemmal calcium transport. After 20 min of ATP-supported calcium uptake, 2 mM EGTA plus 10 microM sodium orthovanadate were added to initiate sarcolemmal calcium efflux. Following an initial rapid phase of calcium release, an extended slow phase of calcium efflux (k = 0.012 min-1) was similar for both MHS and normal sarcolemma vesicles. We conclude that although a number of sarcolemmal properties, including passive calcium permeability, are normal in MH, a small but significant defect in MHS sarcolemmal ATP-dependent calcium transport may contribute to the abnormal calcium homeostasis and altered contractile properties of MHS skeletal muscle.