Antilipolytic activity of viprostol, a transdermally active antihypertensive PGE2 analog

Viprostol, a novel prostaglandin E2 congener, was assessed for in vitro antilipolytic activity in the spontaneously obese rat. In isolated epididymal adipocytes, viprostol exhibited a dose-dependent inhibition of catecholamine-stimulated lipolysis at concentrations ranging from 10 microM to 1 mM, but was ineffective at lower concentrations. Additionally, viprostol exhibited approximately 50% of the antilipolytic activity of naturally-occurring PGE1 and PGE2 at similar concentrations, but was as potent as PGF2 alpha. At 10 microM, viprostol inhibited maximum catecholamine-stimulated lipolysis by approximately 35% of the total, hormone-stimulated glycerol release. The results of these experiments indicate that viprostol exhibits antilipolytic activity in vitro, but is less potent than the naturally-occurring PGE's to which it is most closely related structurally.     

Drought Stress and Elevated CO(2) Effects on Soybean Ribulose Bisphosphate Carboxylase Activity and Canopy Photosynthetic Rates

Soybean (Glycine max [L.] cv Bragg) was grown at 330 or 660 microliters CO₂ per liter in outdoor, controlled-environment chambers. When the plants were 50 days old, drought stress was imposed by gradually reducing irrigation each evening so that plants wilted earlier each succeeding day. On the ninth day, as the pots ran out of water CO₂ exchange rate (CER) decreased rapidly to near zero for the remainder of the day. Both CO₂-enrichment and drought stress reduced the total (HCO₃⁻/Mg²⁺-activated) extractable ribulose-1,5-bisphosphate carboxylase (RuBPCase) activity, as expressed on a chlorophyll basis. In addition, drought stress when canopy CER values and leaf water potentials were lowest, reduced the initial (nonactivated) RuBPCase activity by 50% compared to the corresponding unstressed treatments. This suggests that moderate to severe drought stress reduces the in vivo activation state of RuBPCase, as well as lowers the total activity. It is hypothesized that stromal acidification under drought stress causes the lowered initial RuBPCase activities. The K_m(CO₂) values of activated RuBPCase from stressed and unstressed plants were similar; 15.0 and 12.6 micromolar, respectively. RuBP levels were 10 to 30% lower in drought stressed as compared to unstressed treatments. However, RuBP levels increased from near zero at night to around 150 to 200 nanomoles per milligram chlorophyll during the day, even as water potentials and canopy CERs decreased. This suggests that the rapid decline in canopy CER cannot be attributed to drought stress induced limitations in the RuBP regeneration capability. Thus, in soybean leaves, a nonstomatal limitation of leaf photosynthesis under drought stress conditions appears due, in part, to a reduction of the in vivo activity of RuBPCase. Because initial RuBPCase activities were not reduced as much as canopy CER values, this enzymic effect does not explain entirely the response of soybean photosynthesis to drought stress.     

l-Phenylalanyl-l-Glutamate-Stimulated, Chloride-Dependent Glutamate Binding Represents Glutamate Sequestration Mediated by an Exchange System

Stimulation of glutamate binding by the dipeptide L-phenylalanyl-L-glutamate (Phe-Glu) was inhibited by the peptidase inhibitor bestatin, suggesting that the stimulation was caused by glutamate liberated from the dipeptide and not by the dipeptide itself. It further suggests that this form of glutamate binding should be reinterpreted as glutamate sequestration and that stimulation of binding both by dipeptides and after preincubation with high concentrations of glutamate is likely to be due to counterflow accumulation. Several other criteria indicate that most of glutamate binding stimulated by chloride represents glutamate sequestration: Binding is reduced when the osmolarity of the incubation medium is increased, when membranes incubated with [3H]glutamate are lysed before filtration, and when membranes are made permeable by transient exposure to saponin. Moreover, dissociation of bound glutamate after a 100-fold dilution of the incubation medium is accelerated about 50 times by the addition of glutamate to the dilution medium. This result would be anomalous if glutamate were bound to a receptor site; it suggests instead that glutamate is transported in and out of membrane vesicles by a transport system that preferentially mediates exchange between internal and external glutamate. Glutamate binding contains a component of glutamate sequestration even when measured in the absence of chloride. Sequestration is adequately abolished only after treating membranes with detergents; even extensive lysis, sonication, and freezing/thawing may be insufficient.     

Photosynthetic Activity in the Flower Buds of ;Valencia' Orange (Citrus sinensis [L.] Osbeck)

Flower buds of `Valencia' orange (Citrus sinensis [L.] Osbeck) were able to fix ¹⁴CO₂ into a number of compounds in their own tissues under both light and dark conditions. The total incorporation, however, was about 4-fold higher in the light than in the dark. In the light, 50% of the total ¹⁴C label was found in the neutral fraction (sugars), 22% in the basic fraction (amino acids), and 26% in the acid-1 fraction (organic acids). In the dark, about 95% of the ¹⁴C label was incorporated into the basic and acid-1 fractions. Activities of ribulose bisphosphate carboxylase and phosphoenolpyruvate carboxylase (expressed in micromoles CO₂ per milligram protein per hour) averaged 1.95 and 8.87 for the flower buds, and 28.5 and 3.6 for the leaves, respectively. The ability of orange flower buds to fix ambient CO₂ into different compounds suggests that this CO₂ assimilation may have some regulatory role during the early reproductive stages in determining citrus fruit initiation and setting.     

Intrapleural and intravenous Corynebacterium parvum in patients with resected stage I and II non-small cell carcinoma of the lung

Summary A prospective randomized trial compared the administration of intrapleural plus intravenous Corynebacterium parvum (C. parvum) versus placebo in patients with resected Stage I and Stage II non-small cell bronchogenic carcinoma. Treatment consisted of 7 mg C. parvum injected into the pleural space and 7 mg C. parvum intravenously once between days 6 and 12 postoperatively and 7 mg intravenously every 3rd month during the 1st year. Intrapleural administration of 35 cc of saline served as the placebo and the flush after intrapleural C. parvum.Of the 303 patients entered into this study, 286 were evaluable, with an average follow-up time of 3.5 years. More complications, especially fever, were observed in patients receiving C. parvum. A fever greater than 38 °C was observed in 9% of the patients assigned to placebo and 76% of the patients assigned to C. parvum. There was no significant difference between the treatments with respect to disease-free interval or survival.M. Kaufmann, J. Stjernswärd**, A. Zimmermann (Ludwig Institute for Cancer Research, Bern Branch); K. Stanley**, M. Isley, M. Zelen (Frontier Science & Tech. Research Foundation, Brookline, MA, USA); C. Mouritzen, P. Paulsen, U. Henriques (Dept. of Thoracic and Cardiovascular Surgery and Institute of Pathology, Kommunehospital, Aarhus, Denmark); N. Konietzko, W. Maassen, W. Hartung, W. Wierich (Ruhrland Clinic, Essen-Heidhausen, and Pathology Institute, Ruhr-University, Bochum, FRG); P. Oehl (Innere Klinik und Poliklinik Tumorforschung, Essen, FRG); J. Vogt-Moykopf, H. Toomes, W. Hofmann (Rohrbach Hospital, Clinic for Thoracic Medicine and Pathology Institute, Heidelberg, FRG); F. Krause, R. Rios, R. Spanel (Klinik Löwenstein, Löwenstein, and Pathology Institute, Ulm, FRG); J. Orel, B. Hrabar, D. Ferluga, T. Rott (University Medical Center, Thoracic Surgery and Pathology, Ljubljana, Yugoslavia); H. A. Rostad, J. R. Vale, P. Lexow (Rikshospital, Oslo, Norway); S. Hagen, S. Birkeland (Ulleval Hospital, Oslo, Norway); T. Harbitz, R. Nissen-Meyer (Aker Hospital, Oslo, Norway); E. Aspevik, H. Engedal, A. Mykin (Haukeland Hospital, Bergen, Norway); V. O. Björk, L. Rodriguez, K. Böök, J. Willems (Karolinska Sjukhuset, Thoracic Surgical Clinic and Pathology Department, Stockholm, Sweden); E. Grädel, J. Hasse, P. Dalquen (Kantonsspital, Dept of Surgery, Div. of Cardiac & Thoracic Surgery & Pathology Institute, Basel, Switzerland); L. Eckmann, K. Hänni, K. Zimmermann (Tiefenauspital Surg. Clinic, Univ. of Bern, Switzerland); B. Nachbur, H. U. Würsten, H. Cottier, A. Zimmermann (Inselspital Dept. of Thoracic and Cardiovascular Surg. and Pathology Institute, Bern, Switzerland); W. Maurer, M. Kaufmann (Bürgerspital, Surgical Department, Solothurn, Switzerland); H. Denck, E. Zwintz, St. Wuketich (Krankenhaus der Stadt Wien-Lainz, I. Chir. Dept., and Path. Inst., Vienna, Austria); N. Pridun, H. Hackl (Pulmonologisches Zentrum der Stadt Wien, and Path. Inst., Vienna, Austria); E. Moritz, W. Schlick, H. Holzner (II. Chir. University Clinic and Path. Inst., Vienna, Austria); K. Karrer (Institute for Cancer Research, Vienna, Austria); R. G. Crispen (ITR-Biomedical Research, University of Illinois, Chicago, USA); D. S. Freestone, R. Bomford, M. T. Scott, T. Priestman, L. Toy (The Wellcome Research Laboratories, Beckenham, England)** Present address: Cancer Unit, World Health Organization, Geneva, Switzerland Offprint requests to: K. Stanley, Ludwig Institute for Cancer Research, Inselspital, CH-3010 Bern, SwitzerlandLudwig Lung Cancer Study Group:     

Pharmacological Characteristics and Distributions of σ and Phencyclidine Receptors in the Animal Kingdom

The phylogenetic distributions ofσ- and phencyclidine receptors in neural tissues of 13 species and the pharmacological characteristics of these receptors in whole sea anemone and neural tissues of the guinea pig, chicken, and frog were studied. Specific binding of [³H]haloperidol and [³H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine, ligands that bind with high affinity to σ- and phencyclidine receptors, respectively, was detected in all organisms examined. The order of potencies of various ligands to inhibit 1 nM [³H]haloperidol binding in brains of frogs and guinea pigs or 1 nM [³H]N-[1-(2-thienyl)cyclohexyl]-3,4–piperidine in chicken or guinea pig brain homogenates was very similar. However, the characteristics and stereospecificity of binding of the two radioligands in sea anemone were different than in higher organisms. The results suggest that σ– and phencyclidine binding sites are evolutionarily old, as the characteristics of the two sites are well preserved over a range of vertebrate phyla.     

Human melanoma cells express a novel integrin receptor for laminin

This study sought to determine whether human melanoma cells express integrin-related receptors that mediate their adhesion to laminin. We found that antibodies against the integrin beta 1 chain blocked cell attachment to laminin-coated surfaces. Furthermore, immunofluorescence staining demonstrated beta 1 complexes in vinculin-positive focal adhesion plaques on the basal surface of cells attached to laminin substrates. Chromatography of detergent extracts of 125I-surface-labeled cells on laminin-Sepharose columns recovered two major laminin-binding proteins (100 and 130 kDa, reduced) that bound with high affinity to the columns and were eluted with EDTA. Both proteins were specifically immunoprecipitated from column fractions with monoclonal and polyclonal antibodies to the integrin beta 1 subunit, indicating that they form a noncovalent heterodimer complex. The alpha-like subunit is composed of a 30-kDa light chain that is joined by a disulfide bond to the 100-kDa heavy chain. This complex was not recovered from columns of fibronectin- or collagen type I- or IV-Sepharose. Laminin-binding by the alpha beta 1 complex was independent of Arg-Gly-Asp or Tyr-Ile-Gly-Ser-Arg-like sequences, but required the presence of divalent cations. The 100-kDa alpha-like subunit was electrophoretically and immunochemically distinct from the other known alpha subunits, alpha 1-alpha 6. The results indicate that human melanoma cells express a novel laminin-specific integrin beta 1 complex which may mediate the cells' interactions with this ligand.     

Thiophosphorylation causes Ca2+-independent norepinephrine secretion from permeabilized PC12 cells

Adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) was used to examine the role of phosphorylation in the regulation of norepinephrine secretion by digitonin-permeabilized PC12 cells. While most kinases will use ATP gamma S to thiophosphorylate proteins, thiophosphorylated proteins are relatively resistant to dethiophosphorylation by protein phosphatases. Norepinephrine secretion by permeabilized PC12 cells was ATP- and Ca2+-dependent but resistant to calmodulin antagonists. Half-maximum secretion was obtained in 0.75 microM Ca2+. Permeabilized PC12 cells were incubated with ATP gamma S in the absence of Ca2+, the ATP gamma S was removed, and norepinephrine secretion was determined. Preincubation with ATP gamma S increased the amount of norepinephrine secreted in the absence of Ca2+, but it had no effect on the amount released in the presence of Ca2+. After a 15-min preincubation in 1 mM ATP gamma S, there was almost as much secretion in the absence of Ca2+ as in its presence. Inclusion of ATP in the preincubation inhibited the effect of ATP gamma S. Ca2+ stimulated the rate of modification by ATP gamma S as brief preincubations with ATP gamma S in the presence of Ca2+ resulted in higher levels of Ca2+-independent secretion than did preincubations with ATP gamma S in the absence of Ca2+. Similarly, brief preincubations of permeabilized cells with ATP in the presence of Ca2+ resulted in elevated levels of Ca2+-independent secretion. Secretion of norepinephrine from ATP gamma S-treated cells was ATP-dependent. These results suggest that norepinephrine secretion by PC12 cells is regulated by a Ca2+-dependent phosphorylation. Once this phosphorylation has occurred, secretion is still ATP-dependent, but it no longer requires Ca2+.     

Effects of D2O on permeation and gating in the Ca2+-activated potassium channel fromChara

We studied the effects of H₂O/D₂O substitution on the permeation and gating of the large conductance Ca²⁺-activated K⁺ channels inChara gymnophylla droplet membrane using the patchclamp technique. The selectivity sequence of the channel was: K⁺>Rb⁺≫Li⁺, Na⁺, Cs⁺ and Cl⁻. The conductance of this channel in symmetric 100mm KCl was found to be 130 pS. The single channel conductance was decreased by 15% in D₂O as compared to H₂O. The blockade of channel conductance by cytosolic Ca²⁺ weakened in D₂O as a result of a decrease in zero voltage Ca²⁺ binding affinity by a factor of 1.4. Voltage-dependent channel gating was affected by D₂O primarily due to the change in Ca²⁺ binding to the channel during the activation step. The Hill coefficient for Ca²⁺ binding was 3 in D₂O and around 1 in H₂O. The values of the Ca²⁺ binding constant in the open channel conformation were 0.6 and 6 μm in H₂O and D₂O, respectively, while the binding in the closed conformation was much less affected by D₂O. The H₂O/D₂O substitution did not produce a significant change in the slope of channel voltage dependence but caused a shift as large as 60 mV with 1mm internal Ca²⁺.