Respiration and sodium transport in rabbit urinary bladder

Respiration of rabbit urinary bladder was measured in free-floating pieces and in short-circuited pieces mounted in an Ussing chamber. Ouabain, amiloride, and potassium-free saline inhibited respiration approx. 20%; sodium-free saline depressed respiration approx. 40–50%. The coupling ratio between respiration and transport in short-circuited tissues was about two sodium ions per molecule O₂. Chloride-free saline depressed mean oxygen consumption 21% in free-floating tissue pieces; 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS) and furosemide had no effect. The effect of chloride-free saline in short-circuited tissues was variable; in tissues with low transport rates, respiration was stimulated about 21% while in tissue with high transport rates respiration was reduced about 24%. Nystatin and monensin, both of which markedly increase the conductance of cell membranes with a concomitant increase in sodium entry, stimulated respiration. These data indicate that 50–60% of the total oxygen consumption is not influenced by sodium, 20–25% is linked to (Na⁺ + K⁺)-ATPase transport, while the remaining 25–30% is sodium-dependent but not ouabain-inhibitable.     

The effects of anion transport inhibitors on structural transitions in erythrocyte membranes

Red blood cell membranes have been labeled with several covalent and non-covalent inhibitors of anion transport and their heat capacity profiles determined as a function of temperature. Covalent inhibitors include the amino reactive agents 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid, 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid, pyridoxal phosphate and 1-fluoro-2,4-dinitro benzene. The non-covalent inhibitors include several well known local anesthetics. The study was undertaken in order to identify regions of the membrane involved in anion transport. Covalent modification in all cases resulted in a large upward shift of the C transition, which is believed to involved a localized phospholipid region. Evidence is presented which indicates that Band III protein and this phospholipid region are in close physical proximity on the membrane. Addition of non-covalent inhibitors affects the membrane in either or both of two ways. In some cases, a lowering and broadening of the C transition occurs; in other the B₁ and B₂ transitions are altered. These latter transitions are believed to involve both phospholipid and protein, including Band III. These results may indicate that the non-covalent inhibitors produce their inhibitory effect on anion transport at least in part by interacting with membrane phospholipid.     

Flavonoids from the exudate of Acacia neovernicosa

2′,4′-Dihydroxychalcone, 4′-hydroxy-2′-methoxychalcone and 2′,4′-dihydroxy-3′-methoxychalcone were isolated and characterized from the resinous exudate produced by Acacia neovernicosa. Smaller amounts of isoliquiritigenin, pinocembrin and chrysin were also found and identified by their chromatographic properties and UV spectra. The material of one collection contained galangin, 3-methylkaempferol and 3,3′ -dimethylquercetin.     

Effect of staphylococcal alpha-hemolysin upon anion transport in the rabbit erythrocyte

Equilibrium exchange of SO₄^2? was measured prior to and during hemolysis in rabbit erythrocytes exposed to staphylococcal α-hemolysin. The anion-transport protein of the rabbit erythrocyte has also been identified. Equilibrium exchange of SO₄^2? was measured by both efflux and influx of ³⁵SO₄^2?. The rate of influx of SO₄^2? in rabbit erythrocytes exposed to α-hemolysin was twice that of the untreated cells. The rate of SO₄^2? efflux was unchanged by α-hemolysin. Inhibition of anion exchange with 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) did not inhibit hemolysis, therefore, the increased influx of SO₄^2? may occur through a DIDS-insensitive pathway.     

A relationship between anion transport and a structural transition of the human erythrocyte membrane

Scanning microcalorimetry was employed as an aid in examining some structural features of the anion transport system in red blood cell vesicles. Two structural transitions were previously shown to be sensitive to several covalent and non-covalent inhibitors of anion transport in red cells. In this study, these transitions were selectively removed, either thermally or enzymatically, and the subsequent effect on ³⁵SO₄^2? efflux in red cell vesicles was determined. It is shown that removal of one of these transitions (B₂) has a negligible inhibitory effect on anion transport. Cytoplasmic, intermolecular disulfide linkages between band 3 dimers are known to form during the B₂ transition. The integrity of the 4,4′-diisothiocyanostilbene-2,2′-disulfonate-sensitive C transition, on the other hand, is shown to be a requirement for anion transport. The localized region of the membrane giving rise to this transition contains the transmembrane segment of band 3, as well as membrane phospholipids. The calorimetric results suggest a structure of band 3 which involves independent structural domains, and are consistent with the transmembrane segment playing a direct role in the transport process.     

Cl− transport in apical plasma membrane vesicles isolated from bovine tracheal epithelium

The Cl^? transport properties of the luminal border of bovine tracheal epithelium have been investigated using a highly purified preparation of apical plasma membrane vesicles. Transport of Cl^? into an intravesicular space was demonstrated by (1) a linear inverse correlation between Cl^? uptake and medium osmolarity and (2) complete release of accumulated Cl^? by treatment with detergent. The rate of Cl^? uptake was highly temperature-sensitive and was enhanced by exchange diffusion, providing evidence for a carrier-mediated transport mechanism. Transport of Cl^? was not affected by the ‘loop’ diuretic bumetanide or by the stilbene-derivative anion-exchange inhibitors SITS (4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid) and DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid). In the presence of the impermeant cation, tetramethylammonium (TMA⁺), uptake of Cl^? was minimal; transport was stimulated equally by the substitution of either K⁺ or Na⁺ for TMA⁺. Valinomycin in the presence of K⁺ enhanced further Cl^? uptake, while amiloride reduced Na⁺-stimulated Cl^? uptake towards the minimal level observed with TMA⁺. These results suggest the following conclusions: (1) the tracheal vesicle membrane has a finite permeability to both Na⁺ and K⁺; (2) the membrane permeability to the medium counterion determines the rate of Cl^? uptake; (3) Cl^? transport is not specifically coupled with either Na⁺ or K⁺; and, finally (4) Cl^? crosses the tracheal luminal membrane via an electrogenic transport mechanism.     

Isoflavonoids from Myroxylon balsamum

Myroxylon balsamum (Leguminosae-Lotoideae) trunk wood contains a series of biogenetically related flavonoids, including the novel (±) -7-hydroxy-4′-methoxyisoflavanone, (±)-7,3′-dihydroxy-4′-methoxyisoflavanone and 2-(2′,4′-dihydroxyphenyl)-5,6-dimethoxybenzofuran.     

Labeling of human erythrocyte membranes with eosin probes used for protein diffusion measurements. Inhibition of anion transport and photo-oxidative inactivation of acetylcholinesterase

The binding of eosin-isothiocyanate (eosin-NCS) and iodoacetamido-eosin (IA-eosin) to band 3 proteins in the membrane of human erythrocytes is characterized by studying the effect of these probes on the anion transport system. Although the unbrominated fluorescein precursors do not affect anion transport, both eosin labels are strong inhibitors of sulphate exchange in intact erythrocytes. 50% inhibition is obtained by binding 4.7 · 10⁵ or 6.0 · 10⁵ molecules/cell for eosin-NCS and IA-eosin, respectively. Both eosin probes are irreversibly bound and occupy common binding sites with 4,4′-diisothiocyano-1,2-diphenyl-ethane-2,2′-disulfonic acid (H₂DIDS), although other sites are labeled as well. The inhibition of anion transport is light independent and can therefore not be attributed to a photosensitizing action of the eosin probes. Both eosin derivatives, however, inactivate acetylcholinesterase upon illumination of air-equilibrated samples of hemoglobin-free labeled ghosts. The inactivation of the enzyme is accompanied by the formation of protein aggregates as visualized by polyacrylamide gel electrophoresis. These effects are not observed when intact erythrocytes are illuminated in the presence of eosin probes suggesting a protective effect of hemoglobin during the labeling procedure. Protection of ghosts from photo-oxidation is achieved by displacing air with argon. These results are discussed in relation to the use of these and similar probes to measure protein diffusion in membranes.     

The location of a disulfonic stilbene binding site in band 3, the anion transport protein of the red blood cell membrane

The binding site for 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, a specific, potent, irreversible inhibitor of anion transport in red blood cells is located in a 15 000 dalton transmembrane segment of band 3, produced by chymotrypsin treatment of ghosts stripped of extrinsic proteins. The segment was cleaved into three fragments of 7000, 4000 and 4000 daltons by CNBr. The C-terminus of the segment is located in the 7000 dalton fragment; the N-terminus in one of the 4000 dalton fragments; and the binding site for 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid in the middle 4000 dalton fragment. The latter was cleaved by $\text{N-}\text{bromosuccinimide}$ into two fragments of 2000 daltons. The binding site for 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid was located on the fragment containing the newly formed N-terminus. It is concluded that the binding site is located about 9000 daltons from the C-terminus (at the outside face of the membrane) and 6000 daltons from the N-terminus (at the cytoplasmic face). In view of the existing evidence that the binding site may be located near the outside face of the membrane, it is suggested that the 15 000 dalton segment is folded, so that it crosses the bilayer three times.     

The effects of general anaesthetics on glucose and phosphate transport across the membrane of the human erythrocyte

A study has been carried out into the effects of clinically important general anaesthetics, althesin, thiopentone and propanidid, on the transport of glucose and phosphate across the membrane of the human erythrocyte. In general these three substances all inhibit both transport processes but with characteristic inhibition profiles and varying degrees of efficacy. Glucose transport was more sensitive to the hydrophobic steroids and phosphate transport to propanidid. Some hydrophobic agents, e.g., iodobenzene and its azide, were not inhibitory. Removal of cholesterol to some extent augmented the inhibitory effects of most of these compounds (not propanidid). It is argued that these effects are due to the penetration of the anaesthetics into the lipid bilayer and either subsequent disruption of the lipid annuli surrounding the integral membrane proteins and/or direct anaesthetic-protein interaction.     

Specific drug sensitive transport pathways for chloride and potassium ions in steady-state ehrlich mouse ascites tumor cells

A major aim of this investigation was to determine whether, in steady-state ascites cells, Cl^? transport can be partitioned into a furosemide-sensitive cotransport with K⁺ and a separate 4,4′-isothiocyanostilbene-2,2′-disulfonic acid (DIDS) sensitive self-exchange. Both Cl^? and K⁺ fluxes were studied. The furosemide- and Cl^? sensitive K⁺ fluxes were equivalent, both in normal ionic media and when the external K⁺ concentration, [K⁺]_o, was varied from 4 to 30 mM. The stoichiometry of the furosemide-sensitive Cl^? and K⁺ fluxes was 2 Cl^?: 1 K⁺ at 0.1 and 0.5 mM drug levels but increased to 3 Cl^? : 1 K⁺ at 1.0 mM furosemide. DIDS at 0.1 mM had no effect on the K⁺ exchange rate but inhibited Cl^? exchange by $\text{39\% ± 2}$ (S.E.). The effects of DIDS and 0.5 mM furosemide on Cl^? transport were additive but 1.0 mM furosemide and DIDS had overlapping inhibitory actions. Thus furosemide acts on components of K⁺ and Cl^? transport which are linked to each other, but the drug also inhibits an additional DIDS-sensitive Cl^? pathway, when present at higher concentrations. The dependence of the furosemide-sensitive K⁺ and Cl^? transport on [K⁺]_o was also studied; both fluxes fell as the [K⁺]_o increased. The latter results recall those in an earlier study by Hempling (Hempling, H.G. (1962) J. Cell. Comp. Physiol. 60, 181–198).     

Evaluation of 3-(4′-(4″-fluorophenyl)-6′-phenylpyrimidin-2′-yl)-2-phenylthiazolidin-4-one for in vivo modulation of biomarkers of chemoprevention in the 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis

A new bis heterocycle comprising both bioactive 2-aminopyrimidine and thiazolidin-4-one nuclei namely 3-(4′-(4″-fluorophenyl)-6′-phenylpyrimidin-2′-yl)-2-phenylthiazolidin-4-one 3 was synthesized, characterized with the help of melting point, elemental analysis, FT-IR, MS, one-dimensional NMR (¹H, ¹³C) spectra and we evaluated the chemopreventive potential of 3-(4′-(4″-fluorophenyl)-6′-phenylpyrimidin-2′-yl)-2-phenylthiazolidin-4-one based on in vivo inhibitory effects on 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch carcinogenesis. Administration of 3 effectively suppressed oral carcinogenesis initiated with DMBA as revealed by the reduced incidence of neoplasms. Lipid peroxidation, glutathione (GSH) content, and the activities of glutathione peroxidase (GPx), glutathione S-transferase (GST) were used to biomonitor the chemopreventive potential of 3. Lipid peroxidation was found to be significantly decreased, whereas GSH, GPx, GST, and GGT were elevated in the oral mucosa of tumor-bearing animals. Our data suggest that 3 may exert its chemopreventive effects in the oral mucosa by modulation of lipid peroxidation and enhancing the levels of GSH, GPx, and GST.     

A comparison of the inhibitory potency of reversibly acting inhibitors of anion transport on chloride and sulfate movements across the human red cell membrane

The effects of a variety of chemically diverse, reversibly acting inhibitors have been measured on both Cl^? and SO₄^2? equilibrium exchange across the human red cell membrane. The measurements were carried out under the same conditions (pH 6.3, 8°C) and in the same medium for both the Cl^? and SO₂⁴ tracer fluxes. Under these conditions the rate constant for Cl^?-Cl^? exchange is about 20 000 times larger than that for SO₄^2?-SO₄^2? exchange. Despite this large difference in the rates of transport of the two anions, eight different reversibly acting inhibitors have virtually the same effect on the Cl^? and SO₄^2? transport. The proteolytic enzyme papain also has the same inhibitory effect on both the Cl^? and SO₄^2? self-exchange. In addition, the slowly penetrating disulfonate 2-(4′-aminophenyl)-6-methylbenzenethiazol-3′,7-disulfonic acid (APMB) is 5-fold more effective from the outer than from the inner membrane surface in inhibiting both Cl^? and SO₄^2? self-exchange. We interpret these results as evidence that the rapidly penetrating monovalent anion Cl^? and the slowly penetrating divalent anion SO₄^2? are transported by the same system.     

Nucleosides and Nucleotides. 232. Synthesis of 2′-C-Methyl-4′-thiocytidine: Unexpected Anomerization of the 2′-Keto-4′-thionucleoside Precursor

The synthesis of 2′-C-methyl-4′-thiocytidine (16) is described. Since the 2′-keto-4′-thiocytidine derivative 2β unexpectedly isomerized to 2α and the methylation of 2β proceeded predominantly from the less hindered α-face to give 7, the desired product 16 was synthesized via the Pummerer reaction of the sulfoxide 14 and N ⁴ -benzoylcytosine.     

Pyrenocine C,A phytotoxin-related metabolite produced by onion pink root fungus,Pyrenochaeta terrestris

The structure of pyrenocine C, a new metabolite isolated from onion pink root fungus, Pyrenochaeta terrestris (Hansen) has been elucidated as (±)-(2′E)-5-(1′-hydroxybut-2′-enyl)-4-methoxy-6-methyl-2-pyrone by spectroscopic methods and chemical correlation with pyrenocine A.     

Synthesis of dihydrochalcones of Myrica gale

4,4,6-Trimethyl-2-(3-phenylpropionyl)cyclohexane-1,3,5-trione, 2′-hydroxy-4′,6′-dimethoxy-3′-methyldihydrochalcone, 2′,6′-dihydroxy-4′-methoxy-3′,5′-dimethyldihydrochalcone and 2,2,5-trimethyl-4(3-phenylpropionyl)cyclopent-4-ene-1,3-dione, constituents of Myrica gale, have been synthesized.     

Dihydrochalcones from lindera umbellata

Two dihydrochalcones, 2′,6′-dihydroxy-4′-methoxydihydrochalcone and 2,4′,6′-trihydroxydihydrochalcone have been isolated from leaves of Lindera umbellata.     

Anion transport across the erythrocyte membrane,in situ proteolysis of band 3 protein,and cross-linking of proteolytic fragments by 4,4′-diisothiocyano dihydrostilbene-2,2′-disulfonate

Extracellular chymotrypsin cleaves the 95 000 dalton protein that migrates in band 3 of SDS-polyacrylamide gel electropherograms of the erythrocyte membrane into fragments of 60 000 and 35 000 daltons, but not further. Minor components of band 3 that remain at the original 95 000 dalton location may be eluted from the membrane by 0.1 N NaOH, indicating that, in contrast to the major component and the chymotryptic fragments, they are not integral membrane constituents.Incubation at neutral pH of chymotrypsinized erythrocytes with the bifunctional anion transport inhibitor 4,4′-diisothiocyano dihydrostilbene-2,2′-disulfonic acid results in covalent binding of that inhibitor primarily to the 60 000 dalton fragment and some cross-linking of the 60 000 dalton fragment with the 35 000 dalton fragment. Increasing the pH to 9.5 leads to a crosslinking of virtually all of the pairs of chymotryptic fragments and thus to a reconstitution of band 3 with its typical diffuse appearance in the 95 000 dalton region of the SDS-polyacrylamide gels. This indicates that (1) each integral 95 000 dalton protein molecule is capable of binding at least one 4,4′-diisothiocyano dihydrostilbene-2,2′-disulfonic acid molecule; (2) the 35 000 dalton fragment, though it is only weakly stained with Coomassie blue, is present in an amount that is equimolar with that of the 60 000 dalton fragment. Since the number of 4,4′-diisothiocyano dihydrostilbene-2,2′-disulfonic acid binding sites on the protein in band 3/cell is known to be close to the number of band 3 molecules/cell, it is suggested that the cross-linking takes place at a region of the band 3 molecule that is involved in the control of anion transport.Like chymotrypsin, papain digests the band 3 protein from the outer membrane surface. Unlike chymotrypsin, however, papain digestion results in an inhibition of anion exchange. Papain produces a major fragment of 60 000 daltons that differs from the major chymotryptic fragment by at most six amino acid residues. The only detectable difference between the non-inhibitory action of chymotrypsin and the inhibitory action of papain on the band 3 protein is that papain is capable of partially digesting the 35000 dalton fragment. No reconstitution of band 3 by cross-linking of the fragments with 4,4′-diisothiocyano dihydrostilbene-2,2′-disulfonic acid can be achieved. Since the 35 000 dalton fragment reacts with one of the two reactive groups of 4,4′-diisothiocyano dihydrostilbene-2,2′-disulfonic acid and is also susceptible to digestion by the inhibitory papain, we suggest that a portion of this peptide participates, together with a portion of the 60 000 dalton fragment, in the control of anion transport.     

Macrantaline and macrantoridine,new alkaloids from a turkish sample of Papaver pseudo-orientale

The major alkaloids of the aerial parts of a Turkish sample of Papaver pseudo-orientale are salutaridine and a new alkaloid, macrantaline, UV, IR, PMR, MS and CD have been used to establish the structure of macrantaline as 1-(2′-hydroxymethylene-3′,4′-dimethoxybenzyl)-2-methyl-6,7-methylenedioxy-8-methoxy-1,2,3,4-tetrahydroisoquinoline. The corresponding 2′-methyl substituted analogue prepared from (?)-α-narcotine and also from macrantaline proved to have identical properties, including CD spectra, thus confirming the structure and establishing the absolute configuration of macrantaline. A new minor alkaloid, macrantoridine, yielded macrantaline on lithium aluminium hydride reduction and differs from the latter in that the 2′-substituent is a carboxyl instead of hydroxymethylene. UV, IR, PMR, MS and CD data are reported for macrantoridine.     

Triplex Formation Involving 2′-O,4′-C-Methylene Bridged Nucleic Acid (2′,4′-BNA) with 2-Pyridone Base Analogue: Efficient and Selective Recognition of C:G Interruption

Abstract

For the effective recognition of C:G interruption in homopurine-homopyrimidine duplex DNA, we examined triplex-forming ability and sequence-selectivity of a triplex-forming oligonucleotide (TFO) involving of 2′-O,4′-C-methylene bridged nucleic acid with 2-pyridone base analogue. We found that the modified TFO formed stable triplex with high binding affinity and sequence-selectivity.