Glycine transport by hemolysed and restored pigeon red cells effects of a domnan-induced electrical potential on entry and exit kinetics

The influence of a Donnan effect on the transport of glycine by hemolysed and restored pigeon red cells was examined. The Donnan effect was produced by replacing Cl^? with 2,4-toluenedisulfonate or glutamate. The effects of the associated membrane potential and inside-outside pH difference on glycine entry and exit rates were examined. The effects of pH on entry and exit rates in the absence of a Donnan effect were also examined.In the absence of a Donnan effect, Na⁺-dependent glycine entry requires the protonated form of a group with a pK_app of 7.9 and the depronated form of another group with a pK_app of 6.8. Neither of these are required for exit but the deprotonated form of a group(s) with a pK_app of 6.2 is required. The pK 7.9 group and pK 6.2 group probably react with H⁺ at the inner face of the membrane and the pK 6.8 group probably reacts at the outer face.The V for glycine entry was determined for cells with their Cl^? largely replaced by toluenedisulfonate and without such replacement. Between pH 6.1 and 7, the ratio of the respective V values, V_T/V_Cl, was 1.5–1.7. V_T/V_Cl rose above pH 7 to near 4 at pH 8.3. At pH 6.9, with glutamate replacing cell Cl^?, the analogous ratio (V_Glu/V_Cl) was 1.7. The increase of V_T/V_Cl above pH 7 could be quantitatively accounted for by the increase in cell [H⁺]/medium [H⁺] caused by the Donnan effect together with the assumption that the pK 7.9 group reacts with H⁺ at the inner face of the membrane.When cell Cl^? was replaced by toluenedisulfonate or glutamate there was a drop in the term in the glycine K_m describing Na⁺ dependence of glycine entry. When cell Cl^? was replaced by toluenedisulfonate there was a rise in the Na⁺-independent term in the glycine entry K_m. By replacing varying amounts of cell Cl^? with either toluenedisulfonate or glutamate, plots were obtained of entry rates vs. the cell [Cl^?]/medium [Cl^?] ratio consistent with the assumption that the Donnan-induced membrane potential acts on a “moving” charge. Glycine exit was only slightly accelerated by trans-toluenedisulfonate. The ratio, exit rate into toluenedisulfonate medium/exit rate into Cl^? medium rose with decreasing pH. This rise could be accounted for by a Donnan-induced inside-outside pH difference which affects a pK_app 6.2 group reacting with internal H⁺.The observed influences of the Donnan effect on V(glycine entry), on both components of K_m(glycine entry), on the shape of the plot of glycine entry rate vs. the cell [Cl^?]/medium [Cl^?] ratio and on glycine exit all fit the assumptions that when the empty porter reorients, one unit of negative charge accompanies it “across” the membrane and that no other steps involve charge movement.The properties of the system seem inconsistent with a translational (“ferry boar”) mobile carrier.     

Thermoregulatory effects of purines and caffeine

Purines are putative neurotransmitters which appear to be involved in regulating several vegetative functions. We examined the effect of purines and their antagonist, caffeine, on colonic temperature of rats. Adenosine injected ip lowered colonic temperature in a dose responsive manner at ambient room temperatures. Adenine and AMP also lowered body temperature whereas 7-methylinosine and inosine only slightly influenced colonic temperature. Caffeine (50 mg/kg) injected sc, increased colonic temperature and when injected within 60 seconds of adenosine, counteracted the hypothermic effect of adenosine (50 mg/kg). Low ambient temperature (4°C) accentuated the thermoregulatory effects of adenosine. Thus adenosine appears to have a hypothermic effect on body temperature regulation when administered peripherally which can be reversed by caffeine.     

Properties and characterization of low molecular weight inhibitor of calmodulin-dependent cAMP phosphodiesterase from rat liver

We have identified two different species of inhibitors of calmodulin-dependent cAMP phosphodiesterase: 1) a low molecular weight (LMW) and 2) a high molecular weight (HMW) form. These inhibitors are extracted from rat liver. Both LMW and HMW inhibitors are heat-stable, acidic in nature and lose activity with prolonged storage and/or repeated freezing and thawing. The low molecular weight inhibitor has been purified to about 7,000-fold with 300% recovery. LMW inhibits calmodulin-dependent cAMP phosphodiesterase regardless of the source of calmodulin (e.g. fat, brain, heart, erythrocytes). LMW appears to be lipid in nature with a molecular weight of 1,500-5,000. The role of these inhibitors in diabetes and mechanism of action of insulin is presented.     

Nerve Outgrowth by Dorsal Root Ganglia in vitro: Stimulation by Inhibitors of DNA Metabolism in the Absence of Exogenous Nerve Growth Factor

Dorsal root ganglia from 8-day chick embryos can be stimulated to extend nerve processes in culture by inclusion of cytosine arabinoside (Ara-C) in the culture medium, in the absence of exogenous nerve growth factor (NGF). The degree of stimulation is dose dependent, and is not mimicked by either free cytosine or free arabinose. Since Ara-C is known to inhibit DNA synthesis, other inhibitors of DNA synthesis were tested. Hydroxyurea, fluorodeoxyuridine, and 3 mM thymidine all stimulated nerve outgrowth in the absence of exogenous NGF. In addition, bromodeoxyuridine also stimulated nerve outgrowth. In all cases, stimulation was observable after 24 h of culture, with maximal outgrowth achieved by 72 h of culture. The experimental response was never as large as the response to NGF, but was up to seven times greater than control outgrowth. In all cultures, nerve processes were characterized by growth cones at their distal tips, colchicine-sensitivity, and a high tubulin content visualized by immunofluorescence with anti-tubulin antibody.     

Prostaglandin D2 lowers nuclear DNA polymerase activity in cultured mastocytoma cells

Prostaglandin D₂ strongly inhibited growth of cultured mastocytoma P-815, 2-E-6 cells, which were established and cloned from mouse mast tumor cells. The inhibition was dose-dependent (IC₅₀ = 2.09 × 10⁻⁵ M). Prostaglandin D₂ also inhibited the DNA synthesizing activity of the cells dose-dependently. We next measured the activities of endogenous DNA polymerases extracted from untreated and prostaglandin D₂-treated cells. Prostaglandin D₂ pretreatment reduced DNA polymerase α activity by 52%. The sedimentation coefficients of the enzymes from untreated and prostaglandin D₂-treated cells were the same suggesting there was no gross change in the size of the enzyme. Prostaglandin D₂ pretreatment of the cells reduced endogenous DNA polymerase β activity to 68% of the control value; the sedimentation coefficients of the enzymes from treated and untreated cells were both 3.5 S. Interestingly, prostaglandin D₂ had no direct inhibitory effect on the activity of either DNA polymerase α or β. Our results indicate that the activities of DNA polymerase α and β are lower in prostaglandin D₂-treated mastocytoma cells. This finding account for the lower level of DNA synthesis in these cells.     

Polarized infrared spectra of crystalline glycosaminoglycans

Polarized infrared spectra have been recorded for oriented, crystalline specimens of hyaluronates, chondroitin 4-sulfate and 6-sulfate, dermatan sulfate, and a cartilage proteoglycan, having different known chain conformations as determined by X-ray diffraction. The dichroism data for the vibrational modes of the amide and carboxyl groups have been interpreted with respect to the particular molecular structures.     

Diuretic effects on renal brush border membrane transport and metabolism

Previous studies have indicated that the thiazide diuretics exert effects on proximal electrolyte transport. To determine whether the locus of these effects is at the brush border membrane (BBM) and if renal metabolism is affected, adult female Sprague-Dawley rats were acutely treated with either 1 mg/kg metolazone, 20 mg/kg chlorothiazide followed by a 20 mg/kg/hr maintenance infusion, 10 mg/kg acetazolamide followed by a 10 mg/kg/hr maintenance infusion, or the vehicles only. Administration of these agents resulted in an approximately tenfold increase in sodium excretion. Neither urinary phosphate nor inulin excretion changed significantly in any group. Sodium dependent BBM vesicle phosphate transport was examined at 0.15, 0.5, and 1 and 120 minute incubation periods in the diuretic treated groups and their respective control groups. Decreased uptake was seen in all pre-equilibrium time points in rats treated with metolazone: 0.15 minutes: 221 +/- 24 pmoles/mg protein (pmol/mg prot) in control rats versus (vs) 185 +/- 23 pmoles/mg prot in metolazone-treated animals (P less than .05) ; 0.5 minutes: 463 +/- 54 vs 369 +/- 49 pmol/mg prot (P less than .005); 1 minute: 549 +/- 74 vs 460 +/- 61 pmol/mg prot (P less than .05); no significant difference in phosphate transport was noted at the two hour equilibrium time point. No significant differences in sodium dependent phosphate transport existed between chlorothiazide or acetazolamide treated rats and control animals. Substrate-stimulated renal gluconeogenesis did not differ between metolazone treated and control animals. We therefore conclude that metolazone inhibits phosphate transport through an effect on the BBM and does not affect renal gluconeogenesis in the rat.     

Rapid stepwise solubilization and purification of type C retrovirus structural proteins by extraction with organic solvent

The phosphoprotein p12 and the nucleoprotein p10 of Rauscher murine leukemia virus have been isolated by extraction of aqueous virus suspensions with neutral chloroform-methanol followed by centrifugation to separate the phases. The procedure involves, first, extraction with neutral chloroform-methanol under conditions of low ionic strength. The phosphoprotein p12 and the RNA partition to the aqueous phase and the viral lipids to the organic phase, and the remainder of viral proteins form an interphase layer. In the second step, extraction of the interphase resuspended in high ionic strength buffer selectively partitions p10 to the aqueous phase. The antigenicity of p10 and p12 proteins is preserved and their N-terminal amino acid sequences and compositions were found to be identical with published data. By extraction of the interphase with acidic chloroform-methanol, viral proteins p30, p15, and p15(E) can be solubilized.     

Solubilization of brush borders of hamster small intestine and fractionation of some of the components

About 90% of the protein of hamster intestinal brush borders was solubilised in 0.25% (w/v) sodium dodecyl sulphate without total loss of biological activity. Detergent-polyacrylamide gel electrophoresis of the solubilised protein separated 10–15 bands and partially resolved maltase, lactase, sucrase-maltase, trehalase and alkaline phosphatase activities. The disaccharidases, which were associated with the higher molecular weight proteins, were preferentially solubilised with 0.1%. (w/v) Triton X-100, butanol or papain, whereas Tris and NaI extracted only the lower molecular weight proteins, possible derived from the core filaments.Electrophoresis of brush border proteins metabolically labelled with [¹⁴C] glucosamine suggested that many of the membrane-bound enzymes are glycoproteins. However, chromatography of a papain digest on Sephadex G-200 showed that the sucrase-maltase complex can be separated nearly free of carbohydrate without total loss of activity.The importance of characterizing membrane proteins solubilised by a number of techniques is discussed.     

Effects of culture conditions an hyperoxia on antioxidant enzymes in pig pulmonary artery and aortic endothelium

Because hyperoxia induces early injury to lung endothelial cells and since tolerance to hyperoxia is correlated with increased lung antioxidant enzyme activity, we measured superoxide dismutase, catalase and glutathione peroxidase in both fresh isolates and primary cultures of endothelial cells from pig pulmonary artery and aorta. Cultured endothelial cells were studied at confluency and up to 5 days thereafter under control or hyperoxic conditions. In both types of confluent cell, total and cyanide-insensitive superoxide dismutase increased when compared to fresh cells. The most conspicuous postconfluency change in both types of endothelial cell was a marked decrease in gluthathione peroxidase, which could be prevented by the addition of selenomethionine to culture media. A 5-day exposure to hyperoxia resulted in a 2-fold increase in cyanide-insensitive superoxide dismutase in both aortic and pulmonary artery endothelial cells. In view of a similar decrease in DNA in both types of cells despite some differences in enzyme levels, oxygen cytotoxicity could not be related to a particular antioxidant enzyme profile.     

A comparison of negatively and positively charged liposomes containing entrapped polynosinic · polycytidylic acid for interferon induction in mice

Intravenous injection of negatively charged liposomes containing entrapped poly(I) · poly(C) induced a vigorous interferon response in mice with serum titers of interferon reaching twenty times those observed with comparable dosages of free poly(I) · poly(C). The response did not persist over an extended time period as was observed earlier for enhanced interferon production stimulated by positively charged liposomes containing the inducer. Both negatively and positively charged liposomes containing [¹⁴C]poly(I) · poly(C) were taken up chiefly by the liver when given intravenously. Negatively charged particles were concentrated somewhat preferentially by the spleen (7–9% of the dose compared to 4–6%). Less radioactivity was found in liver and spleen when negatively charged particles were given intraperitoneally than was the case when positively charged particles were injected by this route. Free [¹⁴C]poly(I) · poly(C) was extensively metabolized to low molecular weight materials within four hours of injection, while encapsulation of the polymer provided protection against in vivo degradation. When both preferential localization and protection were considered, from three to five times as much high molecular weight [¹⁴C]poly(I) · poly(C) was recovered from liver at four hours after intravenous injection when the compound was given in encapsulated form compared to the free polymer. Similarly, for spleen, seven times and three times as much polymeric [¹⁴C]poly(I) · poly(C) was recovered following injection of negatively charged liposomes and positively charged liposomes respectively compared to free [¹⁴C]poly(I) · poly(C). At 48 h after an intravenous injection of positively charged liposomes, as much as four percent of the dose remained in high molecular weight form in the liver and one percent in the spleen. Following intraperitoneal injections, polymeric [¹⁴C]poly(I) · poly(C) recovered from the liver never exceeded 4.3% of the dose, showing that most of the radioactivity in the liver consisted of metabolites. These results suggest that elevated and prolonged production of interferon in animals treated with encapsulated inducer results from a combination of factors including preferential tissue location and protection of the inducer from hydrolytic cleavage.     

Collagen and elastin synthesis in the developing chick aorta

Thoracic aortas from 8- to 18-day embryonic chicks were incubated in vitro for 30 min with [³H]glycine and the newly synthesized, labeled proteins were subjected to polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The gels were fractionated and the incorporation of label into procollagen (125,000 M_r) and tropoelastin (70,000 M_r) was estimated by summation of the radioactivity found in the appropriate regions of the gel. The analyses showed that at Day 8 approximately 14% of the incorporated [³H]glycine was found in procollagen and 22% in tropoelastin. In the following 6 days of development, there was a significant decline in the relative incorporation into procollagen and an increase into tropoelastin so that at Days 14–18 less than 10% of the label was found in collagen and 40% was now found in tropoelastin. Since glucocorticoids have been shown to alter the rate of synthesis of other proteins in the developing chick, 150 μg of hydrocortisone was injected into 8-day eggs and 24 h later the aortas were incubated and treated as described above. The pattern of protein synthesis exhibited by the hormone-treated aortas resembled that of 14- to 18-day embryos. Furthermore, incubation of 8-day aortas with 10^?8m hydrocortisone for 24 h produced a significant increase in the rate of elastin synthesis relative to that of other proteins. These results demonstrate that collagen and elastin synthesis vary during development of the chick aorta and they suggest that glucocorticoids may be involved in the control of their synthesis.     

Phosphorylation of lens membrane: identification of the catalytic subunit of Na+, K+-ATPase

Receptor mediated endocytosis appears to depend on the action of a transglutaminase (TGase). Endocytosis can be induced in intact human RBC by the action of several classes of drugs. We tested the hypothesis that drugs acted by stimulating TGase activity. Of the endocytosis inducing drugs tested, neither primaquine nor vinblastine nor chlorpromazine enhanced TGase activity. We next tested the hypothesis that TGase activity was required for drug endocytosis in RBC by adding known TGase inhibitors. Paradoxically, m-Dansyl cadaverine, the most potent TGase inhibitor, produces endocytosis in human RBC. Therefore despite apparent striking morphologic similarities, drug induced endocytosis in RBC appears to proceed via different mechanisms from those involved in receptor mediated endocytosis in other cells.In the receptor-mediated endocytosis of some hormones and growth factors, it appears that the receptor-ligand complex forms clusters over clathrin coated pits which are then internalized as endocytic vacuoles. Both the clustering and internalization of ligands are inhibited by a variety of agents shown to inhibit transglutaminase (TGase) and it is therefore proposed that TGase participates in receptor-mediated endocytosis (1–3). Human erythrocytes undergo endocytosis when exposed to drugs like primaquine, chlorpromazine, and vinblastine (4), all of which are amphipathic cations (4). However, the mechanism of drug action is not known nor is it clear that this is a form of receptor-mediated endocytosis (4). Furthermore, clustering of receptors can occur in neonatal but not adult human RBC (5). TGase has been measured in human red cells (6) although its physiologic role is unknown. Like all TGases, it is calcium dependent (6,7), and primaquine induced red cell endocytosis is enhanced by Ca⁺⁺ addition (8). Therefore, we tested the hypothesis that TGase participates in drug induced endocytosis in intact human red cells.     

A physiological requirement of Na+ for the regulation of cAMP levels in intact NG108-15 cells.

An antigonadotropic compound present in extracts of bovine pineal gland which reduces compensatory ovarian hypertrophy in adult mice was partially purified by gel filtration and further characterized by ion-exchange chromatography and high voltage paper electrophoresis.An acetic acid extract of bovine pineal glands was gel-filtered on Sephadex G-25, from which two antigonadotropic fraction were obtained and designated as F4 and F5. Each of these fractions was further purified by high voltage paper electrophoresis. The antigonadotropic activity of F4 was found in the neutral and acid regions. The F4 fraction was also further purified by cation exchange chromatography. The fraction eluted at pH 4.4 from the cation exchange chromatogram was found to be antigonadotropic. This fraction (pH 4.4) was then further purified by high voltage paper electrophoresis. Antigonadotropic activity was found in the area of the neutral region of the electrophoretogram.The antigonadotropic material is thought not to be melation or arginine vasotocin based on the antigonadotropin being eluted from Sephadex G-25 in a fraction distinct from these two compounds.     

Effect of acriflavine on ultraviolet inactivation of Acholeplasma laidlawii

An increased sensitivity to inactivation was observed when ultraviolet light-irradiated Acholeplasma laidlawiiAn increase sensitivity to inactivation was observed when ultraviolet light-irradiated Acholeplasma laidlawii cells were plated on medium containing either acriflavine or chloramphenicol. Chloramphenicol reduced liquid holding recovery (dark repair) to about 10 percent of that in untreated irradiated cells. In acriflavine treated cells no dark repair could be observed and there was a progressive degradation of cell DNA during holding. While the primary effect of acriflavine may be to inhibit excision repair, since ultraviolet-irradiated Mycoplasma gallisepticum (cells which lack an excision repair mechanism) show a slight increase in inactivation when plated on medium containing acriflavine, the dye must also have some other effects on ultraviolet repair processes. Acriflavine treatment of A. laidlawii cells before ultraviolet irradiation has a protective effect, as seen by an increased cell survival.     

Studies of the metabolism of carbon disulfide by rat liver microsomes

Carbon disulfide has been examined as a substrate for the hepatic mixed function oxidase enzyme system. These studies have revealed that carbonyl sulfide is formed when carbon disulfide is incubated with rat liver microsomes in the presence of NADPH. No carbonyl sulfide is formed in the absence of NADPH. The formation of carbonyl sulfide is inhibited when the reaction is carried out in an atmosphere containing carbon monoxide. The other product of the reaction leading to carbonyl sulfide formation appears to be a highly reactive form of sulfur which covalently binds to the microsomal membrane. A chemical mechanism for the mixed function oxidase catalyzed metabolism of carbon disulfide to carbonyl sulfide is proposed.     

Steroid induction of nerve growth factor synthesis in cell culture

Nerve growth factor (NGF) is a peptide hormone which is necessary for the development of sympathetic neurons. Exposing a rat central nervous system glioma cell line (C-6) to the steroid hormone 17β-estradiol increases the amount of NGF secreted by these cells into the surrounding medium. This induction is highly specific to 17β-estradiol in that similar steroids do not increase NGF levels. Both NGF activity and protein levels increase upon estradiol stimulation and there is a parallel increase in NGF $\text{de}$$\text{novo}$ synthesis. The estradiol effect can be blocked with actinomycin D but not with puromycin or cycloheximide. This is the first report demonstrating regulation of NGF synthesis by a steroid hormone in a clonal cell line of glial origin. We propose this system as a model system for the study of the regulation of NGF synthesis and the isolation and analysis of putative precursors to the NGF molecule.