Collagen in developing chick muscle in vivo and in vitro

Because of the known role of collagen in chick skeletal muscle differentiation the collagen synthesized by embryonic chick muscle was studied. The major collagen synthesized by this muscle was found to be type I collagen. In addition, the effectiveness of types I, II, III and IV collagens in promoting myoblast fusion in vitro was compared. These collagens were found to be equally effective as in vitro substrates.     

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.     

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.     

Vitamin D-dependent calcium-binding protein synthesis by chick kidney and duodenal polysomes

The hormonally active form of vitamin D, 1,25-dihydroxy vitamin D₃, is known to induce in the intestine and kidney of chicks the synthesis of a calcium-binding protein (CaBP). Here we report a correlation between the tissue levels of CaBP and the levels of apparent messenger RNA in total polysomes as determined by the vitamin D and dietary calcium status. Polysomes from pooled duodenal mucosa and kidney were prepared by the Mg²⁺ precipitation method. After translation in a heterologous, rabbit nuclease-treated reticulocyte system, the immunoprecipitated pellet of CaBP was dissolved and the proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gels. When 13 nmol of D₃ was given to 4-week-old rachitic chicks which were sacrificed 48 h later, it was found that the duodenum had eightfold more apparent mRNA for CaBP in the polysomes than the kidney. This was also reflected in the values of CaBP/mg protein in these tissues (duodenum, 7 μg/mg vs kidney, 0.9 μ/mg). Also, after giving D₃, there was a twofold increase in both apparent mRNA levels in the polysomes and in CaBP levels in the duodena of chicks which were raised on low-calcium diets versus chicks raised on high-calcium diets. While apparent mRNA for CaBP was present in polysomes from rachitic chick kidney, it was not detectable in the duodenum. From these studies it appears that the induction of CaBP by 1,25(OH)₂D₃ in both the intestine and kidney is determined by similar control mechanisms.     

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.     

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.     

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.     

Differential expression of aortic and lung elastin genes during chick embryogenesis

The ratios of tropoelastin b to a were measured in chick aorta and lung during embryogenesis. The rates of tropoelastin a and b synthesis were determined in short-term organ culture. The results demonstrated that in lung tissue the ratio of the two tropoelastins remained essentially constant. Each of the tropoelastins comprised 50% of the total elastin synthesis. In the aortic tissue, tropoelastin b represented 70% of the total elastin in the 11- to 13-day embryos and increased to 91% by Day 16. These observations seen in the organ culture system were paralleled in measurements of functional mRNAs coding for the two proteins. Measurements of functional tropoelastin mRNAs from both lung and aortic tissues were performed in a mRNA-dependent rabbit reticulocyte lysate system. Although the changes in the abundance of the tropoelastin mRNAs revealed the same trend as that seen in the organ culture data, the magnitude of the tropoelastin b to a ratio in the aortic organ culture was twice that determined in the cell-free translation of aortic mRNAs. The data obtained from both cell-free translations and organ culture experiments demonstrate that there is a differential expression of elastin genes during aorta development which is significantly different from that found in developing lung.     

High affinity renal [3H]flunitrazepam binding: Characterization,localization, and alteration in hypertension

The binding of [³H]flunitrazepam was studied in membranes prepared from the kidney and cerebral cortex of unilaterally nephrectomized rats made hypertensive by simultaneous deoxycorticosterone acetate (DOCA) and NaCl administration. A significant 35–43% increase in the number of [³H]flunitrazepam binding sites (Bmax) was found in the renal membranes prepared from the hypertensive rats; there was no change in the density of binding sites in the membranes obtained from the cerebral cortex. The K_d of [³H]flunitrazepam binding did not change either in the renal or in the cerebral membranes (～ 12 nM in the kidney and ～2.0 nM in the brain). Drug specificity studies with renal membranes showed that the inhibition of [³H]flunitrazepam binding by various benzodiazepines did not jibe with their pharmacologic potency as anxiolytic agents. An intrarenal distribution of specific [³H]flunitrazepam binding was found in the bovine kidney; specific binding was greatest in the outer cortex and virtually absent in the medulla, the minor calyx and the renal artery. The evidence that the renal benzodiazepine binding site is of high affinity, is specific, has a unique distribution, and is regulated during hypertension suggests that it may be associated with an important pathophysiologic structure.     

Separation of three cyclic-nucleotide-phosphodiesterases from bovine aorta.

Three distinct forms of cyclic-nucleotide-phosphodiesterases were separated and purified from the media layer of bovine aorta using two successive DEAE-cellulose column chromatographies. Form A hydrolyzed both cAMP and cGMP with similar K_m and V_max and was sensitive to the calcium dependent protein activator; its activity on both substrates was inhibited by cIMP. Form B hydrolyzed specifically cGMP, was insensitive to the activator but was slightly stimulated in a proteinaceous medium; it was inhibited by cIMP. Form C was specific for cAMP and was insensitive to the activator and to a proteinaceous medium; it was poorly inhibited by cIMP.     

A control element within a structural gene: the gal operon of Escherichia coli

The gal operon of Escherichia coli is transcribed from two overlapping promoters, PG1 and PG2. Cyclic AMP and its receptor protein (CRP) modulate the two promoters in opposite directions by binding to a single cat locus. Both the promoters are negatively regulated by a single repressor, the product of the galR gene. An operator site, defined by several mutations, has previously been located upstream from the cat locus. We have isolated and characterized a new set of cis-dominant constitutive mutations of the gal operon and determined their locations by DNA sequencing. From these studies, we propose the existence of a second functional gal operator element at an extraordinary site--within galE, the first structural gene. Both the operators, OE (exterior) and OI (interior), are involved in the repression of PG1 and PG2. This would be the first example of the presence of a functional operator element within a structural protein-coding region.     

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.     

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.     

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.     

The behavioral responses in ovariectomized cattle to either estradiol,testosterone, androstenedione,or dihydrotestosterone

Steroid hormone effects on sexual behavior were measured in 15 sexually mature nulliparous cattle which were bilaterally ovariectomized. They were alloted at random to five groups of three animals each (sesame oil vehicle control, estradiol, testosterone, androstenedione, and dihydrotestosterone) in the fall of the year and reassigned at random to replicate the study the following spring. Each experiment was divided into three weekly trials. Animals within treated groups were reassigned each week to receive in random order one of three levels of a particular hormone (200, 400, and 800 μg of estradiol and up to 1000 times these doses of androgens).Estradiol, and to a lesser extent, testosterone were capable of increasing the frequencies of occurrence of most behavioral parameters studied. These were: (1) elicitation of vulval interest; (2) vulval sniffing; (3) agonistic interactions; (4) giving chin rests; (5) receiving chin rests; (6) attempted mounts; (7) successful mounts; and (8) standing when mounted. The mean interval from treatment to first standing to be mounted was 25.4 ± 0.8 and 33.3 ± 5.2 hr for the estradiol-treated and testosterone-treated heifers, respectively. Peak activity generally occurred the second day after initiation of hormone treatment and rapidly declined after the third day. Flehmen lip curl and bellowing were not stimulated by either hormone. Neither androstenedione nor dihydrotestosterone was capable of stimulating sexual behavior in these heifers, as measured by any of the parameters studied.     

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.     

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.