STIMULATION OF COLLAGEN SYNTHESIS IN RAT CARDIAC FIBROBLASTS BY EXPOSURE TO HYPOXIC CULTURE CONDITIONS AND SUPPRESSION OF THE EFFECT BY NATRIURETIC PEPTIDES

Synthesis of type I and type III collagens by rat cardiac fibroblasts was stimulated when the cells were cultured under 95% N₂/5% CO₂for one hour followed by incubation under normoxic conditions for 24 hours. The stimulative effect was attenutated by the presence of atrial natriuretic peptide (ANP, 10⁻⁶m) or brain natriuretic peptide (BNP, 10⁻⁶m) in the culture medium. Northern blot analysis indicated that α1(I) and α1(III) collagen mRNA levels were also increased by hypoxia, and decreased with the addition of ANP or BNP in a dose-dependent manner. These results indicate interaction between intracellular signals of a physical stimulus (hypoxic stress) and those of a chemical one (ANP or BNP) and demonstrate that both signals regulate collagen synthesis by cardiac fibroblasts at the levels of the mRNAs. The results also suggest that natriuretic peptides produced by cardiomyocytesin vivomay function as paracrine factors that play a role in the prevention of cardiac fibrosis in ischaemic heart diseases.     

STIMULATION OF BRAIN SEROTONIN SYNTHESIS BY DIBUTYRYL-CYCLIC AMP IN RATS

Cyclic AMP and dibutyryl-cyclic AMP, a derivative of cyclic AMP resistant to phosphodiesterase inactivation, were injected into the lateral ventricles of rats. These nucleotides did not change the level of brain 5-HT but increased the brain level of its principal metabolite, 5-hydroxyindoleacetic acid. Cyclic AMP was less potent than dibutyryl-cyclic AMP. Butyrate and 5′-AMP were inactive. The effect of dibutyryl cyclic AMP on 5-HT metabolism was studied both in vivo and in vitro. The rate of synthesis of 5-HT was measured by the rate of accumulation of 5-hydroxyindoleacetic acid after the transport of this acid out of the brain was blocked with probenecid. The rate of synthesis of brain 5-HT increased from 0-38 μg/g/h in control rats to 0-65 μg/g/h after dibutyryl-cyclic AMP. In addition cyclic AMP and dibutyryl-cyclic AMP markedly increased brain tryptophan, while AMP was inactive. Since brain tryptophan hydroxylase has a K_m for its substrate that is much higher than the concentrations of tryptophan normally present in the brain, it is likely that the increase in the rate of synthesis of brain 5-HT is secondary to the cyclic AMP induced increase in the levels of brain tryptophan. In vitro studies revealed that dibutyryl-cyclic AMP increased the uptake of radioactive labelled tryptophan into slices of rat brain stem and the formation of 5-HT and 5-hydroxyindoleacetic acid.     

RNA SYNTHESIS IN THE DIFFERENT PHASES OF CELL CYCLE OF CHICK EMBRYO CELLS

RNA synthesis was studied at different phases of the cell cycle of chick embryo fibroblasts, which were synchronized by medium replacement in the confluent phase. The synthesis of DNA started at 4 hr and continued for 8 hr. RNA synthesis increased with time after medium change. The ratio of total amount of radioactivity in nuclear RNA prepared at 0, 2 and 8 hr was 1.0:1.03:5.05. The distribution of radioactive RNA in the sedimentation pattern was similar, showing remarkable incorporation in 45S region of ribosomal precursor RNA. The base composition of newly synthesized RNA, however, varied at different time intervals after medium replacement. Even within the G₁ phase, the molar percentage of G and C was quite different. Treatment with actinomycin D at a concentration of 0.02 μg/ml for 1 hr specifically inhibited ribosomal RNA synthesis. At 2 hr after medium change, ribosomal and AU-rich RNA including larger than 28S were synthesized in about equal amounts.     

STIMULATION OF BRAIN DOPAMINE SYNTHESIS BY GAMMA-HYDROXYBUTYRATE

Abstract— Gamma-hydroxybutyrate administration produces a marked selective increase of brain dopamine in different animal species. Following γ -hydroxybutyrate administration, dopamine accumulated in the basal ganglia of the rat and in the caudate nucleus of the rabbit at a rate which greatly exceeded the normal synthesis rate of the amine in these species. Dopamine accumulation was prevented by α -methyltyrosine. These data indicate that γ -hydroxybutyrate stimulates dopamine synthesis. In addition, γ -hydroxybutyrate increased the homovanillic acid level in the rat basal ganglia to a maximum of about 300 per cent of the normal level indicating that γ -hydroxybutyrate inhibits neither monoamine oxidase nor catechol O -methyltransferase in vivo. The possible mechanisms of dopamine accumulation following γ -hydroxybutyrate administration are discussed.     

RNA SYNTHESIS IN CULTURED CHICK EMBRYO CELLS IN GROWING AND CONFLUENT PHASES

RNA synthesis at the growing phase in monolayer cultures of chick embryo fibroblasts was compared with that at confluent phases by zonal sedimentation, base composition and hybridization experiments. The nuclei were isolated by treatment with Nonidet p-40. The ratio of RNA/DNA in isolated nuclei was higher at the growing phase than that of confluent. The rate of RNA synthesis was reduced in the cells at confluent phase to 15.1% of that at the growing phase. The sucrose density gradient sedimentation pattern of nuclear RNA was on the whole the same in both phases. According to the distribution of ¹⁴C-uridine incorporated into nuclear RNA, 45S ribosomal precursor RNA was more distinct for the growing cell, while the radioactivities were found to be polydispersed, including the RNA which sedimented faster than 28S RNA in the cells at confluent phase. The base compositions and hybridization analyses indicated that ribosomal RNA was synthesized more actively in the growing cells. About 50% of newly synthesized RNA was ribosomal in the growing cells but 35% in the confluent.
It was found that newly synthesized 18S and 28S ribosomal RNAs appeared in cytoplasm after 21 and 33 min lag periods respectively. These times were exactly same in both growing and confluent phases.     

COLLAGEN SYNTHESIS AND CELL GROWTH IN CHICK EMBRYO FIBROBLASTS: INFLUENCE OF COLCHICINE,CYTOCHALASIN B AND CONCANAVALIN A

Culturing of chick embryo fibroblasts in the presence of colchicine or cytochalasin B with and without concanavalin A (Con A) demonstrated that colchicine induces greater neosynthesis of endocellular type I collagen, whereas cytochalasin B boosts secretion. The effects are modified by the addition of Con A, which increases α₂more than a1 chain production.³H-thymidine incorporation is unaffected by cytochalasin B, but stimulated by colchicine. Con A neutralizes the stimulatory action of colchicine. It would therefore seem that Con A exerts transmembrane control of effects induced by colchicine and cytochalasin B by binding to cell surface receptors and so triggering rearrangement of the cytoskeleton.     

INHIBITION OF DNA SYNTHESIS IN CHICK EMBRYO CULTURES BY DEPRIVATION OF EITHER SERUM OR ZINC

The rate of DNA synthesis in cultures of chick embryo cells is proportional to the concentration of serum added. The concentration of serum required to stimulate DNA synthesis increases with cell population density and with the duration of culture after trypsinization. The increase of the serum requirement with population density is not caused by the depletion of serum constituents. The requirement of cells for external zinc in DNA synthesis also increases with population density and duration of culture. The kinetics of inhibition of DNA synthesis by deprivation of serum or zinc are similar. Serum deprivation, however, inhibits 2-deoxyglucose uptake and cell movement, but zinc deprivation does not. The deprivation of either serum or zinc inhibits RNA synthesis about twofold. Very low concentrations of actinomycin D prevent the resumption of RNA and DNA synthesis upon restoration of serum or zinc to deprived cultures.     

STIMULATION OF DNA SYNTHESIS BY ISOPROTERENOL IN RAT SUBMANDIBULAR GLAND DURING POSTNATAL GROWTH

The post-natal growth of rat submandibular gland and the effect of isoproterenol on this process were studied. Between 2 and 42 days of age the DNA content of the gland increased linearly but the increase in RNA and protein content was more rapid after 29 days of age. The RNA: DNA and protein: DNA ratio increased linearly with age. The proliferative activity, measured by the incorporation of tritiated thymidine, was maximum in the gland of 7-day-old rats. It declined steadily to a low level in 42-day-old rats. A single injection of isoproterenol had no effect on thymidine incorporation in 2-day-old rats. The drug, however, stimulated DNA synthesis in older animals and the degree of stimulation was inversely correlated with the proliferative activity in control rats. Small doses of isoproterenol given to rats for 4 days between 2 and 5 days of age produced a hypertrophy of the submandibular gland. The same treatment between 7 and 10 days of age caused both hypertrophy and hyperplasia of the gland. It is concluded that both the regulation of growth and the regulation of induced cell proliferation are a function of cellular differentiation and that cell proliferation can be induced only in cells that reached a certain degree of differentiation.     

ULTRASTRUCTURAL DISTRIBUTION OF CELL SURFACE ANTIGENS IN AVIAN TUMOR VIRUS-INFECTED CHICK EMBRYO FIBROBLASTS

The distribution of neoantigens in the surface membrane of avian tumor virus-infected chicken embryo fibroblasts was examined on carbon replicas of cell cultures using hemocyanin-labeled antibody. New determinants appearing on the cell surface of virally infected but not transformed cells are thought to be common with components of the viral envelope. These antigens were found to exist in a diffuse, random array on the dorsal cell surface, with a denser accumulation along the cell processes. In living cells, surface antigens are capable of several types of redistribution when activated by reaction with antibody. Leukosis virus-infected (non-transformed) cells showed two apparently independent modes of redistribution: a relocation of some antibody-related sites to the cell margin; or an involvement of essentially all sites in randomly dispersed aggregates. Viral antigenic sites on sarcoma virus-infected (transformed) cells, reacted with antibody, were able to produce weak marginal relocation; but revealed a more striking tendency to migrate to some central location. The centripetal coalescence thus formed resembles the "cap" noted in other systems. Prior aggregation into "patches" may not be a prerequisite for such cap formation. Tumor-specific surface antigen detection and mapping was attempted by this technique, but results were equivocal. An antigen possibly characteristic of rapidly dividing cells occurred in a sparse, diffuse fashion over the surface of morphologically distinct "round" cells.     

THE DISTRIBUTION, ULTRASTRUCTURE, AND CHEMISTRY OF MICROFILAMENTS IN CULTURED CHICK EMBRYO FIBROBLASTS

The distribution, ultrastructure, and chemistry of microfilaments in cultured chick embryo fibroblasts were studied by thin sectioning of flat-embedded untreated and glycerol-extracted cells, histochemical and immunological electron microscopic procedures, and the negative staining of cells cultured on electron microscopic grids. In these cultured cells, the microfilaments are arranged into thick bundles that are disposed longitudinally and in looser arrangements in the fusiform-shaped cells. In the latter case, they are concentrated along the margins of the flattened cell, on the dorsal surface, and particularly at the ends of the cell and its ventral surface, where contact is made with the plastic dish or with other cells. Extracellular filaments, presumably originating from within the cell, are found at these points of contact. The microfilaments are composed in part of an actin-like protein. These filaments are between 70 and 90 Å in diameter, they are stable in 50% glycerol, they have an endogenous ATPase (myosin-like?) associated with them, they bind rabbit muscle heavy meromyosin, and they specifically bind antibody directed against isolated actin-like protein. In the cultured chick embryo fibroblasts, the microfilaments are essential for the establishment and maintenance of form, and they are probably critical elements for adhesion and motility. The microfilaments might also serve as stabilizers of intramembranous particle fluidity.     

THE SITE OF SYNTHESIS OF GANGLIOSIDES IN THE CHICK OPTIC SYSTEM

Abstract– In the retinas of 1-day-old chickens that received an intraocular injection of N-[³H]acetylmannosamine the labelling of N-acetylneuraminic acid and CMP-N-acetylneuraminic acid increased for at least 8 h and that of gangliosides for at least 24 h after injection. In the optic tectum contralateral to the injected eye at 8 h after the intraocular injection, the labelling of gangliosides exceeded the labelling of gangliosides in the ipsilateral tectum by approx 20-fold. In the contralateral tectum the highest concentration of labelled gangliosides was in subfractions enriched in synaptosomes and synaptic plasma membranes. No significant contralateral ipsilateral differences were found in the acid soluble substances of the tectum. In the optic tectum, labelled gangliosides appeared earlier in the neuronal perikarya than in synaptosomes when the injection was intracranial. Conversely, when the injection was intraocular the labelling appeared earlier in the synaptosomes than in the neuronal perikarya. The radioactivity pattern of the optic tectum gangliosides resembled the pattern of retina gangliosides when N-[³H]acetylmannosamine was injected intraocularly, but when N-[³H]acetylmannosamine was given intracerebrally the radioactivity pattern resembled that of optic tectum gangliosides. Intraocular injection of colchicine or vinblastine did not affect the labelling of retinal gangliosides from N-[³H]acetylmannosamine injected into the same eye but prevented the appearance of labelled gangliosides in the optic tectum. In vitro the ganglioside glycosylating activity of optic tectum synaptosomes and synaptic plasma membranes was between 6 and 10-fold lower than that found in the optic tectum neuronal perikarya. These findings support the notion that the main subcellular site of synthesis of neuronal gangliosides is in the neuronal perikarya, from which they are translocated to the nerve endings.     

THE SELECTIVE INTERRUPTION OF NUCLEOLAR RNA SYNTHESIS IN HELA CELLS BY CORDYCEPIN

Cordycepin is an analogue of adenosine lacking the 3'-OH. When incorporated into a growing RNA molecule, cordycepin prevents further elongation, thus producing a prematurely terminated RNA molecule. When HeLa cells are exposed to low concentrations of cordycepin, DNA and protein synthesis are unaffected during short exposure periods. The synthesis of completed ribosomal and ribosomal-precursor (45S) RNA is significantly depressed. Partially completed 45S ribosomal precursor molecules accumulate in the nucleolus. 18S ribosomal RNA can be cleaved from these incomplete precursors, while 32S ribosomal precursor cannot be produced from partially snythesized 45S molecules. The synthesis of transfer RNA is also reduced in the presence of cordycepin. The synthesis of the nuclear heterogeneous RNA species is unaffected by the drug while the cytoplasmic heterogeneous RNA is slightly reduced.     

TOTAL SYNTHESIS OF CRYSTALLINE BOVINE INSULIN

<正>We should like to communicate,as a result of our combined efforts for the past five years,this preliminary report on the total synthesis of crystalline bovine insulin,the structure of which was first elucidated by Sanger and coworkers[1]ten years ago.A number of papers on the synthetic work concerning insulin has been published during the past two years.Katsoyannis and coworkers[2,3].synthesized both the A and B chains of sheep insulin and,by combining them together according to the procedure of Dixon and Wardlow[4],observed only traces of insulin activity.Zahn,Meienhofer and co-     

CELL—CELL CONTACTS ACCELERATE CELL COLONY GROWTH IN SPARSE CULTURES OF CHICK EMBRYO FIBROBLASTS

There is no cell proliferation in very sparcely plated chick embryo cell cultures. Substituting conditioned medium or adding of ethanol-fixed homologous cells to the cultures accelerates cell colony growth. The mechanism for the mitogenic action of fixed cells is considered to be the contact stimulation of cell proliferation, and addition of extra cells to sparse culture is believed to mimic the cell micro-environment existing in subconfluent cultures. The role of diverse cell—cell contacts in cultured cell growth regulation is discussed. The procedure used (addition of ethanol-fixed cells) may improve normal cell cloning techniques.     

EXTRACELLULAR STIMULATION OF EPIDERMAL DNA SYNTHESIS

The removal of the topmost cell layers of the epidermal stratum corneum by stripping initiates a series of biochemical events which alters the normal homeostatic control of and results in the acceleration of the cell cycle in basal cells which are ten to twenty cell layers removed from the site of stripping. One measure of accelerated events is a stimulation of thymidine incorporation into epidermal DNA at time intervals following stripping. Two peaks of maximal stimulation occur between 12 and 24 hr and 48 and 54 hr after stripping. Stimulation of thymidine incorporation into epidermal DNA by limited stripping is a useful technique for studying the stripping-initiated signal at the stratum corneum and its subsequent translation at the proliferative cell receptor site.     

RNA SYNTHESIS BY EXOGENOUS RNA POLYMERASE ON CYTOLOGICAL PREPARATIONS OF CHROMOSOMES

Cytological preparations of Drosophila polytene chromosomes serve as templates for RNA synthesis carried out by exogenous RNA polymerase (Escherichia coli). Incorporation of labeled ribonucleoside triphosphates into RNA may be observed directly by autoradiography. Because of the effects of rifampicin, actinomycin D, ribonuclease, high salt, and the requirement for all four nucleoside triphosphates, we conclude that the labeling observed over chromosomes is due to DNA-dependent RNA polymerase activity. Using this method, one can observe RNA synthesis in vitro on specific chromosome regions due to the activity of exogenous RNA polymerase. We find that much of the RNA synthesis in this system occurs on DNA sequences which appear to be in a nondenatured state.     

THE NUTRITION OF ANIMAL TISSUES CULTIVATED IN VITRO : IV. AMINO ACID REQUIREMENTS OF CHICK EMBRYONIC HEART FIBROBLASTS

1. The amino acid requirements of freshly explanted chick embryonic heart tissues cultivated in completely synthetic media have been determined, employing a nutritional depletion technique. Arginine, histidine, lysine, tyrosine, tryptophan, phenylalanine, cystine, methionine, threonine, leucine, and valine were found to be essential. Serine, isoleucine, glycine, and glutamine were found to be non-essential. Glutamic acid, aspartic acid, α-alanine, proline, and hydroxyproline were found to be inhibitory in this test system. 2. A total amino acid level of approximately 100 mg. per cent was found to be optimal and DL-amino acids were found to be non-toxic, unless used in high concentrations. 3. A comparison has been made of the amino acid requirements of various types of tissue cultures, of the chick, and of man and certain differences in these requirements have been discussed.