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PubMed1976

Selective control of fibroblast proliferation and its effect on cardiac muscle differentiation in vitro.

The stability of the differentiated state of cardiac myocytes in vitro was examined under culture conditions which selectively stimulated or inhibited proliferation of fibroblasts. Regulation of fibroblast proliferation in cultures of myocardial cells from 8-day embryonic chicks was achieved by adjustment of the glutamine (Gln) concentration in the culture medium (Ham's F-12 medium containing 2 x amino acids and 5% fetal calf serum). Myocardial cells, when plated at 80 cells/mm2 in Gln- medium, maintained a stable density of approximately 40% of the plating density for more than 30 days. When Gln was added to the medium (292 micrograms/ml) fibroblast proliferation was stimulated, and by 5-6 days after this addition cell densities had increased to confluency. The selective action of glutamine on fibroblast proliferation was determined by labeling cultures with tritiated thymidine ([3H]TdR) and scoring its incorporation into myocytes and fibroblasts by radioautography. After 2 weeks in Gln- medium, the mitotic index was 0.3% and the [3H]TdR-labeling index (1.5-hr pulse) was 6.4%. In addition, the proportion of myocytes in the population was constant at 64.2% for at least 30 days in vitro, and contractile activity was observed for up to 6 months. After 5 days of Gln replacement, the cells exhibited a labeling index of 25%, the proportion of myocytes decreased to less than 10% and contractile activity was rarely observed. Although the [3H]TdR-labeling index of fibroblasts and myocytes was nearly identical in Gln- medium, the addition of Gln produced a fivefold stimulation in the fibroblast labeling index, but did not affect myocyte proliferation or DNA synthesis. A unique phenomenon of myocyte congregation was observed only in Gln- medium which resulted in the formation of myocyte colonies from which fibroblasts were largely absent. It is suggested that this process with the resultant establishment of a functional electrical syncytium plays a significant role in the development and stabilization of myocyte differentiation in vitro.

Animals↗

PubMed1985

Selective expression of high-affinity uptake of catecholamines by transiently catecholaminergic cells of the rat embryo: studies in vivo and in vitro.

Transient expression of catecholaminergic phenotypic traits is a widespread phenomenon during embryonic development in mammals, occurring in cells of the embryonic gut mesenchyme, in ventrolateral portions of the neural tube, cells of cranial sensory and dorsal root ganglia, and in the embryonic pancreas. In the current study the manifestation of the catecholamine (CA) phenotype in these populations has been further defined. Specifically, the existence of the high-affinity uptake process for CAs in these populations has been investigated. By combining the techniques of radioautography following accumulation of [3H]norepinephrine (3H-NE) and [3H]dopamine (3H-DA) with immunohistochemical detection of tyrosine hydroxylase (T-OH), it has been possible to demonstrate simultaneously CA accumulation by T-OH-positive gut cells. Uptake of 3H-NE was first detected in T-OH-positive cells of the gut on gestational day 12.5 (E12.5). By contrast, T-OH immunoreactivity was first detected on E11.5. By E13.5 virtually every T-OH-positive cell oral to the umbilical flexure was radioautographically labeled. Uptake at E13.5 displayed Michaelis-Menten saturation kinetics, had a Vmax of 35 fmole/gut/min, a Km of 1.45 microM, was blocked by desmethylimipramine (DMI), and by incubation at 4 degrees C. On subsequent gestational days, silver grains marking areas of amine concentration were found increasingly over T-OH-negative cells. A similar pattern of uptake was found in guts which had been grown in organotypic tissue culture for the purpose of eliminating extrinsic sympathetic innervation. T-OH-positive gut cells also accumulated 3H-DA. Concentration of 3H-DA was blocked by both benztropine and DMI suggesting that accumulation had properties common to both NE and DA systems. By contrast to cells of the gut, accumulation of CAs was not a property of transiently T-OH-positive cells in other locations. Therefore, specific, high-affinity uptake and retention of CAs is an additional property of transiently catecholaminergic gut cells. Appearance of CA synthetic enzymes precedes the appearance of the CA storage process in cells of the gut. Persistence of the uptake process after the loss of detectable T-OH suggests continued viability of the population. The absence of CA accumulation by other T-OH-positive cells suggests basic molecular differences among the various populations.

Animals↗

PubMed1985

Appearance of nerve growth factor receptors on cultured neural crest cells.

Light microscopic radioautography of differentiating quail neural crest cultures (1 to 2 weeks after explanation) incubated with Iodine-125-labeled nerve growth factor (125I-NGF) revealed that approximately 35% of the cells bound NGF. The binding was specific and saturable; it was blocked by an excess of nonradioactive NGF, and was not detected following incubation with biologically inactive 125I-NGF. In addition, the binding did not appear to be blocked or diminished by insulin. Cell cultures prepared from somites or notochord showed no specific binding of 125I-NGF. Melanocytes comprised approximately 10% of the cell population in these cultures and appeared to be unlabeled. The subpopulation of cells with NGF receptors that were morphologically similar to other non-melanocyte unlabeled cells present in the neural crest cultures are probably the targets of the factor during differentiation and development. In contrast, there was no evidence of 125I-NGF binding by premigratory neural crest (adherent to the isolated neural tube) or by early migratory neural crest cells (24 hr after explantation). Both of these types of neural crest cells are relatively undifferentiated. The cells of the neural tube were also unlabeled. The binding of 125I-NGF to differentiating neural crest cells was not noticeably diminished by a brief pretreatment with trypsin or Dispase, enzymes used in the isolation of neural tubes. Hence, the absence of NGF receptors on premigratory neural crest and early migratory neural crest cultures was not due to enzymatic alterations of the receptor. It seems, therefore, that receptors for NGF appear on neural crest cells during the time when these cells are acquiring their phenotypic characteristics.

Animals↗

PubMed1991

Localization of epidermal growth factor receptors in cells of the enamel organ of the rat incisor.

Epidermal growth factor (EGF) is a peptide shown to effect precocious incisor tooth eruption in rat pups. Binding sites for EGF were visualized in the continuously erupting adult rat incisor by light and electron microscope radioautography after in vivo injection of 125I-EGF. These binding sites represented EGF receptors because of (i) competition between 125I-EGF binding at 2 min after injection and a coinjected excess of unlabeled EGF; (ii) the receptor-mediated endocytosis of 125I-EGF at 15 and 30 min after injection; and (iii) the demonstration of EGF receptor kinase activation in vivo. The stem and the mitotic cells in the epithelial odontogenic organ at the growing end of the tooth develop into two nondividing layers of the enamel organ: (i) ameloblasts which secrete enamel and are subsequently involved in the enamel maturation process, and (ii) papillary layer cells situated between the blood supply and the ameloblasts. Although few EGF receptors were present at the mitotic end, receptor density was highest at the mature end of the enamel organ. High levels of 125I-EGF binding were found on papillary layer cells and ruffle-ended, but not smooth-ended, ameloblasts. This implies a cyclical exteriorization and internalization of receptors during modulations between the two cell types. These data suggest that the EGF receptor mediates a major function of the enamel organ in the formation of enamel.

Animals↗

PubMed1983

Onset of DNA replication in nuclei of proliferating and resting NIH 3T3 fibroblasts following fusion.

NIH 3T3 mouse fibroblasts arrested in medium containing 0.5% serum were fused with stimulated cells taken at 2-h intervals after replacing the medium with one containing 10% serum, and DNA synthesis was studied in mono-, homo- and heterokaryons using radioautography with double-labelling technique. The presence of a resting nucleus in a common cytoplasm with a stimulated nucleus from the prereplicative period has an inhibitory effect on the entry of the stimulated nucleus into the S period in medium containing either 0.5 or 10% serum, but ongoing DNA synthesis continues. After a 24-h stay in a common cytoplasm with resting nuclei the stimulated nuclei return into the state of rest. When resting cells are stimulated by 10% serum, their inhibitory effect on stimulated nuclei in heterokaryons still persists, at least for 2 h following stimulation. Preincubation of resting cells with cycloheximide for 4 h abolishes their ability to suppress DNA synthesis in stimulated nuclei. The data suggest that resting cells produce an endogenous inhibitor of cell proliferation, whose formation depends upon the synthesis of protein. When stimulated, the cells can proliferate only after decreasing the level of this inhibitor. The results obtained are consistent with the idea of a negative control of cell proliferation.

Animals↗

PubMed1983

Declining histone phosphorylation during myogenesis revealed by in vivo and in vitro labeling.

To investigate histone phosphate levels during myogenesis, proliferation (d 1), pre-fusion postmitotic (d 2) and myotube (d 3) stage cultured chicken myoblasts were phosphorylated in vivo with [32P]orthophosphate or in vitro by incubating isolated nuclei with 32P-gamma-ATP. Incorporation of radioactive phosphate into histone was assessed by SDS and acid/urea/Triton-X-100 (AUT) gel electrophoresis and radioautography. During proliferation, in vivo labeling with [32P]orthophosphate revealed that all histones except H2b were phosphorylated in the following order of decreasing modification: H1 a greater than H2a greater than H1 b greater than H3 greater than H4. In pre-fusion post-mitotic cells phosphorylation of histones H1 a, H3 and H4 declined, whereas all histones exhibited significantly decreased modification at the myotube stage. It is unlikely that these changes resulted from decreased specific radioactivity of intracellular inorganic phosphate pools, since uptake of [32P]orthophosphate by myotubes increased six-fold, compared with proliferating cells. Isolated nuclei incubated with 32P-gamma-ATP displayed similar decreases during myogenesis; however, 1 a, H1 b and H3 were the only histones modified by in vitro phosphorylation.

Animals↗

PubMed1984

Spermine-induced phosphorylation of myotube histones by endogenous nuclear protein kinases.

The effects of spermine on phosphorylation of nuclear proteins in isolated nuclei from proliferation and myotube stage cells during differentiation of cultured chicken myoblasts have been investigated. Incorporation of phosphate from 32P-gamma-ATP was assessed by incubating nuclei with and without 2 mM spermine, which caused an approx. 1.5-fold increase in phosphorylation of total nuclear proteins in both cell types. Modification of individual proteins was assessed by extracting basic proteins in dilute acid, followed by SDS-electrophoresis on 18% acrylamide gels and radioautography. Results indicated that whereas most phosphoproteins in both cell types were increased 1.5-2.0-fold, phosphorylation of a 31 000 D band increased several-fold. Most strikingly, myotube nuclei displayed selective 3.5- and 9-fold increases in specific radioactivity of histones Hla and H3, respectively, which normally exhibit little, if any, phosphorylation.

Animals↗

PubMed1985

Recycling of membrane proteins during endo- and exocytosis in amoebae.

The fate of a membrane protein of the amoeba plasmalemma was studied by means of 125I iodination by lactoperoxidase, gel electrophoresis, radioautography and gamma counting. There was only one iodinatable polypeptide group with a molecular weight (MW) of 175 000 on the external surface of the plasmalemma. Two hours or more after induced phagocytosis, isolated phagolysosomal membranes contained two other smaller polypeptides with MWs of 70 000 and 35 000, respectively, suggesting that the 175 000 polypeptide was broken down to these smaller components during endocytosis. After 22 h of induced phagocytosis, isolated plasmalemma contained a 35 000 polypeptide group in addition to the 175 000 polypeptide species. The results suggested that some of the iodinatable membrane proteins were altered and recycled during endo- and exocytosis in amoebae, while others were recycled intact.

Amoeba↗

PubMed1986

Effects of colchicine on cardiac cell function indicate possible role for membrane surface tubulin.

The effects of the tubulin-binding drug colchicine on cultured neonate cardiac cell function were investigated. Application of low doses of colchicine (but not lumicolchicine) caused an early reversible increase in beating rate with a concomitant decrease in amplitude. Treatment of the cells with trypsin at a dose that removes surface tubulin but does not inhibit spontaneous beating, diminished the colchicine effect. Surface radio-iodination of the live cultures followed by two-dimensional gel electrophoresis and radioautography revealed that two spots were heavily labeled. These spots co-migrated with purified brain tubulin. Fibroblasts derived from the cardiac cultures did not label over the tubulin spots. Trypsin treatment removed the presumptive tubulin from the radioautographs but only removed the most basic portion of the alpha-tubulin spot from the stained gel. These results are consistent with a surface membrane role for an iso-form of tubulin in neonate cardiac cells.

Animals↗

PubMed1987

RNA polymerase activity in PtK1 micronuclei containing individual chromosomes. An in vitro and in situ study.

Micronuclei have been induced by colchicine in rat kangaroo (Potorous tridactylis) PtK1 cells. The synthesis of RNA was investigated both in isolated micronuclei by quantifying RNA polymerase activities at different ionic strengths with or without inhibitors, and in micronucleated cells by radioautography after [3H]uridine pulse labeling. In vitro transcription shows that isolated micronuclei are able to take up [3H]UTP. The rate curves of incorporation are close to those of isolated diploid nuclei, though the level of incorporation was relatively lower (65-70%) than control nuclei. This indicates that micronuclei react to the ionic environment and to inhibitors in the same manner as described for many species of isolated diploid nuclei. The labelling distributions plotted from radioautographs show that micronuclei were able to efficiently incorporate the hot precursor. Furthermore, for short pulses there is no homogeneity in the labelling density among the different micronuclei and there is no correlation between the labelling intensity and the size of micronuclei. After 60-min pulse time, there is an enhanced uptake of [3H]uridine and all the micronuclei exhibit considerable labelling, although less than control cells. Thus, the micronuclei exhibit some characteristic RNA transcriptional activity in situ as well as after isolation. This material should be a particular interesting model with which to study the physiological activity and the role of each individual interphasic chromosome.

Animals↗

PubMed1984

The phosphorylation of bovine and human lens polypeptides.

The phosphorylation of bovine and human lens polypeptides was examined using an in vitro labeling assay with [gamma-32P]-ATP, followed by SDS-PAGE and radioautography. The major protein phosphorylation in all lens preparations was cAMP-dependent. The effect of cAMP could be depressed by monovalent ions. Phosphorylated polypeptides were detected in capsule-epithelial and outer cortical but not inner cortical-nuclear preparations. Differences in phosphorylated polypeptide distributions and molecular weight patterns were observed between bovine capsule-epithelium and outer cortex fiber cells and bovine and human preparations. Human cataractous lens preparations had the same pattern of phosphorylation as human normal lens preparations but with much less 32P incorporation. Major phosphorylated polypeptides in the 100 000 dalton, 60 000 dalton and 43 000 dalton range were detected in both bovine and human preparations. A 32 000 dalton phosphorylated polypeptide appeared exclusively in the bovine capsule-epithelium and a phosphorylated 23 000 dalton polypeptide was detected only in outer cortical preparations.

Animals↗

PubMed1983

Axoplasmic incorporation of amino acids in myelinated fibers of the cat.

Incorporation of amino acids into the axoplasm of nerve fibers of the cat was studied with radioautography. The dorsal funiculus of the spinal cord, or the sciatic nerve, was exposed to tritiated leucine, lysine, and proline; the tissue was processed 1 h later or the following day. In all instances, a radioautographic response of widely varying intensity was observed over the axoplasm; this response could not be adscribed to emissions originating from axonal mitochondria or myelin. The axoplasmic response ranged to as much as 56% compared with that over nearby glial cytoplasm and was often conspicuously greater than that over the surrounding myelin. We interpret our findings as axoplasmic synthesis of proteins. This finding has implications in the maintenance of the neuron.

Amino Acids↗

PubMed1983

Axoplasmic incorporation of amino acids in a myelinated fiber exceeds that of its soma: a radioautographic study.

The axoplasmic incorporation of amino acids was studied in the giant Mauthner axon to determine its magnitude, to estimate the half-life of the resulting material, and to establish whether or not it undergoes transport. The incorporation of labeled leucine, lysine, and proline was followed by radioautography. The tracers were injected away from the cell body, either in the floor of the IV ventricle or into the spinal cord. Radioautographic reaction on the Mauthner somata was barely discernible. However, at the site of injection, for survivals to 5.6 days, a peak of reaction was observed on the Mauthner axoplasm, with a sharp and symmetric decrement in both central and distal directions; the intensity of the peak decreased with survival (14% at 5.6 days). In intact fibers, the intensity of the reaction was 1.4% compared with that over the Nissl substance of neighboring somata; by contrast, in severed fibers whose axoplasm had been directly exposed to the tracers the reaction rose to 4.1%. At early times, the intensity of the response of the axoplasm as a function of survival of the fish did not have a lag time. In high-resolution radioautograms, most of the grains overlay the ground axoplasm. Cycloheximide depressed the reaction over the fiber open to the extracellular space by 55% whereas only by 20% over the intact fiber. Adsorption of free amino acids or their binding to tRNA were ruled out as a cause of the radioautographic response. Our results indicated that incorporation of amino acids into macromolecules of the Mauthner fiber occurred locally, and its magnitude over the whole axoplasm was estimated to be one order of magnitude larger than that occurring in the soma. The bulk of the material resulting from this incorporation did not undergo transport and survived some days. We suggest that the amino acids were incorporated by a ribosomal mechanism.

Amino Acids↗

PubMed1989

Trichostrongylus colubriformis: epithelial cell migration in the proximal and distal small intestine of infected rabbits.

In Trichostrongylus colubriformis-infected rabbits, epithelial cell migration rates and cell transit times along the villi were compared by radioautography on histological slides to normal values from noninfected small intestine. Regions of gut with high (upper jejunum) and low (ileum) burdens of worms were both examined. In the control rabbits, the estimated values for the cell migration rates in the proximal and distal parts of gut were respectively 5.8 and 2.8 microns/hr. Seventy-two hours after the thymidine injection, the labeled epithelial cells were near the tip of the villi in the jejunum whereas only 60% of the villous length was labeled in the ileum. In the infected rabbits, the presence of T. colubriformis was associated with a two-fold increase of the cell velocity, in the main site of infection. Although less prominent than in the proximal region, a significant acceleration in the cell migration was also noticed in the ileum. The cell transit time was markedly reduced in the parasitized jejunum, but no variation of this parameter was found in the distal part of gut. These changes in the dynamics of epithelial cells in both regions of the gut appeared to underlie the morphological and enzymological changes of the parasitized mucosa. They particularly contribute to create an adaptive region in the small intestine beyond the main site of infection.

Animals↗

PubMed1991

Phosphorylation of alpha-crystallin B in Alexander's disease brain.

The phosphorylation of alpha-crystallin B was studied in homogenates of autopsy samples of brain tissue from patients with Alexander's disease, a condition characterized by over-expression of this protein. After incubation in the presence of [gamma-32P]ATP and cAMP the homogenates were analyzed by two-dimensional electrophoresis, (isoelectric focusing followed by SDS-PAGE). Three major polypeptides having the same molecular weight as bovine lens alpha-crystallin B and pIs 7.1, 6.9 and 6.7 were detected in the Coomassie blue stained gels. These three polypeptides were recognized by an alpha-crystallin B-specific antiserum in Western blots. The polypeptides with pIs 7.1 and 6.7 co-migrated in isoelectric focusing gels with bovine lens alpha B and its phosphorylated form alpha Bp, respectively. Radioautography of the two-dimensional gels demonstrated the presence of 32P in the most acidic polypeptide. The results demonstrate the occurrence of alpha B phosphorylation in Alexander's disease brain tissue.

Blotting, Western↗

PubMed1985

Tritiated thymidine radioautographic study on the origin and renewal of secretin cells in the rat duodenum.

The origin and renewal of secretin cells in the duodenum were investigated using the unlabeled antibody peroxidase-antiperoxidase technique and radioautography in rats killed at various times after single or multiple injections of [3H]thymidine. Secretin cells were spatially distributed from the upper crypt to the villus tip, being particularly numerous in the upper two-thirds of the duodenal villi. After a single injection of [3H]thymidine, there were no labeled secretin cells, indicating a lack of self-replicating activity. After repeated injections of the isotope, labeled secretin cells appeared and increased in number. They first occurred at the upper part of the crypt and the lower part of the villus, and later at the villus tip. All these cells were found to be labeled after continuous labeling for 120 h, which is considered to be the renewal time for this cell population.

Animals↗

PubMed1986

Growth hormone-releasing factor (somatocrinin) stimulates epithelial cell proliferation in the rat digestive tract.

The possible influence of growth hormone-releasing factor (GHRF) on epithelial cell proliferation in the digestive tract was investigated. Fasted young rats received five hourly subcutaneous injections of either GHRF or saline. They were killed 6, 12, or 18 h after the initial injection and 45 min after [3H]thymidine pulse labeling. At the time of death, blood was taken to determine circulating growth hormone and gastrin levels. After radioautography, DNA synthetic and mitotic activities were estimated in the fundic, antral, duodenal, jejunal, and colonic mucosae. Growth hormone-releasing factor significantly increased labeling indices 6, 12, and 18 h after the initial injection in fundic mucosa, and 6 and 18 h after injection in antral and duodenal mucosae. Furthermore, GHRF significantly increased mitotic indices at 12 h in fundic mucosa and at 12 and 18 h in jejunal mucosa. No effect was seen in the colon. At the three checkpoint times, circulating growth hormone showed no change, but plasma gastrin was increased in the rats treated with GHRF as compared with controls. However, whether the reported stimulatory effect of the GHRF on target cells is direct or indirect remains to be determined.

Animals↗

PubMed1993

Maturation of human fetal stomach in organ culture.

BACKGROUND: This investigation was undertaken to establish a serum-free organ culture technique allowing for the morphological and physiological maintenance of human fetal stomach in vitro. METHODS: Explants from gastric corpuses (12-17 weeks of gestation) were cultured in serum-free medium for periods of up to 15 days. RESULTS: After 15 days of culture, surface mucous cells were more mature, gastric glands were numerous and well developed, and all epithelial cell types were morphologically very well preserved. Morphometric measurements of the glands revealed an accelerated development in culture compared with that found in utero. Even though the incorporation of [3H]thymidine into total DNA decreased, the labeling indices determined by radioautography confirmed that epithelial cell proliferation was maintained especially in the pit/neck portion and at the base of the glandular compartments. A significant increase in total glycoprotein synthesis, as evaluated by the incorporation of [3H]glucosamine, was observed and correlated with the differentiation of the mucous cells. CONCLUSIONS: This investigation establishes for the first time that human gastric mucosa can be maintained up to 15 days in organ culture and that maturation of the gastric mucosa can be reproduced in chemically defined media.

Cell Differentiation↗