Autoradiographische Untersuchungen über das Wachstum des Reissnerschen Fadens von Cyprinus carpio (L.)

Summary The incorporation of³⁵S-l-cysteine into the hypothalamic-hypophyseal neurosecretory system was studied in normal male albino rats and in similar rats which had received thiouracil or excess thyroxine during 18 days. The study was performed by autoradiography. Labelled cysteine was administered by intraperitoneal injections. The animals were sacrificed 30 min, 3 and 24 hours after injection. From the autoradiographs the grain counts were determined in the supraoptic nucleus, paraventricular nucleus, median eminence and infundibular process. The thyroxine-treated rats in the 30 minute group displayed a statistically almost significantly higher grain count in the supraoptic nucleus than the corresponding controls; the same was observed in the 3 hour group in the median eminence and infundibular process. In the 24 hour group, however, the grain count was clearly lower at all sites examined than in the controls. With the rats that had been under thiouracil treatment statistically significant differences were only established in the 24 hour group. Their grain count was higher at all sites than that of the corresponding controls. From the results the inference is drawn that thyroxine accelerates the protein synthesis in the ganglion cells of the supraoptic nucleus and accelerates the flow of NSM to the infundibular process and its release there into the blood flow. Thiouracil, again, retards the flow and the release.This work was supported by a grant from the Sigrid Jusélius Foundation, Helsinki.     

Topographical differences of RNA labelling in rat brain after intraventricular administration of labelled RNA precursors

Summary ¹⁴C-uridine or ¹⁴C-orotic acid was injected into the third or fourth brain ventricle of adult rats. The rate of incorporation of these precursors into the RNA of various brain regions was studied by autoradiography. 0.5 hour, 1 hour, 2 hours and 4 hours after the injection of labelled uridine and/or orotic acid into the third ventricle, a very uneven labelling of different brain regions was observed. The highest grain density was found over the ventricular walls and in the closely adjacent brain tissue; the intensity of labelling decreased sharply with distance from the ventricular lumen. 24 hours after intraventricular injection, a medio-lateral gradient of grain density was no longer observed. An intense labelling of leptomeninx (especially at the base of the brain) and of ependymal cells was observed at all time intervals investigated. At time intervals 0.5–2 hours the grain density of these structures surpassed by a considerable amount the grain density over neurons, glial cells or neuropile.Two hours after the injection of ¹⁴C-orotic acid into the fourth ventricle, the grains were mainly localised over leptomeningeal cells and vessels at the base of the brain and in the adjacent narrow strip of brain tissue. The rest of the brain was only very faintly labelled.     

WOUND HEALING AND COLLAGEN FORMATION : III. A Quantitative Radioautographic Study of the Utilization of Proline-H3 in Wounds from Normal and Scorbutic Guinea Pigs

The sequence of incorporation and utilization of tritium-labeled proline has been examined in healing wounds from normal and scorbutic guinea pigs. Linear incisions in the skin of the animals were allowed to heal for 7 days. Each animal was given proline-H³, and the wounds were excised 30 minutes, 1 and 4 hours, 1, 3 and 7 days after proline administration. The tissues were fixed in osmium tetroxide, fixed again in neutral buffered formalin, embedded in epoxy resin, and sectioned at 1 micron thickness. The sections were coated with nuclear track emulsion, exposed, developed, and stained. The results of grain counts were quantitated as the number of counts per unit area overlying cells, fibers, etc. In both groups the proline reaches a maximum over the fibroblasts within 4 hours and subsequently disappears from the cells. Concomitantly, the proline reaches a maximum over the collagen (in normal animals) and extracellular fibrillar material (in scorbutic animals) by 4 hours, where it remains. The modified technique of radioautography used in this study allows not only resolution of approximately 1 micron, but also minimal background, decreased artifact, and a clear separation of the randomly situated elements within the wounds so that grain counting is facilitated. The results correlated with previous electron microscopic studies are consistent with the utilization of proline by the fibroblasts and its incorporation into collagen (in normal animals) and into the extracellular, fibrillar, non-collagenous material seen in scorbutic animals.     

3H-glucosamine and 3H-fucose incorporation by murine peritoneal macrophages “non-divided” during differentiation in vitro

Normal murine peritoneal macrophages were maintained in culture for 24 hours and 6 days. These cells continuously incorporated ³H-glucosamine and ³H-fucose in their membrane components. For both precursors culture ages, these components may be considered to undergo turnover according to a given rhythm with a 24 hours doubling time. Treatment with neuraminidase did not modify the doubling time, although a slight increase of incorporation of radioactivity was observed. Variations of ³H-fucose and ³H-glucosamine incorporations were observed between 24 hours old and 6 days old cultures: 6 days old cultures incorporated more glucosamine than 24 hours old cultures whereas no difference in fucose incorporation was observed between the two cultures.     

Rat endometrial stromal-epithelial response to estrogen infusion

Morphologic changes at the interface of rat endometrial luminal epithelial cells and the stromal cells immediately adjacent were examined and correlated with hypertrophy of the epithelial cells during estradiol (E2) infusion (1 microgram E2/24 h). While the lamina densa in castrate endometrium was thread-like, it became thicker and apparently more granular in some areas below the luminal epithelium during E2 infusion. However, no changes were seen in the intensity of laminin-like immunoreactivity at various time points up to 96 hours after beginning infusion, suggesting that these alterations were due to changes in nonlaminin components. The stromal cells adjacent to the basal lamina in the castrate state had cell processes extending toward the epithelium that terminated on the basal lamina. Under estrogen infusion, stromal cell bodies migrated close to and became oriented along the basal lamina. No interruptions were seen in the lamina densa or in the laminin-like immunoreactivity in the basal lamina. Thus, there were no direct morphologic interactions between epithelial and stromal cells induced by estrogen. Some of the stromal cells developed a dilated rough endoplasmic reticulum and some developed multiple elaborate processes within 41 hours after minipump implantation. Within 28 hours, nuclear hypertrophy had occurred in 15% of the epithelial cell layer. If interactions occur between stromal and epithelial cells, and morphologic evidence presented here suggests they do, then all such interactions are through an intact lamina densa-laminin layer, and any chemical mediators affecting cells on opposite sides of the lamina densa must migrate through it.     

Studies on the innervation of the endometrium

Summary The innervation of the endometrium of rabbit, rat, mink, mongoose and pig has been investigated electron microscopically. Large bundles of nerve fibers can be observed in the connective tissue spaces within the basal layer of the endometrium. Unmyelinated nerve fibers enter the lamina functionalis, terminal nerve fibers penetrate the basal lamina and make contact with the glandular and the cavum epithelial cells. The terminal axons contain abundant synaptic vesicles, dense core vesicles and mitochondria. To date, no specialized presynaptic or postsynaptic membranes have been found.Supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg (Grants Ku 210/5 and Be 524/4).Dedicated to Prof. Dr. Drs. h.c. Wolfgang Bargmann on his 70th birthday in friendship and admiration.     

Evidence for Two Metabolically Distinct Types of Ribonucleic Acid in Chromatin and Nucleoli

Patterns of radioisotope incorporation are useful characteristics in describing cellular RNA fractions, and have indicated a distinctive "nuclear" RNA. In order to characterize the RNA fractions of the two nuclear components, nucleoli and chromatin, and to determine thereby the precise localization of the RNA typical of isolated nuclei, time-courses of P³² incorporation into nucleolar, chromosomal, and cytoplasmic RNA of Drosophila salivary glands have been determined from autoradiograms. Two experiments are reported which cover 12 and 18 hour periods, including an initial 2 hour feeding on P³². Concentrations of RNA-P³² (identified by ribonuclease digestion) were determined by grain counts. After 1 hour only the nucleolar RNA is labelled. Activity is detectible in chromosomal and cytoplasmic RNA after the 2nd hour. The nucleolar fraction reaches its maximum activity shortly after transfer of the larvae to non-radioactive food, the other fractions several hours later. Maximum activities persist in the chromosomal and cytoplasmic fractions; nucleolar activity decreases after the 9th hour. The observed differences in times at which incorporation begins and maximum activities are reached, and in maintenance of maximum activities indicate that chromosomal and nucleolar RNA are distinct fractions. The metabolic characteristics which have been ascribed to "nuclear" RNA apply only to the nucleolar fraction.     

Intact testosterone receptor complex does not induce RNA synthesis of tfm-nuclei in multinucleated urethral muscle fibres of mosaic mice

Summary The X-linked testicular feminization mutation (Tfm) of the mouse leads to androgen insensitivity of target cells. Through the autosomal sex reversed (Sxr) factor we have converted female heterozygotes into males. Due to X-inactivation, mosaic animals arise which are composed of androgen sensitive wild-type and androgen insensitive Tfm cells. In the androgen dependent striated urethral muscle, Tfm and wild-type cells fuse and form multinucleated muscle fibres. In the muscle fibres the Tfm nuclei are exposed to the intact cytoplasmic testosterone receptor complex coded for by the wildtype nuclei. We ask the question whether under these conditions RNA synthesis can be stimulated in the Tfm nuclei. Castrated mosaic animals were injected with testosterone, and incorporation of ³H-uridine was studied by autoradiography. We found two classes of muscle cell nuclei, those with low grain counts corresponding to the Tfm controls and those with high grain counts corresponding to the stimulated male controls. The results indicate that the Tfm nuclei are not stimulated by the intact testosterone receptor complex.This study is dedicated to Prof. Dr. W. Graumann on occasion of his 65th birthday     

Localization of newly synthesized precursors of basal lamina in the regenerating planarian as revealed by autoradiography

Autoradiography has been carried out to investigate the site of synthesis of the basal lamina in the regenerating planarian, Dugesia japonica. Since the basic collagenous structures of the basal lamina arose from RR-positive amorphous precursor, [³H]proline, [³H]glucose and [³⁵S]sodium sulphate were used as radioactive precursors of collagen, unsulphated and sulphated GAG respectively. Cytoplasm of the most regenerating epidermal cells was heavily labeled with [³H]proline during epithelization. A quantitative uptake analysis of [³H]proline indicates a progressive decline in the amount of labeled precursor in the epidermis with a corresponding increase in deposition of the labeled collagen at the presumptive basal lamina. Several myoblasts at the subepidermal region were highly labeled with both [³H]glucose and [³⁵S]sodium sulphate. Silver grains of these labeled precursors were also present in the presumptive portion of basal lamina. These observations suggest that the regenerating epidermal cell is the only site of synthesis of the basal lamina collagen while the myoblast exclusively secretes extracellular GAG. Some of the GAG may be closely associated with the amorphous zone.     

Effects of adrenalectomy and excess hydrocortisone on the incorporation of 35S-labelled cysteine in the hypothalamic-neurohypophyseal neurosecretory system of the rat

Summary The incorporation of ³⁵S-labelled cysteine in the hypothalamic-neurohypophyseal system was studied in normal and adrenalectomized rats and in rats treated with excess hydrocortisone. Labelled cysteine was intraperitoneally administered and grain counts were made of autoradiographs produced from sections of the supraoptic and paraventricular nucleus, median eminence and neurohypophysis of animals killed 45 min., 4 hours and 24 hours after administration of the labelled substance. On the whole, lower incorporation levels of the label were noted in the adrenalectomized rats, compared with the controls. In the rats treated with excess hydrocortisone, the grain counts at 45 min and 4 hours after injection were higher and those at 24 hours were lower than those of the controls.The findings are discussed, among other things, in terms of rate of uptake vs. time and related to previous reports on the cysteine uptake and neurosecretory activity of the hypothalamic-neurosecretory sytem.This study was financially supported by the Sigrid Jusélius Foundation, Helsinki, Finland and the National Research Council for Medical Sciences, Finland.     

INCORPORATION OF H3-THYMIDINE IN LEAF NUCLEI OF XANTHIUM PENNSYLVANICUM

The basic kinetics and the pattern of incorporation of H³-thymidine was studied in the leaf lamina of Xanthium pennsylvanicum. A method of foliar absorption was used to incorporate the radioisotope into leaf nuclei. The autoradiographic techniques employed provided data on the amount of the isotope incorporated. It was determined that 10 μc/ml (sp. act. 6.7 c/mmole) of H³-thymidine with 1–8 hr of isotopic growth and 4 hr of postisotopic growth gave the most satisfactory results. The percent of labelled nuclei and the number of grains per nucleus were presented as functions of isotopic and postisotopic growth periods. Distribution of grains in the nuclei approximated the Poisson distribution at 1 hr of isotopic growth. Increased time of isotopic growth changed the pattern of grain distribution. No deleterious effects were observed using an 8-hr period of isotopic growth, but prolonged incubation time significantly decreased the proportion of mitotic figures in the lamina. The amount of incorporation of the DNA precursor expressed as percent of labelled nuclei was linear to about 16 hr of isotopic growth and thereafter decreased gradually. As indicated by the average number of grains per nucleus, H³-thymidine incorporation increased to about 16 hr, and soon after reached a saturation level. The percent of labelled nuclei and the number of grains per nucleus decreased as a function of the postisotopic growth period. However, they were significantly greater in the lamina near the vein than in the lamina region at some distance from the vein. The radioactive precursor was initially absorbed by the cells of the lamina and was subsequently translocated into the vascular system. There it was circulated and made available to the dividing cells near veins of the lamina. This region may be a metabolically distinct part of the lamina with significantly higher rates of incorporation and mitotic turnover.     

A study of the two-way transport of horseradish peroxidase across the visceral pleura

Summary The authors report on a study of the transpleural transport of horseradish peroxidase after intrapleural and intracardiac application. Following intrapleural introduction, a retention of the marker on the apical membrane of mesothelial cells was observed, with subsequent transcellular transfer after incorporation into microvesicles. Following intracardiac injection, the marker moved out of the pulmonary capillaries across the endothelial vesicles and progressed to the pleural cavity across the intercellular spaces and mesothelial vesicles. With either route of injection, reaction product was noted in the basal lamina of the mesothelium, elastic membrane, alveolar macrophages and pneumocytes type II.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Effect of L-azetidine 2-carboxylic acid on growth and proline metabolism in Escherichia coli

The effects of L-azetidine 2-carboxylic acid on growth and proline metabolism in a proline-requiring auxotroph of Escherichia coli are described. The homologue inhibited growth of the wild type and it, alone, did not substitute effectively for proline as a growth supplement for the mutant. In medium containing 0.05 mM proline, the addition of increasing amounts of homologue progressively inhibited growth of the wild type but stimulated growth of the mutant at homologue: proline ratios of 10 : 1 and 50 : 1. This suggested that the homologue exerted a “sparing effect” on proline in the mutant.The incorporation of L-[U-¹⁴C]proline and L-[³H]azetidine 2-carboxylic acid into hot trichloroacetic acid-insoluble material in the mutant was measured. Amino acid analysis of the insoluble material from cells incubated with radiolabeled proline alone revealed that proline was partially degraded and metabolized to other amino acids prior to incorporation into protein. The addition of unlabeled homologue to the incubation medium significantly reduced proline catabolism, suggesting that the homologue exerted a sparing effect on proline in this mutant. In medium containing unlabeled proline and radiolabeled L-azetidine 2-carboxylic acid, the homologuewas incorporated both intact and partially degraded prior to incorporation into protein. Alanine was the major L-azetidine 2-carboxylic acid catabolite.     

Effect of L-azetidine 2-carboxylic acid on growth and proline metabolism in Escherichia coli

The effects of L-azetidine 2-carboxylic acid on growth and proline metabolism in a proline-requiring auxotroph of Escherichia coli are described. The homologue inhibited growth of the wild type and it, alone, did not substitute effectively for proline as a growth supplement for the mutant. In medium containing 0.05 mM proline, the addition of increasing amounts of homologue progressively inhibited growth of the wild type but stimulated growth of the mutant at homologue: proline ratios of 10 : 1 and 50 : 1. This suggested that the homologue exerted a “sparing effect” on proline in the mutant.The incorporation of L-[U-¹⁴C]proline and L-[³H]azetidine 2-carboxylic acid into hot trichloroacetic acid-insoluble material in the mutant was measured. Amino acid analysis of the insoluble material from cells incubated with radiolabeled proline alone revealed that proline was partially degraded and metabolized to other amino acids prior to incorporation into protein. The addition of unlabeled homologue to the incubation medium significantly reduced proline catabolism, suggesting that the homologue exerted a sparing effect on proline in this mutant. In medium containing unlabeled proline and radiolabeled L-azetidine 2-carboxylic acid, the homologuewas incorporated both intact and partially degraded prior to incorporation into protein. Alanine was the major L-azetidine 2-carboxylic acid catabolite.     

In Vitro Protein Synthesis by Plastids of Phaseolus vulgaris. I. Localization of Activity in the Chloroplasts of a Chloroplast Containing Fraction from Developing Leaves

The incorporation of uniformly labeled leucine-¹⁴C into protein by a chloroplast containing fraction from developing primary leaves of bean is reported. Chloroplasts, obtained from week old plants grown in darkness, and then illuminated with white light for 12 hours, were shown to be the principal sites of incorporating activity. Incorporation may continue for 2 hours. Rates of up to 50 μμmole leucine incorporated per mg protein per hour are observed when a 1 hour assay period is used. Incorporation is only partially sensitive to ribonuclease.     

An acellular human amnionic membrane model for in vitro culture of type ii pneumocytes: The role of the basement membrane in cell morphology and function

To determine whether a preformed basement membrane contributes to the maintenance of morphology and function of type II pneumocytes, we cultured isolated adult rat type II pneumocytes on the basement membrane and stromal surfaces of an acellular human amnionic membrane and on plastic. The presence of lamellar bodies on transmission electron microscopy and epithelial morphology in culture and a characteristic phospholipid profile after incubation with ³H-acetate identified the cells as type II. When type II cells were cultured on a preexisting basement membrane, they formed a well-organized monolayer with polarity, centrally located surface microvilli, and more basally located nuclei. Individual cells maintained a cuboidal morphology for 8–10 days. Intracellularly, there were numerous mitochondria, endoplasmic reticulum (ER), and lamellar bodies. The cells secreted a new basal lamina of their own. When cultured on the stromal side of the amnion, the cells became flattened within 48–60 hours, formed small lamellar bodies, and had scanty surface microvilli; they formed clumps and appeared less ordered. These cells did not secrete a visible basement membrane, and the majority detached from the stromal surface after 7–8 days in culture. In addition, culture on the basement membrane aspect of the amnion prevented the rapid decline in the percentage of ³H-acetate label incorporated in phosphatidylcholine after 72 hours of culture. We conclude that a preformed basement membrane influences the function and morphology of type II pneumocytes, organizes them into a monolayer in culture, and influences deposition of a visible basal lamina. Thus, the acellular human amnion provides an excellent model for the systematic study of basement membrane influence on the biology and pathology of these cells.     

Studies on the radiosensitivity of the function of the Golgi complex.

The uptake of 3H-glucosamine into primary human-embryo fibroblasts and into the Golgi-rich fraction isolated from liver of mice labelled in vivo was studied, after various doses of X-radiation, by autoradiography and biochemical methods. A dose of 90 rad resulted in an increased precursor uptake in interphase cells at 24 hours and in mitotic cells at 48 hours after irradiation; 226 rad had virtually no effect on the grain counts of interphase cells, but reduced the labelling of mitotic forms. The characteristic intracellular localization of the grains were not influenced by these doses. Although no immediate radiation-induced reaction could be observed in liver cells either, significant stimulation of the 3H-glucosamine incorporation was measured in isolated Golgi-rich fractions 24 hours after whole-body irradiation with 90, 450, or 905 rad. This phenomenon is discussed as a part of the somatic regeneration of membrane structures.     

Ribonucleic acid within the extracellular matrix during embryonic tooth formation

During embryonic tooth formation, interactions between epithelial and mesenchymal cells results in the formation of a metachromatic interface or extracellular matrix. The cervical or germinative region of this epidermal organ system is populated by an increasing gradient of cellular differentiation and an extracellular matrix which is the progenitor for subsequent dentine organic matrix formation. Embryonic rabbit tooth primordia can be maintained in culture enabling kinetic studies of labeled precursor incorporation. Autoradiographs of tooth organ cultures continusly incubated with labeled uridine for periods up to eight hours, demonstrated initial cellular incorporation with subsequent transfer of 2% of the grain density to the extracellular matrix by four hours. The grain density was removed by ribonuclease treatment. No incorporation of tritiated thymidine into the matrix was observed. The incorporation of C¹⁴-uridine during organ culture was inhibited by actinomycin D. Micrurgy was employed to isolate the extracellular matrix free of adherent cells. Electron microscopy demonstrated membrane-bound, electron dense bodies within the matrix, presumably cytoplasmic extensions. No cells per se were observed on the isolated matrix. Several experimental criteria suggested that uridine incorporation into the extracellular matrix was regulated by epithelial and mesenchymal cells. Phenol extraction procedures of labeled cervical matrices demonstrated an ultraviolet absorption maximum at 260 μU. Both spectrophotometric determinations and orcinol assays found RNA to be 0.4–0.5% of the cervical extracellular matrix. These results are interpreted to indicate that RNA is a component of the metachromatic extracellular matrix during epithelio-mensenchymal interactions associated with tooth formation. The functional significance of these observations is premature at this time.     

Attachment of columnar airway epithelial cells in asthma

Shedding of airway epithelial cells is a common finding in asthma. In this study, the attachment of the airway epithelial cells to the basal lamina (BL) was investigated by transmission electron microscopy (TEM) of biopsies from patients with atopic asthma and healthy controls. The following parameters were quantitatively determined: the height of the epithelium and of the columnar cells, the number of basal cells per 100 microm of basal lamina, the contact surfaces of basal cells or columnar cells with the basal lamina, and between basal cells and columnar cells. In order to compare the quantitative method with previous literature data, measurements were also carried out on rat airway epithelium. Compared to the rat, the columnar cell height in the human is increased, basal cells are smaller, and there is a larger contact area between basal cells and basal lamina, as well as between basal and columnar cells. The contact area between columnar cells and basal lamina is hence less in the human airway. The contact area between columnar cells and basal lamina in asthmatics is significantly less than in healthy controls, due to larger intercellular spaces. It is concluded that attachment of columnar cells to the basal lamina occurs mainly indirectly, via desmosomal attachment to basal cells, and that direct attachment of columnar cells to the basal lamina is weakened in asthmatics.     

Nuclear-Cytoplasmic Relationships in Human Cells in Tissue Culture : III. Autoradiographic Study of Interrelation of Nuclear and Cytoplasmic Ribonucleic Acid

The movement of ribonucleic acid (RNA) from nucleus to cytoplasm has been studied, by autoradiographic techniques, in cells of the human amnion grown in tissue culture. Cells were exposed to cytidine-H³ for 1 hour after which time only the RNA of the nuclei was labelled. After this 1 hour exposure the cells were placed in a medium containing an excess amount of unlabelled cytidine. Periodically, cells from this medium were fixed. Autoradiographs showed that there was a progressive movement of the label from nucleus to cytoplasm, such that after 24 hours essentially all the label was in the RNA of the cytoplasm. A study of the incorporation of the cytidine-H³ in deoxyribonucleic acid (DNA), in the same population of cells at the same times, indicated that the presence of excess amounts of unlabelled cytidine almost instantaneously inhibited further utilization of cytidine-H³. It is concluded that RNA moves from nucleus to cytoplasm as a complex polynucleotide structure.