A study of TSH-synthesis of spontaneously hypertensive rats by electron microscopic morphometry and autoradiography

Summary In order to compare the functional state of the anterior pituitary of spontaneously hypertensive rats (SHR) with that of normotensive Wistar Kyoto rats (WKR), the anterior pituitary was examined by morphometry and autoradiography at the level of electron microscopy. The relative number and the relative volume of thyrotrophs in the anterior pituitary were significantly greater in SHR compared with age-matched WKR at 0, 7, 30–33 days, and 10 months of age, while the relative number of somatotrophs in SHR was significantly smaller at 1 and 10 months of age. Electron microscope autoradiographic analysis of uptake of ³H-lysine by thyrotrophs of both strains at the age of approximately one month showed that ³H-lysine was incorporated into protein and transported finally to secretory granules which migrated to near the cell membrane to be discharged. Silver grains were significantly more numerous over the thyrotrophs of SHR than over those of WKR at 30 min, 1 h, and 4h after the injection of ³H-lysine.The present study has ascertained morphologically that a congenital hypersynthesis of TSH by the anterior pituitary occurs in SHR.     

In vivo sterol biosynthesis by pea aphid symbiotes as determined by digitonin and electron microscopic autoradiography

Summary Pea aphid primary symbiotes have previously been shown to synthesize cholesterol in vitro. Two electron microscopic techniques were used here to determine whether the symbiotes also synthesize cholesterol in vivo and whether this cholesterol is made available to the aphid. We also inquired into a possible role of secondary symbiotes in cholesterol biosynthesis. Treatment of aphids with digitonin resulted in significant alteration of ultrastructural sites in primary and secondary symbiote membranes. We concluded that these sites are areas of high cholesterol concentration in the symbiotes.Electron microscopic autoradiography with ³H-mevalonate precursor indicated that both primary and secondary symbiotes synthesize cholesterol; in both cases, the majority of grains were associated with the symbiote membranes. While the frequency of grains on the symbiotes remained constant, irrespective of incubation time in labelled media, the frequency of grains over surrounding tissues increased exponentially as the time of incubation was increased from 30 min to 8 h, indicating that symbiote cholesterol is transported to other tissues. High voltage electron microscopic autoradiography permitted thick section autoradiography, reducing the time of emulsion exposure from 54 days (thin section) to 12 days (0.5 m sections).Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, and by a research grant (PCM 74-2401 A01) from The National Science FoundationThe authors wish to thank Dr. G.A. DeZoeten for his invaluable advice and assistance with the autoradiographic techniques, Mr. Gary Gaard for his help with electron microscopy, and Dr. Dale Johnston and Dr. Damien Neuberger for their generous help in the use of the high voltage EM     

The fine structure of the proliferative cells of the mouse intestine as revealed by electron microscopic autoradiography with 3H-thymidine

Summary The duodenal and colonic epithelia in mice were observed with electron microscopic autoradiography 2, 5 and 24 hours after a single injection of ³H-thymidine. After 2 hours, in the duodenum, silver grains are found in many undifferentiated cells, in a few young goblet cells, in some crystal-containing cells, and in some lymphocytes. In the colon after 2 hours silver grains are seen in some undifferentiated cells, and in many young goblet cells. Undifferentiated cells are characterized by a few short microvilli, poorly developed rough-surfaced endoplasmic reticulum, abundant free ribosomes, and a few apical moderately dense granules. In normal animals, absorptive cells seem to arise from undifferentiated cells, and goblet cells — from younger goblet cells. Undifferentiated cells could also become young goblet cells. Crystal-containing cells, which may not be of epithelial origin, proliferate in the epithelium in the adult animal.     

Uptake of H3-5-hydroxytryptophan by noradrenergic nerves in the mouse pancreas studied with electron microscopic autoradiography

Summary The uptake and distribution of radioactivity in vascular adrenergic nerves in the mouse pancreas following the injection of tritiated 5-hydroxytryptophan was studied by means of electron microscopic autoradiography. Autoradiographic silver grains were found selectively accumulated over axonal profiles. Quantitative analysis revealed a characteristic intraneuronal distribution of the silver grains, most of which probably represent 5-hydroxytryptamine formed by decarboxylation from the labeled precursor. Thus, the grain density over adrenergic nerve terminals, containing a mixed population of vesicles and granules, was about 5 times higher than the grain density recorded over non-terminal axonal parts and at least 20 times higher than the grain density found over surrounding adventitial tissue and smooth muscle cells. This was interpreted as an evidence that 5-hydroxytryptamine was taken up and stored in adrenergic terminals.     

Regeneration of soleus muscles of rat autografted in toto as studied by electron microscopy

Summary Rat soleus muscles were autografted from right to left legs, and regeneration following necrosis of all original myofibres was studied after 7 to 250 days. The best regenerates were from grafts replacing all calf muscles and sutured to the tendon stumps. After 30 days the size of such regenerates was equal to those from minced gastrocnemius muscles: the cross sectional area of muscle tissue was 30% (1.7 mm²) and the number of fibres was 180% (4500) of normal soleus muscles; the fibre diameters were 10 to 40 m. To increase the number of myoblasts before grafting some muscles were injured by Ringer solution of 70° C and transplanted after 2 days. Nevertheless, this did not influence regeneration.After 7 days clusters of myotubes occurred in the periphery of the muscle. These myotubes originated from myoblasts growing like endothelial cells on the inner face of the persisting basal lamina tubes of necrotic fibres. After 30 days the muscles were vascularized. Fibres formed in a common basal lamina detached and so looked split. Satellite cells of new fibres came from undifferentiated cells associated with myotubes, i.e. from myoblasts. After 30 days and more regenerates contained three sorts of fibres. 1. Thin (5 to 20 m) fibres resembling fetal muscle fibres. They were most prominent after 30 days, and probably not yet innervated. 2. Thin (10 m) degenerating fibres as in long-time denervated muscles. 3. Thick (more than 30 m) mature looking fibres which were innervated and revealed end-plates.Half of the grafts studied after 30 and 60 days contained unmyelinated and myelinated axons which had grown along strands of surviving Schwann cells. After 250 days, only two muscles were studied which both lacked innervation. Almost all regenerates contained muscle spindles, which, however, were not innervated. Within the persisting spindle capsules new muscle fibres had been formed from satellite cells of the former intrafusal fibres.This study was supported by grants from the Danish Medical Research Council, and the National Danish Association against Rheumatic Diseases. I wish to thank Miss U. Hellhammer for valuable technical help and Dr. T. Tobias for correcting my English     

Osteoclasts and their relationship to bone as studied by electron microscopy

Summary Osteoclasts in metaphyses from young rats were systematically sectioned at different levels. Two types of osteoclasts were recognized. One type had no ruffled border while the other, and predominant type contained a ruffled border in a part of its length; some of the latter contained two ruffled borders. The closest contact between osteoclast and bone occurred at the level of the ruffled border and this bone under the border showed characteristic changes indicative of resorption. In some osteoclasts the ruffled border consisted of numerous slender cytoplasmic projections separated by very narrow spaces or channels while in other osteoclasts it was more open. The ruffled border was commonly surrounded by a transitional zone containing numerous thin filaments. The osteoclast usually had its greatest dimension at the level of the ruffled border and the cytoplasm here contained many bodies and vacuoles but a sparse endoplasmic reticulum. Away from the level of the ruffled border the cytoplasmic vacuoles and bodies were fewer while the endoplasmic reticulum was often more pronounced. Parts of the osteoclasts were usually situated close to a vessel. It is suggested that there is a correlation between the development of the ruffled border and the degree of bone resorption and that osteoclasts without a ruffled border are, at least temporarily, inactive with respect to bone resorption. The numerous cytoplasmic bodies, interpreted as lysosomes, are presumed to be important in the resorption process. The closely adjacent positioning of osteoclasts and vessels may facilitate the transport of resorption products to the blood.This research was supported by the Danish Research Council. Grant no. 512–727, 512–819 and 512–1545.I wish to thank Professor Arvid B. Maunsbach for valuable discussions.     

Localization of the membrane-bound hydrogenase in Alcaligenes eutrophus by electron microscopic immunocytochemistry

Abstract The membrane-bound hydrogenase was localized in cells of Alcaligenes eutrophus by electron microscopic immunocytochemistry. Post-embedding labeling performed on ultrathin sections revealed that the enzyme was located predominantly (80%) at the cell periphery in autotrophically and heterotrophically grown bacteria harvested from the exponential phase of growth. In the stationary growth phase, however, only 50% of the enzyme was found at the cell periphery; the remaining 50% was distributed over the cytoplasm. The relative amount of electron microscopic label per cell as seen by application of the protein A—gold technique was higher in cells grown autotrophically as compared to cells grown heterotrophically on fructose. Derepression of the enzyme was followed electron microscopically in a substrate-shift experiment (growth on fructose, followed by a shift to glycerol). Major amounts of the enzyme appeared to undergo a reattachment to the cytoplasmic membrane under these conditions, starting with a reduced location of the enzyme in the cytoplasm and an accumulation in cell areas close to the cytoplasmic membrane. These findings indicate that the 'membrane-bound' hydrogenase (i.e., that material enriched as membrane-bound enzyme according to the appropriate activity test) is not, in fact, membrane bound or membrane integrated but membrane associated. It may or may not interact with the cytoplasmic face of the cytoplasmic membrane, depending on the growth phase and conditions.     

Transport of Endocytosed Material Into Melanin Granules in Cultured Choroidal Melanocytes of Cattle—New Insights Into the Relationship of Melanosomes With Lysosomes

Cultured choroidal melanocytes from cattle were incubated with gold labeled albumin. After phagocytosis of the labeled protein, the label appeared inside the melanin granules, as was observed under the electron microscope. Melanin granules associated with gold particles were also exocytosed into the culture medium by the melanocytes. The results of this study show that endosomes or phagosomes are transported from the cell surface of a melanocyte to the melanin granules. Therefore, melanin granules are part of the lysosomal degradation pathway. The possibility that albumin is degraded by proteases present in lysosomes and melanosomes and that the tyrosine released during degradation is used as substrate by tyrosinase and thereby converted to melanin is discussed. The present study additionally shows that the choroidea of cattle can be used as a source for cell culture of melanocytes.     

Electron microscopic autoradiography for demonstration of pineal serotonin in rat

Summary The fine localization of rat pineal serotonin has been studied by means of electron microscopic autoradiography. Two hours after the intravenous injection of tritium labelled 5-hydroxytryptophan, the location of large number of silver tangled threads is seen in the sympathetic nerve terminals. There is also a less specific accumulation of the silver grains in the pinealocytes, some appearing in the cytoplasmic organelles and some in the nucleus.In quantitative terms, 43% of the total count of silver grains were in the nerve endings whereas pinealocytes, which comprise a much larger volume of the section, contain a proportionally much smaller number of silver particles (53%). Furthermore the perivascular spaces, which comprise a larger percentage in volume of the section than the nerve endings has nevertheless only about 4% of the grains counted.Although the precise localization of the silver grains is obscure, the reaction of the granulated vesicles in the nerve terminals to the double fixation used, is similar to that shown by the extremely dense material in vesicles of platelets, which was demonstrated to contain serotonin. The results therefore suggest that the silver grains appearing in the nerve terminals two hours after the administration of 5-hydroxytryptophan are in the serotonin binding site in the axon terminals, containing the granulated vesicles.     

The cell cycle of Bacillus subtilis as studied by electron microscopy

Bacillus subtilis strain Marburg was grown exponentially with a doubling time of 65 min. To follow the time course of various cell cycle events, cells were collected by agar filtration and were then classified according to length. The DNA replication cycle was determined by a quantitative analysis of radioautograms of tritiated thymidine pulse labeled cells. The DNA replication period was found to be 45 min. This period is preceded and followed by periods without DNA synthesis of about 10 min.The morphology and segregation of nucleoplasmic bodies was studied in thin sections. B. subtilis contains two sets of genomes. DNA replication and DNA segregation seem to go hand in hand and DNA segregation is completed shortly after termination of DNA replication.Cell division and cell separation were investigated in whole mount preparations (agar filtration) and in thin sections. Cell division starts about 20 min after cell birth; cell separation starts at about 45 min and before completion of the septum.     

Nature of micellar calcium phosphate in cows' milk as studied by high-resolution electron microscopy

The nature of the inorganic calcium phosphate in the casein micelle of cows' milk has been studied by high-resolution electron microscopy. No periodic lattice spacings could be imaged, and diffraction patterns were of the diffuse amorphous type. Short-range order of less than 15 Å may be present, but the results indicate that there is no long-range order in micellar calcium phosphate.     

Quantitative electron microscopic autoradiography on the mouse thyroid gland after administration of 3H-L-DOPA

Summary The cellular and subcellular distribution of radioactivity in the mouse thyroid gland different times (20 min — 8 hours) after intravenous administration of ³H-L-DOPA was studied by means of quantitative electron microscopic autoradiography.High concentrations of autoradiographic silver grains occur over parafollicular cells and adrenergic nerves while the labelling of follicular cells and lumina is low or absent and similar to the labelling of connective tissue cells at all observation times.Over the parafollicular cells high levels of radioactivity can be recorded already 20 min after administration of the labelled amino acid. The grain counts are highest at 1 hour and decrease then at 2.5 and 8 hours.The intracellular distribution of label is similar at all observation times; thus, the concentration of silver grains over the typical cytoplasmic granules of the parafollicular cells is 4–5 times higher compared to the concentration over the remainder of the cytoplasm and the nucleus.Treatment with a decarboxylase inhibitor prior to the injection of ³H-L-DOPA results in a low and uniform labelling of all thyroid cells. This finding, taken together with the observation that also pretreatment with reserpine abolishes the autoradiographic reaction over the cytoplasmic granules, gives strong support to the idea that the great majority of silver grains observed over parafollicular cells represents dopamine formed by decarboxylation of the labelled precursor.This study was supported by grant K71-12X-3352-01 from the Swedish Medical Research Council. The author wishes to express his gratitude to Mrs. Gunnel Bokhede and Miss Dala Sjögren for expert technical assistance.     

Liposome transport as a model of substance absorption in the small intestine studied by electron microscopic autoradiography and scanning microscopy

Using electron microscope autoradiography and scanning microscopy, evidence was provided on the space-time character of the transport of enterally introduced liposomes containing 125I-lecitin and 3H-cholesterine. Liposomes underwent degradation on the mucous surface of the epithelial cells, followed by the appearance of monolayer vesicles to be transported to the glycocalix area between the microvilli. This is accompanied with the release of the radioactive trait from liposomes and its transporting to enterocytes.     

Structural organization of the membrane-bound hydrogenase isolated from Alcaligenes eutrophus as revealed by electron microscopy

Abstract Electron microscopy of negatively stained samples of the membrane-bound hydrogenase isolated from Alcaligenes eutrophus was used to obtain enzyme images with an estimated resolution of 2.5 nm. The two subunits with shapes similar to the letter 'U' making up the enzyme could be seen to be joined in two planes orthogonal to each other, making contact with their concave sides. In face-on view, the particle exhibited bilateral symmetry.     

Light and electron microscopic studies of the ventricular wall

Summary Electron microscopic data confirm the results gained with rapid Golgi preparations of adult rodent brains that tanycytes occur in clusters along the lateral wall of the third ventricle. The cytoplasmic matrix of these cells is considerably denser than that of typical ependymal cells. They have filaments and microtubules throughout their cytoplasm along with mitochondria and polysomes. At the surface is a compact group of microvilli which suggest that tanycytes might selectively absorb material from the ventricle.The tanycytes are segregated from neuropil by other tanycyte processes, by neighboring ependymal cells and by astrocytes. Yet there are gaps in this sheath. At these points tanycytes either abut upon or surround nonglial components of the neural fabric.Their cytological features and relations with the neuropil suggest that tanycytes selectively absorb material from the ventricle and release it along the basal process, primarily affecting those segments of neurons immediately adjacent to the tanycyte.Supported by: NINDS Grants 5 R01 NS 09001-02 NEUA, 5T01 NB 5309, and GM 00958, and by the Eleanor Roosevelt Cancer Foundation Research Institute.Acknowledgements: This work was initiated in the Anatomy Department of the Harvard Medical School with facilities provided by Prof. S. L. Palay (U.S. Public Health Service Grant No. NB 05591). Dr. R. B. Wuerker kindly and patiently provided the instruction and orientation to electron microscopy. The major portion of the study was completed in the Neurology Department of the University of Utah with the extremely competent, challenging assistance of Dee Lerdahl, Nina Belgarian, Keith Johnson and Lynn Kendricks.     

ATP-induced FliI hexamerization facilitates bacterial flagellar protein export

FliI ATPase forms a homo-hexamer to fully exert its ATPase activity, facilitating bacterial flagellar protein export. However, it remains unknown how FliI hexamerization is linked to protein export. Here, we analyzed the capability of ring formation by FliI and its catalytic mutant variants. Compared to ATP a non-hydrolysable ATP analog increased the probability of FliI hexamerization. In contrast, FliI(E221Q), which retained the affinity for ATP but has lost ATPase activity, efficiently formed the hexamer even in the presence of ATP. The mutations, which reduced the binding affinity for ATP, significantly abolished the ring formation. These results indicate that ATP-binding induces FliI hexamerization and that the release of ADP and Pi destabilizes the ring structure. FliI(E221Q) facilitated flagellar protein export in the absence of the FliH regulator of the export apparatus although not at the wild-type FliI level while the other did not. We propose that FliI couples ATP binding and hydrolysis to its assembly-disassembly cycle to efficiently initiate the flagellar protein export cycle.     

Solution structure of the KdpFABC P-type ATPase from Escherichia coli by electron microscopic single particle analysis

The K⁺-translocating KdpFABC complex from Escherichia coli functions as a high affinity potassium uptake system and belongs to the superfamily of P-type ATPases, although it exhibits some unique features. It comprises four subunits, and the sites of ATP hydrolysis and substrate transport are located on two different polypeptides. No structural data are so far available for elucidating the correspondingly unique mechanism of coupling ion transport and catalysis in this P-type ATPase. By use of electron microscopy and single particle analysis of negatively stained, solubilized KdpFABC complexes, we solved the structure of the complex at a resolution of 19 Å, which allowed us to model the arrangement of subunits within the holoenzyme and, thus, to identify the interfaces between subunits. The model showed that the K⁺-translocating KdpA subunit is in close contact with the transmembrane region of the ATP-hydrolyzing subunit KdpB. The cytosolic C-terminal domain of the KdpC subunit, which is assumed to play a role in cooperative ATP binding together with KdpB, is located in close vicinity to the nucleotide binding domain of KdpB. Overall, the arrangement of subunits agrees with biochemical data and the predictions on subunit interactions.