Production of specific-protein secretion granules by fat body cells of the blowfly,Calliphora erythrocephala

Summary During the first four days of the imaginai stage the fat cells of ovariectomized females of Calliphora develop a protein synthetic apparatus, and produce dense bodies (lysosomes) as do the fat cells of normal females, but apparently they cannot synthesize the protein secretion granules that characterize the productive phase of the fat cells of normal females and that we believe to represent vitellogenin. Injection of ovariectomized females with -ecdysone restored the ability of the fat cells to produce the secretion granules. It is suggested that the ovary gives off a factor which induces the production of the protein secretion granules by the fat cells, and that the factor from the ovary can be substituted by -ecdysone. This, we believe, is the first ultrastructural evidence for an effect of the ovary and of -ecdysone on the synthesis of specific protein.We are grateful to the Carlsberg Foundation and to the Danish Science Research Council for generous grants, and to the latter for placing an electron microscope at our disposal. It is a pleasure to thank Dr. Gareth Griffiths for valuable advice as to the preservation of the fat body tissue. We also thank Mrs. Lotte Bakhoj and Mrs. Elsebeth Lund for skilful technical assistance1896–1976     

Langerhans cells in the lymph node: mirror section and immunoelectron microscopic studies

Cells immunostained with antibodies against both OKT-6 and S-100 protein were observed only in superficial and hilar lymph nodes draining tissues with predominantly squamous epithelia. In contrast, in mesenteric lymph nodes and the spleen, only S-100 protein-positive, but OKT-6-negative cells were found. We suspect that the S-100 and OKT-6-positive cells might be Langerhans cells (LC) and the S-100-positive, OKT-6-negative cells, interdigitating reticulum cells (IDC). We further postulate that the LC in superficial and hilar lymph nodes might migrate from squamous epithelia, with which contact is required for the formation of Birbeck granules.     

Organization of the nematocyst battery in the tentacle of hydra: Arrangement of the complex anchoring junctions between nematocytes,epithelial cells,and basement membrane

Summary Nematocytes (stinging cells) of hydra tentacles are anchored to the basement membrane by peculiar complex junctions in which a flattened tongue of an epithelial cell is interposed between the nematocyte and the basement membrane. In this paper we describe the arrangement of these junctions with emphasis on how they are related to the architecture of the epithelial cell. Each epithelial cell, called a battery cell, harbors 10–20 nematocytes and bears muscle processes that extend along the basement membrane. The epithelial cell component of the complex junction is usually a lateral extension of a muscle process. All nematocytes within a battery cell make junctions with muscle processes of the same (resident) epithelial battery cell despite the presence of numerous muscle processes from adjacent (foreign) cells. Some nematocytes make junctions with several resident processes, spanning the foreign processes to do so. Most junctions reside near the proximal ends of the muscle processes. New findings are reported on the substructure of the junctions. They are composed of aggregates of smaller elements, and the cytoskeleton within the complexes has a pronounced longitudinal organization. These observations are consistent with a suggestion that the complex junctions develop by aggregation of smaller junctional units originating elsewhere on the cells.     

Morphometric evaluation of subcellular changes induced by in vivo TRH treatment in the pituitary gland of Rana perezi: Effects on prolactin and thyrotropic cells

Summary The effects of synthetic thyrotropin-releasing hormone on pituitary prolactin and thyrotropic cells were investigated in adult male Rana perezi (formerly Rana ridibunda) frogs. Animals were given daily injections of synthetic thyrotropin-releasing hormone into the dorsal lymph sac. Prolactin and thyrotropic cells were identified by the colloidal-gold method, using anti-human prolactin and anti-human--thyrotropin hormone as primary antisera. The stereological parameters of the rough endoplasmic reticulum, Golgi complex, and secretory granules of prolactin and thyrotropic cells were evaluated by ultrastructural morphometry (point-counting method). Thyrotropin-releasing hormone caused cytological changes in both cell-types which were consistent with increased synthesis and release of both prolactin and thryrotropin. These changes were still significant after 48 h treatment in the case of thyrotropic cells, while in prolactin cells the thyrotropin-releasing hormone increased the number of secretory granules. After 6 days, the cells resembled essentially those used as controls. These results indicate that thyrotropin-releasing hormone stimulates the synthesis and release of prolactin and thyrotropin, and that the response of each cell type to this hypothalamic stimulus follows a different time-course.This work has been supported by grants no. 2184-83 and PB 86-0095 from the Comisión Interministerial para la Ciencia y Tecnología, Spain     

Immunocytochemical localization of S-100 protein in stellate cells (folliculo-stellate cells) of the adenohypophysis in the monkeys Macaca irus and Cercopithecus aethiops

Summary With the use of an antibody against bovine S-100 protein, it was possible to reveal a characteristic cell type in the pars distalis and the pars tuberalis of the monkey Macaca irus. In the adenohypophysis of Cercopithecus aethiops, labeled cells were present in the pars distalis, pars tuberalis, and pars intermedia. These cells, so-called folliculo-stellate cells, were found in all pituitaries studied. Surprisingly, an antibody against human S-100 protein did not label the stellate cells of the adenohypophysis. However, in Macaca irus, this antibody gave a strong positive reaction with various other cell types (interstitial cells of the pineal gland, Müller cells of the retina, autonomic ganglionic cells, glial cells of the central nervous system, Schwann cells, Bergmann glia of the cerebellum, fat cells, reticular cells of lymphoid organs). By use of double immunoenzymatic labeling, it was evident that stellate cells are spatially related either to somatotropes, prolactin cells, corticotropes, or to glycoprotein-containing cells. Thus, a specific relationship to a particular endocrine-cell type could not be observed.Dr. M.P. Dubois died in Tours (France) on March 30, 1986, aged 65     

Intercellular communications within the rat anterior pituitary. XVI: Postnatal changes of distribution of S-100 protein positive cells,connexin 43 and LH-RH positive sites in the pars tuberalis of the rat pituitary gland. An immunohistochemical and electron microscopic study

The architecture of luteinizing hormone-releasing hormone (LH-RH) nerve ends and the S-100 protein containing folliculo-stellate cells forming gap junctions in the pars tuberalis is basically important in understanding the regulation of the hormone producing mechanism of anterior pituitary glands. In this study, intact male rats 5–60 days old were prepared for immunohistochemistry and electron microscopy. From immunostained sections, the S-100 containing cells in pars tuberalis were first detected on day 30 and increased in number to day 60; this was parallel to the immunohistochemical staining of gap junction protein, connexin 43. LH-RH positive sites were clearly observed on just behind the optic chiasm and on the root of pituitary stalk on day 30. On day 60, the width of layer increased, while follicles and gap junctions were frequently observed between agranular cells in 10 or more layers of pars tuberalis.     

Intercellular communication within the rat anterior pituitary: relationship between LH-RH neurons and folliculo-stellate cells in the pars tuberalis

The distribution of LH-RH-positive nerve fibers in the median eminence was demonstrated in the 1970s and 1980s. A few LH-RH fibers have been reported to be present in the adjacent pars tuberalis of the pituitary, but their functional significance has not been clarified and still remains enigmatic. Adult male Wistar-Imamichi rats were separated into two groups: one for immunohistochemistry of LH-RH and S-100 protein (for the identification of folliculo-stellate cells) and the other for electron microscopy. For both immunohistochemistry and electron microscopy, the specimens obtained contained the pituitary gland connected with the hypothalamus. Numerous LH-RH-positive fibers were observed as tiny lines with several varicosities both on the primary vascular plexus and in the hypothalamus corresponding to the posterior half of the portal vein area. LH-RH-positive fibers were also noted around S-100-positive cells in the pars tuberalis. Weakly reactive S-100 cells were scattered in the pars tuberalis in the midsagittal plane, while clusters of strong reactive elements occurred 100–300 m from the center. Similar observations were made using fluorescence immunohistochemistry for LH-RH and S-100, and at the electron-microscopic level. At the posterior portion of the portal vein system, bundles of the LH-RH-immunoreactive fibers invaded the pars tuberalis and terminated on agranular cells. Gap junctions were clearly seen among agranular cells corresponding to folliculo-stellate cells. It is postulated that the LH-RH message might be transmitted not only by the established hypophyseal portal vein system but also via the folliculo-stellate cells in the pars tuberalis to aid in the modulation of LH release.     

Dendritic cells might be one of key factors for eliciting antitumor effect by chemoimmunotherapy in vivo

In this study, we demonstrated that chemoimmunotherapy using S-1, a novel oral fluoropyrimidine anticancer drug, combined with lentinan (LNT), a (13) glucan, was effective in vivo, and we clarified the augmentation of the function of dendritic cells (DCs) in vivo and in vitro. The survival period of Colon-26–bearing mice treated with S-1+LNT was significantly more prolonged than that of mice treated with S-1 alone (P<0.05). On the other hand, LNT did not prolong the survival period when combined with S-1 in Colon-26–bearing athymic mice. The frequency of CD86⁺ DCs infiltrated into Colon-26 was increased in mice treated with S-1+LNT, and splenic DCs harvested from mice treated with S-1+LNT showed more potent T-cell proliferation activity than that of DCs from mice treated with S-1 alone (P<0.05). Furthermore, the activity of cytotoxic T lymphocytes (CTLs) in splenocytes of S-1+LNT–treated mice was specific and more potent than that of CTLs from mice treated with S-1 alone (P<0.05). These results suggest that modulation of specific immunity with LNT has a significant role in enhanced antitumor effects through the modification of DC function. We demonstrated that DCs might play an important role in chemotherapy, and the combination therapy of S-1 and LNT presents a promising chemoimmunotherapy, which might lead to better survival for cancer patients.     

The pituitary of Aphanius dispar (Rüppell) from hypersaline marshes and freshwater

Summary An ultrastructural study of the rostral pars distalis of the pituitary of Aphanius dispar specimens taken from freshwater or hypersaline marshes revealed significant structural differences which indicate higher activity of the prolactin cells in the hypotonic medium. Prolactin cells from freshwater specimens had larger secretory granules, a higher amount of endoplasmic reticulum, and expanded intercellular spaces with many secretory lakes. These cells contained an unusual cytoplasmic structure, consisting of twisted canals with vesicular lumina, connected to the endoplasmic reticulum of the cell. This structure is about 1–2 m in diameter.Stellate cells are characterized by extracellular spacing junctions which are particularly noticeable at the confluence of the interstellate cell canaliculi and the pericapillary space.Abbreviations FW freshwater - HS hypersaline - NS neurosecretory - PCB paracrystalline body - PNH proximal neurohypophysis - RPD rostral pars distalis - SG secretory granule - SW seawater This paper is dedicated with affectionate respect to Professor Berta Scharrer on the occasion of her 70th birthdayThe assistance of Cynthia Bellon in editing this paper is gratefully acknowledged     

The prolactin cell of the white-crowned sparrow,Zonotrichia leucophrys pugetensis

Summary The anterior pituitaries from a series of female White-crowned Sparrows,Zonotrichia leucophrys pugetensis, in the periods of oviposition, incubation, and brooding under natural conditions, have been investigated by electron microscopy. The prolactin cells occur in cephalic lobe and are characterized by large (ca. 300–600 m), polymorphic electron-dense secretory granules and an extremely well developed, lamellated endoplasmic reticulum. During incubation and brooding it is only these prolactin cells that are in an activated secretory phase, as indicated by increase in number and size, extremely well developed endoplasmic reticulum, decrease in number of mature secretory granules, and by active formation of granules in the enlarged Golgi apparatus. In the late stages of brooding, and post-breeding, the prolactin cells regress with involution of the endoplasmic reticulum and Golgi apparatus, reaccumulation of granules, and the appearance of lysosomes.The gonadotropes of both the cephalic and caudal lobes undergo progressive morphologic changes through the course of the breeding period. They are numerous and active in the ovulating bird. They undergo gradual regression during the periods of incubation and brooding to become typical broody cells.This investigation was supported by Grant No. GF-33334, U.S.-Japan Cooperative Science Program of the National Science Foundation and by Grant No. GB-28080X, also from the National Science Foundation, to Professor Farner; and by Grant No. 5R040 Japan-U.S. Cooperative Science Program of Japan Association of Science Promotion, to Professor Mikami.     

Immunocytochemical and ultrastructural study of the frog (Rana pipiens) pars distalis with special reference to folliculo-stellate cell function during in vitro superfusion

Summary The colloidal gold immunocytochemical technique was used to determine the ultrastructural features of the glandular cells in the pituitaries of male frogs, Rana pipiens, both in vivo and after superfusion in vitro. Specific reactions to antisera against bullfrog gonadotropins, human prolactin, and synthetic 1–39 corticotropin allowed identification of the 3 corresponding types of glandular cells. No immunoreaction was obtained with antisera against human or ovine-growth hormone, human -thyrotropin hormone, and bovine S-100 protein. General morphological features of these immunocytochemically identified glandular cells were similar to those of equivalent cells previously described in other amphibian species. Non-glandular folliculo-stellate cells were distinctive. In freshly removed pituitaries, these folliculo-stellate cells contained lysosome-like structures, but did not show phagocytic vacuoles in the cytoplasm; they contained many mitochondria, and the Golgi complex and endoplasmic reticulum were relatively undeveloped. After 4 or 18 h of superfusion, some immunoreactive gonadotropic, prolactin, and corticotropic cells showed degeneration and destruction. In the same gland, folliculo-stellate cells retained a viable appearance, but showed phagocytic vacuoles containing secretory granule-like structures which were immunoreactive to gonadotropic, prolactin, and corticotropic antibodies. Some folliculo-stellate cells showed phagocytic vacuoles containing complete glandular cells. These results suggest that superfusion causes a destruction of some of the glandular cells, and that folliculo-stellate cells act as phagocytes when cellular debris or moribund cells are present in the intercellular space in the pituitary parenchyma.Supported by grant DCB 8710462 from the National Science Foundation, grant 2148-83 from the CAICYT (Spain) and the Junta de Andalucia (Spain)     

Langerhans cells in the lymph node: mirror section and immunoelectron microscopic studies.

Cells immunostained with antibodies against both OKT-6 and S-100 protein were observed only in superficial and hilar lymph nodes draining tissues with predominantly squamous epithelia. In contrast, in mesenteric lymph nodes and the spleen, only S-100 protein-positive, but OKT-6-negative cells were found. We suspect that the S-100 and OKT-6-positive cells might be Langerhans cells (LC) and the S-100-positive, OKT-6-negative cells, interdigitating reticulum cells (IDC). We further postulate that the LC in superficial and hilar lymph nodes might migrate from squamous epithelia, with which contact is required for the formation of Birbeck granules.     

A quantitative ultrastructural study of Chromatium minus in the bacterial layer of Lake Cisó (Spain)

A quantitative ultrastructural study was performed with samples taken throughout a layer of the purple sulfur bacterium Chromatium minus in Lake Cisó (Spain). Ultrathin sections of cells were analyzed by transmission electron microscopy, in order to study the size, number and volume of intracytoplasmic membranes (ICM), sulfur globules and poly--hydroxybutyrate (PHB) granules per unit volume of cell. Important differences were seen between cells from the top (receiving 60 E · m^–2 · s^–1 at noon) on the one hand, and cells from the peak and bottom parts of the bacterial layer (receiving less than 1 E · m^–2 · s^–1) on the other hand. The amount of ICM per cell increased as a function of depth being about three times higher in bottom cells than in top cells. Neither statistically significant differences in cell size, nor in numbers of sulfur globules were found, but the ultrastructure changed with depth. Finally, the most important changes throughout depth were detected in PHB granules. Top cells had 0.5% of their volume occupied by PHB granules, whereas in the bottom cells the corresponding value was 12.2%. These changes were due to the number of PHB granules per unit volume of cell since globule size was constant.Non-common abbreviations ECM intracytoplasmic membrane systems - PHB poly--hydroxybutyrate - Bchl bacteriochlorophyll - SED sphere equivalent diameter     

Immunohistochemical localization of anterior pituitary hormones in S-100 protein-positive cells in the rat pituitary gland

In the anterior and intermediate lobes of the rat pituitary gland, non-hormone-producing cells that express S-100 protein coexist with various types of hormone-producing cells and are believed to function as phagocytes, supporting and paracrine-controlling cells of hormone-producing cells and stem cells, among other functions; however, their cytological characteristics are not yet fully understood. Using a transgenic rat that expresses green fluorescent protein under the promoter of the S100β protein gene, we immunohistochemically detected expression of the luteinizing hormone, thyroid-stimulating hormone, prolactin, growth hormone and proopiomelanocortin by S-100 protein-positive cells located between clusters of hormone-producing cells in the intermediate lobe. These findings lend support to the hypothesis that S-100 protein-positive cells are capable of differentiating into hormone-producing cells in the adult rat pituitary gland.     

Immunohistochemical localization of S-100 protein,glial fibrillary acidic protein,and neuron-specific enolase in the pars distalis of quail,rat, and human hypophyses

Summary In the present study, we have localized immunohistochemically S-100 protein, glial fibrillary acidic (GFA) protein, and neuron-specific enolase (NSE) by the unlabelled antibody peroxidase-antiperoxidase technique. Special attention was paid to the influence of fixation and of pretreatment of sections with proteolytic enzymes. It appeared that the final immunostaining of a given antigen largely depends on the fixative and on the species used. Moreover, pepsin pretreatment proved to be necessary to unmask S-100 protein in quail and GFA protein in rat. S-100 protein (rat, human) and GFA protein (human) immunoreactivities were detected in the folliculo-stellate (FS) cells. In quail, S-100 protein was also found in cells, which were not arranged around a follicular lumen and, in rat, the endothelial cells were immunostained for GFA protein. Clusters of granular cells were weakly immunostained for NSE in all species. An exclusive relationship between FS cells and S-100 protein could not be ascertained from this study.     

Immunohistochemical localization of S-100 protein, glial fibrillary acidic protein, and neuron-specific enolase in the pars distalis of quail, rat, and human hypophyses

In the present study, we have localized immunohistochemically S-100 protein, glial fibrillary acidic (GFA) protein, and neuron-specific enolase (NSE) by the unlabelled antibody peroxidase-antiperoxidase technique. Special attention was paid to the influence of fixation and of pretreatment of sections with proteolytic enzymes. It appeared that the final immunostaining of a given antigen largely depends on the fixative and on the species used. Moreover, pepsin pretreatment proved to be necessary to unmask S-100 protein in quail and GFA protein in rat. S-100 protein (rat, human) and GFA protein (human) immunoreactivities were detected in the folliculo-stellate (FS) cells. In quail, S-100 protein was also found in cells, which were not arranged around a follicular lumen and, in rat, the endothelial cells were immunostained for GFA protein. Clusters of granular cells were weakly immunostained for NSE in all species. An exclusive relationship between FS cells and S-100 protein could not be ascertained from this study.     

Enhanced plasmid stability through post-segregational killing of plasmid-free cells

Summary In order to develop an extremely stable, inducible host/vector system, the following genetic manipulations were made: a recA mutation was introduced into the chromosome of the host strain, the plasmid selectable marker was changed from ampicillin to kanamycin, and the parB stability locus of plasmid R1 was added to the plasmid. The stability of the new vector, pTKW106, was increased such that the fraction of plasmid-bearing cells present during chemostat fermentations under selective pressure increased from 1.75% to 100% when plasmid protein production was fully induced. At this level of induction, -galactosidase represents 10% of the total cell protein. In addition, the frequency of generation of plasmid-free cells was shown to decrease from 1.0 per generation to less than 10^–11 with full promoter induction under non-selective conditions.     

Seasonal changes in the prolactin cell of the pituitary gland of the freshwater stickleback,Gasterosteus aculeatus,form leiurus

Summary The relative volume of the RPD region of the pituitary gland of Gasterosteus aculeatus form leiurus was greater in animals collected in the spring than in winter. A morphometric analysis of the prolactin cells from spring animals showed only slight changes in cell ultrastructure compared with winter animals. However, in spring the prolactin cells apparently formed and released more secretory granules. Three distinct sites for the release of secretory granules are described. A preliminary study of the response of prolactin cells from spring animals to 70% sea water did not reveal any large scale changes in cell ultrastructure. The significance of this finding is discussed. It is concluded that although the freshwater stickleback cannot be regarded as physiologically hypophysectomised: with regard to prolactin secretion in the winter, it still shows a seasonal change in prolactin cell ultrastructure.This work formed part of a thesis submitted for the degree of Doctor of Philosophy in 1973 and for which the author was in receipt of an S.R.C. studentship. My thanks are due to Dr. M.P. Ireland for his support and supervision throughout the course of the work.     

Immunohistochemistry of opioid peptides in the guinea pig endocrine pancreas

Summary Previous immunochemical investigations have demonstrated various opioid peptides in the pancreas. However, controversies exist related to the cellular localization of these peptides in the endocrine pancreas. Therefore, the guinea pig endocrine pancreas was immunohistochemically investigated for the presence of opioid peptides derived from pro-dynorphin, pro-enkephalin or pro-opiomelanocortin. Immunoreactivities were demonstrated on serial semithin sections by the peroxidase anti-peroxidase technique. In routinely immunostained sections, immunoreactivities for dynorphin A and -neo-endorphin were localized in pancreatic enterochromaffin cells, but not in islet cells. Immunoreactivity for Met-enkephalin was confined exclusively to B-cells and was localized only in some secretory granules. However, pre-treatment of semi-thin sections with trypsin and carboxypeptidase B led to a marked increase of Met-enkephalin immunoreactivity in B-cells. In addition, immunoreactivities for Met-enkephalin-Arg-Gly-Leu and bovine adrenal medulla dodecapeptide could be demonstrated in B-and A-cells, and -endorphin immunoreactivity was localized in A-cells. In no case, however, were immunoreactivities detected for bovine adrenal medulla docosapeptide, peptide F, corticotropin, melanotropin or dynorphin 1–32. The immunohistochemical findings indicate that opioids of different peptide families are present in the guinea pig endocrine pancreas. Since several opioid peptides of the corresponding pro-hormones could be demonstrated in the reference organs but not in the pancreas, it is concluded that the biosynthetic pathways of the respective precursors are different from those in the adrenal medulla or in the pituitary.     

Some features of the fine structure and histochemistry of planarian subepidermal gland cells

Summary The conspicuous subepidermal gland cells, namely eosinophiles, basophiles and rhabdite forming cells, in Planaria vitta, have been studied using a variety of histochemical methods for proteins, amino acids, polysaccharides, lipids, nucleic acids and calcium, in addition to common histological methods. Several fixation methods and freeze-drying have been employed. The eosinophilia of certain of the gland cells has been investigated by blocking amino acid end- and side-groups and by the staining capacity at different pH's. The fine structure of the secretion granules has been elucidated by means of electron microscopy.The eosinophilic granules are very coarse and up to 1.5 long and 0.8 wide. They are predominantly composed of protein and give positive reactions for tyrosine, arginine and cysteine-cystine and a negative reaction for tryptophan. They possibly also contain histidine. The eosinophilia is only slightly affected by acetylation or nitrosation and is only slightly decreased at pH 11.8, so the presence of arginine is probably the basis for the eosinophilia. The granules probably also contain phospholipid. The fine structure of the granules is rather unique for secretion granules. They are not surrounded by a membrane and are built up of light and dark striated structures, very regular in appearance and unperforated. The striation is mainly oriented transverse to the long axis of the granules. Parallel with this axis a much finer striation is found, bounding rectangular compartments. The coarse, dark striations are about 175–250Å wide.The basophilic cells may be divided into at last three types. The most commonly encountered contain granules 0.2–0.3 in diameter. They are selectively stained by aldehyde-fuchsin after permanganate oxidation. The granules are negative to tests for protein and amino acids. They are PAS-positive and become intensely metachromatic and stainable by alcian blue at pH 2.9 after sulfation. These and several other observations point to the presence of neutral and/or acid mucopolysaccharides. Glycogen is not present. Another type of basophilic cells contains RNA in great amounts. The basophilic granules appear homogeneous on the electron micrographs.The rhabdites are up to 5 long and 1 wide. They are negative to tests for lipid, nucleic acid and polysaccharides. They are intensely eosinophilic even at pH 11.8. They are probably composed of proteins, but only one amino acid was found reacting to a noteworthy degree: cysteine. It was not possible to elucidate the basis for the strong eosinophilia. The presence of ionic calcium was ruled out. The rhabdites are very electron-dense and on the electron micrographs appear homogeneous and not invested by a membrane.