Peptide Transporter in the Rat Small Intestine: Ultrastructural Localization and the Effect of Starvation and Administration of Amino Acids

Peptide transporter-1 is a H+/peptide cotransporter responsible for the uptake of small peptides and peptide-like drugs, and is present in the absorptive epithelial cells of the villi in the small intestine (duodenum, jejunum, and ileum). It has been localized to the apical microvillous plasma membrane of the absorptive epithelial cells of the rat small intestine using the immunogold electron microscopic technique. Digital image analysis of the jejunum revealed that the transporter protein was abundant at the tip of the villus and that the amount decreased from the tip of the villus to its base. The effect of dietary administration of amino acids and starvation on the expression of PepT1 in the jejunum was examined by immunoblotting and image analysis of immunofluorescence. Starvation markedly increased the amount of peptide transporter present, whereas dietary administration of amino acids reduced it. The gradient of the transporter protein along the crypt-villus axis was maintained under either condition. These observations show that it is specific to the microvillous plasma membrane and that its expression is regulated by the nutritional condition.     

Immunofluorescent localization of the gastrin-secreting G cells in the pyloric antrum of the pig

Summary An indirect immunofluorescence technique, using anti-human gastrin serum, was applied to formalin-fixed, paraffin-embedded material from antral tissues of porcine stomach. After photographing the result, post-fixation in formalin was followed by the Grimelius silver technique. The argyrophil G cells, situated predominantly in the middle zone of the gastric glands, all showed positive gastrin immunofluorescence. Some weakly fluorescent cells failed to stain by the silver technique, suggesting that the latter is less sensitive than immunofluorescence in that it depends on the presence of the granular storage product.     

Mitosis in the yeast phase of the basidiomycetes Bensingtonia yuccicola and Stilbum vulgare and its phylogenetic implications

Phylogenetic studies of yeasts rely on an extensive molecular and biochemical data set, but structural characters are scarce. Details of mitosis in yeasts have been studied with transmission electron microscopy and immunofluorescence. Of these two methods immunofluorescence is faster and easier and yields sufficient detail for cytological comparisons. Only three basidiomycetous yeasts have been studied thus far with immunofluorescence. Mitosis in budding cells of ascomycetous yeasts occurs in the parent, while in basidiomycetous yeasts, except in Agaricostilbum pulcherrimum, it occurs in the bud. Mitosis in additional yeasts in the Agaricostilbomycetidae of the Urediniomycetes was observed using immunofluorescence localization of freeze-substituted material. In Stilbum vulgare, mitosis occurred in the parent, but in Bensingtonia yuccicola it occurred in the bud as in most other basidiomycetous yeasts. Stilbum vulgare also had predominantly binucleate yeast cells. Nuclear small subunit rDNA sequence data showed that A. pulcherrimum and S. vulgare are more closely related to each other than to B. yuccicola within the Agaricostilbomycetidae. Based on the few taxa examined, mitotic and cytoskeletal characters provide phylogenetic information.     

Localization of L-glutamate decarboxylase immunoreactivity in the major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex of the rat

The localization of L-glutamate decarboxylase (GAD), the GABA-synthesizing enzyme, was studied in the rat major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex by indirect immunofluorescence technique with a specific antiserum raised in rabbits. GAD immunoreactivity was demonstrated in small cells of these ganglia. The GAD-immunoreactive small cells were 10-20 microns in diameter and formed clusters or occurred as solitary cells. The principal neurons were non-reactive but they were surrounded by immunoreactive processes. Studies on colocalization of GAD with tyrosine hydroxylase (TH), the rate-limiting enzyme of the catecholamine synthesis, in the major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex indicated that all GAD-immunoreactive small cells were also labelled with TH. In the major pelvic ganglion all TH-immunoreactive SIF cells were also immunoreactive for GAD. However, in the coeliac-superior mesenteric ganglion complex there occurred TH-immunoreactive small cells which showed no immunoreactivity to GAD. It is suggested that the small GAD-immunoreactive cells represent small intensely fluorescent (SIF) cells.     

Focal contacts of normal and RSV-transformed quail cells : Hypothesis of the transformation-induced deficient maturation of focal contacts

Morphology and distribution of cell-substrate contacts and their association with microfilament bundles in normal and RSV-transformed quail fibroblasts (16Q line) were studied by indirect immunofluorescence. The focal contacts were visualized by antibody exclusion method using monoclonal antibody to 80 kD serum protein adsorbed on the substratum. Embryo quail cells formed focal contacts of two morphological types: (1) small punctate; and (2) elongated large contacts. These two variants of contacts were designated respectively as dot and dash contacts. Both of focal contacts contained vinculin and alpha-actinin. Double immunofluorescence staining with polyclonal antibody to actin and monoclonal antibody to vinculin revealed that the dot contacts, in contrast to the dash ones, were not associated with microfilament bundles. The dot contacts were localized mostly near the active cell edges, while the dash contacts were found near the retracted cell edges and also under the central parts of the cell. We suppose that dot contacts represent initial structures which then can undergo maturation transforming them into dash contacts. RSV-transformed 16Q cells had predominantly the dot contacts which were not only located at the edges but also in the more central parts of the lamella. The dash contacts were present only in the minority of 16Q cells. RSV transformation is assumed to affect not the ability of cells to form initial dot contacts, but the maturation of dot contacts into dash contacts.     

Transduction of satellite cells after prenatal intramuscular administration of lentiviral vectors

BACKGROUND: We have previously reported long-term expression of lacZ in myocytes after in utero intramuscular injection of Mokola and Ebola pseudotyped lentiviral vectors. In further experiments, we have noted that these vectors also transduce small cells at the periphery of the muscle fibers that have the morphology of satellite cells, or muscle stem cells. In this study we performed experiments to further define the morphology and function of these cells. METHODS: Balb/c mice at 14-15 days gestation were injected intramuscularly with Ebola or Mokola pseudotyped lentiviral vectors carrying CMV-lacZ. Animals were harvested at various time points, muscles were stained with X-gal, and processed for electron microscopy (EM) and immunofluorescence. To determine whether transduced satellite cells were functionally capable of regenerating injured muscles, animals were injected with notexin in the same area 8 weeks after the in utero injection of viral vector. RESULTS: Transmission EM of transduced cells confirmed the ultrastructural appearance of satellite cells. Double immunofluorescence for beta-galactosidase and satellite cell markers demonstrated co-localization of these markers in transduced cells. In the notexin-injured animals, small blue cells were seen at the areas of regeneration that co-localized beta-galactosidase with markers of regenerating satellite cells. Central nucleated blue fibers were seen at late time points, indicating regenerated muscle fibers arising from a transduced satellite cell. CONCLUSIONS: This study demonstrates transduction of muscle satellite cells following prenatal viral vector mediated gene transfer. These findings may have important implications for gene therapy strategies directed toward muscular dystrophy.     

UV micro-irradiation of the Chinese hamster cell nucleus and caffeine post-treatment immunocytochemical localization of DNA photolesions in cells with partial and generalized chromosome shattering

UV micro-irradiation of a small part of the Chinese hamster nucleus and caffeine post-incubation often results in shattered chromosomes at the first post-irradiation mitosis. In some of these mitotic cells, chromosome shattering is restricted to a few chromosomes spatially related in a small area of the metaphase spread; in others, shattering includes the whole chromosome complement. These 2 types of damage have been called partial and generalized chromosome shattering (PCS and GCS).Using antisera that specifically react with UV-irradiated DNA, we identified micro-irradiated chromatin in interphase nuclei and in mitotic cells with PCS or GCS by indirect immunofluorescence microscopy. In PCS, immunofluorescence staining was found in the damaged area, while the surrounding intact chromosomes were not stained. In GCS, staining was also restricted to a small region of the shattered chromosome complement. In other experiments, cells synchronized in G1 were micro-irradiated in the nucleus, pulse-labelled with [³H]thymidine and post-incubated with caffeine. Autoradiographs of cells with GCS showed unscheduled DNA synthesis restricted to a small chromatin region.Our data present direct evidence that the distribution of DNA photolesions does not coincide with the sites of chromosomal damage in GCS. As a working hypothesis, we propose that an indirect mechanism is involved in the induction of GCS by which DNA photolesions in a small nuclear segment induce shattering of both micro-irradiated and non-irradiated chromosomes.     

Distribution of the microtubule-dependent motors cytoplasmic dynein and kinesin in rat testis.

To examine the possible role of microtubule-based transport in testicular function, we used immunofluorescent techniques to study the presence and localization of the microtubule mechanoenzymes cytoplasmic dynein (a slow-growing end-directed motor) and kinesin (a fast-growing end-directed motor) within rat testis. Cytoplasmic dynein immunofluorescence was observed in Sertoli cells during all stages of spermatogenesis, with a peak in apical cytoplasm during stages IX-XIV. Cytoplasmic dynein immunofluorescence was also localized within Sertoli cells to steps 9-14 (stages IX-XIV) germ cell-associated ectoplasmic specializations. In germ cells, cytoplasmic dynein immunofluorescence was observed in manchettes of steps 15-17 (stages I-IV) spermatids, and small, hollow circular structures were seen in the cytoplasm of step 17 and step 18 spermatids during stages V and VI. Kinesin immunofluorescence was observed in manchettes of steps 10-18 spermatids (stages X-VI). The stage-dependent apical Sertoli cell cytoplasmic dynein immunofluorescence, in conjunction with the previously reported orientation of Sertoli cell microtubules (slow-growing ends toward the lumen) and peak secretion of androgen-binding protein and transferrin, is consistent with the hypothesis that cytoplasmic dynein is involved in Sertoli cell protein transport and secretion. Further, the localization of cytoplasmic dynein and kinesin to manchettes is consistent with current hypotheses concerning manchette function.     

Localization of l-glutamate decarboxylase immunoreactivity in the major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex of the rat

Summary The localization of l-glutamate decarboxylase (GAD), the GABA-synthesizing enzyme, was studied in the rat major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex by indirect immunofluorescence technique with a specific antiserum raised in rabbits. GAD immunoreactivity was demonstrated in small cells of these ganglia. The GAD-immunoreactive small cells were 10–20 m in diameter and formed clusters or occured as solitary cells. The principal neurons were non-reactive but they were surrounded by immunoreactive processes. Studies on colocalization of GAD with tyrosine hydroxylase (TH), the rate-limiting enzyme of the catecholamine synthesis, in the major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex indicated that all GAD-immunoreactive small cells were also labelled with TH. In the major pelvic ganglion all TH-immunoreactive SIF cells were also immunoreactive for GAD. However, in the coeliac-superior mesenteric ganglion complex there occured TH-immunoreactive small cells which showed no immunoreactivity to GAD. It is suggested that the small GAD-immunoreactive cells represent small intensely fluorescent (SIF) cells.     

Expression and localization of Muc4/sialomucin complex (SMC) in the adult and developing rat intestine: implications for Muc4/SMC function

Muc4/sialomucin complex (SMC) is a high molecular mass heterodimeric membrane mucin, encoded by a single gene, and originally discovered in a highly metastatic ascites rat mammary adenocarcinoma. Subsequent studies have shown that it is a prominent component of many accessible and vulnerable epithelia, including the gastrointestinal tract. Immunoblot and immunofluorescence analyses demonstrated that Muc4/SMC expression in the rat small intestine increases from proximal to distal regions and is located predominantly in cells at the base of the crypts. These cells were postulated to be Paneth cells, based on their location, morphology, and secretory granule content. Immunohistochemistry indicated the presence of Muc4/SMC in these granules. Muc4/SMC expression was higher in the rat colon than small intestine and was abundantly present in colonic goblet cells, but not in goblet cells in the small intestine. Immunohistochemistry also suggested the presence of MUC4 in human colonic goblet cells. Biochemical analyses indicated that rat colonic Muc4/SMC is primarily the soluble form of the membrane mucin. Analyses of Muc4/SMC during development of the rat gastrointestinal tract showed its appearance at embryonic day 14 of the esophagus and at day 15 at the surface of the undifferentiated stratified epithelium at the gastroduodenal junction, then later at cell surfaces in the more distal regions of the differentiated epithelium of the small intestine, culminating in expression as an intracellular form in the crypts of the small intestine at about day 21. Limited expression in the colon was observed during development before birth at cell surfaces, with expression as an intracellular form in the goblet cells arising during the second week after birth. These results suggest that membrane mucin Muc4/SMC serves different functions during development of the intestine in the rat, but is primarily a secreted product in the adult animal.     

Immunological detection of an analogue of the erythroid protein 4.1 in endothelial cells

Endothelial cells (EC) of arterial and venous origin were investigated by indirect immunofluorescence and immunoautoradiography for the presence of red cell membrane 4.1-like protein. By immunofluorescence, EC exhibited a relatively uniform fluorescent staining sometimes of a reticular pattern, distributed over the entire cell. All controls were negative. Immunoblot analysis of EC revealed a cross reactive band of a molecular weight comparable to that of the erythrocyte band 4.1. These findings indicate that endothelial cells of arterial and venous origin express a polypeptide immunologically related to the erythrocyte protein 4.1, which may play an important role in membrane-cytoskeleton interactions.     

Quantitative and qualitative immunofluorescence studies of neoplastic cells transfected with a construct encoding p53-EGFP.

The p53 protein is a major regulator of cell cycle progression and apoptosis. We used a p53-enhanced green fluorescent protein (EGFP) construct for transfections into human breast cancer (MCF-7) cells. Cells expressing p53-EGFP showed an increased apoptotic index compared to cells transfected with EGFP alone. Interestingly, apoptotic cells showed localization of p53-EGFP to both nuclei and cytoplasm, whereas non-apoptotic cells usually only showed nuclear localization of p53-EGFP. This result is in agreement with the hypothesis that p53 induces apoptosis by interaction with both nuclear and cytoplasmic targets. Transfected p53-deficient osteosarcoma cells were used for immunofluorescence quantitation. The intensity of immunofluorescence for either p53 or EGFP showed excellent linear correlation to the EGFP autofluorescence, proving that measurements of immunofluorescence intensities can be used for determining endogenous protein levels.     

Ultrastructural and immunofluorescent investigations on the secretin cell in the dog intestinal mucosa

Summary Besides 5-hydroxytryptamine-storing EC cells, four types of endocrine non-EC cells have been identified in the dog intestinal mucosa: the S and D cells, mainly concentrated in the upper tracts of the small intestine, the L cell, concentrated in the lower tracts, and the I cell showing intermediate patterns between S and L cells. In immunofluorescence tests epithelial endocrine-like cells reacting to antisecretin sera were found in the duodenal mucosa. Only the S cell seems to exactly reproduce the distribution of secretin activity in different portions of gastrointestinal mucosa.     

Studies on the development and maintenance of epithelial cell surface polarity with monoclonal antibodies

We examined epithelial cell surface polarity in subconfluent and confluent Madin-Darby canine kidney (MDCK) cells with monoclonal antibodies directed against plasma membrane glycoproteins of 35,000, 50,000, and 60,000 mol wt. The cell surface distribution of these glycoproteins was studied by immunofluorescence and immunoelectron microscopy. At the ultrastructural level, the electron-dense reaction product localizing all three glycoproteins was determined to be uniformly distributed over the apical and basal cell surfaces of subconfluent MDCK cells as well as on the lateral surfaces between contacted cells; however, after formation of a confluent monolayer, these glycoproteins could only be localized on the basal-lateral plasma membrane. The development of cell surface polarity was followed by assessing glycoprotein distribution with immunofluorescence microscopy at selected time intervals during growth of MDCK cells to form a confluent monolayer. These results were correlated with transepithelial electrical resistance measurements of tight junction permeability and it was determined by immunofluorescence that polarized distributions of cell surface glycoproteins were established just after electrical resistance could be detected, but before the development of maximal resistance. Our observations provide evidence that intact tight junctions are required for the establishment of the apical and basal- lateral plasma membrane domains and that development of epithelial cell surface polarity is a continuous process.     

Investigations on the lectin-producing cells in the sponge Axinella polypoides (Schmidt)

Summary The localization of two carbohydrate binding proteins, so-called lectins, was studied in the sponge tissue of Axinella polypoides by light and immunofluorescence microscopy. They do not occur at the cellular surface of any cell type, but they are stored in vesicles of the spherulous cells. After short formaldehyde fixation spherulous cells can be isolated and they release the active lectins upon lysis in distilled water.Electron microscopical studies of spherulous cells show that they contain almost nothing else but a small nucleus and vesicles of different size and number. Small vesicles are full of an electron dense material, whereas the content of large vesicles has a fluffy and fibrillar structure. Spherulous cells are large and tightly packed in the outer layer of the ectosome and in the mesh work of the spongin fibres of the central axis. They are small and scattered in the inner layer of the ectosome, and they are found throughout the choanosome. The function of the lectins is not clearly defined, and different alternatives such as participation in glycoprotein synthesis, immunological defense, or carbohydrate transport are possible.This study was supported by a grant from the Deutsche ForschungsgemeinschaftWe are gratefully indebted to Dr. D. Keyser for his help in our electron microscopical studies     

Two distinct regulatory steps in cartilage differentiation

The effect of developmental stage on chondrogenic capacity in high-density cell cultures of chick embryonic wing bud mesenchyme is examined. Mesenchyme from stage 19 embryos forms aggregates of closely associated cells which do not form cartilage matrix, nor contain significant levels of type II collagen that are detectable by immunofluorescence, unless they are treated with dibutyryl cyclic AMP. Mesenchyme from stage 24 embryonic wing buds in high-density cell cultures will spontaneously form cartilage, as defined by electron microscopy and immunofluorescence with antibody to type II collagen. Cultures prepared from stage 26 wings form numerous aggregates which fail to accumulate an Alcian blue-staining matrix and which resemble mesenchyme cells morphologically. However, because these cells show considerable intracellular immunofluorescence for type II collagen, they are actually unexpressed cartilage cells. Several treatments, including exposure to dibutyryl cyclic AMP, ascorbic acid and an atmosphere of 5% oxygen, or mixture with small numbers of stage 24 wing mesenchyme cells, stimulate expression, as determined by the accumulation of an Alcian blue-staining matrix and an ultrastructurally recognizable cartilage matrix. Since the addition of similar numbers of differentiated cartilage cells does not stimulate expression of stage 26 cells, it is proposed that initial cartilage expression is dependent on a mesenchyme-specific influence which might be removed by cell dissociation. These studies demonstrate that there are at least two distinct transitions in cartilage differentiation: one involves the conversion of mesenchyme to unexpressed chondrocytes and the second involves mesenchyme-dependent expression of chondrogenic differentiation.     

Immunoflurorescence demonstration of cells reacting with an antipancreatic polypeptide antibody in the intestine of the monkey Macacus irus

Indirect immunofluorescence technique using an antibody against bovine pancreatic polypeptide has allowed us to detect immunoreactive cells in the small intestine of the monkey Macaca irus. These cells are located almost exclusively in the wall of Lieberkuhn's glands, especially in the jejunum and, in a smaller number, in the duodenum; these cells are scattered, always isolated, never in clusters; immunoreactive cells were never seen in the colon. There were no sex-linked differences in the morphology as well as in the distribution of these immunoreactive cells.     

Subcellular distribution of secretogranins I and II in GH3 rat tumoral prolactin (PRL) cells as revealed by electron microscopic immunocytochemistry

The GH3 rat pituitary cell line which secretes prolactin (PRL) is characterized by the paucity and small size of secretory granules. We looked for the presence, in these cells and in normal PRL cells, of two acidic tyrosine-sulfated proteins which are widely distributed in dense-core secretory granules of endocrine and neuronal cells, secretogranins I and II, using immunofluorescence and electron microscope immunoperoxidase techniques. Both secretogranins were detected in secretory granules of GH3 cells and of normal cells. Moreover, with our pre-embedding approach, secretogranins were localized within some RER cisternae and within all sacules of the Golgi stacks in both PRL cell models. A few small vesicles, large dilated vacuolar or multivesicular structures, and some lysosome-like structures were also immunoreactive. Double localization of secretogranins and PRL performed on GH3 cells by immunofluorescence indicated that all cells contained secretogranins I and II, whereas only 50-70% of the cells contained PRL. Moreover, in the case of hormone treatment known to increase the number of secretory granules, most if not all mature secretory granules were immunoreactive for secretogranins, whereas in certain cells some of the granules were apparently not immunoreactive for PRL. These immunocytochemical observations show that GH3 cells, which under normal conditions form only a small number of secretory granules, produce secretogranins and package them into these granules.     

Identification by immunofluorescence of prolactin-and somatotropin-producing cells in the pituitary gland of the Mexican axolotl (Ambystoma mexicanum,Shaw)

Summary The indirect immunofluorescence procedure was used to identify prolactin (LTH)-and somatotropin (STH)-producing cells in the pituitary of the Mexican axolotl. Histological staining techniques were employed to corroborate immunocytological results. The LTH cells are large, orange-staining cells (acidophils 1) distributed in the posterior two-thirds of the pars distalis. The STH cells are small, erythrosinophilic elements (acidophils 2) principally concentrated in the dorsal part of the pars distalis.     

Age-dependent increase of extrathymic T cells in the liver and their appearance in the periphery of older mice.

The liver is a major site of generation of extrathymic T cells with unique properties (e.g., expressing intermediate TCR and containing self-reactive clones). We investigated herein whether the levels of extrathymic alpha beta T cells varied in various organs as a function of age. A systematic examination of the number of mononuclear cells in various organs of BALB/c mice revealed that the number of hepatic MNC increased with age whereas the number of thymocytes decreased. These changes were more striking in mice fed under conventional conditions than under specific pathogen-free condition. The age-dependent changes in the number of mononuclear cells in the spleen and lymph nodes were minimal. Although the total proportion of alpha beta T cells in each organ remained constant, the staining patterns of TCR-alpha beta as shown by immunofluorescence profiles varied. The most prominent change was that intermediate TCR-alpha beta cells, which constituted a small population in the liver of young mice, expanded in the liver of older mice. Intermediate TCR cells appeared even in the periphery of older mice. These findings were confirmed by the appearance of extrathymic T cells with other unique properties, e.g., double-negative CD4-8- phenotype and CD44 expression. In athymic nude mice, only intermediate TCR cells were present in the liver and periphery. An age-dependent increase of intermediate TCR cells was also seen in these mice. Taken together with the result of bromodeoxyuridine-injection experiment, which showed an intensive in vivo proliferation of cells in the hepatic sinusoids, extrathymic T cells may differentiate predominantly in the liver and appeared even to the periphery in older mice.