Immunoelectron microscopic localization of partially purified antigens in adult Paragonimus iloktsuenesis.

An immunoelectron microscopy employing immunogold labeling method was performed to detect tissue origin of D1 fraction (D1A) among 5 antigenic protein fractions partially purified by DEAE-anion exchange chromatography from water-soluble crude antigen (PIWA) of adult Paragonimus iloktsuenensis. Immune reactions of adult worm tissues with rabbit serum immunoglobulin immunized with crude antigen (PI-Ig) and D1 antigen (D1-Ig), as well as rat serum immunoglobulin infected with P. iloktsuenensis were observed. D1A showed strong antigenicity in the intestinal epithelium of the worms during the early infection period of 2-4 weeks after infection. The vitellaria also showed stronger antigenicity than the other tissue sites in immune reaction of tissues against all immunoglobulins from 4 to 33 weeks after vitelline development. Therefore, it is suggested that D1A was mainly originated from the intestinal epithelial tissues before the development of vitelline gland of the parasites. Immuno-reactivity of two immunoglobulins (PI-Ig, D1-Ig) was significantly different in intestinal epithelial cytoplasmic protrusions (CP) and intestinal epithelial secretory granules (SG). In the experimental group with D1-Ig, gold particles were labeled significantly in CP than in SG when compared to the PI-Ig group. Thus, the major antigenic materials in D1 antigen having a strong antigenicity in the early infection period was considered to be originated from the intestinal epithelial tissue.     

Differential Silver Enhanced Double Labeling in Immunoelectron Microscopy

A two-sided double labeling method using protein A gold was used to demonstrate the presence of two hormones within the same anterior pituitary cell granule. A single probe size was used for both section faces but one side of the grid was silver enhanced. The use of a single probe size reduced the cost of the study and eliminated the variations in labeling efficiency that result from the use of different probe sizes.     

The Determination of Comparable Labeling Densities in Quantitative Immunoelectron Microscopic Double Labeling Studies

In quantitative ultrastructural studies using colloidal gold immunocytochemical techniques, labeling intensities vary according to the size of the probe used. Using postembedded indirect two-sided double labeling and single labeling protocols, the labeling characteristics of four antigens were studied using two probe sizes commonly used in double labeling studies. It was determined that the labeling intensity variation resulting from the use of different probe sizes was unpredictable after correcting for the increased probe size alone. It was possible, however, to obtain comparable labeling densities by first determining the labeling intensities for each probe size with its antigen in single label studies on serial sections and using the same procedure as the double labeling studies. A probe size correction factor for each antigen was calculated from these data. This factor was used to obtain comparable measurements of the relative abundance of each label.     

Immunoelectron microscopic study of polyamines in the gastrointestinal tract of rat

Polyamines (PAs) are ubiquitous polycationic metabolites in the eukaryotic and prokaryotic cells and are believed to be intimately involved in the regulation of DNA, RNA, and protein biosynthesis. However, the subcellular localization of PAs has not yet been fully elucidated in a variety of cell types. In the present study, a pre-embedding indirect immunoperoxidase approach was used to define the fine structural localization of PAs in the gastrointestinal tract of rat, which was fixed with glutaraldehyde and the monoclonal antibody ASPM-29 specific for spermine (Spm) and spermidine (Spd). Examination by a transmission electron microscopy showed that the peroxidase end products were commonly and predominantly localized in the free and attached ribosomes of the rough endoplasmic reticulum (rER) in the active protein- or peptide-secreting cells, and in rapidly proliferating cells including the gastric chief cells, mucous neck cells, and intestinal crypt cells. The nuclei, mitochondria, and secretory vesicles were devoid of PAs. Of note is the new finding that PAs are also located even on the small number of ribosomes in the cytoplasm of the parietal cells and of the villus-tip cells, because these were the cell types that were found to be almost PA-negative at the light microscopic level. These results seem to be completely consistent with those recently obtained for rat neurons. Thus, the present study generalized the subcellular localization of PAs on the ribosomes, and demonstrated that PAs are one of the components of biologically active ribosomes, possibly in any type of cell, that are closely involved in the translation processes of protein biosynthesis.     

Use of immunocytochemical staining of somatostatin for correlative light and electron microscopic investigation of D cells in the pancreatic islet of Xiphophorus helleri H. (Teleostei)

Summary Somatostatin-containing cells have been demonstrated by immunocytochemistry in semithin sections of the pancreatic islet of the teleost fish, Xiphophorus helleri. These cells were shown by correlative light and electron microscopy to be identical with D cells previously defined in this species by the silver impregnation method of Hellman and Hellerström.Supported in part by grants from the British Council and from the Medical Research Council of Great Britain     

Characterization of somatostatin receptors and associated signaling pathways in pancreas of R6/2 transgenic mice

The present study describes the status of somatostatin receptors (SSTRs) and their colocalization with insulin (β), glucagon (α) and somatostatin (δ) producing cells in the pancreatic islets of 11 weeks old R6/2 Huntington's Disease transgenic (HD tg) and age-matched wild type (wt) mice. We also determined expression of tyrosine hydroxylase (TH), glutamic acid decarboxylase (GAD) and presynaptic marker synaptophysin (SYP) in addition to signal transduction pathways associated with diabetes. In R6/2 mice, islets are relatively smaller in size, exhibit enhanced expression and nuclear inclusion of mHtt along with the loss of insulin, glucagon and somatostatin expression. In comparison to wt, R6/2 mice display enhanced mRNA for all SSTRs except SSTR2. In the pancreatic lysate, SSTR1, 4 and 5 immunoreactivity decreases whereas SSTR3 immunoreactivity increases with no discernible changes in SSTR2 immunoreactivity. Furthermore, at the cellular level, R6/2 mice exhibit a receptor specific distributional pattern of SSTRs like immunoreactivity and colocalization with β, α and δ cells. While GAD expression is increased, TH and SYP immunoreactivity was decreased in R6/2 mice, anticipating a cross-talk between the CNS and pancreas in diabetes pathophysiology. We also dissected out the changes in signaling pathway and found decreased activation and expression of PKA, AKT, ERK1/2 and STAT3 in R6/2 mice pancreas. These findings suggest that the impaired organization of SSTRs within islets may lead to perturbed hormonal regulation and signaling. These interconnected complex events might shed new light on the pathogenesis of diabetes in neurodegenerative diseases and the role of SSTRs in potential therapeutic intervention.     

Immunoelectron microscopic study of the distribution of porin on outer membranes of rat heart mitochondria

The distribution of porin on the outer membranes of rat heart mitochondria has been studied by means of immunogold labelling with antibodies to the N-terminal part of the human protein. It was found that only a minority of isolated, unfixed mitochondria are labelled by these antibodies, with the gold particles frequently organized in threads or bands. Extensive immunogold labelling is frequently observed on regions of outer membranes stripped away from mitochondria and on regions separating two mitochondrial compartments whose cristae display different configurations (possibly representing two mitoplasts covered by a common outer membrane). Also, pairs of connected mitochondria are sometimes heavily labelled in the neck regions, which may represent the junctions involved in electrical communication between mitochondria in cardiac tissue.     

Development of immunoreactive somatostatin in C-cell complexes in the thyroid gland of the dog

Summary In connection with our previous finding that an intense immunoreaction to somatostatin transiently appears in thyroid C cells of the dog during early fetal periods, the present study investigated C-cell complexes in thyroid glands from early fetuses to adults in an attempt to clarify whether the transient appearance of immunoreactivity to somatostatin is dependent on the degree of differentiation of C cells. C-cell complexes retain their fetal characteristics; even in the complexes of postnatal dogs, there are numerous undifferentiated cells, immature C cells and primitive follicular cells, which are not yet organized into follicles. Neither the degree of differentiation of C cells nor that of other constituent elements of the complexes affected the developmental pattern of somatostatin immunoreactivity in C cells. The C cells located in complexes displayed the same pattern of developmental changes in immunoreactivity to somatostatin as the cells in thyroid parenchyma. In the C-cell complexes of early fetal dogs a very intense immunoreactivity for somatostatin was observed; almost all calcitonin-positive cells were also somatostatin positive. The immunoreactivity to somatostatin progressively decreased with age. In the postnatal complexes the number of somatostatin-positive cells was very small compared with that of calcitoninpositive cells.     

Ubiquitin signals in the developing acrosome during spermatogenesis of rat testis: an immunoelectron microscopic study.

The localization of ubiquitin (UB) signals in the acrosomes of rat spermiogenic cells was investigated by immunoelectron microscopy using two anti-UB antibodies: UB1, reacting with ubiquitinated proteins and free UB; and FK1, recognizing polyubiquitinated proteins but not monoubiquitinated proteins or free UB. Labeling of UB by UB1 (UB1 signal) was detected in the acrosomes at any stage of differentiation. In step 1 spermatids, UB1 signals were detected on the cytoplasmic surface and in the matrix of transport vesicles located between the trans-Golgi network and the acrosome. Weak signals were detected in acrosomal granules within acrosome vesicles that had not yet attached to the nucleus. In step 4-5 spermatids, the acrosome vesicles had enlarged and attached to the nucleus. Strong gold labeling was noted in a narrow space between the outer acrosomal membrane and the developing acrosomal granule, where a dense fibrous material was observed on routine electron microscopy, whereas the acrosomal granule was weakly stained by UB1 antibody. In step 6-8 spermatids, UB1 signals were detected in the fibrous material that expanded laterally to form a narrow electronless dense zone between the acrosomal granule and the outer acrosomal membrane. Labeling in the acrosomal granule increased. In step 9-11 spermatids, UB1 signals were confined to the narrow zone from the tip of the head to the periphery of the ventral fin. The matrix of the acrosome was weakly stained. In epididymal sperm, UB1 labeling in the acrosome decreased without any pretreatment, whereas staining was noted in a spot in the neck region and in the dorsal fin after trypsin digestion. On the other hand, the staining pattern with FK1 was quite different from that with UB1. The trans-Golgi network was weakly stained but the cis-Golgi network was strongly stained. The dense fibrous material just beneath the outer membrane was never stained with FK1. The results suggest that UB on the surface of transport vesicles is involved in anterograde transport from the Golgi apparatus to the acrosome. The physiological role of UB in acrosomes is not clear. Two candidates for monoubiquitinated proteins in the acrosome, which have a UB-interacting motif, were found by cyber screening.     

A,B, D cells and A fourth cell type in longterm cultures of fetal rat pancreas

Summary Whole pancreases from fetal rats of 13 days and 18 days gestation were explanted onto rayon grids and grown in organ culture. Cultures were fixed in Bouin’s fluid, sectioned and stained with the fluorescent antibody techniques for glucagon and insulin, aldehyde fuchsin for B cells, pseudoisocyanin for D cells and a silver technique for the fourth cell type. The 13-day explants were fixed after 10 days in culture. A, B and D and the fourth cell type were seen, indicating that precursors of all four endocrine cell types must be present in the fetal pancreas shortly after the formation of the pancreatic bud (11 days). Further, the presence of these four cell types in the walls of tubules in these cultures indicates the tubules as the site of origin of all the endocrine tissue. The 18-day explants were collected every other day of culture from 2 to 30 days in a long-term experiment. A number of large islets with well granulated B cells was still present after 30 days of culture. The relative abundance of cell types at different stages was estimated as follows: 18-day fetal controls, A>B=4>D; after 2 to 10 days in culture, B>A⩾4>D; after 18 to 30 days in culture, B>D>A>4.     

Suppression of ANP secretion by somatostatin through somatostatin receptor type 2

Somatostatin is a cyclic-14 amino acid peptide which mainly distributed in digestive system and brain. Somatostatin receptor (SSTR) is a G-protein coupled receptor and all five SSTR subtypes are expressed in cardiomyocytes. The aim of this study was to investigate the effect of somatostatin on atrial natriuretic peptide (ANP) secretion and its signaling pathway. Somatostatin (0.01 and 0.1 nM) decreased ANP secretion in isolated beating rat atrium in a dose-dependent manner. But atrial contractility and translocation of extracellular fluid were not changed. Somatostatin-induced decrease in ANP secretion was significantly attenuated by the pretreatment with CYN 154806 (SSTR type 2 antagonist; 0.1 μM), but not by BIM 23056 (SSTR type 5 antagonist; 0.1 μM) and urantide (urotensin II receptor antagonist; 0.1 μM). When pretreated with an agonist for SSTR type 2 (Seglitide, 0.1 nM) and SSTR type 5 (L 817818, 0.1 nM), only Seglitide reduced ANP secretion similar to that of somatostatin. The suppressive effect of somatostatin on ANP secretion was attenuated by the pretreatment with an inhibitor for adenylyl cyclase (MDL-12330A, 5 μM) or protein kinase A (KT 5720, 0.1 μM). In diabetic rat atria, the suppressive effect of somatostatin on ANP secretion and concentration was attenuated. Real time-PCR and western blot shows the decreased level of SSTR type 2 mRNA and protein in diabetic rat atria. These data suggest that somatostatin decreased ANP secretion through SSTR type 2 and an attenuation of suppressive effect of somatostatin on ANP secretion in diabetic rat atria is due to a down-regulation of SSTR type 2.     

Distribution,ontogeny and ultrastructure of somatostatin immunoreactive cells in the pancreas and gut

Summary Somatostatin cells are numerous in the pancreas and digestive tract of mammals as well as birds. In the pancreas of chicken, cat and dog they occur in both the exocrine parenchyma and in the islets. In the rat and rabbit, somatostatin cells have a peripheral location in the islets, whereas in the cat, dog and man the cells are usually more randomly distributed. In the stomach of rabbits and pigs, somatostatin cells are more numerous in the oxyntic gland area than in the pyloric gland area, whereas the reverse is true for the cat, dog and man. In the cat, pig and man, somatostatin cells are fairly numerous in the duodenum, whereas in the rat, rabbit and dog they are few in this location. In the remainder of the intestines somatostatin cells are few but regularly observed. Somatostatin cells are numerous in the human fetal pancreas and gut. In the fetal rat, somatostatin cells first appear in the pancreas and duodenum (at about the 16–17th day of gestation) and subsequently in the remainder of the intestine. Somatostatin cells do not appear in the gastric mucosa until after birth. Three weeks after birth, somatostatin cells show the adult frequency of occurrence and pattern of distribution. In the chicken, somatostatin cells are numerous in the proventriculus, absent from the gizzard, abundant in the gizzard-duodenal junction (antrum), infrequent in the duodenum and virtually absent from the remainder of the intestines. No immunoreactive cells can be observed in the thyroid of any species nor in the ultimobranchial gland of the chicken. In the chick embryo, somatostatin cells are first detected in the pancreas and proventriculus (at about the 12th day of incubation). They appear in the remainder of the gut much later, in the duodenum at the 16th day, in the antrum at about the 19th day and still later in the lower small intestine. The ultrastructure of the somatostatin cells was studied in the chicken, rat, cat and man; the cells were identified by the consecutive semithin/ultrathin section technique. The somatostatin cells display the properties of the D cell. There was no difference in granule ultrastructure between somatostatin cells in the gut and the pancreas. The granules, which are the storage site of the peptide, are round, supplied with a tightly fitting membrane and have a moderately electron-dense, fine-granulated core. The mean diameter of the somatostatin granules is smallest in rat (155–170 nm) and largest in the chicken (270–290 nm).     

Distribution of somatostatin receptor 5 in mouse and bullfrog retinas

Somatostatin (SRIF), as a neuroactive peptide in the CNS, may act as a neuromodulator through activation of five specific receptor subtypes (sst(1)-sst(5)). In this work we conducted a comparative study of the expression of sst(5) in mouse and bullfrog retinas by immunofluorescence double labeling. Basically, the expression profiles of sst(5) in the retinas of the two species were similar. That is, in the inner retina sst(5) was localized to dopaminergic and cholinergic amacrine cells, stained by tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) respectively, and cells in the ganglion cell layer, whereas in the outer retina immunostaining for sst(5) was observed in horizontal cells. However, a more widespread, abundant distribution of labeling for sst(5), as compared to mouse retina, was seen in bullfrog retina: strong labeling for sst(5) was diffusely distributed in both outer and inner plexiform layers (OPL and IPL) in the bullfrog retina, but the labeling was only observed in the IPL of the mouse retina. In addition, bullfrog photoreceptors, both rods and cones, but not mouse ones, were labeled by sst(5). In combination with the experiments showing that SRIF-immunoreactivity was mainly found in the inner retina, our results suggest that SRIF, released from SRIF-containing cells in the inner retina, may play a neuromodulatory role in both outer and inner retina mediated by volume transmission via sst(5) in bullfrog retina, while the SRIF action may be largely restricted to the mouse inner retina.     

Cryptosporidiosis of liver and pancreas in rhesus monkeys with experimental SIV infection

Following an experimental SIV infection, 11 rhesus monkeys were evaluated to determine the presence of opportunistic infections. Five animals had severe alterations of the hepatobiliary tree, three of which were associated with the presence of numerous Cryptosporidium spp. Subacute to chronic inflammatory changes were observed in the pancreatic ducts of four animals, one without histologic evidence of parasites. In one animal, the inflamed ducts were associated with a chronic interstitial pancreatitis. The rate of Cryptosporidium infection together with hepatic and pancreatic involvement (36%) supports the hypothesis that systemic cryptosporidiosis is the result of a loss of protective mucosal immunity.     

Resin Embedding for Quantitative Immunoelectron Microscopy. A Comparative Computerized Image Analysis

Quantitative immunoelectron microscopy (QIEM) is dependent on the reliability of preparative techniques for both efficient immunolabeling and consistent quantitative results among series of immunostained sections. The present study compared Lowicryl K4M and Epon embedding after identical fixation and dehydration of rat somatotrophic secretory granules. Labeling intensity, diameter, roundness, uptake of uranyl acetate, and gray value were measured with computer assisted image analysis. Lowicryl-embedded granules showed the highest labeling densities after conventional fixation and Progressively Lowering Temperature (PLT) dehydration, but values were not consistent in a series of immunostained sections. A lower but more uniform level of immunostaining was seen in Eponembedded sections when tissue was cryofixed and physically dehydrated. Gray value measurements from micrographs from both embedding media confirmed the better contrast of Epon sections and the different reliefs of the granule surfaces. This study emphasizes the importance of complete comparisons of preparative techniques for QIEM for reliability and reproducibility.     

Immunoelectron microscopic study for histamine in the gastric enterochromaffin-like cells of rats treated with the proton pump inhibitor lansoprazole

The enterochromaffin-like (ECL) cells of the gastric mucosa in animals play an important role in gastric acid secretion. They contain few granules and numerous secretory vesicles and microvesicles. They operate under the control of circulating gastrin. In the present study, we conducted an immunoelectron microscopic study for histamine (HA) in the ECL cells of rats given the proton pump inhibitor lansoprazole (LP), which is known to induce hypergastrinemia. The pre-embedding indirect immunoperoxidase procedure utilized a mouse monoclonal antibody AHA-2 against glutaraldehyde-conjugated HA. Rats received LP (50 g/kg per day, subcutaneously) over a period of a month, and developed hypertrophy of the ECL cells in the stomach. It was clearly demonstrated that HA was located to a much higher degree in the cytoplasm of ECL cells of LP-treated rats than in normal rats. HA immunoreactivity was observed in the cores of the granules and secretory vesicles of the ECL cells in all the rats, but in the LP-treated rats it was observed in the cores of the newly developed vacuoles as well. These results may suggest that HA may be actively generated in the cytoplasm of the hypertrophic ECL cells of LP-treated rats. Also suggested in the present study is that HA is instrumental in the transformation of granules into secretory vesicles and in their consequent enlargement, and that vacuoles are formed by the fusion of large secretory vesicles. Furthermore, the finding that relatively little HA immunoreactivity existed in the vacuoles may suggest that the vacuoles actively degrade superfluous secretory products (for example, HA) through enhanced autophagocytosis and/or oxidative stress. Another possibility may be that the membrane-bounded structure regarded as the vacuoles in this study might actually be an invagination structure produced as a result of successive series of exocytosis through which the secretory vesicles actively and rapidly release HA.     

Orientation of the 19S regulator relative to the 20S core proteasome: an immunoelectron microscopic study

Specific labelling with monoclonal antibodies reveals that in regulator-proteasome complexes the asymmetric 19S regulator (PA700) binds to one or both terminal alpha-disks of the cylinder-shaped 20S core proteasome in such a way that its reclining front part is positioned in the vicinity of proteasome subunit alpha6. The protruding rear part of the regulator appears to be situated distal to the sites occupied by the subunits alpha2 and alpha3, respectively. When viewed from beta1/beta1' to beta4/beta4' along the polar 2-fold axis of the 20S proteasome core, the rear part of each 19S regulator cap appears to protrude clockwise. Thus, a defined alignment of the 19S regulator with respect to the single polar 2-fold rotational axis of the 20S core proteasome is obtained.     

Light and electron microscopic study on the endocrine cells of the pancreas in a marine teleost,Fugu rubripes rubripes

Summary The pancreatic endocrine tissue of Fugu rubripes rubripes consists of numerous round principal islets (Brockmann bodies) of various sizes scattered around the gall-bladder. The endocrine cells are divided into A-, B-, D-, and F_f-cells. Each cell type was identified by comparing thick and thin sections in both light and electron microscopy. Aldehyde-fuchsin positive B-cells contain numerous round secretory granules (average diameter 300 nm) each of which has a round compact core of moderate density; a narrow space exists between this core and the limiting membrane. Grimelius' silver positive A cells contain round secretory granules (average diameter 360 nm) with a hexagonal or tetragonal crystalline core (average diameter 170 nm) of high density; the silver grains preferentially appear in the space between the limiting membrane and the core. The crystalline core of each -granule often contains an appendix-like structure of variable shape. D cells blackened by the silver impregnation method of Hellman and Hellerström (1960) have round secretory granules (average diameter 320 nm) filled with a flocculent material of low density. The fourth cell type (F_f-cell) has a clear cytoplasm after differential staining for light microscopy. By electron microscopy, this cell has elongated fusiform secretory granules (520 nm average length × 230 nm average width) filled with numerous filaments arranged in parallel with the longitudinal axis. Figures suggesting granule formation in the sacs of the Golgi apparatus were obtained in all of islet cell types. Equivalents of emiocytotic release of secretory granules were encountered in the A and F_f cells.     

Effect of glucagon antiserum on somatostatin,glucagon and insulin release from the isolated perfused rat pancreas

In order to elucidate the effect of glucagon antiserum on the endocrine pancreas, the release of somatostatin, glucagon, and insulin from the isolated perfused rat pancreas was studied following the infusion of arginine both with and without pretreatment by glucagon antiserum. Various concentrations of arginine in the presence of 5.5 mM glucose stimulated both somatostatin and glucagon secretion. However, the responses of somatostatin and glucagon were different at different doses of arginine. The infusion of glucagon antiserum strongly stimulated basal secretion in the perfusate total glucagon (free + antibody bound glucagon) and also enhanced its response to arginine, but free glucagon was undetectable in the perfusate during the infusion. On the other hand, the glucagon antiserum had no significant effect on either insulin or somatostatin secretion. Moreover, electron microscopic study revealed degrannulation and vacuolization in the cytoplasm of the A cells after exposure to glucagon antiserum, suggesting a hypersecretion of glucagon, but no significant change was found in the B cells or the D cells. We conclude that in a single pass perfusion system glucagon antiserum does not affect somatostatin or insulin secretion, although it enhances glucagon secretion.