Ultrastructural immunocytochemical localization of the transferrin receptor using a monoclonal antibody in human KB cells

Using a monoclonal antibody (HB21) against the human transferrin receptor, we have localized this receptor in cultured KB human carcinoma cells by fluorescence and ultrastructural immunocytochemistry. The receptor was found diffusely distributed on the cell surface, concentrated in clathrin-coated pits of the cell surface, in intracellular endocytic vesicles (receptosomes) derived from coated pits, in tubular elements of the trans-reticular Golgi system, and in microtubule-associated membranous elements thought to be part of the constitutive exocytic system. This distribution is the same as that previously shown for labeled transferrin in these same cells (Willingham MC, Hanover JA, Dickson BB, Pastan J: Proc Natl Acad Sci USA 81:175, 1984). No significant amounts of receptor were found in lysosomes. An aggregation of membranous elements containing this receptor was found in the pericentriolar region of cells during mitosis. Together with the previous data on the immunocytochemical localization of transferrin, these results suggest that the transferrin receptor may constitutively enter and exit KB cells by endocytosis and exocytosis, carrying bound transferrin into and out of the cell for the purpose of supplying iron from the extracellular environment for cell growth.     

A global view of protein expression in human cells,tissues, and organs

Defining the protein profiles of tissues and organs is critical to understanding the unique characteristics of the various cell types in the human body. In this study, we report on an anatomically comprehensive analysis of 4842 protein profiles in 48 human tissues and 45 human cell lines. A detailed analysis of over 2 million manually annotated, high‐resolution, immunohistochemistry‐based images showed a high fraction (>65%) of expressed proteins in most cells and tissues, with very few proteins (<2%) detected in any single cell type. Similarly, confocal microscopy in three human cell lines detected expression of more than 70% of the analyzed proteins. Despite this ubiquitous expression, hierarchical clustering analysis, based on global protein expression patterns, shows that the analyzed cells can be still subdivided into groups according to the current concepts of histology and cellular differentiation. This study suggests that tissue specificity is achieved by precise regulation of protein levels in space and time, and that different tissues in the body acquire their unique characteristics by controlling not which proteins are expressed but how much of each is produced.     

Identification and immunocytochemical localization of proctolin in pericardial organs of the shore crab, Carcinus maenas

The occurrence of proctolin (Arg-Tyr-Leu-Pro-Thr) in crab neurohemal pericardial organs (POs) has been demonstrated by isolation of the pentapeptide by HPLC and manual microsequencing according to the DABITC-PITC double coupling technique. From one pair of POs approximately 5.4 pmol were obtained (= 45 pmol/mg protein). Immunocytochemically, an extensive system of positive structures was found in both whole mount preparations and semithin sections, consisting of numerous varicose fibres of varying diameter and many knoblike neurosecretory terminals abutting upon the epineurium of the PO trunks. The relatively high concentration in the POs as well as the pattern of proctolin-positive fibres and terminals clearly suggest a neurohormonal role of the pentapeptide in decapod crustaceans.     

Ultrastructural immunocytochemical localization of the phosphomannosyl receptor in Chinese hamster ovary (CHO) cells

Using ultrastructural immunocytochemistry and antibodies directed against bovine liver phosphomannosyl (PM) receptor, we have localized the receptor in Chinese hamster ovary (CHO) cells. The majority of the receptor was found within the cell. Only a small fraction of the receptor was found on the surface and most of it was clustered in coated pits. Because these cells contain endogenous ligands for the receptor, it was not possible to determine if this clustered state was dependent on occupancy of the receptor. The bulk of the cell's receptor was found in the endoplasmic reticulum, nuclear envelope, and in the Golgi system. Most of the Golgi localization was associated with peripheral Golgi elements, suggesting a possible concentration of receptor in GERL. Very little receptor was found associated with mature lysosomes. PM receptor was also localized in structures that were identified as receptosomes by the presence of alpha 2-macroglobulin (alpha 2M)-gold, a ligand previously shown to enter CHO cells by the coated pit-receptosome pathway. This finding is consistent with the notion that during receptor-mediated endocytosis, receptors accompany ligand from the coated pit into the receptosome. The observation that the majority of the receptor was found in the endoplasmic reticulum and structures similar to GERL raises the possibility that the PM receptor plays an important role in compartmentalization of lysosomal enzymes in the GERL system.     

Light and electron microscopical immunocytochemical localization of pancreastatin-like immunoreactivity in porcine tissues

Pancreastatin is a 49 amino acid comprising peptide isolated from porcine pancreas that is derived by proteolytic processing from chromogranin A. Using an antibody against the synthetic C-terminal fragment pancreastatin (33-49), we examined the light and electron microscopical immunocytochemical localization of this peptide in porcine tissues. Pancreastatin-like immunoreactivity (PLI) was found in pancreatic somatostatin-, insulin- and glucagon cells in varying intensities; pancreatic polypeptide cells were always negative. At the electron microscopical (EM) level the immunoreactivity was confined to the electron dense core of the secretory granules in the case of somatostatin and insulin cells or to the less electron dense "halo" of the glucagon granules. In the antrum PLI positive cells represented gastrin (G), somatostatin (D) and enterochromaffin (EC) cells, in the duodenum in addition to EC- and G-cells a small number of PLI positive cells showed a positive immunoreaction for glucagon-like peptide (GLP) I and secretin in serial sections. Both norepinephrine and epinephrine containing cells of the adrenal medulla exhibited a strong reaction for PLI. In the pituitary several cell populations stained with varying intensities, including gonadotrophs and thyrotrophys. PLI is present in a distinct and characteristic subpopulation of neuroendocrine cells in various organs. The subcellular localization may indicate a function in the granular concentration, packaging and storage of peptides and amines in the brain-gut endocrine system.     

Light and electron microscopical immunocytochemical localization of pancreastatin-like immunoreactivity in porcine tissues

Summary Pancreastatin is a 49 amino acid comprising peptide isolated from porcine pancreas that is derived by proteolytic processing from chromogranin A. Using an antibody against the synthetic C-terminal fragment pancreastatin (33–49), we examined the light and electron microscopical immunocytochemical localization of this peptide in porcine tissues. Pancreastatin-like immunoreactivity (PLI) was found in pancreatic somatostatin-, insulin- and glucagon cells in varying intensities; pancreatic polypeptide cells were always negative. At the electron microscopical (EM) level the immunoreactivity was confined to the electron dense core of the secretory granules in the case of somatostatin and insulin cells or to the less electron dense halo of the glucagon granules. In the antrum PLI positive cells represented gastrin (G), somatostatin (D) and enterochromaffin (EC) cells, in the duodenum in addition to EC- and G-cells a small number of PLI positive cells showed a positive immunoreaction for glucagon-like peptide (GLP) I and secretin in serial sections. Both norepinephrine and epinephrine containing cells of the adrenal medulla exhibited a strong reaction for PLI. In the pituitary several cell populations stained with varying intensities, including gonadotrophs and thyrotrophs. PLI is present in a distinct and characteristic subpopulation of neuroendocrine cells in various organs. The subcellular localization may indicate a function in the granular concentration, packaging and storage of peptides and amines in the brain-gut endocrine system.     

Production and characterization of monoclonal antibodies against the polyamine,spermine: immunocytochemical localization in rat tissues

Two monoclonal antibodies of types IgG_2b and IgG_2a, anti-spermine-(Spm)-1 (ASPM-1) and anti-Spm-2 (ASPM-2) respectively were found among five clones of murine monoclonal antibodies, which were raised against Spm conjugated with bovine serum albumin via the cross-linker N-(-maleimidobutyryloxy) succinimide (GMBS). Antibody specificity was evaluated by a recently developed ELISA binding test, and led to the study of tissue sections by immunocytochemistry (ICC). ASPM-1 showed exclusive immunoreactivity with Spm, with the exception of a negligible cross-reactivity (2.0%) with spermidine (Spd). ASPM-2, on the other hand, reacted almost equally with acetylspermine (Ac-Spm) and N ¹-acetylspermidine (N¹-Ac-Spd) but with none of the other polyamine-related compounds tested. Complete agreement was obtained with the results of immunoblot analysis. Furthermore, results for antibody specificity obtained with the ELISA inhibition test and ICC model experiments using Sepharose gel beads strongly suggested that ASPM-1 recognizes the Spm molecule possessing at least a free terminal primary amino group, while ASPM-2 recognizes the Spm molecule acylated at both the terminal primary amino groups. An ICC method using ASPM-2 produced strong staining for polyamines (PAs) in the cytoplasm (but very few in the nuclei) of two different tumor cell lines and protein- or peptide-secreting cell systems, including exocrine and endocrine cell types; ASPM-1 showed immunoreactivity only with the tumor cell lines. These results strongly suggest that ASPM-2 may be useful for studies on actively proliferating and neoplastic cells, supporting our previously proposed idea that in immunocytochemistry PAs were converted to a variety of PA derivatives during the fixation process.     

Rho localization in cells and tissues

Rho family small GTPases regulate cytoskeletal organization. Although their spatiotemporal activities appear to be important for cellular morphogenesis, there has been little characterization of the localization of Rho family GTPases in cells and tissues. Here we show precise localization of Rho subfamily proteins in mammalian cultured cells and tissues through evaluation of anti-Rho antibodies and fixation protocols. Although Rho is not a structural protein but functions as a switching molecule, it often localizes at several distinct domains or structures of cells. In cultured epithelial cells, Rho was highly accumulated at lateral membranes. However, in fibroblastic cells, Rho appeared to be distributed evenly in the cytoplasm. Rho concentration at the cleavage furrow at cytokinesis was generally observed. In A431 cells, Rho translocation from the cytoplasm to elongating microvilli at the apical membrane within 30 s after EGF stimulation was clearly demonstrated. Also, Myc- or GFP-tagged RhoA did not always reflect the localization of endogenous Rho, indicating a drawback of protein-tagging methods for localization research. In mouse tissues, Rho localization differed depending on cell type, probably reflecting the functional differences of each cell type.     

Ultrastructural immunocytochemical localization of lysozyme in human monocytes and macrophages

Summary In order to investigate the mechanism of synthesis and secretion of lysozyme (LZ) by human mononuclear phagocytes, the ultrastructural localization of LZ was studied by a pre-embedding direct immunoperoxidase method. Blood monocytes showed a reaction product for LZ in cytoplasmic granules, whereas cultured monocytes showed the reaction product in phagosomes as well as granules at 5 h of culture and in numerous large granules at 3 days of culture. In Kupffer cells, LZ was present in cytoplasmic granules, vacuoles and phagosomes. Some Kupffer cells showed a positive reaction for LZ in the rough endoplasmic reticulum, perinuclear cisterna and Golgi apparatus. Macrophages in the lymph nodes contained LZ in cytoplasmic granules. Bone marrow macrophages contained numerous phagosomes with electron-dense degradation products of erythrocytes, but the reaction product for LZ could not be clearly identified. The present study demonstrated that LZ is present in the granules of human mononuclear phagocytes and released into phagosomes. An in-vitro culture study, furthermore, demonstrated that macrophages produce LZ-containing large granules distinct from those of monocytes. However, findings that indicate the synthesis and secretion of LZ by cultured monocytes, as suggested previously by other investigators, were not observed in this study.     

An immunocytochemical study on the occurrence of liver fatty-acid-binding protein in the digestive organs of rats: specific localization in the D cells and brush cells

Rat liver fatty-acid-binding protein (L-FABP) was originally isolated from the liver parenchymal cells and later found also in the intestinal absorptive cells. By light- and electron-microscopic immunocytochemistry we examined the distribution of L-FABP in the entire digestive system of the rat and revealed two other cell types, i.e. the endocrine D cell and the brush cell, to be specifically immunoreactive for L-FABP. The immunoreactive D cells, identified by the simultaneous immunoreactivity for somatostatin and by characteristic endocrine granules, were found in the stomach epithelium and pancreatic islets. The immunoreactive brush cells, identified by the ultrastructural features of cell apex, were found primarily in the stomach epithelium and also in the epithelia of the rectum and common bile duct. Almost all immunoreactive brush cells had a thin process in contact with the basement membrane. No secretory granules with dense cores similar to those of the endocrine cells were observed in the brush cells. The present findings reveal L-FABP to be a useful marker of the gastrointestinal D cells and brush cells, especially of the latter, confirming that the brush cell is a distinct entity different from any other cell types in the gastrointestinal epithelia. Furthermore, FABP may be involved in the specific functions of these cell types related to fatty acid metabolism.     

Immunocytochemical localization of the urotensin-II receptor, UT, to rat and human tissues: relevance to function

We have examined whether differential expression of UT receptors in cardiovascular tissues from rats and humans may account for the diverse vascular actions reported for urotensin-II. We found UT immunoreactivity ubiquitously expressed in arterial and venous smooth muscle and cardiomyocytes in both species, however, compared to human, levels of UT immunoreactivity in rat vascular endothelial cells was below the level for detection. In rat skeletal muscle cells UT receptor localized to the sarcolemma, a pattern comparable to that for isoforms of nitric oxide synthase suggesting that urotensin-II mediated hindquarter vasodilatation may involve release of nitric oxide from skeletal muscle fibers.     

Ultrastructural localization of myocilin in human trabecular meshwork cells and tissues.

We examined ultrastructurally the localization of myocilin (formerly called trabecular meshwork inducible glucocorticoid response, or TIGR) protein in cultured human trabecular meshwork (TM) cells and in normal human TM tissues. The TM, a specialized tissue located at the chamber angle of the eye, is believed to be responsible for the development of glaucoma. The myocilin gene has been directly linked to both juvenile and primary open-angle glaucomas, and multiple mutations have been identified. Human TM cells were treated with 0.1 mM of dexamethasone (DEX) to induce myocilin expression. This protein was immunolocalized by colloidal gold electron microscopy using an anti-human myocilin polyclonal antibody. Double labeling with different sizes of gold particles was also performed with additional monoclonal antibodies specific for cell organelles and structures. In both DEX-treated and untreated cultured cells, myocilin was associated with mitochondria, cytoplasmic filaments, and vesicles. In TM tissues, myocilin was localized to mitochondria and cytoplasmic filaments of TM cells, elastic-like fibers in trabecular beams, and extracellular matrices in the juxtacanalicular region. These results indicate that myocilin is localized both intracellularly and extracellularly at multiple sites. This protein may exert diverse biological functions at different sites.     

Purification of GP57, and auxin-regulated extracellular glycoprotein of carrots,and its immunocytochemical localization in dermal tissues

A glycoprotein (GP57) was purified by ion-exchange and hydroxylapatite column chromatography from the 70%-ethanol precipitate of culture medium of non-embryogenic carrot cells (Daucus carota L.) grown with 2,4-dichlorophen-oxyacetic acid (2,4-D). Its apparent molecular mass (M _r) was estimated to be 57000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis and 50000 by gel filtration. GP57 contained 14% (w/w) carbohydrate; the M _r of the peptide portion was estimated to be 55000 after deglycosylation by trifluoromethanesulfonic acid. GP57 is composed of two polypeptides with the same M_r and with very similar amino-acid composition but different pI values, 8.8 and 9.5. Both are rich in aspartic acid, serine and threonine, and may possess N-linked oligosaccharide chains, including fucose and xylose. A monoclonal antibody (MAb) against the purified GP57 reacted with both the pI 8.8 and the 9.5 components, as well as the deglycosylated GP57. Immunoblotting with the MAb indicated that GP57 is synthesized in and released from cultured cells which have been supplied with auxin. In immunocytochemical studies, GP57 was found in the space between the embryo and the endosperm of dry seeds, and its content decreased during germination. GP57 was also found in the endodermis and epidermis of young roots, the periderm of mature taproots, and the epidermis of petioles and young leaves.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GP57 M _r-57000 glycoprotein - GP65 M _r-65000 glycoprotein - MAb monoclonal antibody - M _r apparent molecular mass - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - TFMS trifluoromethanesulfonic acid     

Light-microscopic immunocytochemical localization of growth hormone-releasing factor in the human hypothalamus

Summary The distribution of growth hormone-releasing factor (GRF)-like immunoreactivity in the human hypothalamus was studied by light-microscopic immunocytochemistry. With antibodies that we developed against synthetic human pancreatic GRF (hpGRF), we localized GRF immunoreactivity in neuronal cell bodies that were observed only in the infundibular (arcuate) nucleus. Immunostained nerve fibers were found in large numbers in the neurovascular zone of the median eminence, in the proximal portion of the pituitary stalk and in periventricular areas. These localizations are in agreement with those of studies recently performed in other species and strongly suggest that GRF can be released into the capillaries of the pituitary portal plexus to reach the anterior pituitary gland. The projections of GRF neurons in extra-infundibular regions suggest that GRF can also act as a neuromodulator or neurotransmitter in the hypothalamus.     

Electron immunocytochemical localization of pepsinogen I (PgI) in chief cells,mucous-neck cells and transitional mucous-neck/chief cells,of the human fundic mucosa

Summary Specific PgI antibodies devoid of PgII cross reactivity have been applied to aldehyde-osmium fixed human, fundic-type, gastric mucosa investigated with the protein A-immunogold technique. PgI immunoreactivity has been detected in the homogeneous secretory granules of glandular chief cells, in bipartite granules of mucous-neck cells, in the granules of cells showing intermediate patterns and topography in between chief and mucous-neck cells (transitional cells), as well as in the granules of a few cells in the foveolar/mucous-neck boundary zone showing mixed foveolar/mucous-neck granule populations. The findings support progressive transformation of mucous-neck cells into chief cells.     

Immuno-electron microscopic localization of estradiol receptor in cells of male and female reproductive and non-reproductive organs

Summary— The localization of estradiol receptor (ER) in various tissues and their distribution in sub-cellular compartments were studied by means of immunogold-electron microscopic methods using a site-directed polyclonal antibody developed against a peptide from the DNA binding site of ER. This method was used to determine the presence and localization of ER in tissues and cells of male and female reproductive and non-reproductive organs. In the female reproductive tract, endometrial cells and the cells of the corpus luteum were found to contain ER. In non-reproductive organs of both sexes the following cell types showed significant labeling: hepatocytes, epithelial duodenal cells, striated muscle fibers, cells of the proximal convoluted tubules of the kidney, lymphocytes, neurons, and adipose cells. Alveolar epithelial cells were studied only in female specimens and were labeled by the anti-ER. Prostatic and epididymal epithelial cells were found to be labeled in the male reproductive organs. In all these cells a higher density of label was found in the nucleus, especially in the space between the clumps of compact chromatin, as was previously found in epithelial endometrial cells. These results suggest that estradiol exerts its effects through a common nuclear mechanism in cells of male and female reproductive and non-reproductive organs.     

Epithelial proteomics in multiple organs and tissues: similarities and variations between cells, organs, and diseases

Epithelial cells play an important role in physiological and pathophysiological situations, with organ-, tissue-, type-, and function-specific patterns. Proteome analysis has been used to study epithelial-origin diseases and identify novel prognostic, diagnostic, and therapeutic markers. The present review compares the variation of sample preparation for epithelial proteomic analysis, search similarities, and differences of epithelial proteomics between different cells, locations, and diseases. We focus on specificity of proteomic markers for epithelial-involved diseases. Proteomic alterations in epithelial cell lines were mapped to understand protein patterns, differentiation, oncogenesis, and pathogenesis of epithelial-origin diseases. Changes of proteomic patterns depend on different epithelial cell lines, challenges, and preparation. Epithelial protein profiles associated with intracellular locations and protein function. Epithelial proteomics has been greatly developed to link clinical questions, e.g., disease severity, biomarkers for disease diagnosis, and drug targets. There is an exciting and attractive start to link epithelial proteomics with histology of clinical samples. From the present review, we can find that most of disease-associated investigation of epithelial proteomics has been focused on epithelial-origin cancer. There is a significant gap of epithelial proteomics between acute and chronic organ injury, inflammation, and multiple organ dysfunction. Epithelial proteomics will provide powerful information on the relationships between biological molecules and disease mechanisms. Epithelial proteomics strategies and approaches should become more global, multidimensional, and systemic.     

Different subclass distribution of IgA-producing cells in human lymphoid organs and various secretory tissues

A highly reproducible paired immunofluorescence staining method was used to map the relative distribution of IgA1- and IgA2-producing cells in peripheral lymphoid organs and various secretory tissues. Spleen, peripheral lymph nodes, and tonsils all contained a marked predominance (91 to 95%) of IgA1 immunocytes. However, striking variations were demonstrated among the secretory tissues with regard to the median proportion of IgA1-producing cells: nasal mucosa, 96%; lacrimal glands, 81%; major salivary glands, 66%; mammary glands, 63%; gastric and proximal small intestinal mucosa, 84 to 77%; ileum, 55%; and large bowel, 41%. Thus, IgA2 production is relatively enhanced mainly in the distal gut and in mammary and salivary glands, in that order.