Immunoelectron microscopic studies of glycosaminoglycans in the metaphyseal bone trabeculae of growing rats

Summary The types and distribution of glycosaminoglycans (GAGs) were studied immunocytochemically in osteoid, mineralized bone matrix, and cartilage matrix of growing rat metaphyseal bone after aldehyde fixation and EDTA demineralization, using four monoclonal antibodies (mAbs 1-B-5, 2-B-6, 3-B-3 and 5-D-4). These mAbs specifically recognize epitopes in non-sulphated chondroitin (C0-S); chondroitin 4-sulphate (C4-S) and dermatan sulphate (DS); chondroitin 6-sulphate (C6-S) and C0-S; and keratan sulphate (KS) respectively. In osteoid, all mAbs except 1-B-5 weakly stained matrix material on and between collagen fibrils, and moderately stained organic material corresponding to bone nodules, which are known sites of mineralization. However, the staining of osteoid abruptly decreased at the mineralization front; weak staining was confined mostly to the organic material of bone nodules in mineralized bone matrix, with very weak or no staining of the rest of the bone matrix. This staining progressively decreased toward the mineralized cartilage matrix and became negative. The mineralized cartilage matrix and lamina limitans reacted strongly with all mAbs except 5-D-4. These results indicate that osteoid contains sulphated proteoglycans containing C4-S and/or DS, C6-S and KS, and subsequent bone matrix mineralization appears to require accumulation of these macromolecules within bone nodules and eventual loss of these substances for complete mineralization, whereas proteoglycans containing C0-S, C4-S and/or DS, and C6-S, still exist in mineralized cartilage matrix and lamina limitants.     

Immunoelectron microscopic demonstration of tissue antigens with monoclonal antibodies

Two methods of demonstrating tissue antigens by ultrastructural enzyme immunohistochemistry were tested. The monoclonal antibodies Ki-M1 and Ki-M4 were chosen for testing the methods because Ki-M1 identifies a relatively stable, and Ki-M4 a very unstable antigen. The two antibodies react selectively with human macrophages and interdigitating reticulum cells or dendritic reticulum cells of lymphoid follicles. The Ki-M1 reaction product is confined to the surface membrane. Ki-M4 reactivity is located on the surface membrane and, less often and to a lesser extent, in the cytoplasm. The technical prerequisites for reliable conservation of the antigens identified by these two antibodies were standardized. The results indicated that prior fixation in 4% paraformaldehyde is preferable for optimum preservation of stable antigens. Application of the primary antibody prior to fixation was found to be the best procedure for demonstrating unstable antigens, although nonspecific reactions were seen more often with this method.     

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.     

Immunoelectron microscopic localization of herpes simplex virus antigens in infected cells using the unlabeled antibody-enzyme method.

Subcellular localization of viral antigens was demonstrated during viral morphogenesis using herpes simplex virus type 1 (HSV-1) infected monolayers of rabbit cornea cells. The localization was done by immunoelectron microscopy employing the peroxidase-antiperoxidase (PAP) immunocytochemical technique and the postembedding staining method. The localization of viral antigens was followed at time intervals during infection from 2 to 19 hr. After exposure of sections to either polyspecific antibodies against total HSV-1 antigens or monospecific antibodies against HSV-1 antigen No. 8, specific immunological reaction products were identified both in the cytoplasm and nucleus after 2 hr. The distribution and quantity of reaction products varied in the infected cells during the viral morphogenesis. The present results on the subcellular distribution of the HSV-1 antigens are related to current biochemical findings.     

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.     

Immunoelectron microscopic localization of calmodulin in corn root cells

Methods for the localization of plant calmodulin by immuno-gold and immuno-peroxidase electron microscopy have been developed. In both corn root-cap cells and meristematic cells, calmodulin was found to be localized in the nucleus, cytoplasm, mitochondria as well as in the cell wall, In the meristematic cells, calmodulin was distinctly localized on the plasma membrane, cytoplasmic face of rough endoplasmic rcticulum and polyribosomes. Characteristically, calmodulin was present in the amyloplasts of root-cap cells. The widespread distribution of calmodulin may reflect its plciotropic functions in plant cellular activities.     

Immunoelectron microscopic localization of ubiquitin in hepatoma cells.

Ubiquitin, a 76 amino acid protein, is covalently attached to abnormal and short-lived proteins, thus marking them for ATP-dependent proteolysis in eukaryotic cells. Free (unconjugated) ubiquitin was localized in hepatoma cells using affinity purified anti-ubiquitin antibodies and colloidal gold immunoelectron microscopy. The anti-ubiquitin antibodies recognize only unconjugated ubiquitin. Ubiquitin is found within the cytoplasm, nucleus, the microvilli, autophagic vacuoles and lysosomes.     

Immunoelectron microscopic study of somatostatin biosynthesis in dog pancreas

The biosynthesis of somatostatin has been studied at the ultrastructural level in pancreatic islets by using rabbit antiserum against synthetic somatostatin. To document that the antiserum specifically bound preprosomatostatin, we have tested the ability of the antiserum to precipitate the product synthesized in vitro. Poly(A) enriched RNA isolated from catfish islets was translated in both the wheat germ extract and nuclease-treated reticulocyte lysate systems. It was found that the in vitro translation product, preprosomatostatin, could be recognized by the antibody against synthetic somatostatin. The morphological study was then performed by immunoelectron microscopy by using the Fab-peroxidase conjugate technique. In dog pancreatic islets, somatostatin immunoreactive reaction product was seen only in the delta cells. In these cells, they were detected on bound ribosomes, in the cisternae of the rough endoplasmic reticulum (ER) and Golgi apparatus, in the Golgi associated vesicles, and in secretory vesicles. These findings suggest that somatostatin precursor molecules are synthesized on bound ribosomes and discharged into the cisternae of the rough ER. They are then transported to the Golgi apparatus and transferred to the secretory vesicles for secretion. The different staining intensities in the secretory vesicles would suggest that the processing of the precursor molecules of somatostatin probably takes place in the secretory vesicles.     

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.     

Immunoelectron microscopic localization of acidic intracellular compartments in hepatoma cells.

Using protein A-colloidal gold immunoelectron microscopy and monospecific antibodies to the weak base primaquine, we have delineated acidic intracellular compartments in the human hepatoma cell line, HepG2. Primaquine specifically accumulated within endocytotic compartments (including CURL vesicles, multivesicular bodies and lysosomes). In addition, the Golgi cisternae were positive. However, the CURL tubules, which contain recycling asialoglycoprotein receptor, did not accumulate primaquine. Thus, there may be a gradient of acidification within the endocytotic pathway.     

Stromal cells of the regenerating bone marrow in rats (an immunocytochemical electron microscopy study)

Process of the bone marrow regeneration has been studied after its removal out of the rat femoral bone cavity. The stage of stroma formation precedes hemopoiesis. The stromal cells during its reconstruction (the 4th-5th day after removal of the bone marrow) are analyzed by means of the indirect immune-peroxidase electron microscopical method with antiserum applied against insoluble antigens of the rat bone marrow cells. Most of the stromal cells do not fix the antiserum used, as do the hemopoietic cells, macrophages and preosteoclasts. Some part of the stromal cells (not more than 30%) demonstrate the immune-peroxidase label. The labelled stromal cells have some ultrastructural signs of poorly differentiated elements of fibroblastic and osteoblastic raws. In the regeneration area, there are non-labelled poorly differentiated cells, which do not differ, at the ultrastructural level, from labelled poorly differentiated stromal elements. Possible causes of the difference revealed among the poorly differentiated stromal cells concerning their fixing of the anti-bone-marrow antiserum are discussed.     

Liproprotein inhibitor of bone marrow cells in tumor-bearing rats.

The role of a plasma inhibitor of erythropoiesis is evaluated in rats with Walker-256 carcinoma (W-256). Plasma from tumor-bearing rats was treated by gel filtration chromatography (Sephadex G-150) and fractions were combined into four pools on the basis of mol. wt. Inhibitory activity was assayed by adding an aliquot of the plasma fractions to normal rat marrow cells which were cultured for 24 hr with and without erythropoietin. 59Fe-heme synthesis, [3H]thymidine DNA synthesis, and 14C-leucine protein synthesis were studied. The results indicated that cultures containing the high mol. wt. pool (greater than 400,000 daltons) had significantly decreased heme, DNA and protein synthesis. This inhibitor also diminished the response to erythropoietin in polycythemic mice. The lower mol. wt. pool stimulated heme synthesis in vitro. To identify the inhibitor further, plasma lipoprotein classes were isolated by density gradient ultracentrifugation. The very low density lipoprotein (VLDL) and chylomicron fractions markedly inhibited DNA, protein and heme synthesis. Low density and high density lipoprotein fractions were inactive. A lipoprotein inhibitor of erythropoiesis was also identified in cancerous ascitic fluid, and to a lesser degree, in normal rat plasma. We suggest that this VLDL inhibitor of marrow erythropoiesis is a contributing factor in the anaemia of cancer.     

Cytogenetic changes in the bone marrow cells of long-term irradiated rats]

The occurrence and characteristics of the chromosome structural changes in femur bone marrow cells under continuous irradiation with the exposure rate of 50 R/day within 90 days was followed. The 25% increase in the chromosome aberration frequency was observed within 7 days of the irradiation, and then the aberration rate was constant up to the end of the irradiation (90 days).     

Effects of aluminum on erythroidal cells in bone marrow in rats

The study has been carried out on Wistar rats. The aim of the present study was to trace the effect of aluminum on erythroidal cells in bone marrow in rats. The number of proerythroblast after 10 days of experiment with aluminum slowly decreased up to 80 days of experiments. However, the number of basophilic erythroblasts after 10 days insignificantly increased but after 20 days gradually decreased up to 80 days of experiments. The bone marrow polichromatic erythroblasts after 10 days of experiment slightly decreased, however after 20, 40 and 80 days of experiments the values decreased significantly. The quality of orthochromatic erythroblasts after 10 days of experiments dropped and after 20, 40 and 80 days of experiments significantly decreased compared to the control value. Aluminum also brings about histological changes in the bone marrow. The statistical significant reductions of hemoglobin and hematocrit levels were found in the aluminum exposed rats.