Journal of Histochemistry & Cytochemistry

Pringle, G A; Dodd, C M

doi: 10.1177/38.10.1698203pmid: 1698203

Two monoclonal antibodies, 6D6 and 7B1, previously shown to recognize different epitopes on different regions of the protein core of decorin were used to localize the protein core in relation to the positively stained bands in the D period of bovine tendon collagen fibrils. Peroxidase-antiperoxidase staining revealed that the antigen is associated with the surface of all fibrils and suggested that the axial distance between antigens is D-periodic. Immunoferritin labeling with each antibody produced a distribution of ferritin particles that showed that both epitopes of the protein core are localized near the d and e bands in the D period. The data indicate that the decorin protein core binding site(s) on tendon collagen fibrils is/are located near these bands, axially, within the D period.

Jonges, G N; Van Noorden, C J; Gossrau, R

doi: 10.1177/38.10.2169492pmid: 2169492

We have optimized a cerium-diaminobenzidine-based method for histochemical analysis of glucose-6-phosphatase (G6Pase) activity and have determined quantitative data on the zonal distribution pattern in the liver acinus of fasted male rats. In the cerium-diaminobenzidine technique, cerium instead of lead ions is used as capturing reagent for the enzymatically liberated phosphate. For light microscopy, the primary reaction product, cerium phosphate, is then visualized by conversion into cerium perhydroxide using hydrogen peroxide and subsequent oxidative polymerization of diaminobenzidine to diaminobenzidine brown as the final reaction product. Variation of the substrate (glucose-6-phosphate) concentration in the incubation medium yielded in periportal zones a KM value of 2.3 +/- 0.7 mM and a Vmax value of 0.96 +/- 0.18 (expressed as mean integrated absorbance). In perivenous zones a KM value of 1.1 +/- 0.4 mM and a Vmax value of 0.51 +/- 0.08 were calculated. The cytophotometric analysis performed in this study demonstrated for the first time that a functional difference of G6Pase, the key enzyme for gluconeogenesis, exists in the periportal and perivenous zones of the liver acinus. Periportal zones contain twice as many enzyme molecules (high Vmax) as perivenous zones, but the affinity for the substrate is twice as low. This may have important implications for the concept of metabolic zonation of the liver and also for glucose homeostasis in the blood.

Torrisi, M R; Pavan, A; Lotti, L V; Migliaccio, G; Pascale, M C; Covelli, E; Leone, A; Bonatti, S

doi: 10.1177/38.10.2401782pmid: 2401782

We have compared in the same cell type the surface distribution and partition in freeze-fractured plasma membranes of Sindbis virus glycoproteins in three different situations: (i) in permanently transformed cells that express the glycoproteins as the only viral product; (ii) in cells in which prebound viruses were forced to fuse with the plasma membrane by low pH treatment; (iii) in virus-infected cells. We report here that the viral proteins expressed on the surface of transfected cells show a uniform and unclustered distribution; conversely, in Sindbis virus-infected cells they appear clustered, regionally distributed, and always associated with budding viruses (i.e., interacting with the nucleocapsid on the cytosolic side of the membrane). Furthermore, the viral proteins expressed on transfected cells or implanted by low pH-mediated fusion partition during freeze-fracture with the exoplasmic faces of the cell plasma membranes, whereas an opposite partition is observed in infected cells. These results strongly suggest that in infected cells the clustering and the partition with the protoplasmic faces of the plasma membrane depend only on the strong "anchorage" of the glycoproteins to the nucleocapsid.

Naganuma, H; Ohtani, H; Harada, N; Nagura, H

doi: 10.1177/38.10.2401783pmid: 2401783

In this study we investigated the immunohistochemical localization of a unique aromatase, a single protein of 51,000 daltons, in the human placenta and ovary at light and electron microscopic levels. Microwave fixation was adopted for the immunoelectron microscopic study because it is an excellent method for preserving antigenicity and subcellular structures in frozen sections. Tissue samples from four immature human placentas, four full-term human placentas, and two human ovaries fixed in 10% formalin were examined by light microscopy. In addition, tissues from three full-term human placentas and one immature human placenta fixed in 4% paraformaldehyde were examined by electron microscopy. By light microscopy, immunoreactivity for this aromatase was located in the syncytiotrophoblast and a part of the cytotrophoblast of the placenta and in the lutein and granulosa cells of the ovary. Immunoelectron microscopy revealed that the aromatase antigen was localized on the surface of the microvilli, the lateral plasma membrane, and in the endoplasmic reticulum (ER) in the syncytiotrophoblast of the placenta. The positive immunoreactivity in the syncytiotrophoblast ER is consistent with previous results using antibodies for other types of aromatase, whereas the reactivity on the microvilli has not been previously described. The present report describes the fine localization of this unique aromatase in placental and ovarian tissues; its localization on the plasma membranes requires further physiological investigation.

Yasuda, K; Shiozawa, M; Yamashita, S; Aiso, S

doi: 10.1177/38.10.1976129pmid: 1976129

We studied the distribution of gamma-glutamyl transpeptidase (gamma-GT) by use of a monoclonal antibody (MAb) against human kidney gamma-GT in human sweat glands. In the eccrine sweat gland, the enzyme was localized along the luminal membrane and small apocrine extrusions of the superficial cells of the secretory portion. The intercellular canaliculi between basal cells were occasionally immunoreactive. In the secretory portion of the apocrine gland, luminal membrane and apocrine extrusions of various sizes and stages at the apices of the secretory cells exhibited positive reactions. Immunoreaction was also seen in the Golgi area of the cuboidal secretory cells. No positive reaction was observed in the myoepithelial cells of either gland or in the excretory duct cells.

Quatacker, J R; Annaert, W G; De Potter, W P

doi: 10.1177/38.10.2401784pmid: 2401784

Highly glycosylated compounds have been demonstrated in the axonal reticulum elements of the superior cervical ganglion cells of the rat, and this is considered to suggest a connection of the reticulum with the trans Golgi side. In the present study, the axonal reticulum and the Golgi elements were further characterized by post-embedding methods of lectin-gold cytochemistry to determine their carbohydrate residues and to see, more specifically, if sialic acid residues could be detected in the axonal reticulum elements. Therefore, the affinity of neuronal cell structures for Limax flavus agglutinin (LFA), wheat germ agglutinin (WGA), and Ricinus communis agglutinin I (RCA-I) was tested in ultra-thin sections of glycolmethacrylate-embedded material, counterstained with phosphotungstic acid (PTA) at low pH. The trans Golgi network, the Golgi-associated axonal reticulum, the reticulum within axons, the large dense-cored vesicles, and the plasma membranes were reactive for all three lectins used. We conclude that the axonal reticulum elements carry sialic acid residues, relating them to the trans Golgi network. The present results support the concept that the axonal reticulum is an extension of the trans network of the Golgi apparatus specialized for neurosecretion.

Lautier, D; Anthelme, B; Salmon, J M; Vigo, J; Viallet, P

doi: 10.1177/38.10.2401785pmid: 2401785

PAH metabolism is known to proceed in two successive steps, the first step resulting in the production of activated metabolites which are subsequently transformed by the different pathways involved in the second step. Microspectrofluorometry enables the study of the kinetics of these steps in living intact cells into which no imbalance has been artificially introduced. We used this technique to check the influence of pre-incubation with D-galactosamine on the kinetics of the detoxification step. 9- and 3-hydroxybenzo(a)pyrene (OH-B(a)P) were selected as fluorescent substrates because they are potential substrates for the different pathways of the second step. The physiological cell status was controlled at the level of the intrinsic cellular fluorescence. Pre-incubation with D-galactosamine results in a strong decrease of the experimental rate constants characteristic of the metabolism of 9- and 3-OH-B(a)P in both RTG2 and 3T3 cells. Moreover, such pre-incubation leads to a strong decrease of the transitory intracellular accumulation of 3-O-glucuronide when 3-OH-B(a)P is used as substrate for 3T3 cells. Nevertheless, it cannot be said that both phenols cannot be used as substrates by MFOs and STase, at least in rigorous experimental conditions.

Kogaya, Y; Kim, S; Haruna, S; Akisaka, T

doi: 10.1177/38.10.1698204pmid: 1698204

Using the high-iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining technique and enzymatic digestion, we investigated the ultrastructural distribution pattern of heparan sulfate side chains of heparan sulfate proteoglycan (HSPG) in various basement membranes (nerve, capillary, oral epithelial, muscle, and dental basement membranes). Four different distribution patterns of stain deposits were identified as heparan sulfate on the basis of enzymatic degradation by heparitinase. In some basement membranes associated with tooth germs and oral epithelium, HID-TCH-SP stain deposits were regularly located at both sides of the lamina densa, but few were observed in the lamina densa itself. In nerve, muscle, and capillary basement membranes, the stain deposits were localized at the external side of the lamina densa adjacent to the underlying connective tissue, but were not found in the laminae lucida and densa. In the internal basal lamina of junctional epithelium of gingiva, the stain deposits were detected mainly in the lamina lucida region. Finally, in some dental and oral epithelial basement membranes, the stain deposits were randomly distributed throughout both laminae lucida and densa. Thus, the present study demonstrated distinct differences in heparan sulfate distribution pattern among various basement membranes, suggesting their architectural heterogeneity.

Eisenmann, D R; Salama, A H; Zaki, A M; Ashrafi, S H

doi: 10.1177/38.10.2144864pmid: 2144864

Colchicine is known to affect secretory, transport, and degradative functions of ameloblasts. The effects of colchicine on membrane-associated calcium and Ca2+,Mg2(+)-ATPase in secretory and maturation ameloblasts were investigated cytochemically. The pyroantimonate (PPA) method was used for localizing calcium and a modified Wachstein-Meisel medium was used to localize Ca2+,Mg2(+)-ATPase. Sections representing secretory and early maturation stages were examined by transmission electron microscopy. Morphological changes induced by colchicine included dislocated organelles and other well-established reactions to such anti-microtubule drugs. Calcium pyroantimonate (Ca-PA) deposits in most ameloblast types were markedly reduced, with the greater reduction occurring in those cells more severely altered morphologically. However, the cell membranes of both control and experimental smooth-ended maturation ameloblasts were essentially devoid of Ca-PA. The normal distribution and intensity of Ca2+,Mg2(+)-ATPase was not affected by colchicine. Because the observed reduction of membrane-associated calcium is apparently not mediated by Ca2+,Mg2(+)-ATPase in this case, other aspects of the calcium regulating system of ameloblasts are apparently targeted by colchicine.

McCarthy, K J; Couchman, J R

doi: 10.1177/38.10.2401786pmid: 2401786

Heparan sulfate proteoglycans have been described as the major proteoglycan component of basement membranes. However, previous investigators have also provided evidence for the presence of chondroitin sulfate glycosaminoglycan in these structures. Recently we described the production and characterization of core protein-specific monoclonal antibodies (MAb) against a chondroitin sulfate proteoglycan (CSPG) present in Reichert's membrane, a transient extra-embryonic structure of rodents. This CSPG was also demonstrated to be present in adult rat kidney. We report here the tissue distribution of epitopes recognized by these MAb. The ubiquitous presence of these epitopes in the basement membranes of nearly all adult rat tissues demonstrates that at least one CSPG is a constituent of most basement membranes, and by virtue of its unique distribution is distinct from other chondroitin and dermatan sulfate proteoglycans previously described.