In the epidermis of the fish Monopterus cuchia, the mucous and sacciform cells have been studied by means of a series of histochemical procedures for localizing and characterizing glycoprotein (GP) classes involving oxidizable vicinal diols, O-acyl sugars, O-sulfate esters and sialic acid residues without or with O-acyl substituents. GPs secreted by these gland cells belong to three general categories. These are (i) GPs synthesized by type A mucous cells and predominantly containing O-sulfate esters and small amounts oxidizable vicinal diols, O-acyl sugars and sialic acid residues without or with O-acyl substitution at C7, C8 (or which are di- or tri- substituted) or C9, (ii) GPs elaborated by type B mucous cells primarily with Osulfate esters, and relatively small amounts of oxidizable vicinal diols and sialic acid residues predominantly without or with O-acyl substitution at C7 as well, and (iii) GPs secreted by sacciform cells with exceedingly small amounts of oxidizable vicinal diols and O-sulfate esters. Mixed GPs on the surface mucous layer are considered to play an important function for lubrication, adherence of micro-organisms and inhibition of their invasion and proliferation in the epidermis, hydration and viscoelastic properties, in relation to an adaptation for the maintenance of this semi-terristrial fish frequently exposed to semi-arid conditions.
Using calcyclin-specific antibodies we show that in rat tissues calcyclin is present at high level in two types of cells: epithelial and fibroblasts. However, not all epithelial cells and fibroblasts were calcyclin-immunopositive. The increased number of epithelial cells in experimentally induced rat liver cirrhosis biliaris can be easily detected by immunohistochemistry. Consistently with these observations the level of calcyclin (protein and mRNA) is increased in this pathological tissue as shown by immunoblotting, ELISA, and Northern blotting. The results suggest that calcyclin might be a marker of some diseases where an increase in number of epithelial cells takes place.
The purpose of this study was to demonstrate the distribution of α1-adrenoceptors in the human hypertrophied prostate with [3H]YM617 (a newly synthesized α1-blocker), and to determine the receptor subtype of [3H]YM617 binding sites by the use of chlorethylclonidine dihydrochloride (CEC). The slices of prostate, removed en bloc in 7 patients with benign prostatic hypertrophy and sectioned vertically to the urethra, were preincubated with or without CEC (10μM). The slices were then incubated with [3H]YM617 (1nM) in the presence or absence of phentolamine (100μM). Specific binding of [3H]YM617 was seen relatively homogeneous, and distributed in the periglandular interstitium (mainly corresponding to smooth muscles) of the prostate. The CEC-pretreated specimens showed a decrease in density of specific binding sites (30.9% mean decrease compared with the CEC non-pretreated specimens on quantitative autoradiogram) (p<0.05), but localization was almost the same as in the CEC non-pretreated specimens. Membrane assay gave similar results. These experiments indicate that ca 70% of α1-adrenoceptor binding sites in the hypertrophied prostate are insensitive to CEC, while ca 30% of that are sensitive to CEC.
The process of heterotopic bone formation by bone morphogenetic protein (BMP) implanted in mouse muscle tissue was immunohistochemically studied in terms of the detection of glycosaminoglycans (GAGs) (chondroitin 4 sulfate, C4S; chondroitin 6 sulfate, C6S; keratan sulfate, KS; dermatan sulfate, DS; chondroitin sulfate, CS). Following BMP implantation into mouse muscle tissues, undifferentiated mesenchymal cells were found to infiltrate in situ, and C4S and KS were highly expressed at the initial phase. On day 5, undifferentiated cells expressed positive immunoreactivity for C4S, C6S, KS, CS, and DS, and chondroid matrices stained positively for C4S, C6S, KS, and DS, but weakly for CS. On day 7, they resemble hyalin cartilage tissues with positive reaction for all GAGs. In the cartilage cells, the expression of KS was particularly prominent. On day 14 to 21, chondro-osseous transformation was progressed with gradual calcification and with reduced stainings for all GAGs in their matrices. The calcified bone tissue indicated negative or trace positive reaction for GAGs. During BMP induced chondro-osteogenesis, the expression and accumulation of GAGs in the intercellular matrix play significant roles in chondrogenesis preceding endochondral ossification.
Localization of membrane-bound proteases, glutamyl aminopeptidase (EAP), microsomal alanyl aminopeptidase (mAAP), dipeptidyl peptidase IV (DPP-IV), and γ-glutamyl transpeptidase (γ-GTP), which may participate in the metabolism of various peptides, was studied in guinea pig subfornical organ (SFO). The reaction products of these enzymes were localized in both small vessels located in the central part of SFO and large vessels present in the peripheral part of SFO. The small vessels showed positive enzyme activity for mAAP and γ-GTP, traces for EAP, but a negative reaction for DPP-IV. The large vessels showed positivity in various intensities for mAAP, EAP, and DPP-IV; however, γ-GTP enzyme activity was not present. In the ependyma which cover the SFO, the enzyme-histochemical reactions for mAAP, EAP and DPP-IV were negative, and γ-GTP activity was not clearly demonstrated. In vessels of the choroid plexus in the vicinity of SFO, the reactions for mAAP and EAP were positive, but negative for DPP-IV and γ-GTP. Epithelium of this part of the choroid plexus was positive for γ-GTP but negative for mAAP, EPA, and DPP-IV. Differences in the location and enzyme activity of mAAP, EAP, DPP-IV, and γ-GTP in different vascular compartments, ie., differences among small and large vessels of guinea pig SFO, and vessels of choroid plexus in the vicinity of the SFO, suggest different roles for vascular endothelium in influencing the blood-borne peptides which would be dependent on the spectrum of membrane-bound peptidases present on cells in these structures.
Cysteine endoprotease activity was demonstrated in chicken osteoclasts using an azo-dye method. The activity was also observed in cartilage matrix where an active osteoclast attacked, but not in bone matrix. No significant activity was seen in osteoblasts and osteocytes. The enzyme hydrolyzed benzyloxycarbonyl-L-phenylalanyl-L-arginine-4-methoxy-2-naphthylamide (Z-Phe-Arg-MNA), but not Z-Arg-Arg- nor Z-Ala-Arg-Arg-MNA. Furthermore, the activity was dependent on mercaptoethylamine, an SH-reagent. A glutaraldehyde-containing fixative was needed for demonstration of the activity. There was no significant activity in 4% paraformaldehyde-fixed samples, suggesting that this soluble enzyme protein might diffuse out from the cell during histochemical procedures in mild fixed samples. This is the first report showing the presence of SH-reagent dependent Z-Phe-Arg-MNA hydrolyzing activity in osteoclasts in vivo.
We have studied the cytokeratin expression in subcolumnar reserve cells of the human uterine cervix using the immunohistochemical method. Five commercially available cytokeratin antibodies (AE-3, M630, M717, LL002, and CAM5.2) were used to stain a normal uterine cervix. Endocervical columnar cells stained strongly with CAM5.2, which reacts with low molecular-weight keratins (simple epitheliumtype). The stratified squamous epithelium stained strongly in all layers with M630, which reacts with high molecular-weight keratins (squamous-type). Subcolumnar reserve cells stained not only simple epithelium-type cytokeratin (CAM5.2) but also squamous-type cytokeratins (M630, LL002). This data presented so far indicates that subcolumnar reserve cells exhibit squamous characteristics. In addition, they also exhibit the cytokeratins typically found in non-stratified (simple) epithelia including endocervical columnar cells. This supports the notion that subcolumnar reserve cells have the potential to give rise not only to squamous epithelial cells but also to columnar cells.
Purified polyclonal antibodies have been obtained against 100KD and 36KD Ag-NOR proteins. These two antibodies have been used to study the immunolocalization of both proteins on different cell types subjected to various conditions of cell activity and to observe a nucleolar labelling with anti-36kDa Ag-NOR protein antibody evenly distributed over the dense fibrillar component and granular component, and with anti-100kDa mainly localized to the dense fibrillar component. After actinomycin D treatment, immunolabelling with both antibodies was found restricted to the granular zone of segregated nucleoli. Moreover, to further study the functional role of these proteins, we have used an electroporation system to introduce both antibodies into the nuclei of living cells, and have clearly detected a different nucleolar behaviour. The combination of these techniques has been shown to be an important tool to deepen the knowledge of the nucleolus and has allowed us to propose that the difference in nucleolar localization of the major 36kDa and 100kDa Ag-NOR proteins is the consequence of a different role of both proteins in the synthesis and processing of rRNA.