Concanavalin A binding sites of rough endoplasmic reticulum containing intracisternal microtubules in canine neurones

Lectin histochemistry was used to identify sugar residues of IM-containing RER in elderly canine sympathetic ganglionic neurones. IM-inclusions stained with ConA-peroxidase conjugate, but not with soybean agglutinin (SBA), wheat germ agglutinin (WGA), peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA), Ricinus communis agglutinin (RCA I) and Ulex europaeus agglutinin (UEA I). ConA-binding sites were visualized within cisternae of RER containing IM; reaction product was localized in IM-containing RER cisternae and IM. Inhibition with specific sugars (0.1 M alpha-methyl-D-mannoside or 0.5 M D-glucose) blocked the binding of ConA to IM-inclusions and normal Nissl substance. When a low sugar concentration (5 x 10(-3) M alpha-methyl-D-mannoside or 0.2 M D-glucose) was employed, IM-inclusions were still strongly ConA-positive, but normal Nissl substance was not. These results demonstrate that IM-containing RER have an excessive amount of carbohydrates (mannose or glucose-rich sugars) which are essentially detected in flattened RER under normal conditions and further indicate that glycoproteins in IM differ from those in cytoplasmic microtubules.     

Histochemical and morphologic studies of mucosa bordering rectosigmoid carcinomas: comparisons with normal, diseased, and malignant colonic epithelium

Surgically obtained rectosigmoid mucosa ("transitional" mucosa, TM) adjacent to eight primary carcinomas was compared with diseased mucosa (DM) from eight patients without primary carcinoma and mucosa from two normal control subjects by mucin histochemical and morphologic techniques. No differences were found between TM and DM that might have suggested premalignant changes unique to TM. An excess of sialidase-susceptible sialomucins was found in both TM and DM, as was loss of the sulfomucin-sialomucin gradient usually found between normal crypts and surface cells. Increased sialic acid in TM and DM may represent a nonspecific response to injury or inflammation and has been found in other epithelia under similar circumstances. Sialidase also induced substantial reduction of periodic acid-Schiff (PAS) staining, probably due to loss of sialic acid since no other sugars were released during sialidase digestion, as determined by thin-layer chromatography analysis of post-digestion supernatants. Carcinomas generally showed more staining with PAS than with basic dyes; PAS staining was minimally reduced by diastase and sialidase but markedly reduced by phenylhydrazine interposition, suggesting that some type of neutral glycoprotein was responsible. Finally, it was found that overreliance on the high-iron diamine-Alcian blue technique as a single procedure is unwise; this procedure should be accompanied by the use of singly applied dyes, especially high-iron diamine, together with other enzymatic and staining procedures.     

Peanut lectin binding sites in human fetal colon

We studied peanut lectin (PNA) binding sites in human fetal colons (9 to 19 weeks' gestational age). Peanut lectin has a specificity for beta-D-galactose(1--3)N-acetyl-D-galactosamine (beta-D-Gal [1----3]-D-GalNac) that is the purported determinant for the T-blood-group antigen (TAg) and a precursor of MN-blood-group substance, lacking only in a terminal sialic acid. In the normal adult human colon, PNA fails to bind to the actual goblet theca but does have binding sites localized to the supranuclear portion of both columnar and goblet cells. This represents the detection of nascent oligosaccharides prior to addition of terminal sialic acid. Neuraminidase treatment of adult colons localized PNA to the goblet theca itself and to the apical and/or glycocalyx region of columnar cells. Fetal colons localized PNA to the region of the glycocalyx of columnar cells while the goblet theca itself failed to express PNA binding sites. After treatment of fetal colon sections with neuraminidase, PNA binding was noted in the goblet theca itself. Goblet cells of the human fetal colon exhibit a PNA binding pattern somewhat similar to adult goblets; however, fetal columnar cells have a PNA binding pattern as reported in colonic adenocarcinomas. This pattern of complete and incomplete synthesis of MN-blood-group substances in goblet and columnar cells, respectively, has also been demonstrated in adenomas of the human colon.     

A morphometric assessment of transitional mucosa in the colon

Quantitative morphometric analysis was used in 10 resection specimens to assess so-called transitional mucosa immediately adjacent to colorectal carcinoma. Eleven nuclear and cellular variables were measured from the malignant epithelial area and from zones of increasing distance (1 cm) from the lesion. In addition, mean mucosal height was assessed for each zone. Morphometrical differences between the mucosa immediately adjacent to the malignant epithelium and that taken at some distance from it were determined by Mann-Whitney U tests. Transitional mucosa showed increased mucosal height but no nuclear differences from normal mucosa. Other work has shown that there are nuclear morphometric differences associated with premalignant conditions in the colon. Thus, the suggestion that transitional mucosa represents early neoplastic change cannot be supported.     

Concanavalin a binding and neuronal differentiation

Summary Concanavalin A (Con A) acceptors have been demonstrated in large differentiated neurons in a previous paper. In order to elucidate the correlation between Con A binding in normal and neoplastic neurons and lectin binding dependence upon the differentiation grade, 26 tumours of the neuronal series were examined using formalin fixed and paraffin embedded biopsy specimen. The neoplasms included 3 gangliocytomas, 7 gangliogliomas, 1 central neuroblastoma, 11 medulloblastomas, 2 retinoblastomas, and 2 sympathicoblastomas. Well differentiated neurons in gangliocytomas and gangliogliomas expressed a high intracytoplasmic Con A acceptor density comparable to the feature in large non-neoplastic neurons. Less differentiated neurons and neuroblasts showed a weak perinuclear fine granular binding or an absolute lack of binding molecules, respectively.Our results suggest that in a variety of tumours, Concanavalin A receptor density in neurons depends upon the degree of differentiation of the cell. Well differentiated cells have a higher density than poorly differentiated neoplastic neurons.Supported by a grant of the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg     

Concanavalin A receptor sites on lymph node cells in vivo and in vitro

The distribution and density of receptors for concanavalin A (Con A) on the surfaces of cells of intact and isolated popliteal and axillary lymph nodes were investigated in the rabbit. Intact lymph nodes were perfused via the subcapsular (marginal) sinus with either Con A peroxidase or Con A ferritin, fixed with glutaraldehyde, and processed for electron microscopy. Both Con A peroxidase and Con A ferritin were distributed on the plasmalemma of lymphocytes, macrophages, neutrophils, plasma cells, reticular endothelial cells, and the vascular endothelium. Counts of Con A-conjugated ferritin particles indicated that the density of Con A receptors was generally similar for lymphocytes, macrophages, and neutrophils but lower on plasma cells. When lymph node cells were isolated by mechanical methods and exposed to Con A ferritin, the label was homogenously distributed on the cell surfaces of most cells. However, Con A binding was significantly higher on the surface of isolated cells than in the intact node. It is suggested that the increase in density of Con A binding sites on isolated cells may possibly be due to an unmasking of cell surface moieties in which additional Con A receptor sites become available as a result of the isolation procedure. The density of Con A ferritin binding sites was also significantly lower on the surface of isolated plasma cells than the lymphocyte and macrophage, suggesting that the density distribution of cell surface saccharides is different for various lymphoid cells.     

Effect of cysteamine on cytosolic somatostatin binding sites in rabbit duodenal mucosa

Administration of cysteamine in rabbits elicited a rapid depletion of both duodenal mucosa and plasma somatostatin. A significant reduction was observed within 5 min, returning toward control values by 150 min. The depletion of somatostatin was associated with an increase in the binding capacity and a decrease in the affinity of both high- and low-affinity binding sites present in cytosol of duodenal mucosa. Incubation of cytosolic fraction from control rabbits with 1 mM cysteamine did not modify somatostatin binding. Furthermore, addition of cysteamine at the time of binding assay did not affect the integrity of 125I-Tyr11-somatostatin. It is concluded that in vivo administration of cysteamine to rabbits depletes both duodenal mucosa and plasma somatostatin and leads to up-regulation of duodenal somatostatin binding sites.     

GABAB binding sites in early adult and aging rat brain

The effect of aging on GABA_B binding was investigated in rat brain. Receptor autoradiography was used to investigate both GABA_B and GABA_A binding at 2 months, 3 months, 13 months, and 23 months. GABA_B binding decreases significantly between 2 months and 23 months of age, as does GABA_A binding, with was investigated in rat brain. Receptor autoradiography was used to investigate both GABA_B and GABA_A binding at 2 months, 3 months, 13 months, and 23 months. GABA_B binding decreases significantly between 2 months and 23 months of age, as does GABA_A binding, with greatest decrease between 2 and 3 months. The decrease in GABA_B binding appears to be due to a decrease in binding site affinity rather than a decrease in receptor density. The noncompetitive GABA_B antagonist zinc, the competitive GABA_B antagonist CGP 35348, and the guanyl nucleotide analogue GTP-γ-S all inhibit GABA_B binding identically in 2 month and 23 month brain. These data indicate subtle age-related changes in the GABA_B binding in early adult life but little change with senescence.     

[3H]Nicotine binding sites in developing fetal brains in rats

[³H]Nicotine binding sites were examined in developing fetal brains in rats. The fetal brain membranes bound [³H]nicotine with a similar affinity to that of adult brain membranes. This binding was displaced by unlabelled nicotine or carbamylcholine, the inhibition concentrations being approximately the same for fetal and adult brain preparations. α-Bungarotoxin had no effect on [³H]nicotine binding to fetal brain membranes as well as to adult brain preparations. The specific [³H]nicotine binding was first detectable on day 16 of gestation and it increased several folds until birth.     

Localization and quantification of [125I]-endothelin binding sites in human fetal and adult kidneys — Relevance to renal ontogeny and pathophysiology

Summary Endothelins, 21 amino acid peptides, produced by endothelial cells are potent vasoconstrictors and mitogens. According to experimental studies in animals, endothelins seem to be involved in the regulation of renal hemodynamics. In order to gain insight into its potential effects in man, a quantitative analysis of its binding sites was performed in human kidneys. Because of the proliferative action of endothelin in cell culture we also compared binding sites in fetal and adult kidneys. Binding sites for [¹²⁵I]-endothelin-1,2,3 were visualized by in-vitro autoradiography and quantified by densitometry. In both adult and fetal tissue, specific binding sites occurred in the cortex, medulla, and renal vessels. Unlabeled endothelins and sarafotoxin, a peptide with a high sequence homology to endothelins, inhibited [¹²⁵I]-endothelin-1 binding with IC₅₀ in the 9.8 to 0.023 nM range, whereas unrelated peptides (angiotensin II, atrial natriuretic peptide) and the calcium antagonist nitrendipine failed to compete for [¹²⁵I]-endothelin-1 binding sites. Linear Scatchard analysis revealed that the number of binding sites (expressed per tissue equivalent: TE) were consistently higher in fetal than in adult kidneys, while affinities did not differ significantly in cortex, medulla, and vessels (fetal/adult: cortex K_D 43.4±19.6/55.9±16.7 nM; B_Max13.5±7.8/2.7±1.3 fmol/mg TE; medulla K_D 26.3±10.9/34.6±7.4 nM; B_Max 10.1±0.9/ 3.7±1.1 fmol/mg TE; vessels K_D 41.1±22.9/ 23.7±8.1 nM; B_Max 12.9±3.9/4.1±1.2 fmol/mg TE). Medullary capillaries and veins showed strong binding in human and rat kidneys which may be important for the pathophysiology of acute renal failure. Human adult and fetal glomeruli had only a few binding sites. This contrasts to findings in the rat kidney in which glomeruli have a high concentration of endothelin binding sites; although this does not role out an influence per se, it does point out the need to subject the assumption of a relevant glomerular effect of endothelin in man to closer scrutiny. The diffuse and strong binding in fetal kidney may indicate a role for endothelin in the process of renal maturation.Abbreviations ET Endothelin - TE Tissue equivalent This work is dedicated to Prof. Dr. med. F. Scheler, head of the Department of Nephrology, University Hospitals, Göttingen on the occasion of his 65th birthday.     

Regional distribution of glycoconjugates in normal,transitional and neoplastic human colonic mucosa

Summary Regional distribution of glycoconjugates in normal and neoplastic colonic mucosa was studied by means of eight lectins:Dolichos biflorus (DBA),Glycine max (SBA),Triticum vulgare (WGA),Arachis hypogaea (PNA),Griffonia simplicifolia-I (GS-I),Canavalia ensiformis (Con A),Limax flavus (LFA), andUlex europaeus-I (UEA-I). The lectin binding patterns were examined in 40 normal colonic mucosa (NM) (12 proximal (P) and 28 distal (D)), 38 carcinomas (15 P and 23 D), and 31 transitional mucosa (TM) (9 P and 22 D). Sections of NM located 5 cm and 10 cm distant from the tumour and sections from the resection margins (more than 10 cm from the tumour) of the surgical specimens were also studied in 19 cases (6 P and 13 D). In NM, regional differences between the proximal and distal colon were detected with most lectins. DBA, SBA and LFA bound mainly to the goblet cell mucin of the distal colon, while GS-I and UEA-I labelling predominated in proximal colonic mucosa. The lectin reactivity in carcinomas was: DBA 26%, SBA 63%, PNA 95%, GS-I 66%, UEA-I 76%, WGA 100%, Con A 92% and LFA 42%. No regional differences were observed in the lectin patterns of proximal and distal colonic carcinomas nor was any relationship detected between lectin reactivities and Dukes stage, size or histological type of tumours. Transitional mucosa of both the proximal and distal colon showed an increase in PNA-binding and loss of DBA and SBA. LFA and UEA-I reactivity in proximal TM was similar to that observed in proximal NM. Distal TM showed a decrease inLFA labelling and the appearance of UEA-I reactivity in goblet cell mucin in 5 cases (23%). The reactivity of the other lectins was as with NM. The only change in normal mucosa distant from tumours was a focal increase in PNA reactivity in 4 cases. These findings suggest that carcinomas from different colonic regions have a more uniform distribution of carbohydrates than the respective NM.     

Localization of luteinizing-hormone releasing hormone binding sites in the gastric mucosa of suckling rats

Luteinizing-hormone releasing hormone (LHRH) is a hypothalamic and milk-borne hormone that inhibits the cell proliferation of gastric epithelium in developing rats, although the mechanism of such action is unknown. We investigated the presence of binding sites for LHRH in the stomach of suckling rats after the injection of the hormone. Immunofluorescence at the confocal microscopy level revealed that LHRH binds to gastric cells, being particularly abundant over the gland. Different fluorescent lectins were used to identify gastric cell types and determine which were labeled by the hormone. Colocalization studies in these double-labeling experiments showed that LHRH staining colocalizes with parietal cells, suggesting the presence of binding sites in these cells. The three-dimensional (3-D) reconstruction of isolated parietal cells revealed the localization of the signal, which appears to be in the membrane of the canalicular region. These results suggest that there are binding sites for LHRH in the gastric epithelium, specifically in parietal cells, and they might play a role in the control of cell proliferation during suckling.     

Chromogranin positive cells in colorectal carcinoma and transitional mucosa.

AIMS--Immunostaining of chromogranin identifies gastrointestinal mucosal endocrine cells. The detailed distribution and significance of chromogranin positive cells in colorectal carcinomas and in transitional mucosa remain unclear. The aim of this study was to clarify these aspects. METHODS--The distribution of chromogranin positive cells was studied by immunohistochemical methods in normal epithelium remote from carcinoma, in transitional mucosa, and in carcinomas of the colorectum. In selected cases northern or western blot analyses were performed. RESULTS--Chromogranin positive cells were seen in the lower third of the normal crypts and less frequently in transitional mucosa. Thirty five per cent (n = 38) of colorectal carcinomas showed immunohistochemically positive carcinoma cells in the tumour tissue. Northern and western blot analyses showed similar results. There was no difference in clinicopathological factors, including prognosis, between chromogranin positive cases of colorectal carcinoma (n = 38) and chromogranin negative cases (n = 70). CONCLUSIONS--Neuroendocrine cell differentiation is controlled in transitional mucosa and the presence of chromogranin positive cells in carcinoma tissue does not influence the patient's prognosis.     

L-CAM expression in normal, premalignant, and malignant colon mucosa.

L-CAM is a cell adhesion molecule which is expressed at the intercellular borders of most epithelial cells. L-CAM has been demonstrated to act as an invasion suppressor in carcinoma cell lines. In order to determine whether or not L-CAM expression might distinguish between invasive and non-invasive or metastatic and non-metastatic colon neoplasms, we studied L-CAM expression in normal colon mucosa, colon adenomas with various degrees of dysplasia, and colon carcinomas by immunohistochemistry, using the 6F9 monoclonal anti-L-CAM antibody. Normal mucosa showed evenly distributed distinct L-CAM immunoreactivity along intercellular borders. In adenomas and carcinomas, a similar though weaker expression was observed. This pattern showed a tendency to decrease in parallel with decreasing differentiation. However, no correlation was found with Dukes stage or area within the tumour. In some carcinomas, L-CAM was expressed at the luminal surface of the cells. In others, L-CAM expression was not found. These results suggest that L-CAM expression is disregulated or lost as an early event in the development of colon neoplasia and indicate that L-CAM expression does not correlate with invasive or metastatic potential.     

Differential binding sites of peroxidase-labelled lectins in the submandibular gland of sucking and adult cats

The glycosidic residues in the submandibular gland of cat at different ages were studied using a battery of 6 lectins conjugated with horseradish peroxidase as histochemical reagents. The submandibular glands of sucking and adult cats showed diversities in the reaction intensity or in the localization of binding sites. The morphologically distinct secretory tracts appear to produce different types of oligosaccharides in sucking and adult subjects. The sugars that can be defined as growth markers in the cat submandibular gland seem to be alpha-L-fucose, N-acetyl-D-glycosamine and beta-galactose. The data originated from this research have been compared with biochemical data previously obtained by the authors on the cat submandibular gland during growth.     

Altered expression of Lewis blood group and related antigens in fetal,normal adult and malignant tissues of the uterine endometrium

Summary The expression of the Lewis blood group and its related antigens in fetal, normal adult and malignant tissues of the uterine endometrium was examined immunohistochemically using a panel of mouse monoclonal antibodies with specificities for Lewis-a (La), Sialyl Lewis-a (SLa), Lewis-b (Lb), Lewis-X (LX), Sialyl Lewis-X (SLX) and Lewis-Y (LY) antigens. La, SLa and SLX having one fucose residue were detected in a small number of fetal tissues, while Lb and LY having two fucose residues were found in most cases. In the adult endometrium, expression of Lb and LY was considerably lower than those in fetal tissues, although expression of La and SLa was not different between these two tissues. Expression of LX and SLX was pronouned in adult when compared with fetal tissues. Malignant endometrial glands expressed La, SLa, Lb and LY, extensively, while LX and SLX were expressed less than in normal tissues.Lb and LY can thus be considered oncofetal antigens, extensively expressed in fetal and malignant tissues but not in normal adult tissues. Expression of Lb and LY was greater than that of La and SLA in carcinoma; an increase in the activity of fucose transferase might be associated with malignant transformation in the uterine endometrium.Supported in part by a Grant-in-Aid for Scientific Research (62480346) from the Ministry of Health and Welfare     

Cellular immunophenotypes in human embryonic, fetal and adult heart

The cellular immunoprofile of cardiac dysfunctions and lesions of ischemic etiology are insufficiently studied to date, especially regarding the contribution of non-cardiomyocytic structures. Aiming to explore this immunoprofile, we used immunohistochemistry applied on embryonic, fetal and adult normal or ischemic myocardium. We observed a decrease of smooth muscle alpha-actin expression in fetal vs. embryonic cardiomyocytes, its absence in normal adult myocardium and its intense expression in the fibrotic scars of ischemic myocardium. DDR2 and vimentin, which are present in the interstitial cells and cardiomyocytes of the embryo, fetus and normal adult heart, are absent in the fibrotic scar tissue and cicatricial infarction, the latter expressing smooth muscle alpha-actin and CD34. This suggested that myofibroblasts and not local fibroblasts that participate in ischemic remodeling. An EGFR-positive vascular network was better represented in the ischemic heart than in the adult normal one, a fact possibly related to EGFR implication in cardiac ischemic pre- and post-conditioning. Therefore, cardiomyocytes and non-cardiomyocytic cells have an undulating immunoprofile according to the intrauterine life stage or age after birth, and a variable contribution in cardiac lesions, mostly in ischemic ones.