Quantitative immunocytochemical studies on the gonadotrophs isolated from the pituitary of the male rat

Summary Quantitative studies on the population of the gonadotropic cells in the pituitary of adult male rats were performed after trypsic dissociation of the pituitary glands and immunoenzymatic staining with anti--LH or anti--FSH antisera. Number, area and extinction of labelled cells were measured by use of an image analyser and a cytophotometer. The gonadotrophs represent approximately 14% of the pituitary cells. The mean area of gonadotrophs is significantly larger after staining with anti--LH serum than after staining with anti--FSH serum. Planimetric measurement of the gonadotrophs reveals their variability in size ranging between 30 and 160 m². Moreover, the size distribution depends on the staining serum used: more numerous small-sized cells (<75 m²) are stained with anti--FSH serum than with anti--LH serum, which conversely stains more numerous large-sized cells. Cytophotometric measurements indicate that immunostaining varies greatly among cells of the same size class and that the staining intensity appears to increase according to the cell size. These results emphasize the morphofunctional heterogeneity of the gonadotropic cell population.     

Comparative residual toxicities of pesticides to the predator Agistemus industani (Acari: Stigmaeidae) on citrus in Florida

Residual toxicities of registered and selected experimental pesticides used on citrus against Agistemus industani Gonzalez (Acari: Stigmaeidae) were compared. Pesticides considered highly toxic to A. industani were: abamectin 0.15 EC at 731ml/ha+FC 435-66 petroleum oil at 46.8l/ha, pyridaben 75WP at 469g/ha, ethion 4EC at 7.01l/ha+FC 435-66 petroleum oil at 46.8l/ha, propargite 6.55 EC at 3.51l/ha, chlorfenapyr 2SC at 1.46l/ha applied alone or in combination with FC 435-66 petroleum oil at 46.8l/ha, sulphur 80DF at 16.81kg/ha, dicofol 4EC at 7.01l/ha, fenbutatin oxide 50WP at 2.24kg/ha, benomyl 50WP at 2.24kg/ha, benomyl 50WP at 1.68kg/ha+ferbam 76 GF at 5.60kg/ha, ferbam 76GF at 11.21kg/ha, neem oil 90EC at 46.8l/ha, and copper hydroxide DF (40% metallic copper) at 4.48kg metallic copper/ha+FC 435-66 petroleum oil at 46.8l/ha. Pesticides that were moderately to slightly toxic included: copper sulphate 98% at 4.48kg metallic copper/ha+FC 435-66 petroleum oil at 46.8l/ha, fenbuconazole 2F at 280ml/ha+FC 435-66 petroleum oil at 46.8l/ha, FC 435-66 petroleum oil applied alone at 46.8l/ha or 23.4l/ha, and diflubenzuron 25WP at 1.40kg/ha. Pesticides that were non-toxic included: fenbuconazole 2F at 585ml/ha, malathion 57EC at 5.85l/ha, FC 435-66 petroleum oil at 46.8l/ha, carbaryl 80S at 3.36kg/ha, chlorpyrifos 4EC at 4.68l/ha, and formetanate 92SP at 1.12kg/ha. Understanding the toxic effects of field weathered pesticides against key predacious mite species is important for effective IPM. The results of this study provide a comparison of direct and indirect toxic effects of various pesticides to A. industani under field conditions.     

Laminins, tenascin and type VII collagen in colorectal mucosa

Summary The distribution of different laminin polypeptides, type VII collagen and tenascin has been studied in adult and foetal colorectal mucosa by using the indirect immunofluorescence technique. Immunoreactivity for laminin 1 chain was located to basement membranes of epithelia, muscularis mucosae, and blood vessels, respectively in different segments of adult colon and rectum. Laminin 1 and 1 chains were additionally expressed in lamina propria. Laminin 2 chain was also found in lamina propria around the pericyptal fibrollasts. Immunoreactivity for laminin 2 chain was restricted to basement membranes in the muscularis mucosae and arteries. Laminin 3 and 3 chains, suggestive for laminin-5, were confined especially to surface epithelial basement membranes. Immunoreactivity for type VII collagen was confined to basement membrane of surface epithelium in a punctate manner, while that for tenascin was seen slightly more broadly in the basement membrane zone and also in the muscular layer. The distribution of laminin chains in 16-week-foetal colon mostly resembled that of corresponding adult tissue, although immunoreactivities for laminin 2 and 2 chains were lacking. Type VII collagen and the high molecular weight isoform of tenascin also absent from the foetal colon. The results show that the basement membrane of the surface epithelium of colon and rectum express the components of epithelial adhesion complex, laminin-5 (3-3-2) and type VII collagen, resembling in this respect small intestine and stomach while laminin-2 (2-1-1) appears to be associated with pericryptal fibroblasts, and laminin-1 (1-1-1) widely in most basement membranes.     

Characterization of the fibrillar layer at the epithelial-mesenchymal junction in tooth germs

A characteristic layer containing numerous fibrils is associated with the basement membrane of the inner enamel epithelium during the early stages of odontogenesis. However, its nature is not well understood. In this study, the layer was examined with high-resolution electron microscopy and immuno-histochemical staining. Tooth germs of monkeys (Macaca fuscata) were studied and each fibril in the layer was found to be a tubular structure, 8–9 nm in width, resembling a basotubule, the tubular structure previously observed in various basement membranes. The space between the fibrils was filled with a network formed by irregular anastomosing strands with an average thickness of 4 nm; these strands resembled the cords forming the network in the lamina densa of basement membranes. After immunoperoxidase staining, fine threads immunoreactive for laminin staining were seen winding along the strands of the network, and 1.5-nm wide filaments, immunoreactive for type IV collagen, took the form of a network arrangement. The 5-nm-wide ribbon-like structures associated with the strands were identified as heparan sulfate proteoglycan by immunostaining. These results are similar to those obtained for the cord network of the lamina densa. The fibrillar layer therefore represents a highly specialized lamina fibroreticularis of the basement membrane of the inner enamel epithelium, and rich in basotubules.     

Changes in the distribution of integrins and their basement membrane ligands during development of human thyroid follicular epithelium

Interactions between cells and basement membrane components are crucial for the regulation of epithelial cell differentiation and polarization. We have studied by immunohistochemical methods the distribution of integrin adhesion proteins and some of their basement membrane ligands in foetal (16--19 weeks) and adult thyroid follicular epithelia. A diffuse immunoreactivity for only 3, v and 1 integrins was found in foetal follicular epithelium, whereas in adult follicular epithelium these integrins were expressed basally in a polarized manner. Additionally, 3 integrin was seen in a more basolaterally confined manner in adult follicular epithelium. Among basement membrane components, laminin 1, 1, 1 and 2 chains were found in epithelial basement membranes of the foetal thyroid gland, suggestive of the presence of laminins-1 and -3. In contrast, the basement membranes of adult follicular epithelium presented a much weaker immunoreactivity for the laminin 2 chain. Furthermore, immunoreactivity for the laminin 2 chain was occasionally seen in adult thyroid glands, apparently confined to myofibroblasts. Immunoreactivity for type IV collagen 1 and 2 (IV) chains was found in follicular basement membranes of foetal as well as adult thyroid gland. The results suggest that during maturation of foetal thyroid follicular epithelium a distinct polarization of integrins takes place. In mature thyroid follicular epithelium, the presumable adhesion-mediating integrin complexes are 31, v1 and/or v3 mediating adhesion to laminin-1 (1-1- 1) and type IV collagen trimer 12 (IV)     

γδ T-cell receptor repertoire in human peripheral blood and thymus

T-cell clones expressing the T-cell receptor (Tcr) were generated from peripheral blood lymphocytes (PBLs) and from a thymus sample. In the panel of ten thymus-derived clones, four Tcr phenotypes [as defined by the reaction of monoclonal antibodies (mAbs) directed against known V and V regions] were identified. All the clones lacked expression of the V3 V region, while seven clones were V1+ . V1 was found in combination with V9 or with undefined VVregions. In addition, two other Tcr phenotypes were identified on these clones: V9⁺ V1^– V3^– and V9^– V1^– V3^– One of the clones expressed CD4 and another was CD8positive. The remaining clones were CD4^– CD8^–. In the panel of 76 PBL-derived, Tcr-bearing clones, five Tcr phenotypes could be identified. In contrast to the thymus-derived clones, 30% of the clones were V3⁺ whereas V1 was expressed by a minority of the clones only. One clone was CD4-positive and approximately 30% of the clones were CD8-positive. Four of the five mAb-defined Tcr phenotypes could be identified on both thymus and PBL-derived T-cell clones. However, biochemical analysis of the Tcrs demonstrates differences in the usage of Ct- and C2-encoded y chains by T cells derived from the thymus and PBLs. The results therefore indicate that, at the clonal level, similarities and differences exist between the Tcr repertoires expressed in the thymus and by PBLs. Furthermore, they indicate that combinatorial Tcr heterogeneity is larger than has so far been described. The receptor diversity, combined with the potential of Tcr+ cells to express CD4 or CD8, indicates that these cells are a heterogeneous population that might mediate a number of immune functions.     

Lectin cytochemistry of cell types in human and canine pituitary

Summary Labeled lectins specific for different sugars were employed to identify different cell types in pituitaries from six human autopsies and seven dogs. To determine the lectins bound by each cell type, fixed-paraffin embedded sections serial to those stained with lectins were immunostained for specific hormones and the serial pairs were examined in a comparison microscope. In human pituitaries corticotrophs stained selectively with lectins having affinity for -l-fucose and the core region of complex type N-glycosyl-proteins. Some corticotrophs also stained for the presence of terminal -galactose. Thyrotrophs stained selectively with a periodate oxidation-borohydride reduction-concanavalin A sequence. Some mammotrophs evidenced content of glycoconjugate with terminal -galactose. Dendritic cells stained selectively for abundant glycogen with the periodate-reduction-concanavalin A sequence and a lectin from Griffonia simplicifolia. Adenohypophyseal cells of dog pituitary differed in showing absence of terminal -galactose in corticotrophs, presence of terminal -galactose in thyrotrophs, presence of glycoconjugate with N-glycosidically bound oligosaccharide in thyrotrophs and gonadotrophs and presence of terminal -galactose with a different lectin affinity in mammotrophs. The main contributions of lectin histochemistry applied to the pituitary include providing an additional histologic method for identification of some cell types, and localizing glycosylated prohormone or other biochemically unrecognized non-hormone glycoconjugates whose function in pituitary cells remains to be explained.This research was supported by NIH Grants AM-10956 and HL-29775 and United Health and Medical Research Foundation of South Carolina, Inc. Grant #79     

A morphometric analysis of the subcellular distribution of LHβ and FSHβ in secretory granules in the pituitary gonadotrophs of the frog (Rana japonica)

Immunohistochemical localization of lutropin (LH) and follitropin (FSH) in the pituitary gland of the frog Rana japonica was studied by the peroxidase-anti-peroxidase method and the two-face, double-labeling method with different-sized gold particles at the light-and electron-microscopic levels, respectively, using monoclonal antibodies against bullfrog LH and FSH. Light-microscopic immunohistochemistry indicated that approximately 66.0% of all the gonadotrophs in the pituitary contained both LH and FSH, whereas 33.4% of gonadotrophs contained only LH, and 0.6% contained only FSH. The staining intensity of LH and FSH varied from cell to cell. The gonadotrophs were classified into four types (Types I–IV) in terms of their ultrastructural and immunolabeling characteristics. Moreover, several secretory granule types were recognized according to differences in their shape and electron density. In all the cell types, both LH and FSH were often seen in the same secretory granules, but the proportion of granules bearing both hormones ranged from 5.5% in Type I to 32.7% in Type IV. Most secretory granules in Types I and II were immunolabeled with LH alone, whereas a small number of granules were immunolabeled with FSH alone. More immunolabeled FSH granules were present in Types III and IV than in Types I and II.     

The linear tetrasaccharide,Galβ1-4GlcNAcβ1-6Galβ1-4GlcNAc,isolated from radiolabeled teratocarcinoma poly-N-acetyllactosaminoglycan resists the action ofE. freundii endo-β-galactosidase

A novel linear tetrasaccharide, Gal1-4GlcNAc1-6Gal1-4GlcNAc, was isolated from partial acid hydrolysates of metabolically labeled poly-N-acetyllactosaminoglycans of murine teratocarcinoma cells. It was characterized by exo-glycosidase sequencing and by mild acid hydrolysis followed by identification of all partial cleavage products. The tetrasaccharide, and likewise labelled GlcNAc1-6Gal1-4GlcNAc, resisted the action of endo--galactosidase (EC 3.2.1.103) fromE. freundii at a concentration of 125 mU/ml, while the isomeric, radioactive teratocarcinoma saccharides Gal1-4GlcNAc1-3Gal1-4GlcNAc and GlcNAc1-3Gal1-4GlcNAc were cleaved in the expected manner.Abbreviations WGA wheat germ agglutinin - BSA bovine serum albumin - [³H]GlcNAc1-4-GlcNAc1-4GlcNAcOL N,N,NN'-triacetylchitotriose reduced with NaB³H₄     

Binding of the galactose-specificPseudomonas aeruginosa lectin,PA-I,to glycosphingolipids and other glycoconjugates

The carbohydrate-binding specificity ofPseudomonas aeruginosa lectin I (PA-I) in iodinated or biotinylated form was studied. A large number of glycosphingolipids, as well as some glycoproteins and neoglycoproteins were used as ligands. Also, inhibition by free saccharides of PA-I binding to glycosphingolipids was tested. It was found that the lectin binds most strongly to terminal and nonsubstituted Gal3Gal- or Gal4Gal-structures.Abbreviations PA-I Pseudomonas aeruginosa lectin I - Cer ceramide - lactosylceramide Gal4GlcCer - iso globotriaosylcerami Gal3Gal4GlcCer - globotriaosylceramide Gal4Gal4GlcCer - globoside or globotetraosylceramide GalNAc3Gal4Gal4GlcCer - Forssman glycolipid GalNAc3GalNAc3Gal4Gal4GlcCer - P1 glycolipid Gal4Gal4GlcNAc3Gal4GlcCer - lactoneotetraosylceramide Gal4GlcNAc3Gal4GlcCer - B5 glycolipid Gal3Gal4GlcNAc3Gal4GlcCer - gangliotetraosylceramide Gal3GalNAc4Gal4GlcCer - GM1 Gal3GalNAc4(NeuAc3)Gal4GlcCer - RBC red blood cells - BSA bovine serum albumin - PBS phosphate-buffered saline - SDS sodium dodecyl sulfate - TLC thin-layer chromatography - HPLC high pressure liquid chromatography - MS mass spectrometry - FAB fast-atom bombardment - EI electron impact     

Electron microscopic and cytochemical study on the role of Golgi elements and plasma membrane of enterocytes in the intestinal lipid transport

Summary Intestinal lipid absorption and transport were investigated in albino rats. The observations point towards the existence of a continuity between plasma membrane invaginations and elements of the Golgi complex on its mature face. They also suggest a segregation of lipid droplets by paired Golgi membranes and plasma membrane invaginations. The following way for lipid transport is deduced: lipid droplets moving inside the smooth endoplasmic reticulum accumulate progressively and are condensed in Golgi cisternae of the forming face. Their limiting membrane ruptures and liberated lipid droplets are segregated by paired Golgi membranes of the mature face or by plasma membrane invaginations. Subsequently the inner of the two segregating membranes disappears while the lipid droplet is moved towards the intercellular space inside a canal communicating with this space. The suggestion is made that the Golgi apparatus is of double origin: one component representing a terminal plication of the endoplasmic reticulum; the second one—a terminal plication of the plasma membrane invagination. This concept explains the ultrastructural and histochemical differences between Golgi membranes of the forming and mature faces of the complex.     

Carbohydrate antigen expression in murine embryonic stem cells and embryos. II. Sialylated antigens and glycolipid analysis

Summary A mouse embryonic stem (ES) cell line E14 and early mouse embryos were stained with a panel of 15 monoclonal antibodies recognizing sialylated or potentially sialylated carbohydrate determinants, Sialyl Le-x and sialyl Le-a were detected on the pre-implantation embryo from the 8-cell stage, and sialyl Le-a weakly on undifferentiated ES cells. Changes in cell surface carbohydrates occurred after induction of ES cell differentiation with retinoic acid (RA) and dibutyryl cAMP. Qualitative analysis of the neutral glycolipids of untreated and RA-treated ES cells using high-performance thin-layer chromatography (HPTLC) revealed few differences between the two types of culture. The major gangliosides in both cultures were indicative of an active a ganglioside synthesis pathway. GD₃, a precursor of the b synthesis pathway, previously reported to be characteristic of embryonal carcinoma (EC) cells, was absent. RA-induced differentiation caused a shift in the spectrum to more complex gangliosides. Application of fast atom bombardment mass spectrometry (FAB-MS) to permethylated derivatives of individual bands permitted partial characterization of an unusual sialylated glycolipid and a rare ganglioside with the suggested structure of GalNAc-GD1a.Abbreviations NeuAc N-acetylneuraminic acid - Cer ceramide - CMH monohexosylceramide - CDH lactosylceramide (Gal1-4Glc1-Cer) - CTH ceramide trihexoside (Gal1-4Gal1-4Glc1-Cer) - globoside (GalNAc1-3 Gal1-4Gal1-4Glc1-Cer) - Forssman antigen (GalNAc1-3GalNAc1-3Gal1-4Gal1-4Glc1-Cer) - GM₃ (NeuAc2-3Gal1-4Glc1-Cer) - GD₃ (NeuAc2-8NeuAc2-3Gal1-4Glc1-Cer) - GM1 (Gal1-3GalNAc1-4[NeuAca2-3]Gal1-4Glc1-Cer) - GD1a (NeuAc2-3Gal1-3GalNAc1-4[NeuAc2-3]Gal1-4Glc1-Cer) - GT1b (Neu5Ac2-3Gal1-3GalNAc1-4[Neu5Ac2-8Neu5Ac2-3]Gal1-4Glc1-Cer) The glycolipids are named according to Svennerholm (1963) and the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (1978).     

Ovarian development in Culex nigripalpus and its implication for disease transmission

Ovarian development was investigated in wild and colonized Culex nigripalpus Theobald females. Blood-seeking females with morphologically distinct nulliparous, parous and resorbed ovarian follicles (as opposed to females with degenerating follicles) were collected from the field all year, indicating year around gonoactivity. During the cooler months a larger number of females with resorbed follicles were collected from the field than in warmer months. Laboratory experiments showed that resorbed ovarian follicles occurred in sucrose fed females when deprived of a blood meal after emergence. These resorbed ovarian follicles were functional, since most of them began to develop and mature after the females took a blood meal to repletion. Among females fed a partial blood meal, some did not begin ovarian development and, therefore, exhibited gonotrophic dissociation. Females fed partial blood meals had similar patterns of ovarian development, irrespective of temperature at which they were maintained. Implications of occurrence of females with resorbed ovarian follicles and disease transmission are discussed.Supported in part by National Institutes of Health Research Grants No. A1-06587 and RR-07021. A contribution of the Florida Medical Entomology Laboratory, Vero Beach, Florida, Florida Agriculture Experimental Station Journal Series No. 2367     

In vitro comparison of ceftazidime,aztreonam, cefpirome,gentamicin, imipenem,enoxacin, and ticarcillin plus clavulanic acid against 108 strains ofPseudomonas maltophilia

Pseudomonas maltophilia is an uncommon cause of hospital-acquired infection and is resistant to most of the antimicrobial agents used in the treatment of gram-negative infections. Susceptibility of 108 isolates ofP. maltophilia to ceftazidime, aztreonam, defpirome, gentamicin, imipenem, enoxacin, and ticarcillin plus clavulanic acid was determined by an agar dilution method. The isolates were in general resistant to the antibiotics. Imipenem and cefpirome were not active at clinically achievable levels. Of the isolates, 20% were susceptible to 16 g/ml ceftazidime, 53% were susceptible to 4 g/ml enoxacin, 10% were susceptible to 4 g/ml gentamicin, and 25% were susceptible to 64 g/ml ticarcillin plus 2 g/ml clavulanic acid.     

α-Thalassemia and the production of different α chain variants in heterozygotes

The production of five chain variants (Hb G-Georgia, Hb St. Luke's, Hb Lloyd, Hb Montgomery, and Hb G-Philadelphia) in heterozygotes was evaluated through hematological observations, hemoglobin quantification, and biosynthetic studies. All heterozygotes for Hb St. Luke's and Hb Lloyd and most heterozygotes with Hb G-Georgia and Hb Montgomery had normal hematology and average / values of about 1.1. They were assigned a normal genotype (^G/), although the proportions of Hb St. Luke's and Hb G-Georgia were low (10 to 13%) and those of Hb Lloyd and Hb Montgomery twice as high (20%). Data from short-term incubations confirmed this genotype for some of these heterozygotes. Isolated Hb St. Luke's and Hb G-Georgia gave low ^G/ values (0.2 and 0.3) indicating that these Hb variants were defective at the level of Hb assembly. Isolated Hb Montgomery and Hb G-Philadelphia, however, gave higher ^G/ values of 0.6 and 0.8, respectively. A second type of variability existed among Hb G-Georgia (20 vs. 13%), Hb Montgomery (28 vs. 20%), and Hb G-Philadelphia (47 vs. 34%) heterozygotes, in whom the levels of Hb G differed. The occurrence of higher levels of these three chain heterozygosities was associated with hematological or biosynthetic evidence of a mild or moderate chain deficiency due to an -thalassemia-2 heterozygosity (^G/⁰ or ^0G/) or a homozygosity (^0G/⁰), respectively.This study was supported in part by USPHS Research Grants HLB-05168 and HLB-15158.     

Expression of a chimeric stilbene synthase gene in transgenic wheat lines

A chimeric stilbene synthase (sts)gene was transferred into wheat. Stilbene synthases play a role in the defence against fungal diseases in some plant species (e.g. groundnut or grapevine) by producing stilbenetype phytoalexins like resveratrol. Resveratrol is also claimed to have positive effects to human health. Embryogenic scutellar calli derived from immature embryos of the two commercial German spring wheat cultivars Combi and Hanno were used as target tissue for cotransformation by microprojectile delivery. The selectable marker/reporter gene constructs contained the bargene either driven by the ubiquitinpromoter from maize (pAHC 25, also containing the uidAgene driven by the ubiquitinpromoter), or by the actinpromoter (pDM 302) from rice. The cotransferred plasmid pStil 2 consisted of a grapevine stscoding region driven by the ubiquitin promoter. Eight transgenic Combi and one Hanno T_Oplant were obtained and, except one Combi T_Oplant, found to be cotransformants due to the integration of both the stsgene and the selectable marker or reporter genes. Expression of the stsgene was proven by RTPCR, and, for the first time, by detection of the stilbene synthase product resveratrol by HPLC and mass spectrometry. The stsgene was expressed in four of the seven transgenic Combi T_{_o}plants. Two of the respective T₁progenies segregated in a Mendelian manner were still expressing the gene. Investigations into methylation of the stsgene showed that in three nonexpressing progenies inactivation was paralleled by methylation.     

Molecular complexity of the cutaneous basement membrane zone

Ultrastructural examination of the cutaneous basement membrane zone (BMZ) reveals the presence of several attachment structures, which are critical for integrity of the stable association of epidermis and dermis. These include hemidesmosomes which extend from the intracellular compartment of the basal keratinocyte to the underlying basement membrane where they complex with anchoring filaments, thread-like structures traversing the lamina lucida. At the lower portion of dermal-epidermal attachment zone, anchoring fibrils extend from the lamina densa to the papillary dermis, where they associate with basement membrane-like structures, known as anchoring plaques. Molecular cloning of the cutaneous BMZ components has allowed elucidation of the structural features of the proteins which constitute these attachment structures. Specifically, hemidesmosomes have been shown to consist of at least four distinct proteins. The intracellular hemidesmosomal inner plaque is comprised of the 230-kD bullous pemphigoid antigen (BPAG1), and plectin, a high-molecular weight cytomatrix protein, encoded by the corresponding gene, PLEC1. The transmembrane component of the hemidesmosomes consists of the 180-kD bullous pemphigoid antigen (BPAG2), a collagenous protein also known as type XVII collagen (COL17A1), as well as of the basal keratinocyte-specific integrin 64. The anchoring filaments consist predominantly of laminin 5 with three constitutive subunit polypeptides, the 3, 3 and 2 chains, which is associated with laminin 6 with the chain composition 3, 1 and 1. Also associated with anchoring filaments is a novel protein, ladinin, which serves as autoantigen in the linear IgA disease, and the corresponding gene, LAD1, has been mapped to human chromosome 1. Finally, the major, if not the exclusive, component of anchoring fibrils is type VII collagen, encoded by the gene (COL7A1) which consists of 118 distinct exons, the largest number of exons in any gene published thus far. Collectively, the cutaneous basement membrane zone is a complex continuum of macromolecules which form a network providing the stable association of the epidermis to the underlying dermis. Thus, genetic lesions resulting in abnormalities in any part of this network could result in a blistering skin disease, such as epidermolysis bullosa.Abbreviations BMZ basement membrane zone - EB epidermolysis bullosa - JEB junctional EB - GABEB generalized atrophic benign EB - EB-MD epidermolysis bullosa with muscular dystrophy - EB-PA epidermolysis bullosa with pyloric atresia     

Benzyl-N-acetyl-α-d-galactosaminide inhibits the sialylation and the secretion of mucins by a mucin secreting HT-29 cell subpopulation

We have analysed the mucins synthesized by the HT-29 MTX cell subpopulation, derived from the HT-29 human colon carcinoma cells through a selective pressure with methotrexate (Lesuffleuret al., 1990,Cancer Res 50: 6334–43), in the presence of benzyl-N-acetyl--galactosaminide (GalNAc-O-benzyl), which is a potential competitive inhibitor of the 1,3-galactosyltransferase that synthesizes the T-antigen. The main observation was a 13-fold decrease in the sialic acid content of mucins after 24 h of exposure to 5mm GalNAc-O-benzyl. This effect was accompanied by an increased reactivity of these mucins to peanut lectin, testifying to the higher amount of T-antigen. The second observation was a decrease in the secretion of the mucins by GalNAc-O-benzyl treated cells. The decrease in mucin sialyation was achieved through thein situ -galactosylation of GalNAc-O-benzyl into Gal1–3GalNAc-O-benzyl, which acts as a competitive substrate of Gal1–3GalNAc 2,3-sialyltransferase, as shown by the intracellular accumulation of NeuAc2–3Gal1–3GalNAc-O-benzyl in treated cells.Abbreviations BSM bovine submaxillary mucin - MTX methotrexate - PBS sodium phosphate 10mm, NaCl 0.15m, pH 7.4 buffer - pNp p-nitrophenol - TBS Tris/HCl 10mm, NaCl 0.15m, pH 7.4 buffer Enzymes: CMP-NeuAc: Gal1–3/4GlcNAc 2,3-sialyltransferase, ST3(N), EC 2.4.99.6; CMP-NeuAc: Gal1–4GlcNAc 2,6-sialyltransferase, ST6(N), EC 2.4.99.1; CMP-NeuAc: Gal1–3GalNAc 2,3-sialyltransferase, ST3(O), EC 2.4.99.4; CMP-NeuAc: R-GalNAc1-O-Ser 2,6-sialyltransferase, ST6(O)-I, EC 2.4.99.3; CMP-NeuAc: NeuAc2–3Gal1–3GalNAc 2,6-sialyltransferase, ST6(O)-II, EC 2.4.99.7; UDP-GlcNAc: Gal1–3GalNAc-R·(GlcNAc to GalNAc) 1,6-N-acetylglucosaminyltransferase, EC 2.4.1.102; UDP-GlcNAc: GalNAc-R 1,3-N-acetylglucosaminyltransferase, EC 2.4.1.147; UDP-Gal: GalNAc-R 1,3-galactosyltransferase, EC 2.4.1.122.