Distribution of S-100 protein and its subunits in bovine exocrine glands

 The distribution of S-100 protein and its α- and β-subunits in bovine exocrine glands was studied by indirect immunohistochemistry. The entire spectrum of salivary glands, glands of the respiratory tract, intestinal glands, male and female genital glands, and skin glands was examined. S-100 and its β-subunit were identified in most serous secretory cells of mixed salivary glands, although secretory acini in some serous glands remained unreactive for these antigens. Mucous cells were constantly negative; mucoid cells were positive in the lacrimal and Harderian gland. The α-subunit of S-100 protein was identified in serous cells but the staining reaction was faint. Subunits of S-100 showed a characteristic distribution along the excretory duct systems of compound glands: S-100 and the β-subunit were present in intercalated duct epithelium, while striated duct epithelium stained for S100-α. Therefore, it is suggested that S100-α is related to resorption and secretion in striated ducts, while S100-β may govern acinar exocytosis and probably regulates proliferation and differentiation of glandular cells. Differing staining intensities for S-100 and its subunits in secretory cells of exocrine glands most probably indicate functional differences with regard to secretory activity and the cell cycle. Accepted: 11 February 1997     

Immunohistochemical studies of S-100 protein α and β subunits in adenoid cystic carcinoma of salivary glands

Immunohistochemical studies were performed to explore the distribution of S-100 protein and its α,β subunits in 76 adenoid cystic carcinomas (ACC) of the salivary glands. Histopathologically, ACC was divided into cribriform, tubular, basaloid and trabecular types which could be mixed in the same tumor. S-100 protein was usually positive in tumor cells forming cribriform structures; foci of strongly positive tumor cells were also distributed in the luminal layer of tubular structures, and in areas transitional between cribriform and tubular patterns. S-100 α staining was confined to some tumor cells in cribriform areas, to luminal tumor cells in tubular structures and to few tumor cells in basaloid structures. S-100β reaction was usually localized to luminal surfaces in a fine granular pattern in tubular and microtubular structures in a distribution somewhat similar to that in the normal salivary gland. Great heterogeneity in the immunohistochemical distribution of S-100, S-100 a and S-100β proteins was found in the various histologic types of ACC and the pattern was different from that seen in pleomorphic adenomas. It is possible that the ACC tumor cells positive for S-100 protein may be closely related to true or modified myoepithelial cells.     

Secretory cells of the airway express molecules of the chemoreceptive cascade

Airway secretion is maintained by specialized non-ciliated epithelial cells whose phenotype varies with their topographical location. In addition, specialized epithelial cells located in the airway contain the molecular machinery of chemoreceptive elements. Our aim has been to evaluate whether the secretory cells themselves possess a chemoreceptive capability, which requires the simultaneous presence of chemosensory and secretory mechanisms. We performed immunohistochemical analysis with antibodies against the Clara-cell-specific secretory proteins, CC10 and CC26, as secretory markers. As chemoreceptive markers, we employed antibodies against α-gustducin and phospholipase C beta 2 (PLCβ2), two components of the taste transduction pathway. We also attempted to characterize further the secretory cell type by using a marker of chloride secretion, cystic fibrosis transmembrane regulator (CFTR). We found α-gustducin localized in non-ciliated cells of the epithelium lining the trachea and bronchioles of adult rats, where it was also co-expressed with CC10 and CC26. Ultrastructural immunohistochemistry revealed α-gustducin in the apical cytoplasm of secretory cells, concentrated around and inside the granules. CFTR was also observed in a subpopulation of non-ciliated epithelial cells, co-localized with some α-gustducin- and PLCβ2-immunoreactive cells, at all levels of the airway epithelium. We conclude that non-ciliated epithelial cells of the rat airway express components of distinct signaling mechanisms and suggest that secretory events are driven by a molecular mechanism activated by the binding of luminal substances to G-protein-coupled receptors. This work was supported by the Italian Cystic Fibrosis Research Foundation (grant #2/2004).     

TGFβ1 induces growth arrest and apoptosis but not ciliated cell differentiation in rat tracheal epithelial cell cultures

Summary We are studying the regulation of ciliated cell differentiation using an in vitro model of tracheal regeneration. Previously, we reported that removal of growth stimulating compounds such as epidermal growth factor (EGF) and cholera toxin reduced DNA synthesis and cell number while increasing ciliated cell differentiation (Clark et al., 1995). This result suggested that the induction of growth arrest may stimulate terminal differentiation of airway epithelial cells into ciliated cells. Transforming growth factor βs (TGFβs) inhibit epithelial cell proliferation and have also been shown to stimulate epithelial cell differentiation. In this study, the effect of TGFβ₁ on growth and ciliated cell differentiation of rat tracheal epithelial (RTE) cells was examined. TGFβ₁ inhibited [³H]thymidine incorporation by RTE cells in a dose-dependent manner. A 40% inhibition was observed after a 24-h incubation with 10 pM TGFβ₁. Continuous treatment with TGFβ₁ (1–50 pM) also reduced cell number during the time when ciliogenesis occurs. This reduction resulted in part from a loss of cells through exfoliation, in addition to the inhibition of proliferation. The exfoliated cells exhibited several morphological features characteristic of apoptosis, including shrunken cells, condensed and fragmented nuclei, and intact organelles. In addition, electrophoretic analysis of genomic DNA analysis isolated from exfoliated cells demonstrated the presence of a nucleosomal ladder. However, in contrast to the removal of EGF, treatment with TGFβ₁ for 7 d did not increase ciliated cell differentiation. TGFβ1 is, therefore, capable of inhibiting proliferation and increasing apoptosis in RTE cells without stimulating ciliated cell differentiation.     

TGF-β induces the expression of SAP30L, a novel nuclear protein

Background  

We have previously set up an in vitro mesenchymal-epithelial cell co-culture model which mimics the intestinal crypt villus axis biology in terms of epithelial cell differentiation. In this model the fibroblast-induced epithelial cell differentiation from secretory crypt cells to absorptive enterocytes is mediated via transforming growth factor-β (TGF-β), the major inhibitory regulator of epithelial cell proliferation known to induce differentiation in intestinal epithelial cells. The aim of this study was to identify novel genes whose products would play a role in this TGF-β-induced differentiation.     

Natural killer (NK) activity of cultured S100β-positive T-leukemia cells

In order to clarify the function of human S100β- positive T-cells, S100β-positive T-leukemia cells (S100β TLC) were examined in vitro. S100β TLC were obtained from the peripheral blood of a patient with S100β-positive T-cell leukemia and enriched by an E-rosetting method. Two dimensional flow cytometric analysis indicated that the vast majority of the E-positive fraction were S100β TLC expressing CD3 and CD8 antigens. Although S100β TLC expressed CD3 antigen, they were negative for the α/β and γ/δ T-cell antigen receptor (TCR) defined by monoclonal antibodies (mabs) WT-31 and δ TCS-1, respectively. It was speculated that S100β TLC initially expressed α/β TCR but lost it during malignant transformation. When S100β TLC were cultured for 24 h, they acquired cytotoxic activity towards various NK-sensitive cell lines including K-562, Molt-3 and CEM-CCLF, but did not exhibit lysing activity towards NK-resistant cell lines including Raji, Daudi and MT-1. Despite the NK-activity of cultured S100β TLC, they lacked the morphological features of large granular lymphocytes (LGL). S100β TLC did not exhibit lymphokine-activated killer (LAK) activity. When S100β TLC were cocultivated with NK-sensitive cells or NK-resistant cells, they selectively bound to NK-sensitive cells, indicating that they lysed target cells by cell-to-cell contact. The finding that S100 β TLC lacked TCR molecules and their NK activity was not inhibited by mabs reactive with the CD3-TCR complex indicated that the CD3-TCR complex was not involved in their target recognition. These findings suggest that S100 β-positive T-cells are functionally similar to NK cells. We discuss the roles of S100 β-positive T-cells in the human immune system.     

The effect of TGF-β1 and Smad7 gene transfer on the phenotypic changes of rat alveolar epithelial cells

The aim of this study was to investigate whether transforming growth factor-β1 (TGF-β1) could induce alveolar epithelial-mesenchymal transition (EMT) in vitro, and whether Smad7 gene transfer could block this transition. We also aimed to elucidate the possible mechanisms of these processes. The Smad7 gene was transfected to the rat type II alveolar epithelial cell line (RLE-6TN). Expression of the EMT-associated markers was assayed by Western Blot and Real-time PCR. Morphological alterations were examined via phase-contrast microscope and fluorescence microscope, while ultrastructural changes were examined via electron microscope. TGF-β1 treatment induced a fibrotic phenotype of RLE-6TN with increased expression of fibronectin (FN), α-smooth muscle actin (α-SMA) and vimentin, and decreased expression of E-cadherin (E-cad) and cytokeratin19 (CK19). After transfecting the RLE-6TN with the Smad7 gene, the expression of the mesenchymal markers was downregulated while that of the epithelial markers was upregulated. TGF-β1 treatment for 48 h resulted in the separation of RLE-6TN from one another and a change into elongated, myofibroblast-like cells. After the RLE-6TN had been transfected with the Smad7 gene, TGF-β1 treatment had no effect on the morphology of the RLE-6TN. TGF-β1 treatment for 48 h resulted in an abundant expression of α-SMA in the RLE-6TN. If the RLE-6TN were transfected with the Smad7 gene, TGF-β1 treatment for 48 h could only induce a low level of α-SMA expression. Furthermore, TGF-β1 treatment for 12 h resulted in the degeneration and swelling of the osmiophilic multilamellar bodies, which were the markers of type II alveolar epithelial cells. TGF-β1 can induce alveolar epithelialmesenchymal transition in vitro, which is dependent on the Smads signaling pathway to a certain extent. Overexpression of the Smad7 gene can partially block this process     

The use of flow cytometry and fluorescein-labeled antibodies to measure specific milk proteins in bovine mammary epithelial cells

Summary A flow cytometric technique was developed to measure the relative concentration of whey protein and β-casein in individual fixed and permeabilized bovine mammary epithelial cells. Primary bovine mammary epithelial cells were compared to mammary cells isolated from explants after a 24-h incubation and a bovine mammary epithelial transfected cell line (MAC-T). Cells were incubated with rabbit anti-bovine whey protein (α-lactalbumin + β-lactoglobulin) or β-casein primary antibodies followed by a fluorescein-labeled goat anti-rabbit IgG second antibody. The number and intensity of fluorescing cells were measured using an EPICS Profile Flow Cytometer. Primary and explant cells contained 3.3 and 2.8 times more whey protein than MAC-T cells. Explant epithelial cells contained 2.9 and 5.1 times more β-casein than primary or MAC-T cells. The higher concentrations of specific proteins within the cells was attributed to either greater synthesis or reduced secretion. These data show that flow cytometry is capable of detecting differences in milk protein concentration in different mammary epithelial cell types.     

Establishment of an abalone digestive gland cell line secreting various glycosidases in protein-free culture

A cell line designated as ADG was established from an abalone digestive gland using ERDF medium supplemented with 8% fetal bovine serum (FBS), 8% abalone hemolymph, and high concentrations of NaCl, KCl, MgCl₂, MgSO₄, and CaCl₂. ADG cells proliferated better in protein-free medium than in FBS-supplemented medium. Among 9 kinds of media examined, ERDF medium was shown to be optimal for cell growth. ADG cells secreted 13 different kinds of glycosidases in protein-free medium: α-L-fucosidase, β-L-fucosidase, α-D-galactosidase, β-D-galactosidase, N-acetyl-α-D-galactosaminidase, N-acetyl-β-D-galactosaminidase, α-D-glucosidase, β-D-glucosidase, N-acetyl-α-D-glucosaminidase, N-acetyl-β-D-glucosaminidase, α-D-mannosidase, β-D-mannosidase, β-D-xylosidase, and 1-3 xylanase. When ADG cells were cultured in Grace’s insect cell medium, the activity of some secreted glycosidases increased 25-fold to 65-fold per cell as compared with control cells cultured in ERDF medium. ADG - abalone digestive gland; ERDF - enriched RDF; FBS - fetal bovine serum; L-15 - Leibovitz’s L-15 media; DME - Dulbecco’s modified Eagle medium; F-12 - nutrient mixture (Ham); LDF - L-15; DME: F-12 = 10 : 7 : 3; MEM - minimum essential medium; RPMI - RPMI medium 1640; 199 - media 199; GIC - Grace’s insect cell medium; pNP -p -nitrophenol. This revised version was published online in July 2006 with corrections to the Cover Date.     

Generation of novel chimeric LacdiNAcS by gene fusion of α-lactalbumin and β1,4-galactosyltransferase 1

Novel chimeric lacdiNAc (GalNAc(β1-4)GlcNAc) synthase (c-LacdiNAcS) was generated by gene fusion of α-lactalbumin (α-LA) and β1,4-galactosyltransferase 1 (β1,4-GalT1). c-LacdiNAcS was expressed in Lec8 Chinese hamster ovary (Lec8 CHO) cells and exhibited N-acetylgalactosaminyltransferase (GalNAcT) activity in the absence of exogenous α-LA as well as other glycosyltransferase activities including lactose synthase (LacS), and β1,4-GalT. These glycosyltransferase activities of c-LacdiNAcS were compared to those activities induced in LacS system under the co-presence of bovine β1,4-GalT1 and α-LA, indicating that each domain of α-LA and β1,4-GalT1 on c-LacdiNAcS is not only folding correctly, but also interacting together. Furthermore, c-LacdiNAcS was found to be auto-lacdiNAcylated and can synthesize lacdiNAc structures on cellular glycoproteins, demonstrating that GalNAcT activity of c-LacdiNAcS is functional in Lec8 CHO cells.     

Mast cell activation and its relation to proinflammatory cytokine production in the rheumatoid lesion

Mast cell (MC) activation in the rheumatoid lesion provides numerous mediators that contribute to inflammatory and degradative processes, especially at sites of cartilage erosion. MC activation in rheumatoid synovial tissue has often been associated with tumour necrosis factor (TNF)-α and interleukin (IL)-1β production by adjacent cell types. By contrast, our in situ and in vitro studies have shown that the production of IL-15 was independent of MC activation, and was not related to TNF-α and IL-1β expression. Primary cultures of dissociated rheumatoid synovial cells produced all three proinflammatory cytokines, with production of IL-1β exceeding that of TNF-α, which in turn exceeded that of IL-15. In vitro cultures of synovial macrophages, synovial fibroblasts and articular chondrocytes all produced detectable amounts of free IL-15, macrophages being the most effective.     

Establishment and characterization of an immortalized but non-transformed human prostate epithelial cell line: BPH-1

Summary This report describes the development and characterization of an epithelial cell line (BPH-1) from human prostate tissue obtained by transurethral resection. Primary epithelial cell cultures were immortalized with SV40 large T antigen. One of the isolated clones was designated BPH-1. These cells have a cobblestone appearance in monolayer culture and are non-tumorigenic in nude mice following subcutaneous injection or subrenal capsule grafting. They express the SV40 large T antigen and exhibit increased levels of p53, as determined by immunocytochemistry. Cytogenetic analysis by G-banding demonstrated an aneuploid karyotype with a modal chromosome number of 76 (range 71 to 79,n=28) and 6 to 8 marker chromosomes. Some structurally rearranged chromosomes were observed, but the Y chromosome was normal. The expressed cytokeratin profile was consistent with a prostatic luminal epithelial cell. This profile was the same as that of primary prostatic epithelial cultures from which the BPH-1 cells were derived. In serum-free culture in plastic dishes epidermal growth factor (EGF), transforming growth factor (TGF)-α, fibroblast growth factor (FGF) 1 (aFGF), and FGF 7 (KGF) induced increased proliferation in these cells whereas FGF 2 (bFGF), TGF-β1, and TGF-β2 inhibited proliferative activity. Testosterone had no direct effect on the proliferative rate of BPH-1 cells. 5α-Reductase, 3α-hydroxysteroid oxidoreductase, and 17β-hydroxy-steroid oxidoreductase activities were detected in BPH-1 cells. Expression of androgen receptors and the secretory markers, prostate specific antigen and prostatic acid phosphatase, were not detectable by immunocytochemistry, biochemical assay, or RT-PCR analysis.     

Over-expressed estrogen receptor-α up-regulates hTNF-α gene expression and down-regulates β-catenin signaling activity to induce the apoptosis and inhibit proliferation of LoVo colon cancer cells

TAK-778 has been shown to stimulate osteogenesis both in vitro and in vivo. However, the mechanism by which TAK-778 exerts its effects is still unclear. There is evidence that TAK-778 acts via estrogen-receptor (ER)-mediated signaling; this study therefore aimed to investigate the roles that ERα, ERβ, and membrane ER play in the osteogenic effect of TAK-778. To this end, human bone marrow mesenchymal cells were cultured with TAK-778 in the presence of either ICI182,780 (ERα and ERβ antagonist) or MPP (ERα antagonist) or PD98059 (an extracellular-regulated kinase inhibitor that acts on the membrane ER pathway). The following parameters were evaluated: cell proliferation, collagen content, alkaline phosphatase (ALP) activity and bone-like formation. Data were compared using ANOVA. The effect of TAK-778 on expression of ERα and ERβ was investigated by immunolabeling. In order to investigate whether TAK-778 binds to ER, an ER binding assay was performed. Both immunolabeling and binding assays were conducted using cells from human alveolar bone. The osteogenic effect of TAK-778 was inhibited by ICI182,780 and MPP; however, it was not affected by PD98059. The expression of both ERα and ERβ was not affected by TAK-778. The competition curve obtained from the binding assay using TAK-778 showed maximal displacement when 10⁻⁵ M TAK-778 was used. This study's results show that TAK-778 enhances osteoblast differentiation through an ERα-dependent pathway by binding to this receptor and not by increasing the expression of ER. (Mol Cell Biochem xxx: 1–9, 2005)     

Establishment of human parotid pleomorphic adenoma cells in culture: Morphological and biochemical characterization

Summary This study reports the establishment ofα-amylase-producing human parotid pleomorphic adenoma cell lines (2HP and 2HP₁) which have been maintained in culture for over 1 yr. The procedures required preparation of cellular clumps from tumor tissue and plating them on plasma clot or precoated dishes. During the initial phase of growth they required modified MCDB-153 medium without serum. When cells showed signs of degeneration they were changed to MCDB-153 medium containing first 2% and then 10% heat inactivated fetal bovine serum. Although cells grew well in MCDB-153 containing 10% serum, the epithelial cell morphology was not distinct. Therefore, the growth and morphology of cells grown in MCDB-10% serum were compared with those in RPMI growth medium containing 10% fetal bovine serum and F12 containing 10% agammaglobulin newborn bovine serum. Although the growth of cells was a little slower in F12 medium than those in MCDB and RPMI, the epithelial cell morphology was maintained better than in other growth media. The cells of 2HP and 2HP₁ produce low levels ofα-amylase and relatively high levels ofα-amylase mRNAs of 1176 and 702 bp and contain neurofilament-160, a neuronal-specific marker. The cells of 2HP₁ are tumorigenic when tested in athymic mice, but the cells of 2HP are not. The establishment of amylase-producing human parotid adenoma cell lines of different characteristics in culture provides a new opportunity to study the mechanisms of differentiation and transformation, and regulation ofα-amylase in these cells.     

Interactions between IL-32 and tumor necrosis factor alpha contribute to the exacerbation of immune-inflammatory diseases

IL-32 is a newly described cytokine in the human found to be an in vitro inducer of tumor necrosis factor alpha (TNFα). We examined the in vivo relationship between IL-32 and TNFα, and the pathologic role of IL-32 in the TNFα-related diseases – arthritis and colitis. We demonstrated by quantitative PCR assay that IL-32 mRNA was expressed in the lymphoid tissues, and in stimulated peripheral T cells, monocytes, and B cells. Activated T cells were important for IL-32 mRNA expression in monocytes and B cells. Interestingly, TNFα reciprocally induced IL-32 mRNA expression in T cells, monocyte-derived dendritic cells, and synovial fibroblasts. Moreover, IL-32 mRNA expression was prominent in the synovial tissues of rheumatoid arthritis patients, especially in synovial-infiltrated lymphocytes by in situ hybridization. To examine the in vivo relationship of IL-32 and TNFα, we prepared an overexpression model mouse of human IL-32β (BM-hIL-32) by bone marrow transplantation. Splenocytes of BM-hIL-32 mice showed increased expression and secretion of TNFα, IL-1β, and IL-6 especially in response to lipopolysaccharide stimulation. Moreover, serum TNFα concentration showed a clear increase in BM-hIL-32 mice. Cell-sorting analysis of splenocytes showed that the expression of TNFα was increased in resting F4/80⁺ macrophages, and the expression of TNFα, IL-1β and IL-6 was increased in lipopolysaccharide-stimulated F4/80⁺ macrophages and CD11c⁺ dendritic cells. In fact, BM-hIL-32 mice showed exacerbation of collagen-antibody-induced arthritis and trinitrobenzen sulfonic acid-induced colitis. In addition, the transfer of hIL-32β-producing CD4⁺ T cells significantly exacerbated collagen-induced arthritis, and a TNFα blockade cancelled the exacerbating effects of hIL-32β. We therefore conclude that IL-32 is closely associated with TNFα, and contributes to the exacerbation of TNFα-related inflammatory arthritis and colitis.     

Expression and function of heregulin-α and its receptors in the mouse mammary gland

Heregulin-α (HRGα) is a cytokine secreted by the mammary mesenchyme, adjacent to lobuloalveolar structures. To understand the role of HRGα and its receptors in mammary glands, and the underlying mechanisms, we performed this study to determine the expression and localization of HRGα and its receptors ErbB2 and ErbB3. We also determined the role of HRGα in the development of mammary glands, β-casein expression and secretion, Rab3A protein expression and the phosphorylation of HRGα signaling molecules using confocal laser scanning microscopy, tissue culture, capillary electrophoresis, Western blotting and enzyme-linked immunosorbent assays. We found that a peak was on pregnancy day 15. Changes of ErbB2 and ErbB3 expression were positively and linearly correlated with HRGα, indicating that HRGα positively regulates ErbB2 and ErbB3 expression. During pregnancy, HRGα enhanced the phosphorylation of STAT5, p42/p44, p38, PKC and Rab3A protein expression, stimulated the proliferation and differentiation of the ductal epithelial cells of mammary glands, and increased and maintained the expression and secretion of β-casein. During lactation, HRGα enhanced the phosphorylation of STAT5 and p38, inhibited the phosphorylation of PKC and Rab3A protein expression, maintained the morphology of the mammary glands and increased the secretion of lactoprotein to reduce the expression of β-casein in mammary epithelial cells. During involution, HRGα induced the phosphorylation of STAT3 and Rab3A protein expression, and inhibited the phosphorylation of PKC to stimulate the degeneration of mammary epithelial cells. It also inhibited the secretion of β-casein, resulting in increased levels of β-casein in mammary epithelial cells.     

Change of Morphology and Cytoskeletal Protein Gene Expression during Dibutyryl cAMP-induced Differentiation in C6 Glioma Cells

Elevation of the intracellular cAMP level induces morphological changes of astrocyte-like differentiation in C6 glioma cells. Such changes may be accompanied with expression of cytoskeletal protein genes. We therefore analyzed morphological changes after a treatment with dibutyryl cAMP (dbcAMP) and then assessed the expression of cytoskeletal protein genes by a quantitative real-time polymerase chain reaction. The cell number remained unaltered upon incubation with 1 mM dbcAMP in medium supplemented with 0.1% fetal bovine serum (FBS), whereas the number and lengths of processes increased, when compared with those of cells incubated in medium supplemented with 0.1% or 10% FBS only. The amounts of β-actin, γ-actin, and β-tubulin mRNAs in C6 cells, but not α-tubulin mRNA, increased during the early proliferation in DMEM containing 10% FBS. The expression of cytoskeletal protein genes decreased when incubated with 0.1% FBS or 1 mM dbcAMP in 0.1% FBS, compared with those of cells cultured in 10% FBS. These results indicated that, during the early proliferation in normal culture condition, the expression of cytoskeletal protein genes in C6 cells, except α-tubulin, increased, while in differentiating or differentiated C6 glioma cells, cAMP-induced morphological changes were not accompanied with elevation of gene expression for cytoskeletal proteins, such as actin and tubulin.     

Human trachea primary epithelial cells express both sialyl(α2-3)Gal receptor for human parainfluenza virus type 1 and avian influenza viruses, and sialyl(α2-6)Gal receptor for human influenza viruses

We reported previously that the dominant receptors of influenza A and B viruses, and human and murine respiroviruses, were sialylglycoproteins and gangliosides containing monosialo-lactosamine type I-and II-residues, such as sialic acid-α2-3(6)-Galβ1-3(4)-GlcNAcβ1-. In addition, the Siaα2-3Gal linkage was predominantly recognized by avian and horse influenza viruses, and human parainfluenza virus type 1 (hPIV-1), whereas the Siaα2-6Gal linkage was mainly recognized by human influenza viruses (Paulson JC in “The Receptors' [Conn M Ed] 2, 131–219 (1985); Suzuki Y, Prog Lipid Res 33, 429–57 (1994); Ito T, J Virol 73, 6743–51 (2000); Suzuki Y, J Virol 74, 11825–31 (2000); Suzuki T, J. Virol 75, 4604–4613 (2001); Suzuki Y, Biol. Pharm. Bull. 28, 399–408 (2005)). To clarify the distribution of influenza virus receptors on the human bronchial epithelium cell surface, we investigated a primary culture of normal human bronchial epithelial (NHBE) cells using two types of lectin (MAA and SNA), which recognize sialyl linkages (α2-3 and α2-6), using fluorescence-activated cell-sorting analysis. The results showed that both α2-3- and α2-6-linked Sias were expressed on the surface of primary human bronchial epithelial cells. The cells infected by hPIV-1 bound to MAA, confirming that cells targeted by hPIV-1 have α2-3-linked oligosaccharides. We also compared the ability of hPIV-1 and human influenza A virus to infect primary human bronchial epithelial cells pre-treated with Siaα2-3Gal-specific sialidase from Salmonella typhimurium. No difference was observed in the number of sialidase pre-treated and non-treated cells infected with human influenza A virus, which binds to Siaα2-6Gal-linked oligosaccharides. By contrast, the number of cells infected with hPIV-1 decreased significantly upon sialidase treatment. Thus, cultured NHBE cells showed both α2-3-linked Sias recognized by hPIV-1 and avian influenza virus receptors, and α2-6-linked Sias recognized by human influenza virus receptors.     

The substrate specificity of the enzyme Endo-α-N-acetyl-D-galactosaminidase from Diplococcus pneumonia

The substrate specificity of the enzyme endo-α-N-acetyl-D-galactosaminidase from Diplococcus pneumonia was re-examined using bovine submaxillary mucin and remodelled antifreeze glycoprotein as substrates. Incubation with desialylated bovine submaxillary mucin, which contains six O-linked core types, indicated that the disaccharide Galβ1-3GalNAc, which is present in very small amount, was the only glycan released, while the disaccharide GlcNAcβ1-3GalNAc, which is the major structure present, and other disaccharides, were not released. To test whether the core disaccharide Galβ1-3GalNAc with sialic acid linked α2-3 to the Gal or linked α2-6 to the GalNAc was released, the enzyme was incubated with remodelled antifreeze glycoprotein containing (1) [3H]NeuAcα2-3Galβ1-3GalNAc and (2) Galβ1-3[[14C]NeuAcα2-6]GalNAc as substrates. No NeuAc-containing trisaccharide was released. These results serve to clarify the doubts of many researchers regarding the activity of this enzyme on some newly-described core types and on sialylated substrates. This revised version was published online in November 2006 with corrections to the Cover Date.