T-cadherin (CDH13, H-cadherin) expression downregulated surfactant protein D in bronchioloalveolar cells.

T cadherin is a unique cadherin cell adhesion molecule that is anchored to the surface membrane through a glycosyl phosphatidyl inositol (GPI) moiety. In the present study, we postulated that T cadherin could regulate surfactant protein (SP)-D gene expression in human bronchioloalveolar type-II cells. We transfected A549 cells (human lung cancer cell line with alveolar type-II cell characteristics) with the T-cadherin expression vector. Both original and control plasmid-transfected A549 cells expressed SP-D; however, neither human nor murine T-cadherin-transfected A549 cells expressed SP-D mRNA. The downregulation of SP-D production in human T-cadherin-expressed A549 cells was also demonstrated using Western immunoblotting techniques. Control vector-transfected A549 cells showed a positive band of SP-D but not of T cadherin. In contrast, T-cadherin-transfected A549 cells, which expressed T-cadherin protein, did not produce SP-D. We further examined the relationship of T cadherin and SP-D expression in secondary pulmonary alveolar proteinosis associated with hematolymphoid malignancies. SP-D was detected in bronchioloalveolar type-II cells in alveolar proteinosis. However, little or no T-cadherin expression was detected in alveolar type-II cells in these patients. To our knowledge, this is the first report describing an effect of cadherin on SP production in bronchioloalveolar cells.

Carbohydrate binding properties of banana (Musa acuminata) lectin I. Novel recognition of internal alpha1,3-linked glucosyl residues.

Examination of lectins of banana (Musa acuminata) and the closely related plantain (Musa spp.) by the techniques of quantitative precipitation, hapten inhibition of precipitation, and isothermal titration calorimetry showed that they are mannose/glucose binding proteins with a preference for the alpha-anomeric form of these sugars. Both generate precipitin curves with branched chain alpha-mannans (yeast mannans) and alpha-glucans (glycogens, dextrans, and starches), but not with linear alpha-glucans containing only alpha1,4- and alpha1,6-glucosidic bonds (isolichenan and pullulan). The novel observation was made that banana and plantain lectins recognize internal alpha1,3-linked glucosyl residues, which occur in the linear polysaccharides elsinan and nigeran. Concanavalin A and lectins from pea and lentil, also mannose/glucose binding lectins, did not precipitate with any of these linear alpha-glucans. This is, the authors believe, the first report of the recognition of internal alpha1,3-glucosidic bonds by a plant lectin. It is possible that these lectins are present in the pulp of their respective fruit, complexed with starch.

Carbohydrate binding properties of banana (Musa acuminata) lectin II. Binding of laminaribiose oligosaccharides and beta-glucans containing beta1,6-glucosyl end groups.

This paper extends our knowledge of the rather bizarre carbohydrate binding poperties of the banana lectin (Musa acuminata). Although a glucose/mannose binding protein which recognizes alpha-linked gluco-and manno-pyranosyl groups of polysaccharide chain ends, the banana lectin was shown to bind to internal 3-O-alpha-D-glucopyranosyl units. Now we report that this lectin also binds to the reducing glucosyl groups of beta-1,3-linked glucosyl oligosaccharides (e.g. laminaribiose oligomers). Additionally, banana lectin also recognizes beta1,6-linked glucosyl end groups (gentiobiosyl groups) as occur in many fungal beta1,3/1,6-linked polysaccharides. This behavior clearly distinguishes the banana lectin from other mannose/glucose binding lectins, such as concanavalin A and the pea, lentil and Calystegia sepium lectins.

Degradation of human brain natriuretic peptide (BNP) by contact activation of blood coagulation system.

Brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) were added to venous blood samples from healthy volunteers, and incubated in tubes made of various materials. The residual immunoreactivity was measured with radioimmunoassay for BNP and ANP. In blood samples stored in glass tubes, immunoreactivity of ANP was more stable than that of BNP. In siliconized glass or PET tubes, however, BNP immunoreactivity was more stable than ANP. The activation of blood coagulation factors was evaluated from the kallikrein activity in plasma. Kallikrein activity was increased in plasma stored in glass tube while it was negligible in plasma stored in siliconized glass or PET tubes. In kaolin-activated plasma, more rapid BNP degradation and higher kallikrein activity were observed. Our results indicated that the blood coagulation factors, especially kallikrein, played an important role in digestion of BNP.

Expression of SMARCF1, a truncated form of SWI1, in neuroblastoma.

Previously we cloned and mapped a B120 gene to human chromosome 1p35-36.1 where possible suppressor genes for various neuroendocrine tumors including neuroblastoma have been mapped. Very recently, B120 was identified as a truncated form of p270, a putative human counterpart of SWI1. In the present study, expression of the B120 gene product was immunohistochemically investigated in 23 neuroblastomas. We also examined B120 expression in neural stem cells in developing brain and intact adrenal medulla. Four of 23 neuroblastomas strongly expressed B120 gene product in both cytoplasm and nucleus. The other neuroblastomas expressed B120 gene product in the nucleus; however, the intensity of staining was much weaker and equivalent to that in developing human brain stem cells in the subventricular region. B120 gene product was less strongly expressed in intact adrenal medulla. Subsequently, we performed loss of heterozygosity studies on 19 neuroblastomas using the polymorphic markers D1S195 and D1S511 located near the B120 gene. Loss of heterozygosity was observed in three of 19 tumors that abundantly expressed B120 protein. Furthermore, neuroblastoma cells were transfected with B120 expression vector. These transfected neuroblastoma cells adhered to each other and aggregated. Differential display experiments followed by reverse transcriptase-polymerase chain reaction and Northern blot analysis were performed and three molecules with altered expression in B120-transfected neuroblastoma cells were identified. One of three genes seemed to be a proliferation-related and cell cycle-related nucleolar protein, p120, encoding gene. We further characterized the genomic structure of B120. B120 appeared to be encoded by 17 exons in more than 20-kbp genomic DNA. The present findings contribute to understanding of the B120 gene, a truncated form of human SWII1, an approved term for which is SMARCF1, in normal cells and neuroblastomas.

Expression of T-cadherin (CDH13, H-Cadherin) in human brain and its characteristics as a negative growth regulator of epidermal growth factor in neuroblastoma cells.

In the present study, we first examined the expression of T-cadherin in human CNS by northern blot analysis, immunohistochemical staining, and in situ hybridization. Northern blot analysis demonstrated expression of T-cadherin in human adult cerebral cortex, medulla, thalamus, and midbrain. Immunohistochemical staining with a newly generated monoclonal antibody, designated MA-511, revealed strong expression of T-cadherin in neural cell surface membrane and neurites in adult cerebral cortex, medulla oblongata, and nucleus olivaris. Little or no expression of T-cadherin was found in spinal cord. We further examined T-cadherin expression in various developing nervous systems, and found that T-cadherin expression was lower in developing brain than in adult brain. In situ hybridization revealed that neural cells in medulla oblongata and nucleus olivaris, but not in spinal cord, possessed T-cadherin molecules. We transfected T-cadherin-negative TGW and NH-12 neuroblastoma cells with a T-cadherin cDNA-containing expression vector. T-cadherin-expressing neuroblastoma cells lost mitogenic proliferative response to epidermal growth factor. Epidermal growth factor is known to be required for proliferation of neural stem cells. This finding, together with those of the present study, suggests that T-cadherin functions as a negative regulator of neural cell growth.

Stability of brain natriuretic peptide (BNP) in human blood samples.

Stability of immunoreactivity of human brain natriuretic peptide (BNP) in blood samples was investigated. After storage of the whole blood samples in the blood collecting tubes made of glass or polyethylene terephthalate (PET), residual immunoreactivity of BNP in the plasma was measured by sandwich radioimmunoassay for human BNP. BNP in the blood samples collected in the PET tubes were kept more stable than that in the glass tubes. The results suggested that commercially available PET tubes would enable more accurate BNP values and this would also help to simplify the sample preparation.

Purification and characterization of the alpha-1,3-mannosylmannose-recognizing lectin of Crocus vernus bulbs.

A unique mannose-binding lectin, highly specific for terminal Man(alpha1,3)Man groups, was isolated from bulbs of crocus (Crocus vernus All.). The lectin failed to bind to a mannose affinity column and was purified by simple gel permeation chromatography (Sephacryl S200). The purified lectin, obtained in crystalline form, had a molecular mass of 44 kDa on gel filtration and showed a single peptide band with a molecular mass of 11 kDa on SDS-polyacrylamide gel electrophoresis, indicating it to be a tetrameric protein composed of four identical subunits. The N-terminal amino acid sequence analysis of the crocus lectin showed essentially no homology with that of other mannose-binding bulb lectins. The crocus lectin selectively interacted with the wild type Saccharomyces cerevisiae and other mannans carrying terminal Man(alpha1,3)Man but not with those lacking this disaccharide unit. In hapten inhibition studies, methyl alpha-mannopyranoside did not inhibit the mannan-lectin interaction. Of various alpha-mannooligosaccharides, those having the Man(alpha1,3)Man sequence showed the highest inhibitory potency, confirming the strict requirement of lectin for terminal alpha1,3-linked mannosylmannose units. An affinity column of immobilized lectin enabled the complete resolution of yeast mannan and glycogen. The immobilized lectin may provide a useful tool for purification and analysis of biologically important polysaccharides and glycoproteins.

X-ray study of beijeran sodium salts, a new galacturonic acid-containing exo-polysaccharide.

X-Ray fiber diffraction patterns were obtained from oriented films of sodium salts of a new uronic acid-containing polysaccharide (beijeran) both in its native, poly [-->3)-alpha-D-GalA-(1-->3)-beta-L-Rha-(1-->3)-alpha-D-Glc-O6Ac-(1 -->], and deacetylated forms. Initially the stretched films of both polysaccharides were amorphous, but the crystallinity was much improved by annealing at high temperature. The deacetylated specimen had higher crystallinity than the native. Both films showed similar X-ray fiber patterns indicating that these polysaccharides had similar unit cell dimensions and that the O-acetyl groups in the native beijeran chain did not disturb the regular array in the crystal having space group P21. All the visible reflections could be indexed in terms of a monoclinic unit cell with dimensions a = 1.277, b = 1.611, c (fiber axis) = 2.437 nm, and gamma = 96.79 degrees. The fiber axis length and the presence of (002) and (006) reflections indicated that the conformation was made up of two trisaccharide residues, in an extended two-fold helix.

Studies on the metabolism and disposition of the new retinoid 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carbamoyl] benzoic acid. 4th communication: absorption, metabolism, excretion and plasma protein binding in various animals and man.

4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carbamoyl]benzoic acid (CAS 94497-51-5, Am-80) is a new synthetic retinoid which has been shown to have a potent topical antipsoriatic activity. Pharmaco-kinetic profiles of Am-80 were studied in dogs, mice and rabbits after percutaneous or subcutaneous administration of 14C-Am-80. Plasma protein binding of 14C-Am-80 was also studied in rats, dogs and humans. After topical application of 14C-labeled Am-80 by occlusive dressing technique at a dose of 1 mg 14C-Am-80/1,000 mg ointment/kg, the blood and plasma levels of radioactivity were below the detection limit in normal-skin dogs. In normal skin mice and rabbits, the plasma radioactivity peaked at 8 h (40.8 ng eq./ml) and at 12 h (34.0 ng eq./ml) after application, respectively. Percutaneous absorption of 14C-Am-80 was less than 2% of the dose for dogs, 34% for mice and 23% for rabbits. After subcutaneous administration at a dose of 1 mg/kg to mice, dogs and rabbits, plasma levels of radioactivity peaked at 1, 4 and 4 h after dosing with a concentration of 614.0, 902.9 and 757.7 ng eq./ml and then it declined with half-lives of 2.4, 7.2 and 4.1 h, respectively. Urinary and fecal excretion of radioactivity after subcutaneous administration at a dose of 1 mg/kg was 3.5 and 94.7% of the dose in dogs, 27.0 and 73.2% in mice and 43.5 and 45.6% in rabbits. A possible gastrointestinal secretion, which might lead to excretion into feces, was suggested from the results with bile-duct-cannulated dogs. Unchanged Am-80 was present in high amounts in the plasma and bile or feces of all animal species tested except in rat bile, in which Am-80 was predominantly detected in the form of its taurine conjugate (M-6). Hydroxylation of Am-80 to yield 7-hydroxy-Am-80 (M-4) and 6-hydroxy-Am-80 (M-3), which lead to the formation of 6-oxo-Am-80 (M-5), were commonly observed in all animal species. Taurine conjugation reaction of unchanged Am-80 and hydroxy-Am-80 (to form M-6 and both M-1 and M-2, respectively) was distinct in rats and dogs, but, hardly detected in mice and rabbits. The presence of tetrahydro-tetramethyl-naphtylamine (TTNA) was most marked in mice, followed by rabbits and rats, but it was almost absent in dogs. HPLC-RIA analysis of human samples obtained from the phase II and phase III clinical trials of Am-80 ointment suggested that fecal excretion was the major elimination route, and that hydroxylation and taurine conjugation reaction of unchanged and hydroxy-Am-80 also occurred. Unchanged Am-80 was predominant in human plasma as compared with metabolites M-1 to M-6. In vitro binding of 14C-Am-80 to the plasma protein was found to be more than 99% in rats, dogs and humans. In vivo plasma protein binding of 14C-Am-80 and/or its radioactive metabolites was also found to be more than 98% in rats and dogs after subcutaneous administration of 14C-Am-80. In both dogs and humans, in vitro. 14C-Am-80 appeared to be bound predominantly to serum albumin. The binding of 14C-Am-80 to human serum albumin was scarcely affected in the presence of diazepam, digitoxin or warfarin, indicating that there are no specific binding sites for Am-80 on serum albumin.

Purification and characterization of a new mannose-specific lectin from Sternbergia lutea bulbs.

A new mannose-binding lectin was isolated from Sternbergia lutea bulbs by affinity chromatography on an alpha(1-2)mannobiose-Synsorb column and purified further by gel filtration. This lectin (S. lutea agglutinin; SLA) appeared homogeneous by native-gel electrophoresis at pH 4.3, gel filtration chromatography on a Sephadex G-75 column, and SDS-polyacrylamide gel electrophoresis, These data indicate that SLA is a dimeric protein (20 kDa) composed of two identical subunits of 10 kDa which are linked by non-covalent interactions. The carbohydrate binding specificity of the lectin was investigated by quantitative precipitation and hapten inhibition assays. It is an alpha-D-mannose-specific lectin that interacts to form precipitates with various alpha-mannans, galactomannan and asialo-thyroglobulin, but not with alpha-glucans and thyroglobulin. Of the monosaccharides tested only D-mannose was a hapten inhibitor of the SLA-asialothyroglobulin precipitation system, whereas D-glucose, D-galactose and L-arabinose were not. The lectin appears to be highly specific for terminal alpha(1-3)-mannooligosaccharides. The primary structure of SLA appears to be quite similar to that of the snow drop (Galanthus nivalis) bulb lectin which is a mannose-binding lectin from the same plant family Amaryllidaceae. The N-terminal 46 amino acid sequence SLA showed 76% homology with that of GNA.

Radioimmunoassay for a novel lignan-related hypocholesterolemic agent, S-8921, in human plasma after high-performance liquid chromatography purification and in human urine after immunoaffinity extraction.

The novel compound methyl-1-(3,4-dimethoxyphenyl)-3-(3-ethylvaleryl)-4-hydroxy-6,7,8- trimethoxy-2-naphthoate (S-8921) has hypocholesterolemic activity in animals and is expected to exhibit a similar activity in human. Reversed-phase high-performance liquid chromatography (HPLC) separation followed by radioimmunoassay (RIA) for human plasma samples (HPLC-RIA) and immunoaffinity extraction (IAE) followed by RIA for human urine samples (IAE-RIA) were developed for investigation of S-8921 behavior in clinical studies. For the RIA, antisera from rabbit and a radioiodine-labelled S-8921 were prepared by immunizing a conjugate of S-8921 with bovine serum albumin and by the Bolton and Hunter method, respectively. HPLC-RIA using a semi-micro column was very sensitive, that is a 0.05 ng/ml limit of quantitation in human plasma, and specific for unchanged form of S-8921. IAE-RIA using a centrifugal filtration tube completely eliminated the matrix effect of human urine, and was very feasible. The limit of quantitation was 0.10 ng/ml. RIA detection following HPLC or IAE proved to be very useful for the pharmaceutical analysis of extremely low drug concentrations in body fluids.

Chain conformation of deacetylated beijeran calcium salt.

A well-defined X-ray fiber diffraction pattern was obtained from a stretched film of the calcium salt of a new uronic acid containing polysaccharide designated as beijeran in the deacetylated form, poly[-->3)-alpha-D-GalUA-(1-->3)-beta-L-Rham-(1-->3)-alpha-D -Glc-(1-->]. The oriented film showed no diffraction spots, indicating it to be amorphous. However, when annealed at high temperature, the film exhibited high crystallinity. All the visible reflections could be indexed in terms of a monoclinic unit cell with the following dimensions: a = 1.297; b = 1.676; c (fiber axis) = 2.509 nm; and gamma = 106.50 degrees. The length of the fiber axis and the absence of meridional reflections at any odd layer line indicate that an extended two-fold helical conformation was made up of two trisaccharide residues.

Isolation and chemical characterization of polysaccharides from Iwatake, Gyrophora esculenta Miyoshi.

A heteropolysaccharide and a beta-D-glucan were isolated from a lichen, Gyrophora esculenta Miyoshi (Iwatake), by cold water and alkali extractions, respectively. The chemical properties of purified polysaccharides were examined by acid hydrolysis, methylation, and GC-MS. The heteropolysaccharide is a highly branched galactomannan-type polysaccharide, containing an alpha-(1->6)-linked D-mannan backbone, and the glucan is a linear (1->6)-beta-D-glucan. With regard to the antitumor activity, both the galactomannan and (1->6)-beta-D-glucan had moderate inhibition activities on Sarcoma 180, but lower than those of branched (1->3)-beta-D-glucans.

Characterization of an acidic polysaccharide isolated from the leaves of Corchorus olitorius (Moroheiya).

An acidic polysaccharide was isolated from the water-soluble mucilage extracted from dried leaves of Corchorus olitorius, known as Moroheiya in Japan (3.0 g per 100 g). This polysaccharide showed a single peak in a Sepharose CL-6B column, and the specific rotation in H2O at 25 degrees C was +250 degrees. The polysaccharide was rich in uronic acid (65%), and consisted of rhamnose, glucose, galacturonic acid, and glucuronic acid in a molar ratio of 1.0:0.2:0.2:0.9:1.7, in addition to 3.7% of the acetyl group. A methylation analysis, Smith degradation study and fragmentation analysis suggested that this polysaccharide mainly consisted of O-4 substituted galacturonic acid and glucuronic acid, and O-2 substituted rhamnose residues, and that most of the (1-->4)-linked uronic acid residues were substituted at the O-3 position with glucuronic acid residues. This polysaccharide showed proliferative activity toward the murine splenocyte.

Purification and characterization of the serotype-specific polysaccharide antigen of Trichosporon cutaneum serotype II: a disease-related antigen of Japanese summer-type hypersensitivity pneumonitis.

Summer-type hypersensitivity pneumonitis (SHP) is a unique type of hypersensitivity pneumonitis and the most prevalent in Japan. Our previous study clarified that the causative agent of the disease is Trichosporon cutaneum, and that the patients with SHP have high titres of antibodies against the serotype-specific antigen of polysaccharide nature which exist in the high molecular weight fraction of the culture supernatant of the yeast. In this study, we purified the serotype-specific antigen of serotype II T. cutaneum by gel filtration and affinity chromatography using a monoclonal antibody, D-8, specific for a high molecular weight antigen of serotype II T. cutaneum, and elucidated the structure of the antigen. This affinity-purified antigen was shown to be an essentially acidic polysaccharide comprising mannose, xylose, and glucuronic acid (6:44:4.7). Chemical analysis showed that this polysaccharide antigen contains a (1-3)-linked mannan backbone attached with short side chains of (1-4)-linked mannose and a small proportion of (1-2)-linked xylose residues by substituting the 2- or 4-positions of the (1-3)-linked mannose residues of the main chain. Approximately one-fifth of the side chains were terminated with glucuronic acid residues. The antigenic epitope of the serotype-specific antigen was shown to involve the terminal glucoronic acid residues as revealed by immunodiffusion test and sandwich enzyme-linked immunosorbent assay using monoclonal antibody D-8.

Antitumor activities and immunochemical properties of the cell-wall polysaccharides from Aureobasidium pullulans.

Delipidated cell walls from Aureobasidium pullulans were fractionated systematically. The cell surface heteropolysaccharide contains D-mannose, D-galactose, D-glucose, and D-glucuronic acid (ratio, 8.5:3.9:1.0:1.0). It consists of a backbone of (1-->6)-alpha-linked D-mannose residues, some of which are substituted at 0-3 with single or beta-(1-->6)-linked D-galactofuranosyl side chains, some terminated with a D-glucuronic acid residue, and also with single residues of D-glucopyranose, D-galactopyranose, and D-mannopyranose. This glucurono-gluco-galactomannan interacted with antiserum against Elsinoe leucospila, which also reacted with its galactomannan, indicating that both polysaccharides contain a common epitope, i.e., at least terminal beta-galactofuranosyl groups and also possibly internal beta-(1-->6)-linked galactofuranose residues. It was further separated by DEAE-Sephacel column chromatography to gluco-galactomannan and glucurono-gluco-galactomannan. The alkali-extracted beta-D-glucan was purified by DEAE-cellulose chromatography to afford two antitumor-active (1-->3)-beta-D-glucans. One of the glucans (M(r), 1-2 x 10(5)) was a O-6-branched (1-->3)-beta-D-glucan with a single beta-D-glucosyl residue, d.b., 1/7, and the other (M(r), 3.5-4.5 x 10(5)) had similar branched structure, but having d.b., 1/5. Side chains of both glucans contain small proportions of beta-(1-->6)- and beta-(1-->4)-D-glucosidic linkages.

Alteration of beta-D-glucan from edible mushroom after injection into mouse peritoneal cavity.

Tritium-labeled antitumor beta-D-glucan derivative (T-labeled glucan) was prepared from the branched beta-1,3-D-glucan of an edible mushroom, Volvariella volvacea, by its periodate oxidation followed by reduction with NaBT4. Twenty-three hours after T-labeled glucan had been injected into the mouse peritoneal cavity, about 3% of the total radioactivity injected was found in the mouse serum. In spite of the fact that T-labeled glucan had no affinity to the anion-exchange column before injection, about 50% of the labeled beta-D-glucan in the serum thus obtained was adsorbed onto the column. The labeled beta-D-glucan fraction eluted from the column by salt gradient showed antitumor activity in vivo.