Changes in ganglioside contents, plasma sialic acid and cAMP levels in experimental hepatoma in mice

The present study was designed to assess whether changes in glycolipids and cyclic AMP contents might serve as markers for the diagnosis of malignancy in the liver. The experimental model was a transplantable murine hepatoma. Experimental mice were divided into three groups: (1) a therapeutic group, which had been transplanted with hepatoma and treated with the antimetabolism drug 5-flurouracil (0.2 mg/day i.p.), (2) a control group, which had been transplanted with hepatoma and treated with 0.2 ml 0.9% NaCl/day and (3) a normal group of mice. The ganglioside and cAMP contents in the hepatoma tissue, plasma cAMP, total- and lipid-bound sialic acid levels and red blood cell membrane sialic acid levels were determined. Results showed that the ganglioside content, total and lipid-bound sialic acid levels in the control group were significantly higher than those in the livers of normal mice (p < 0.01) while these respective values in the therapeutic group were significantly lower than those in the control group (p < 0.01). The cAMP levels of tumor tissues and plasma in the control group were lower than those in normal mice. No significant difference in red blood cell membrane sialic acid content was observed between the therapeutic and control groups though levels for both were higher than those in normal mice. These results indicate that ganglioside content and sialic acid levels in hepatoma tissues were significantly elevated, and cAMP levels in hepatoma tissues were significantly decreased during proliferation and abnormal differentiation.     

The structure of the oligosaccharides of N-cadherin from human melanoma cell lines

N-cadherin is calcium-dependent cell adhesion molecule that mediates cell-cell adhesion and also modulates cell migration and tumor invasion. N-cadherin is a heavily glycosylated protein. Many studies have demonstrated that malignant transformation of a number of cell types correlates with changes of cell surface N-linked oligosacharides. We have studied the carbohydrate profile of N-cadherin synthesized in human melanoma cell lines and the effect of this protein and complex N-glycans on in vitro migration of melanoma cells from the primary tumor site--WM35 and from different metastatic sites WM239 (skin), WM9 (lymph node), and A375 (solid tumor). N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterization of its carbohydrate moieties was carried out by SDS-PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and on-blot deglycosylation using PNGase F for glycan release. N-glycans were separated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and their structures identified by the computer matching of the resulting masses with those derived from a sequence database. The assay of in vitro chemotaxic cell migration was performed using QCM Cell Invasion Assay (Chemicon). N-cadherin from WM35 (primary tumor site) possessed high-mannose and biantennary complex type glycans with alpha2-6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines possessed mostly tri- or tetra-antennary complex type glycans. In addition, N-cadherin from WM9 (lymph node metastatic site) and A375 (solid tumor metastatic site) contained heavily alpha-fucosylated complex type chains with alpha2,3 linked sialic acid. Blocking of N-cadherin-mediated intercellular interaction by N-cadherin-specific antibodies significantly (of about 40%) inhibited migration of melanoma cells. Inhibition of synthesis of complex type N-glycans by swainsonine (mannosidase II inhibitor) led to 50% decrease of cell migration. The results indicated differences between N-cadherin glycans from primary and metastatic sites and confirmed influence of N-cadherin and complex -type N-glycans on in vitro migration of melanoma cells.     

Carbohydrate moieties of N-cadherin from human melanoma cell lines

Expression of N-cadherin an adhesion molecule of the cadherin family, in tumor cells is associated with their increased invasive potential. Many studies suggested the role of N-linked oligosaccharides as important factors that contribute to metastasis by influencing tumor cell invasion and adhesion. N-cadherin is a heavily glycosylated protein. We have analysed the carbohydrate profile of this protein synthesized in human melanoma cell lines: WM35 from the primary tumor site and WM239, WM9, and A375 from different metastatic sites. N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterisation of its carbohydrate moieties was carried out by SDS/PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and analysis of their glycans using highly specific digoxigenin or biotin labelled lectins. The positive reaction of N-cadherin from the WM35 cell line with Galanthus nivalis agglutinin (GNA), Datura stramonium agglutinin (DSA) and Sambucus nigra agglutinin (SNA) indicated the presence of high-mannose type glycans and biantennary complex type oligosaccharides with alpha2-6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines gave a positive reaction with Phaseolus vulgaris leukoagglutinin (L-PHA) and lotus Tetragonolobus purpureas agglutinin (LTA). This indicated the presence of tri- or tetra-antennary complex type glycans with alpha-fucose. In addition, N-cadherin from WM9 (lymphomodus metastatic site) and A375 (solid tumor metastatic site) contained complex type chains with alpha2-3 sialic acid (positive reaction with Maackia amurensis agglutinin--MAA). The results demonstrated that N-glycans of N-cadherin are altered in metastatic melanomas in a way characteristic for invasive tumor cells.     

The structure of the oligosaccharides of N-cadherin from human melanoma cell lines

N-cadherin is calcium-dependent cell adhesion molecule that mediates cell-cell adhesion and also modulates cell migration and tumor invasion. N-cadherin is a heavily glycosylated protein. Many studies have demonstrated that malignant transformation of a number of cell types correlates with changes of cell surface N-linked oligosacharides. We have studied the carbohydrate profile of N-cadherin synthesized in human melanoma cell lines and the effect of this protein and complex N-glycans on in vitro migration of melanoma cells from the primary tumor site—WM35 and from different metastatic sites WM239 (skin), WM9 (lymph node), and A375 (solid tumor). N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterization of its carbohydrate moieties was carried out by SDS-PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and on-blot deglycosylation using PNGase F for glycan release. N-glycans were separated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and their structures identified by the computer matching of the resulting masses with those derived from a sequence database. The assay of in vitro chemotaxic cell migration was performed using QCM? Cell Invasion Assay (Chemicon). N-cadherin from WM35 (primary tumor site) possessed high-mannose and biantennary complex type glycans with α2–6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines possessed mostly tri- or tetra-antennary complex type glycans. In addition, N-cadherin from WM9 (lymph node metastatic site) and A375 (solid tumor metastatic site) contained heavily α-fucosylated complex type chains with α2,3 linked sialic acid. Blocking of N-cadherin-mediated intercellular interaction by N-cadherin-specific antibodies significantly (of about 40%) inhibited migration of melanoma cells. Inhibition of synthesis of complex type N-glycans by swainsonine (mannosidase II inhibitor) led to 50% decrease of cell migration. The results indicated differences between N-cadherin glycans from primary and metastatic sites and confirmed influence of N-cadherin and complex -type N-glycans on in vitro migration of melanoma cells. Published in 2004.     

Localization of glycoconjugates on the surfaces of pronephric tumor cells in vitro

Summary Differential localization of glycoconjugates was detected on microvilli and microridges of the intact cell surface of frog pronephric tumor cells in tissue culture. Alcian blue and Alcian blue/PAS staining showed a heavy concentration of dye limited to the unique short microvilli and extensive microridges of the tumor cells as previously seen with SEM (Tweedell and Williams 1976). Staining was absent or greatly reduced on microvilli of the normal pronephric cell surface. Previous exposure of each kind of cells to neuraminidase or extraction by mild hydrolysis removed the active staining sites but Alcian blue uptake was unaffected by prior digestion with testicular hyaluronidase. Fluorescein isothyocyanate (FITC) bound wheat germ agglutinin (WGA) produced a similar pattern of fluorescence on the microvilli of the tumor cells and a limited distribution on the normal cells. Digestion with neuraminidase preferentially removed but did not completely eliminate the surface binding of WGA on both the normal and tumor cells. Exposure of tumor cell monolayers to FITC bound limulin, a lectin specific for sialic acid, also produced an intense surface fluorescence on the microvilli and ridges of tumor cells. Prior treatment with neuraminidase prevented the surface fluorescence but not internal binding. Normal pronephric cells gave sparse surface fluorescence but extensive internal binding. Each procedure indicates a preferential localization of complex carbohydrates, including sialic acid, on the unique microvilli of the tumor cells. Concurrent assays for sialic acid recovered from the tumor cells indicated that lectin bound surface sialic acid was removable with neuraminidase.This study was supported by a grant from the Cancer Society of Saint Joseph County, Indiana and from the Phi Beta Psi Sorority     

Localization of glycoconjugates on the surfaces of pronephric tumor cells in vitro

Differential localization of glycoconjugates was detected on microvilli and microridges of the intact cell surface of frog pronephric tumor cells in tissue culture. Alcian blue and Alcian blue/PAS staining showed a heavy concentration of dye limited to the unique short microvilli and extensive microridges of the tumor cells as previously seen with SEM (Tweedell and Williams 1976). Staining was absent or greatly reduced on microvilli of the normal pronephric cell surface. Previous exposure of each kind of cells to neuraminidase or extraction by mild hydrolysis removed the active staining sites but Alcian blue uptake was unaffected by prior digestion with testicular hyaluronidase. Fluorescein isothiocyanate (FITC) bound wheat germ agglutinin (WGA) produced a similar pattern of fluorescence on the microvilli of the tumor cells and a limited distribution on the normal cells. Digestion with neuraminidase preferentially removed but did not completely eliminate the surface binding of WGA on both the normal and tumor cells. Exposure of tumor cell monolayers to FITC bound limulin, a lectin specific for sialic acid, also produced an intense surface fluorescence on the microvilli and ridges of tumor cells. Prior treatment with neuraminidase prevented the surface fluorescence but not internal binding. Normal pronephric cells gave sparse surface fluorescence but extensive internal binding. Each procedure indicates a preferential localization of complex carbohydrates, including sialic acid, on the unique microvilli of the tumor cells. Concurrent assays for sialic acid recovered from the tumor cells indicated that lectin bound surface sialic acid was removable with neuraminidase.     

Cell surface sialylation and tumor metastasis. Metastatic potential of B16 melanoma variants correlates with their relative numbers of specific penultimate oligosaccharide structures

Numerous investigations suggest that cell surface glycoconjugates, and in particular sialic acids, are directly involved in determining the metastatic phenotype. To further evaluate this hypothesis, we have used a variety of techniques to probe the cell surfaces of several metastatic variants of the murine B16 melanoma that were selected for experimental lung-colonizing ability (Fidler, I. (1973) Nature 242, 148-149) or for their ability to spontaneously metastasize from the site of a subcutaneous injection (Stackpole, C. W., Alterman, A. L., and Fornabaio, D. M. (1985) Invasion & Metastasis 5, 125-142). Using a highly sensitive high performance liquid chromatography sialic acid assay in conjunction with Vibrio cholerae sialidase, we find that none of these metastatic variants differ significantly in their overall levels of cell surface sialic acid. Using highly purified, linkage-specific sialyltransferases, in conjunction with specific glycosidases, to probe the cell surface saccharide topography of specific penultimate oligosaccharides, we also find no significant differences between the efficient lung-colonizing variant, B16-F10 and the poorly-colonizing B16-F1 or B16-Flr variants. In contrast, the spontaneously metastatic variants examined contain substantially different levels of specific penultimate sialylation sites. The tumorigenic but nonmetastatic B16-LM3/G3.26 variant contains 4-fold more penultimate Gal beta 1-3GalNAc sialylation sites than the tumorigenic and highly metastatic B16-LM3/G3.12 variant when CMP[3H]NeuAc and the alpha 2-3Gal beta 1-3GalNAc sialyltransferase are used to probe the melanoma cell surfaces. Several prominent glycoconjugates of apparent Mr 43,000, 40,000, and 30,000 are especially evident upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the nonmetastatic cells. The nonmetastatic variant also contains 2-fold more Gal beta 1-4GlcNAc sialylation sites than the metastatic variant when the alpha 2-6Gal beta 1-4GlcNAc sialyltransferase is used as a cell surface probe. In this case, glycoconjugates of apparent Mr 74,000, 45,000, and 43,000 are more prominently observed on the cell surfaces of the nonmetastatic variant. These data indicate that the differences in lung-colonizing abilities of B16 melanoma metastatic variants do not correlate with the numbers or sialylation states of specific penultimate oligosaccharide structures on their surfaces. However, the relative levels of specific penultimate saccharide structures do correlate with the ability of the cells to undergo spontaneous metastasis from a subcutaneous tumor.     

Oxygenation alters ganglioside expression in rat liver following partial hepatectomy

Gangliosides from livers of weanling rats were analyzed after 15% partial hepatectomy (PH) and different pre- and post-operative hyberbaric oxygenation (pre- and postHBO). Neu5Ac was the predominant ganglioside-derived sialic acid (>85%) compared to Neu5Gc. Almost identical low total sialic acid content (Neu5Ac+Neu5Gc) of the control and operated nonHBO animals opposed a 6.4- to 7.6-fold increase in pre- and postHBO animals (69.26 and 81.64pmol/mg wet weight, respectively). NanoESI-QTOF mass spectrometry combined with HPTLC immunostaining revealed GM3(Neu5Ac) and GM3(Neu5Gc) as major gangliosides, correlating with the respective sialic acid concentrations. Minor neolacto-series gangliosides were enhanced in preHBO and postHBO, but GM1-core gangliosides only in preHBO rats. GM2 and GalNAc-GM1b were clearly detectable in oxygenated rats compared to traces in the control and nonHBO animals. These results point at a functional role of gangliosides in liver growth regulation and reconstitution after PH combined with pre- and post-operative HBO treatment.     

Metabolism of sialic acids from exogenously administered sialyllactose and mucin in mouse and rat

A mixture of N-acetyl-[4,5,6,7,8,9-14C]neuraminosyl-alpha (2-3(6]-galactosyl-beta (1-4-glucose[( 14C]sialyl-lactose) and N-acetylneuraminosyl-alpha (2-3(6]-galactosyl-beta(1-4)-glucit-1-[3H]ol(sialyl-[3H]lactitol) as well as porcine submandibular gland mucin labeled with N-acetyl- and N-glycoloyl-[9-(3)H]neuraminic acid were administered orally to mice. The distribution of the different isotopes was followed in blood, tissues and excretion products of the animals. One half of the [14C]sialyl-lactose/sialyl-[3H]lactitol mixture given orally was excreted unchanged in the urine. The other half was hydrolysed by sialidase and partly metabolized further, followed by the excretion of 30% of the 14C-radioactivity as free N-acetyl-[4,5,6,7,8,9-14C]neuraminic acid and 60% of this radioactivity in the form of non-anionic compounds including expired 14CO2 within 24 h. The 14C-radioactivity derived from the [14C]sialyl-lactose/sialyl-[3H]lactitol mixture which remained in the bodies of fasted mice after 24 h was less than 1%. In the case of well-fed mice, a higher amount of the sialic acid residues was metabolized. The bulk of radioactivity of the mucin was resorbed within 24 h. About 40% of the radioactivity administered was excreted by the urine within 48 h; 30% of this radioactivity represented sialic acid and 70% other anionic and non-anionic metabolic products. 60% of the radioactivity administered remained in the body, and bound 3H-labeled sialic acids were isolated from liver. Sialyl-alpha (2-3)-[3H]lactitol was injected intravenously into rats; the substance was rapidly excreted in the urine without decomposition. These studies show that part of the sialic acids bound to oligosaccharides and glycoproteins can be hydrolysed in intestine by sialidase and be resorbed. This is followed either by excretion as free sialic acid or by metabolization at variable degrees, which apparently depends on the compound fed and on the retention time in the digestive tract.     

Sialic acid metabolism in sialuria fibroblasts

Sialuria is a rare inborn error of metabolism caused by excessive synthesis of sialic acid (N-acetylneuraminic acid, NeuAc). Fibroblasts cultured from the three known cases of sialuria contained 70-200-fold increases in soluble sialic acid, but normal concentrations of bound sialic acid. The sialic acid appeared in the cytosolic fraction of the cells on differential centrifugation, and was susceptible to borohydride reduction, suggesting that accumulated sialic acid was in the form of NeuAc and not CMP-NeuAc. In biochemical studies, CMP-NeuAc (50 microM) inhibited the UDP-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase of normal fibroblasts by 84-100%, but inhibited the epimerase from sialuria cells by only 19-31%. Feeding sialuria cells up to 5 mM D-glucosamine for 72 h increased free sialic acid content 20-60%, but normal cells were unaffected by this treatment. Cytidine feeding (5 mM, 72 h) reduced the NeuAc content of sialuria cells, initially 112, 104, and 266 nmol/mg protein, by 63-71 nmol/mg protein; CMP-NeuAc concentrations, initially 4, 2, and 5 nmol/mg protein, increased by 14-33 nmol/mg protein. Consequently, the total cellular content of soluble sialic acid (NeuAc + CMP-NeuAc) was lowered 14-46% by cytidine feeding. The inheritance pattern of sialuria has not been determined. However, cells from both parents of one sialuria patient contained normal concentrations of free sialic acid, and the parental epimerase activity also responded normally to CMP-NeuAc. We conclude that the basic biochemical defect in all known cases of sialuria is a failure of CMP-NeuAc to feedback-inhibit UDP-GlcNAc 2-epimerase and cytidine feeding can lower the intracellular soluble sialic acid concentration of sialuria cells.     

Sialic acid metabolism in rat liver: effect of carbon tetrachloride

Sialic acid metabolism was investigated in the livers of control rats and of rats treated with a single oral dose (1.5 ml/kg body weight) of carbon tetrachloride. The main change observed during the necrotic stage of CCl4 poisoning (18 h after treatment) was a highly significant reduction in sialyltransferase activity. Slight reciprocal changes in neuraminidase activities, i.e., a small decrease in cytosolic neuraminidase and a small increase in the membrane bound enzyme were also observed. At 72 h after CCl4 treatment, during the stage of liver regeneration, the main change was a marked elevation in membrane-bound neuraminidase (two fold above control values). Moderate increases in the specific activities of CMP-N-acetylneuraminic acid synthetase and sialyltransferase were also observed. A considerable decrease in the sialic acid content of the isolated smooth endoplasmic reticulum (one half of control values) was detected at 72 h after CCl4 administration. The sialic acid content of the rough endoplasmic reticulum, on the other hand, remained at control levels.     

Accessibility of sialo components in a murine tumor cell to extracellular N-acetylneuraminate glycohydrolase (sialidase).

Lipid-bound sialic acid in the murine melanoma cell is not totally inaccessible to an exogenous macromolecular probe, as formerly believed. Roughly 30% of the dialic acid bound to lipid, and an equal proportion of the sialic acid bound to protein is cleaved by the action of Clostridium perfringens N-acetylneuraminate glycohydrolase (neuraminidase, sialidase) when the purified enzyme is added to the suspenion medium of intact murine melanoma cells freshly derived from the tumor. Cleavage of lipid-bound sialic acid is indifferent to the presence of Ca (2+) in the medium. However, maximum release from protein requires a physiological concentration of this divalent cation. Variation in ionic strength has no effect on release of sialic acid. These findings show that restricted portion of the bound sialic acid may be released from the intact murine melanama cell by the extracellularly supplied enzyme acting topographically.     

Effects of carbohydrates on the pharmacokinetics and biological activity of equine chorionic gonadotropin in vivo.

The sialylation of eCG was examined to determine its influence on the in vivo metabolism and biological activity of the molecule. Sialic acid was decrementally removed from eCG by incubation with agarose-linked neuraminidase for varying time periods. Pharmacokinetic parameters for the disappearance of 4,000 IU (267 micrograms) of three desialylated eCG preparations (20%, 53%, and 80% sialic acid removed) and control eCG were determined in sheep. The clearance rate of eCG increased (p less than 0.05) with each decrement of sialic acid. The removal of 53% sialic acid enhanced the distribution of eCG into the tissues, compared to control and 20% desialylated eCG (p less than 0.05), presumably because of increased lipid solubility and decreased molecular size. Desialylation to 53% did not alter the elimination half-life of eCG. The removal of 80% sialic acid resulted in the disappearance of eCG from the serum within 1 h, whereas control eCG was still present at 120 h. In vivo trials in rats disclosed that the control eCG preparation increased ovulatory rate at doses of 10-100 IU and ovarian weight at doses of 10-300 IU relative to saline-treated rats (p less than 0.01). The 20% desialylated eCG induced superovulatory and ovarian weight responses, but 100-500 IU were required to achieve the same result as that produced by control eCG. The 53% and 80% desialylated eCG preparations induced a mild superovulatory response (p less than 0.01) but no ovarian weight response. It was concluded that sialic acid was significant to the distribution and disappearance of eCG. The effects of carbohydrate removal on biological activity (e.g., superovulation) are primarily a function of clearance rate rather than tissue-specific phenomena.     

Cytochrome 3A and 2E1 in human liver tissue: Individual variations among normal Japanese subjects

AimsThe metabolism of drugs, xenobiotic compounds, and other endogenous/exogenous substrates generally begins with their oxidation through cytochrome P450 (CYP). The results of recent pharmacogenetic analyses have demonstrated CYP's polymorphisms to be related to individual differences in metabolism, but only a limited number of CYP3A4 and CYP2E1 variant alleles influence enzymatic activities. Therefore, CYP gene expression profiling of both normal and pathological human livers should provide critical information for an evaluation of the biological significance of CYPs.Main methodsIn our present study, we first characterized the individual differences in CYP3A4 and CYP2E1 expression levels among Japanese normal or non-pathological liver tissue obtained from autopsy or surgery using immunohistochemistry and quantitative RT-PCR array of phase I metabolic enzymes with combined laser capture microscopy and qPCR analysis.Key findingsBoth CYP3A4 and CYP2E1 mRNA and proteins were predominantly detected in hepatocytes surrounding central veins in normal liver, but there were marked individual differences in both CYP3A4 and CYP2E1 mRNA and proteins among the 23 Japanese subjects examined. Individual differences in CYP3A and CYP2E1 subtypes were also detected in the livers obtained from monozygotic neonatal Japanese female twins with different survival periods. CYP3A and CYP2E1-positive cells were decreased in number in non-pathological hepatocytes of diseased livers compared to those in disease-free livers from autopsy.SignificanceThe above results suggest that individual differences in CYP3A4 and CYP2E1 exist among normal human liver tissues and in non-pathological hepatocytes between diseased and normal liver, and these differences may be important in evaluating the pharmacodynamics of various substances.     

自发性可移植性615小鼠肺癌和肝癌组织神经节苷脂高效薄层层析图谱的分析

本文研究了615近交系小鼠的自发性可移植性肺癌(P_(615))和肝癌(H_(615))组织中神经节苷脂结合唾液酸含量和神经节苷脂图谱。P_(615)和H_(615)癌组织中GLS结合唾液酸含量均比正常对照为高,分别为对照组织的2.9倍和1.9倍。615小鼠的正常肺组织和肝组织的GLS主要成分均为GM_3。在P_(615)和H_(615)癌组织中GM_3含量均明显减少。肺癌组织中GM_2大量增加,肝癌组织中不仅GM_2明显增加,GM_1和GD_(18)也明显增加。P_(615)和H_(615)这两种分化程度较高、恶性程度较低的癌组织GD_3的百分含量比正常对照组织略有降低。本文结果提示,自发性可移植性P_(615)和H_(615)肿瘤组织中不仅神经节苷脂含量(以唾液酸量计)升高,而且GLS的组分也发生改变。GM_3含量减少和GM_2含量增高可能与肿瘤的恶性生长和分化程度有关。     

Accessibility of sialo components in a murine tumor cell to extracellular N-acetylneuraminate glycohydrolase (sialidase)

Lipid-bound sialic acid in the murine melanoma cell is not totally inaccessible to an exogenous macromolecular probe, as formerly believed. Roughly 30% of the sialic acid bound to lipid, and an equal proportion of the sialic acid bound to protein is cleaved by the action of Clostridium perfringens$\text{N-}\text{acetylneuraminate}$ glycohydrolase (neuraminidase, sialidase) when the purified enzyme is added to the suspension medium of intact murine melanoma cells freshly derived from the tumor. Cleavage of lipid-bound sialic acid is indifferent to the presence of Ca²⁺ in the medium. However, maximum release from protein requires a physiological concentration of this divalent cation. Variation in ionic strength has no effect on release of sialic acid. These findings show that a restricted portion of the bound sialic acid may be released from the intact murine melanama cell by the extracellularly supplied enzyme acting topographically.     

Immunopositivity for Siglec-15 in gastric cancer and its association with clinical and pathological parameters

The sialic acid-binding immunoglobulin-type lectin Siglec-15 is a promising target for cancer immunotherapy in several tumor types. The present study aimed to investigate Siglec-15 expression in gastric cancer (GC) patient tissues and to evaluate its clinical value. Siglec-15 expression was evaluated by immunohistochemistry in 71 patients. Siglec-15 staining was observed in tumor cells of 53 (74.64%) patients, with significant association with histologic classification and angiolymphatic invasion (p<0.05). Immunohistochemistry analysis also detected Siglec-15 in tumor-associated stroma cells (macrophages/myeloid cells). There was no significant association with outcome parameters. Siglec-15 expression in well differentiated histological GC tissues and in the tumor microenvironment are potential targets to be further investigated as a novel prognostic factor for GC.Key words: Siglec, sialic acid, immunohistochemistry, clinical-pathological analyses     

Interaction with lectin of kappa opioid binding sites solubilized from human placenta

Kappa opioid binding sites from human placenta, prelabeled with 3H-etorphine and solubilized, were retained on wheat germ agglutinin (WGA) agarose and specifically eluted with N-acetylglucosamine. No significant retention was found with other immobilized lectins, including Concanavalin A (Con A), soybean seed lectin (SBA), Pisum sativum lectin (PsA), Lens culinaris Medik. lectin (LcA), and Lathyrus tingitanus lectin(LtA). About 23% of applied kappa sites were specifically eluted from WGA agarose, less than half of the proportion of rat brain opioid binding sites eluted from the same lectin (55%). Receptors from placental extracts were compared with those from other tissues enriched in either kappa or mu sites. The proportion of applied kappa sites from guinea pig cerebellum eluted specifically from WGA agarose was 36%, whereas elution of binding sites from rat thalamus and rabbit cerebellum (enriched in mu sites) was at a level of 55%. This difference in the level of retention on and elution from WGA may reflect differences in the sugar composition of the glycoproteins of the two types of receptors. Succinylation of WGA abolished its ability to retain opioid binding sites, consistent with involvement of sialic acid. However, currently available evidence suggests that differences in retention on WGA between kappa and mu sites may be due to differences in either sialic acid or N-acetylglucosamine content or both.     

Sialic acid concentration of brain gangliosides: variation among eight mammalian species

Sialic acid is a vital component of brain gangliosides which play an essential role in the transmission and storage of information in the brain. The concentration of bound sialic acid in gangliosides and free sialic acid in the brain cortex of eight different mammals [human, chimpanzee (Pan troglodytes), rat (Rattus norvegicus), mouse (Mus musculus), rabbit (Oryctolagus cuniculus), sheep (Ovis aries), cow (Bos indicus) and pig (Sus scrofa)] were compared. Total sialic acid concentration (890+/-103 microg/g wet weight tissue, mean+/-SE, n = 6) was 2-4 times higher in the human brain compared with the other species studied (0.001 < p < 0.05). There was no significant difference between human males and females. The rank order of adult brain sialic acid after humans (in microg/g) was rat (493+/-23, n = 12), mouse (445+/-29, n = 16), rabbit (380+/-18, n = 6), sheep (323+/-43, n = 6), cow (304+/-14, n = 6) and pig (252+/-14, n = 6). Apart from the cow vs the sheep, the differences between species were statistically significant (p < 0.05). In the mouse, cow and sheep, total sialic acid concentration increased during maturation by 18-32% (p < 0.05). In a 2-year-old chimpanzee, the sialic acid concentration in the left lobe of the brain cortex was 25% higher than that of right lobe at 6 weeks of age (p < 0.05). Free sialic acid was higher in the human brain cortex (41+/-3 microg/g) than that of the rat and mouse (32+/-3 and 25+/-5 microg/g respectively) and absent from other species. Variation in brain sialic acid concentration among different animals has implications for the evolution of the brain and may affect learning ability in animals.     

在HBV感染的肝癌组织中Fas/FasL表达的作用

目的探讨Fas／FasL（Fas配体）在原发性肝细胞肝癌（HCC）组织中的表达及其与乙肝病毒（HBV）感染的关系。方法用免疫组化S-P法检测21例肝癌组织及其10例癌旁组织Fas／FasL的表达。结果在肝癌和癌旁组织中Fas表达的阳性率分别为52．38％和80．00％（P〈0．05）,FasL分别为66．67％和40．00％（P〈0．05）。在HBsAg阳性和阴性组Fas表达的阳性率分别为50．00％和66．67％（P〉0．05）;FasL分别为61．11％和100％（P〈0．05）。肝癌组织中Fas、FasL表达与性别、年龄、肿瘤大小无关,与癌栓转移呈负相关,FasL与分化程度有关。结论肝癌细胞能下调Fas及上调FasL的表达而使凋亡受阻,HBV感染能抑制肝癌组织FasL的表达,可能是HBV在HCC的发牛及发展讨稃中的致痛机制。