Biomedical subjects

Minoru Fukuda

Publications and source records attributed to Minoru Fukuda.

At least 73 records · Page 4Linked to original sources

PubMed2004

The core 2 beta-1,6-N-acetylglucosaminyltransferase-M encoded by bovine herpesvirus 4 is not essential for virus replication despite contributing to post-translational modifications of structural proteins.

The Bo17 gene of bovine herpesvirus 4 (BoHV-4) is the only virus gene known to date that encodes a homologue of the cellular core 2 beta-1,6-N-acetylglucosaminyltransferase-mucine type (C2GnT-M). Recently, our phylogenetic study revealed that the Bo17 gene has been acquired from an ancestor of the African buffalo around 1.5 million years ago. Despite this recent origin, the Bo17 sequence has spread to fixation in the virus population possibly by natural selection. Supporting the latter hypothesis, it has been shown by our group for the V. test strain that Bo17 is expressed during BoHV-4 replication in vitro, and that Bo17 expression product (pBo17) has all three enzymic activities exhibited by cellular C2GnT-M, i.e. core 2, core 4 and I branching activities. In the present study, firstly it was investigated whether encoding a functional C2GnT-M is a general property of BoHV-4 strains. Analysis of nine representative strains of the BoHV-4 species revealed that all of them express the Bo17 gene and the associated core 2 branching activity during virus replication in vitro. Secondly, in order to investigate the roles of Bo17, its kinetic class of expression was analysed and a deleted recombinant strain was produced. These experiments revealed that Bo17 is expressed as an early gene which is not essential for virus replication in vitro. However, comparison of the structural proteins, produced by the wild-type, the revertant and the deleted viruses, by 2D gels demonstrated that pBo17 contributes to the post-translational modifications of structural proteins. Possible roles of Bo17 in vivo are discussed.

Enzyme Induction↗

PubMed2004

The histone deacetylase inhibitor FR901228 induces caspase-dependent apoptosis via the mitochondrial pathway in small cell lung cancer cells.

Histone deacetylase inhibitors modulate the transcription of target genes and represent a new class of anticancer agents. The histone deacetylase inhibitor FR901228 has been reported to show antiproliferative and apoptotic effects in various malignancies including small cell lung cancer (SCLC) in vitro; however, the underlying mechanism is not fully understood. BCL-2 and BCL-XL are antiapoptotic proteins, of which overexpression has been reported to confer resistance to anticancer agents. High levels of BCL-2 and BCL-XL are frequently expressed in SCLC tumors. The present study was designed to clarify the apoptotic pathway of FR901228 in SCLC cells in vitro. FR901228 induced apoptosis in three SCLC cell lines after 24 hours of treatment. FR901228 activated caspase-9 and caspase-3 but not caspase-8, and the caspase-3 inhibitor Z-DEVD-fmk blocked the cytotoxicity of FR901228. FR901228 down-regulated the expression of bcl-2 and bcl-xL mRNA through de novo protein synthesis and suppressed the expression of BCL-2 and BCL-XL proteins. In addition, the combination of bcl-2 antisense oligonucleotides with FR901228 enhanced FR901228-induced caspase-3 activity and cytotoxicity. These findings suggest that FR901228 induces caspase-dependent apoptosis via the mitochondrial pathway rather than the death receptor pathway. Considering the possible contributions of BCL-2 and BCL-XL to multidrug resistance, FR901228 is a promising agent in the treatment of refractory as well as primary SCLC tumors.

Apoptosis↗

PubMed2004

[Application of preoperative risk severity evaluation system (EuroSCORE=European system for cardiac operative risk evaluation) for cardiac operative patients].

BACKGROUND: There is no widespread, well-accepted preoperative risk evaluation system for cardiac operative patients in Japan. We evaluated a scoring system for the prediction of postoperative morbidity and mortality in Japanese cardiac surgical patients. METHODS: Between August 1, 2002 and July 31, 2003 154 patients undergoing heart surgery with cardiopulmonary bypass were scored according to EuroSCORE (European system for cardiac operative risk evaluation). All patients were divided into three risk groups, low risk group (score 0-2), medium risk group (score 3-5), and high risk group (score 6 plus). We studied correlations between score points and postoperative morbidity and mortality. Data were analyzed using chi2 test, with a P-value of less than 0.05 as significant. RESULTS: The mean preoperative risk score was 5.6. The increase in preoperative risk score was associated with an increase in postoperative length of ventilation support and ICU stay. The low risk group had 35 patients with 0 death (0%), 5 morbidity (14.2%). The medium risk group had 57 patients with 2 death (3.5%), 14 morbidity (24.6%). The high risk group had 62 patients with 9 death (14.5%), 35 morbidity (56.5%). Overall, there were 11 death (7.1%), 54 morbidity (35.1%) in 154 patients. The risk score point correlated with postoperative morbidity and mortality. CONCLUSIONS: In spite of the limitation of total patient's number (N = 154) in this study, we recommend EuroSCORE as a simple and essential tool for the risk assesment of cardiac surgery and prediction of postoperative morbidity and mortality.

Aged↗

PubMed2003

Gene transfer of alpha1,3-fucosyltransferase increases tumor growth of the PC-3 human prostate cancer cell line through enhanced adhesion to prostatic stromal cells.

Elevated expression of sialyl Lewis X has been postulated to be a prognostic indicator of prostate cancer. However, direct evidence for the relationship between increased expression of sialyl Lewis X and malignancy of prostate cancer is still lacking. To determine whether increased levels of sialyl Lewis X leads to malignancy in prostate tumor, we transfected the human prostate cancer cell line PC-3 with alpha1,3-fucosyltransferase III (FTIII) to obtain stable transfectants, PC-3-FTIII lines, that highly express sialyl Lewis X. When inoculated in the prostate of nude mice, PC-3-FTIII cells produced large prostate tumors, while mock-transfected PC-3 cells, which are negative for sialyl Lewis X antigen, produced small prostate tumors. The aggressive tumor formation by PC-3-FTIII cells was inhibited by preincubation of the tumor cells with anti-sialyl Lewis X antibody, by the presence of sialyl Lewis X oligosaccharide or by selectin ligand mimic peptide but not by control peptide. PC-3-FTIII cells and mock-transfected PC-3 cells exhibited no significant difference in cell numbers when cultured in vitro. Remarkably, PC-3-FTIII adhered to prostatic stromal cells in vitro with higher affinity than mock-transfected PC-3. Such adhesion was inhibited by preincubation of PC-3-FTIII cells with antisialyl Lewis X antibody, by the addition of sialyl Lewis X oligosaccharide or by selectin ligand mimic peptide. However, anti-E-selectin, anti-P-selectin or anti-L-selectin antibodies did not inhibit the adhesion of PC-3-FTIII cells to the stromal cells. These results suggest that prostate cancer cells gain aggressiveness through adhesive interaction with prostatic stromal cells by a novel mechanism involving sialyl Lewis X.

Adenocarcinoma↗

PubMed2003

Core 2 branching beta1,6-N-acetylglucosaminyltransferase and high endothelial venule-restricted sulfotransferase collaboratively control lymphocyte homing.

L-selectin mediates lymphocyte homing by facilitating lymphocyte adhesion to carbohydrate ligands expressed on high endothelial venules (HEV) of the secondary lymphoid organs. Previous studies demonstrated that L-selectin ligand sulfotransferase (LSST) forms 6-sulfo sialyl Lewis x (sLe(x)) on both core 2 branch and MECA-79-positive extended core 1 O-glycans, but the chemical nature and roles of HEV ligands elaborated by LSST and core 2 beta1,6-N-acetylglucosaminyltransferase-1 (Core2GlcNAcT) have been undefined. In the present study, we have generated mutant mice with deficient LSST and show that inactivation of LSST gene alone leads to only partial impairment of lymphocyte homing to peripheral lymph nodes and moderate reduction in lymphocyte counts in the peripheral lymph nodes, despite the fact that L-selectin ligands that contain 6-sulfo sLe(x) are reduced at HEV. By contrast, LSST/Core2GlcNAcT double null mice exhibited a markedly reduced lymphocyte homing and reduced lymphocyte counts as a result of significantly decreased 6-sulfo sLe(x) on HEV L-selectin counterreceptors, relative to LSST- or Core2GlcNAcT-single null mice. Moreover, induction of LSST and Core2GlcNAcT transcripts was observed in HEV-like structure formed in the salivary gland of the non-obese diabetic mouse, which displays chronic inflammation. These results indicate that LSST and Core2GlcNAcT cooperatively synthesize HEV-specific L-selectin ligands required for lymphocyte homing and suggest that LSST and Core2GlcNAcT play a critical role in lymphocyte trafficking during chronic inflammation.

Animals↗

PubMed2003

Different glycosyltransferases are differentially processed for secretion, dimerization, and autoglycosylation.

Modification of Golgi glycosyltransferases, such as formation of disulfide-bonded dimers and proteolytical release from cells as a soluble form, are important processes to regulate the activity of glycosyltransferases. To better understand these processes, six glycosyltransferases were selected on the basis of the donor sugars, including two N-acetylglucosaminyltransferases, core 1 beta1,3-N-acetylglucosaminyltransferase (C1-beta3GnT) and core 2 beta1,6-N-acetylglucosaminyltransferase (C2GnT-I); two fucosyltransferases, alpha1,2-fucosyltransferase-I (FucT-I) and alpha1,3-fucosyltransferase-VII (FucT-VII); and two sialyltransferases, alpha2,3-sialyltransferase-I (ST3Gal-I) and alpha2,6-sialyltransferase-I (ST6Gal-I). These enzymes were fused with enhanced green fluorescence protein and stably expressed in Chinese hamster ovary cells. Spectrofluorimetric detection and immunoblotting analyses showed that all of these glycosyltransferases except FucT-VII were secreted in the medium. By examining dimers formed in cells and culture media, we found that all of the enzymes, except ST3Gal-I, form a combination of monomers and dimers in cells, whereas the molecules released in the media are either exclusively monomers (C2GnT-I and ST6Gal-I), dimers (FucT-I) or a mixture of both (C1-beta3GnT). These results indicate that dimerization does not always lead to Golgi retention. Analysis of the N-glycosylation status of the enzymes revealed that the secreted proteins are generally more heavily N-glycosylated and sialylated than their membrane-associated counterparts, suggesting that the proteolytic cleavage occurs before the glycosylation is completed. Using FucT-I and ST6Gal-I as a model, we also show that these glycosyltransferases are able to perform autoglycosylation in the dimeric forms. These results indicate that different glycosyltranferases differ significantly in dimerization, proteolytic digestion and secretion, and autoglycosylation. These results strongly suggest that disulfide-bonded dimerization and secretion differentially plays a role in the processing and function of different glycosyltransferases in the Golgi apparatus.

Animals↗

PubMed2003

Polysialic acid and mucin type o-glycans on the neural cell adhesion molecule differentially regulate myoblast fusion.

Polysialic acid attached to the neural cell adhesion molecule (NCAM) is thought to play a critical role in development. NCAM in muscle tissue contains a muscle-specific domain (MSD) to which mucin type O-glycans are attached. In the present study, using the C2C12 myoblast system, we show that NCAM containing MSD is increasingly expressed on the cell surface as myotubes form. Polysialic acid is primarily attached to N-glycans of NCAM, and polysialylated NCAM is expressed on the outer surface of myotube bundles. By transfecting cDNAs encoding wild type and mutant forms of NCAM, we found that NCAM containing MSD facilitates myoblast fusion, and this effect is diminished by mutating O-glycosylation sites at MSD. By contrast, forced expression of polysialic acid in early differentiation stages reduces myotube formation and delays the expression of NCAM containing the MSD domain. Strikingly, inhibition of polysialic acid synthesis by antisense DNA approach induced differentiation in both human rhabdomyosarcoma cells, which overexpress polysialic acid, and C2C12 cells. These results indicate that polysialic acid and mucin type O-glycans on NCAM differentially regulate myoblast fusion, playing critical roles in muscle development.

Base Sequence↗

PubMed2003

Diabetes and the accompanying hyperglycemia impairs cardiomyocyte calcium cycling through increased nuclear O-GlcNAcylation.

Diabetic cardiomyopathy is characterized by impaired cardiac contractility leading to poor myocardial performance. We investigated the role that the hexosamine pathway, and especially altered nuclear O-Glc-NAcylation, plays in the development of diabetic cardiomyopathy. Incubating neonatal rat cardiomyocytes in high glucose (25 mM) resulted in prolonged calcium transients when compared with myocytes incubated in normal glucose (5.5 mM), which is consistent with delayed myocardial relaxation. High glucose-treated myocytes also exhibited reduced sarcoendoplasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) mRNA and protein expression, decreased SERCA2a promoter activity, and increased O-GlcNAcylation of nuclear proteins compared with myocytes treated with normal glucose. Exposure of myocytes to 8 mM glucosamine or an adenovirus expressing O-GlcNAc-transferase (OGT) resulted in prolonged calcium transient decays and significantly reduced SERCA2a protein levels, whereas treatment with an adenovirus encoding O-GlcNAcase (GCA) resulted in improved calcium transients and SERCA2a protein levels in myocytes exposed to high glucose. Effects of elevated glucose or altered O-GlcNAcylation were also observed on essential transcription factors involved in cardiomyocyte function. High glucose-treated myocytes (with or without OGT adenovirus) exhibited increased levels of O-GlcNAcylated specificity protein 1 compared with control myocytes, whereas infecting high glucose-treated myocytes with GCA adenovirus reduced the degree of specificity protein 1 Glc-NAcylation. Treatment of myocytes with 25 mM glucose, 8 mM glucosamine, or OGT adenovirus also significantly reduced levels of myocytes enhancer factor-2A protein compared with control myocytes, whereas infection with GCA adenovirus resulted in improved myocytes enhancer factor-2 expression. Our results suggest that the hexosamine pathway, and O-GlcNAcylation in particular, is important in impaired cardiac myocyte function and the development of diabetic cardiomyopathy.

Acetylglucosaminidase↗

PubMed2003

Altered T cell surface glycosylation in HIV-1 infection results in increased susceptibility to galectin-1-induced cell death.

The massive T cell death that occurs in HIV type 1 (HIV-1) infection contributes profoundly to the pathophysiology associated with AIDS. The mechanisms controlling cell death of both infected and uninfected T cells ("bystander" death) are not completely understood. We have shown that HIV-1 infection of T cells results in altered glycosylation of cell surface glycoproteins; specifically, it decreased sialylation and increased expression of core 2 O-glycans. Galectin-1 is an endogenous human lectin that recognizes these types of glycosylation changes and induces cell death of activated lymphocytes. Therefore we studied the possible contribution of galectin-1 in the pathophysiology of AIDS. O-glycan modifications were investigated on peripheral lymphocytes from AIDS patients. Oligosaccharides from CD43 and CD45 of CEM cells latently infected with HIV-1 were chemically analyzed. Consistent with our previous results, we show that HIV-1 infection results in accumulation of exposed lactosamine residues, oligosaccharides recognized by galectin-1 on cell surface glycoproteins. Both latently HIV-1-infected T cell lines and peripheral CD4 and CD8 T cells from AIDS patients exhibited exposed lactosamine residues and demonstrated marked susceptibility to galectin-1-induced cell death, in contrast to control cultures or cells from uninfected donors. The fraction of cells that died in response to galectin-1 exceeded the fraction of infected cells, indicating that death of uninfected cells occurred. Altered cell surface glycosylation of T cells during HIV-1 infection increases the susceptibility to galectin-1-induced cell death, and this death pathway can contribute to loss of both infected and uninfected T cells in AIDS.

Amino Sugars↗

PubMed2003

Extended core 1 and core 2 branched O-glycans differentially modulate sialyl Lewis X-type L-selectin ligand activity.

It has been established that sialyl Lewis x in core 2 branched O-glycans serves as an E- and P-selectin ligand. Recently, it was discovered that 6-sulfosialyl Lewis x in extended core 1 O-glycans, NeuNAcalpha2-->3Galbeta1-->4(Fucalpha1-->3(sulfo-->6))GlcNAcbeta1--> 3Galbeta1-->3GalNAcalpha1-->Ser/Thr, functions as an L-selectin ligand in high endothelial venules. Extended core 1 O-glycans can be synthesized when a core 1 extension enzyme is present. In this study, we first show that beta1,3-N-acetylglucosaminyltransferase-3 (beta3GlcNAcT-3) is almost exclusively responsible for core 1 extension among seven different beta3GlcNAcTs and thus acts on core 1 O-glycans attached to PSGL-1. We found that transcripts encoding beta3GlcNAcT-3 were expressed in human neutrophils and lymphocytes but that their levels were lower than those of transcripts encoding core 2 beta1,6-N-acetylglucosaminyltransferase I (Core2GlcNAcT-I). Neutrophils also expressed transcripts encoding fucosyltransferase VII (FucT-VII) and Core2GlcNAcT-I, whereas lymphocytes expressed only small amounts of transcripts encoding FucT-VII. To determine the roles of sialyl Lewis x in extended core 1 O-glycans, Chinese hamster ovary (CHO) cells were stably transfected to express PSGL-1, FucT-VII, and either beta3GlcNAcT-3 or Core2GlcNAcT-I. Glycan structural analyses disclosed that PSGL-1 expressed in these transfected cells carried comparable amounts of sialyl Lewis x in extended core 1 and core 2 branched O-glycans. In a rolling assay, CHO cells expressing sialyl Lewis x in extended core 1 O-glycans supported a significant degree of shear-dependent tethering and rolling of neutrophils and lymphocytes, although less than CHO cells expressing sialyl Lewis x in core 2 branched O-glycans. These results indicate that sialyl Lewis x in extended core 1 O-glycans can function as an L-selectin ligand and is potentially involved in neutrophil adhesion on neutrophils bound to activated endothelial cells.

Animals↗

PubMed2003

Possible involvement of gelatinases in basement membrane damage and wrinkle formation in chronically ultraviolet B-exposed hairless mouse.

A number of studies indicate that matrix metalloproteinase might be involved in photoaging, but little is known about their direct contribution to ultraviolet-induced histologic and morphologic changes in the skin in vivo. This study reports the relationship between changes of matrix metalloproteinase activities and ultraviolet B-induced skin changes in hairless mouse. The role of matrix metalloproteinase in the skin changes was studied by topical application of a specific matrix metalloproteinase inhibitor. The backs of mice were exposed to ultraviolet B three times a week for 10 wk. Histologic studies showed that the basement membrane structure was damaged, with epidermal hyperplasia, in the first 2 wk of ultraviolet B irradiation, followed by the appearance of wrinkles, which gradually extended in the latter half of the ultraviolet B irradiation period. We observed enhancement of type IV collagen degradation activity, but not collagenase or matrix metalloproteinase-3 activity, in extracts of ultraviolet B-irradiated, wrinkle-bearing skin. Gelatin zymographic analysis revealed that gelatinases, matrix metalloproteinase-9 and matrix metalloproteinase-2, were significantly increased in the extract. In situ zymographic study clarified that the activity was specifically localized in whole epidermis of ultraviolet B-irradiated, wrinkled skin in comparison with normal skin. The activity was induced around the basal layer of the epidermis by a single ultraviolet exposure of at least one minimal erythema dose. Furthermore, topical application of a specific matrix metalloproteinase inhibitor, CGS27023A, inhibited ultraviolet B-induced gelatinase activity in the epidermis, and its repeated application prevented ultraviolet B-induced damage to the basement membrane, as well as epidermal hyperplasia and dermal collagen degradation. Ultraviolet B-induced wrinkles were also prevented by administration of the inhibitor. These results, taken together, suggest that ultraviolet B-induced enhancement of gelatinase activity in the skin contributes to wrinkle formation through the destruction of basement membrane structure and dermal collagen in chronically ultraviolet B-exposed hairless mouse, and thus topical application of matrix metalloproteinase inhibitors may be an effective way to prevent ultraviolet B-induced wrinkle formation.

Animals↗

PubMed2003

Usefulness of the real-time reverse transcription-polymerase chain reaction assay targeted to alpha1,4-N-acetylglucosaminyltransferase for the detection of gastric cancer.

alpha1,4-N-acetylglucosaminyltransferase (alpha4GnT) is a glycosyltransferase that forms a unique glycan, GlcNAcalpha1-->4Galbeta-->R, specifically present in gastric gland mucous cell-type mucin. Recently, we molecularly cloned human alpha4GnT and showed that alpha4GnT is expressed in the mucous cells that secrete this particular mucin. In the present study, we first demonstrated that alpha4GnT was frequently expressed in gastric cancer cells but not in peripheral blood cells using immunohistochemistry. To detect gastric cancer cells circulating in the peripheral blood of gastric cancer patients, we quantitatively analyzed the expression level of alpha4GnT mRNA in the mononuclear cell fraction of peripheral blood using real-time reverse transcription polymerase chain reaction. The transcripts of alpha4GnT were detected in the mononuclear cell fraction isolated from 62.2% of 37 gastric cancer patients but not from any of 23 healthy individuals. Significant correlation was found in the expression levels of alpha4GnT mRNA in peripheral blood and alpha4GnT protein in gastric cancer cells. Surprisingly, alpha4GnT mRNA was detectable in 80% of five patients with an early stage of gastric cancer when the cancer cells were limited to the gastric mucosa, and the expression levels of alpha4GnT mRNA were increased in association with tumor progression. In three patients with gastric cancer, during postsurgical follow-up, the expression levels of alpha4GnT mRNA were decreased after surgical removal of gastric cancer. However, significant amounts of the alpha4GnT transcripts were again detected in two patients, who eventually developed to the recurrence of gastric cancer. Although alpha4GnT was detected in 33.3% of nine patients with Helicobacter pylori-infected chronic active gastritis as well as all of four patients with peptic ulcer, the mean expression level of alpha4GnT mRNA in these benign disorders was lower than that in gastric cancer. These results altogether indicate that the quantitative analysis of alpha4GnT mRNA expressed in the peripheral blood is useful for the detection and, possibly, monitoring of gastric cancer.

Adult↗

PubMed2002

Roles of mucin-type O-glycans in cell adhesion.

Mucin-type O-glycans containing Core2 branches have distinctly different functions from those O-glycans that contain Core1 structures. Core2 branched O-glycans can have terminal structures that function as ligands for carbohydrate binding proteins. However, sialylated Core2 branched O-glycans without additional modifications exhibit anti-adhesive properties. These results demonstrate that certain mucin-type O-glycans can either facilitate or attenuate cell adhesion depending on the core structures and the structures of the non-reducing termini.

Amino Acid Sequence↗

PubMed2002

Natural killer cells attack tumor cells expressing high levels of sialyl Lewis x oligosaccharides.

Epithelial carcinoma and leukemia cells express sialyl Lewis x oligosaccharides as tumor-associated carbohydrate antigens. To determine the role of sialyl Lewis x oligosaccharides in tumor dissemination, human melanoma MeWo cells, which do not express sialyl Lewis x, were transfected with alpha1,3-fucosyltransferase III (FTIII), and cell lines expressing different amounts of sialyl Lewis x were isolated. When these cells were injected into the tail vein of nude mice, cells expressing moderate amounts of sialyl Lewis x (MeWo-FTIII.M) produced a significantly greater number of lung tumor foci than did parental MeWo cells. In contrast, cells expressing large amounts of sialyl Lewis x (MeWo-FTIII.H) produced few lung tumor foci in nude mice but were highly tumorigenic in beige mice, which have defective natural killer (NK) cells. In vitro assays demonstrated that MeWo-FTIII.H cells are much more sensitive to NK cell-mediated cytotoxicity than are MeWo-FTIII.M cells or parental MeWo cells and the susceptibility of MeWo-FTIII.H cells to NK cell-mediated cytolysis can be inhibited by preincubating MeWo-FTIII.H cells with anti-sialyl Lewis x antibody. Moreover, we discovered that NK cell-mediated cytolysis of MeWo-FTIII.H cells can be inhibited by the addition of an antibody against the NK cell receptor CD94 or sialyl Lewis x oligosaccharides. These results, combined with structural analysis of MeWo-FTIII.H cell carbohydrates, indicate that moderate amounts of sialyl Lewis x lead to tumor metastasis, whereas expression of high levels of sialyl Lewis x leads to an NK cell attack on tumor cells, demonstrating that expression of different amounts of sialyl Lewis x results in entirely different biological consequences.

Animals↗

PubMed2002

Sialyl Lewis X-dependent lung colonization of B16 melanoma cells through a selectin-like endothelial receptor distinct from E- or P-selectin.

Endothelial carbohydrate binding proteins, E- and P-selectins, are thought to mediate sialyl Lewis A/X-dependent hematogenous cancer metastasis. We tested this hypothesis using sialyl Lewis X-dependent B16 melanoma lung targeting and its inhibition with selectin ligand mimicry peptide, IELLQAR. In E/P-selectin doubly deficient mutant mice, sialyl Lewis X-expressing B16 melanoma cells colonized the lung, and IELLQAR inhibited this colonization. However, tumors grown in E/P-selectin-deficient mice were significantly smaller than those grown in wild-type mice. These results indicate that the IELLQAR peptide receptor expressed in the lung vasculature plays a major role in sialyl Lewis X-dependent cancer cells targeting to the lung.

Animals↗

PubMed2002

ST8Sia II and ST8Sia IV polysialyltransferases exhibit marked differences in utilizing various acceptors containing oligosialic acid and short polysialic acid. The basis for cooperative polysialylation by two enzymes.

Polysialylation of the neural cell adhesion molecule (NCAM) is thought to play a critical role in neural development. Two polysialyltransferases, ST8Sia II and ST8Sia IV, play dominant roles in polysialic acid synthesis on NCAM. However, the individual roles and mechanisms by which these two enzymes form large amounts of polysialic acid on NCAM were heretofore unknown. Previous studies indicate that ST8Sia IV forms more highly polysialylated N-glycans on NCAM than ST8Sia II in vitro. In the present study, we first demonstrated that a combination of ST8Sia II and ST8Sia IV cooperatively polysialylated NCAM, resulting in NCAM N-glycans containing more, and thus longer, polysialic acid than when the enzymes were used individually. There was also an increase in polysialylated NCAM when we used ST8Sia II and ST8Sia IV sequentially, whereas there appeared to be a subtle increase when the enzymes were used in the reverse order. Furthermore, ST8Sia IV was able to add polysialic acid to oligosialylated oligosaccharides and unpolysialylated antennas in N-glycans attached to NCAM, even when polysialic acid was attached to at least one of the other antennas. By contrast, ST8Sia II added little polysialic acid to the same acceptors. On the other hand, neither ST8Sia II nor ST8Sia IV could add polysialic acid to a polysialylated antenna of NCAM N-glycans. These combined results indicate that the synergistic effect of ST8Sia II and ST8Sia IV is caused by: 1) the ability of ST8Sia IV to add polysialic acid to oligosialic acid formed by ST8Sia II, 2) the potential of ST8Sia IV to act on more antennas of N-glycans than ST8Sia II, and 3) the ability of ST8Sia II and ST8Sia IV in combination to act on the fifth and sixth N-glycosylation sites of NCAM.

Amino Sugars↗

PubMed2002

A peptide mimic of selectin ligands abolishes in vivo inflammation but has no effect on the rat heart allograft survival1.

Acute heart allograft rejection is characterized by leukocyte infiltration and myocyte damage, key elements in the histological grading of rejection. The induction of selectins and their ligands on the graft postcapillary venular endothelium increases leukocyte tethering to, rolling on, and extravasation through the endothelium into graft parenchyma. We have characterized peptide mimicking selectin ligands by screening phage peptide libraries using anti-Lewis A antibodies and E-selectin as target molecules. The effect of this selectin- binding peptide, IELLQAR, on the prevention of inflammation and tissue damage and on the prolongation of graft survival in inbred DA (RT1a) rat heart allografts transplanted to WF (RT1v) recipients was tested. Bovine serum albumin (0.1%, solvent), VTSIAQA (control peptide), or IELLQAR were either continuously infused into the peritoneum via osmotic mini pumps or injected twice daily IV. Treatment with bovine serum albumin and VTSIAQA did not alter the number of graft infiltrating leukocytes or the histological grade of acute rejection, all scored as grade 4. On the contrary, the selectin binding peptide, IELLQAR, dose-dependently reduced inflammation and at the highest dose (6.0 mg/kg per day) eliminated the majority of graft infiltrating leukocytes, reduced the histological grade from 4 to 1B, but had no effect on graft survival. These data indicate that the intensity of inflammation related to the allograft rejection does not correlate to the graft survival.

Animals↗

PubMed2002

Enlargement of secretory vesicles by protein tyrosine phosphatase PTP-MEG2 in rat basophilic leukemia mast cells and Jurkat T cells.

Stimulus-induced secretion of bioactive polypeptides is a fundamental aspect of the immune system. Secretory proteins are synthesized in the endoplasmic reticulum and are transported through the Golgi apparatus to the trans-Golgi network, where they are sorted into transport vesicles that bud off and fuse into condensing vacuoles, which subsequently undergo an editing and concentration process to become mature secretory vesicles. In this study, we report that the PTP-MEG2 protein tyrosine phosphatase is located on these vesicles in mast cells. Expression of PTP-MEG2 caused a striking enlargement of these vesicles in both rat basophilic leukemia mast cells and Jurkat T leukemia cells into giant vesicles with diameters of up to several micrometers. The fused vesicles did not acquire markers for other compartments and were adjacent to the trans-Golgi network, contained carboxypeptidase E, chromogranin C, and IL-2, and had an electron-dense core typical of secretory vesicles. Expression of PTP-MEG2 also caused a reduction in the secretion of IL-2 from stimulated Jurkat cells. The effects of PTP-MEG2 on secretory vesicles required the catalytic activity of PTP-MEG2 and was rapidly reversed by pervanadate. We propose that PTP-MEG2 represents a novel connection between tyrosine dephosphorylation and the regulation of secretory vesicles in hematopoietic cells.

Animals↗