Suguru Akamatsu

Altered mRNA expression of specific molecular species of fucosyl- and sialyl-transferases in human colorectal cancer tissues

Human colorectal cancers express various cancer‐associated carbohydrate determinants such as Lewis Y or sialyl Lewis A, suggesting a considerable alteration in glycosyltransferase activities occurring upon malignant transformation. We investigated the mRNA amounts of fucosyltransferase (Fuc‐T) and sialyltransferase (ST) isoenzymes, including Fuc‐T III, IV, V, VI and VII and ST‐3N, ST‐30 and ST‐4, in human colorectal cancer tissues by Northern blotting and RT‐PCR. Regarding fucosyltransferases, mRNA of Fuc‐T III and VI was not significantly altered, and only Fuc‐T IV mRNA showed a moderate increase in cancer tissues when compared with adjacent non‐malignant colonic epithelia taken from the same patient (273 ± 96%; p < 0.001). The moderate increase of Fuc‐T IV message may be related to an enhanced expression of Lewis Y in colon cancer tissues. In the ST isoenzymes, mRNA for ST‐3N remained unchanged, whereas that for ST‐4 decreased significantly in cancer tissues, to 32 ± 29%, (p < 0.005). The most remarkable finding was that the message of ST‐30 was prominently increased in cancer tissues compared with non‐malignant colorectal mucosa. When further investigated by quantitative RT‐PCR assays on a larger series of patients with colorectal cancers, the average increase in mRNA for ST‐30 was 459 ± 200% compared with that in adjacent non‐malignant epithelium (significant at p < 0.0001). The increase of ST‐30 message was more prominent in the cancer tissues strongly expressing sialyl Lewis A than in the cancer tissues expressing sialyl Lewis A only weakly or moderately (significant at p < 0.05). The marked increase in the message of ST‐30 is suggested to be related to an enhanced expression of sialylated carbohydrate determinants in colon cancer tissues including sialyl Lewis A, since the enzyme exhibited a significant activity against the type I chain carbohydrate substrate and produced the precursors for sialyl Lewis A synthesis, when its cDNA was expressed in Cos‐7 cells. Int. J. Cancer 71:556‐564, 1997

Probucol markedly reduces HDL phospholipids and elevated preβ1-HDL without delayed conversion into α-migrating HDL: Putative role of angiopoietin-like protein 3 in probucol-induced HDL remodeling

Probucol is a unique hypolipidemic agent that increases cholesteryl ester transfer protein (CETP) activity. Enhanced CETP-mediated conversion of high-density lipoprotein (HDL) partly explains the probucol-induced decrease in HDL cholesterol and increase in plasma prebeta1-HDL (native lipid-poor HDL) concentrations. However, HDL cholesterol is reduced in patients that are completely deficient in CETP. Angiopoietin-like protein 3 (ANGPTL3) is an endogenous suppressor of endothelial lipase that promotes the hydrolysis of HDL phospholipids and may generate prebeta1-HDL. To determine whether probucol decreases ANGPTL3 and HDL phospholipids while increasing prebeta1-HDL, we measured these parameters before and after a 4-week probucol treatment in 39 hypercholesterolemic patients and age- and sex-matched controls. The median ANGPTL3 had decreased from 143 to 113 microg/L by week 4 (p<0.05). High-performance liquid chromatography revealed that probucol decreased the phospholipid content of very large (13.5-15 nm) and large (12.1 nm) HDL particles predominantly by 65% (p<0.01) and 53% (p<0.001), respectively. The change in ANGPTL3, but not CETP mass, was positively correlated with that in large HDL phospholipids (r=0.455, p<0.05). The absolute and relative concentrations of prebeta1-HDL increased by 14% (p<0.01) and 60% (p<0.001), respectively. The conversion rate of prebeta1-HDL into alpha-migrating HDL by lecithin-cholesterol acyltransferase did not change significantly. In conclusion, probucol decreases plasma ANGPTL3 and HDL phospholipids while increasing prebeta1-HDL. We speculate that probucol induces HDL remodeling via an endothelial lipase-mediated pathway.

ELEVATION OF AN ALPHA (1,3)FUCOSYLTRANSFERASE ACTIVITY CORRELATED WITH APOPTOSIS IN THE HUMAN COLON ADENOCARCINOMA CELL LINE, HT-29

We studied changes in the carbohydrate expression following apoptotic cell death induced by treatment with interferon (IFN)-γ and anti-Fas antibody using human colon adenocarcinoma HT-29 cells. An apoptotic cell death of HT-29 accompanied with typical DNA fragmentation was observed when the cells were cultured sequentially with IFN-γ and anti-Fas antibody. In flow cytometric analyses, the expression of Lex and Ley antigen was strongly and slightly enhanced, respectively, on the cell surface in accordance with the apoptosis. When the fucosyltransferase (Fuc-T) activities of the lysates from the treated cells were examined relative to those from untreated cells, a 2.5-fold increase of α(1,3)-Fuc-T activities and a slight increase of α(1,2)-Fuc-T activities were observed, but little or no increase of α(1,4)-Fuc-T activity was detected. In Northern blot analyses using probes for Fuc-T III, IV, V, VI and VII genes, strong RNA messages for Fuc-T III, V and/or VI and a weak RNA message for Fuc-T IV were detected in the untreated HT-29 cells. On the other hand, in the treated cells, the messages for Fuc-T III, V and/or VI were found to almost disappear and the 2.3 kb message for Fuc-T IV was observed to elevate 2.8-fold. Therefore, we suggest that the strongly increased expression of Lex antigen found on the HT-29 cell surface might be involved in the process of apoptosis, and that the enhancement of the antigen expression seems to result from the increased activity of α(1,3)-Fuc-T encoded mainly by the Fuc-T IV gene.

Binding of adiponectin and C1q in human serum, and clinical significance of the measurement of C1q-adiponectin / total adiponectin ratio

OBJECTIVE Adiponectin and C1q have similar sequences, exist abundantly in blood, and are produced by adipose tissues. The aim of this study was to examine whether adiponectin and C1q form protein-complex in blood and to know the clinical significance of the C1q-adiponectin (C1q-APN) complex in serum. METHODS The direct interaction between adiponectin and C1q was investigated by far western blotting and co-immunoprecipitation. The relationship between serum C1q-APN and various clinical features was analyzed in 329 Japanese men who underwent health check-up, including measurements of visceral (VFA) and subcutaneous fat area (SFA) by computed tomography (Victor-J study). RESULTS Adiponectin bound to C1q in vitro and C1q-APN complex existed in human blood. C1q-APN complexes were identified in high- and middle-molecular weight forms of adiponectin in human serum by gel-filtration chromatography. Stepwise multiple regression analysis identified body mass index, VFA and SFA as significant determinants of serum C1q-APN level. Serum C1q-APN/Total-APN ratio correlated positively with cardiovascular risk factor accumulation in subjects with VFA ≥100 cm(2). CONCLUSIONS These results indicate that high- and middle-molecular forms of adiponectin partly consist of adiponectin-complex with other proteins including C1q and that the blood C1q-APN/Total-APN ratio may serve as a biomarker of the metabolic syndrome in general male subjects.

URB is abundantly expressed in adipose tissue and dysregulated in obesity.

Dysregulated production of adipocytokines in obesity is involved in the development of metabolic syndrome. URB/DRO1 contains N-terminal signal sequence and is thought to play a role in apoptosis of tumor cells. In the present study, we investigated the expression pattern of URB mRNA in adipose tissue and secretion from cultured adipocytes. In human and mouse, URB mRNA was predominantly expressed in adipose tissue and was downregulated in obese mouse models, such as ob/ob, KKAy, and diet-induced obese mice. In 3T3L1 adipocytes, insulin, TNF-alpha, H(2)O(2) and hypoxia decreased URB mRNA level. This regulation was similar to that for adiponectin and opposite to MCP-1. URB protein was secreted in media of URB cDNA-stably transfected cells and endogenous URB was detected in media of cultured human adipocytes. In conclusion, the expression pattern of URB suggests its role in obesity and the results suggest that URB is secreted, at least in part, from adipocytes.

α2→3Sialyltransferase associated with the synthesis of CA 19‐9 in colorectal tumors

A novel assay method specific for α2→3sialyltransferase that seems to be responsible for the synthesis of CA 19‐9 antigen was developed and the levels of the enzyme in colorectal tumor tissues were measured and compared with the levels of α1→4fucosyltransferase and the CA 19‐9 antigen.

Development and Characterization of a Novel Anti-fucosylated Antigen Monoclonal Antibody YB-2 and Its Usefulness in the Immunohistochemical Diagnosis of Colorectal Cancer

A novel monoclonal antibody, YB‐2 was obtained after immunization of mice with fucosylated antigens isolated from human saliva. The antibody was demonstrated to react with Y (Fucα1→2Gal‐ β1→4[Fucα1→3]GlcNAcβ),Leb(Fucα1→2Galβ1→3[Fucα→4]GlcNAcβ)andHtype2 (Fucα→2Gal‐βl→4GlcNAcβ) antigens, but not with H type 1 (Fucα→2Galβ1→3GlcNAcβ), Le→ (Galβ1→3[Fucα1→4]GlcNAcβ), X (Galβ1→4[Fucα1→3]GlcNAcβ) or with non‐fucosylated antigens. Inhibition assays of YB‐2 antibody with such reactive antigens showed that YB‐2 antibody preferentially reacted with Y antigen. Formalin‐fixed and paraffin‐embedded sections prepared from normal and malignant colorectal tissues were examined immunohistochemically with YB‐2. The positive rates of staining with YB‐2 antibody were 88.6% in malignant and 12.0% in normal tissues. The expression of fucosylated antigens detected by YB‐2 antibody seemed to be correlated with survival among patients with primary colorectal cancer. Therefore, YB‐2 antibody could be useful as an immunochemical tool for diagnosis and evaluation of the prognosis of colorectal cancer.

A novel method for rapid detection of Streptococcus pneumoniae antigen in sputum and its application in adult respiratory tract infections

A highly sensitive immunochromatography test kit, ODK0501, was developed using specific polyclonal antibodies against the C-polysaccharide moiety of Streptococcus pneumoniae for the rapid detection of S. pneumoniae antigen in sputum samples. The clinical utility of ODK0501 for this detection was evaluated prospectively in 52 adult patients with respiratory infections and compared with that of a urinary antigen detection kit. Overall, 21 patients (40.4 %) showed positive results with ODK0501, compared with 16 patients (30.8 %) using the urinary antigen detection kit, and S. pneumoniae was cultured from 18 patients. ODK0501 and the urinary antigen detection kit exhibited a sensitivity of 94.4 and 55.6 % (P<0.01), respectively, and a specificity of 88.2 and 82.4 %, respectively. Eleven of thirteen patients with conflicting results between the two test kits exhibited consistent results for sputum cultures. Moreover, eight out of nine patients positive for ODK0501 and negative for the urinary antigen detection kit were S. pneumoniae culture-positive, including five who exhibited phagocytosis, indicating S. pneumoniae as a causative agent of infection, in Gram staining of sputum samples. These results suggest that the ODK0501 direct sputum detection kit is more clinically useful than the urinary antigen detection kit in adult patients with respiratory infections.

On-site diagnosis of H. pylori infection by urine

We have recently developed an on-site diagnostic kit for H. pylori infection using urine (utilizing immunochromatographic method employing a nitrocellulose membrane coated by extracted H. pylori antigen). Accordingly, we investigated its usefulness in 155 consecutive dyspeptic patients using the 13C urea breath test as a gold standard and further compared its performance with two commercially available rapid diagnostic kits that use whole blood (Helisal Rapid Blood, and ImmunoCard H. pylori). As the results, the urine based on-site diagnostic kit provided 95.9% sensitivity and 87.9% specificity with 92.9% accuracy, which were comparable or even better than that of both rapid whole blood tests, suggesting its usefulness in screening of H. pylori infection.

Increased plasma alpha (1 --> 3)-L-fucosyltransferase activities in patients with hepatocellular carcinoma.

Alpha(1 → 3)-l-fucosyltransferase (α1,3FT) activity was determined in plasma of patients with chronic liver diseases, namely, chronic hepatitis (CH), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). The plasma α1,3FT activity was significantly higher (p<0.01) in chronic liver diseases than that in normal controls. The enzyme activity in plasma of patients with HCC was also significantly higher than that in LC (p<0.05) or that in CH (p<0.01). However, no significant difference was observed in the enzyme activity between LC and CH. Plasma α1,3FT activity in patients with HCC was not significantly changed before and after transcatheter arterial embolization. In addition, the enzyme activity in the homogenate of the cirrhotic liver tissue was higher than that in the preparation of the hepatoma tissue in the same patient. These results suggest that the increased plasma α1,3FT activity in patients with HCC reflects mainly the enzyme activity of cirrhotic liver tissue, not that of hepatoma tissue. The significance of the elevated levels of plasma α1,3FT and its decreased hepatoma tissue activity in patients with HCC, compared with that in LC, remains to be clarified.