Quantitative differences in GlcNAc:beta1-->3 and GlcNAc:beta1-->4 galactosyltransferase activities between human colonic adenocarcinomas and normal colonic mucosa.

The activities of GlcNAc:beta1-->3 and GlcNAc:beta1->4 galactosyltransferases in normal human colonic mucosa and well or moderately differentiated colonic adenocarcinomas and their enzyme-kinetic characteristics were investigated. After UDP-[3H]galactose and N-linked type monoantennary oligosaccharides GlcNAc beta1-->2Man alpha1-->3(6)Man beta1-->4GlcNAc) had been incubated with microsome fractions prepared from these tissues, the synthesized [3H]galactose-labeled oligosaccharides were analyzed by Ricinus communis agglutinin-I agarose chromatography, Streptococcus 6646K beta-galactosidase, Gal beta1-->4-specific diplococcal beta-galactosidase, and Gal beta1-->3GlcNAc-specific lacto-N-biosidase digestion. The beta-galactosyltransferases from normal mucosa synthesized both type 1 and type 2 chains at comparable levels, whereas those from adenocarcinomas predominantly synthesized type 2 chains. To our knowledge, this is the first quantitative estimation of GlcNAc:beta1-->3 galactosyltransferase activity toward N-linked sugar chains. Furthermore, we compared the two galactosyltransferase activities in 10 normal mucosa and adenocarcinoma samples and found that while there existed similar levels of GlcNAc:beta1-->4 galactosyltransferase activity in normal mucosa and adenocarcinomas, GlcNAc:beta1-->3 galactosyltransferase activity apparently decreased from 0.67 +/- 0.26 (normal mucosa) to 0.18 +/- 0.11 nmol/min/mg of protein (adenocarcinomas). These results are consistent with those of comparative structural studies on N-linked sugar chains of carcinoembryonic antigen and its normal counterparts and suggest that in the process of differentiated carcinogenesis of human colonic tissues, the expression of GlcNAc:beta1-->3 galactosyltransferase is negatively regulated.

Elevation of alpha2-->6 sialyltransferase and alpha1-->2 fucosyltransferase activities in human choriocarcinoma.

Structures of N-linked sugar chains are species and tissue specific and change in the course of tumorigenesis. Sialyl linkages of human placental glycoproteins are exclusively Neu5Ac alpha2-->3Gal, whereas Fuc alpha1-->2Gal and Neu5Ac alpha2-->6Gal residues are expressed in human chorionic gonadotropin and alkaline phosphatase, which are produced in human choriocarcinoma JEG-3 and BeWo cells. In the present study, to elucidate the enzymological and molecular biological basis of the structural changes that occur in the course of tumorigenesis, alpha1-->2 fucosyltransferase, alpha2-->3 and alpha2-->6 sialyltransferase activities, and the expression levels of the corresponding mRNAs were measured. The alpha2-->3 sialyltransferase activity did not change as a result of tumorigenesis; however, the alpha2-->6 siayltransferase activity and alpha1-->2 fucosyltransferase activity in JEG-3 and BeWo cells increased to levels several times higher than those in placenta Competitive PCR analysis showed that the expression levels of mRNA encoding alpha1-->2 fucosyltransferase and mRNA encoding alpha2-->6 sialyltransferase increased significantly as a result of tumorigenesis, indicating that such structural changes are regulated at the level of transcription of these glycosyltransferase genes.

Occurence of a sialylglycopeptide and free sialylglycans in hen's egg yolk.

Free sialylglycans (FSGs) and a sialylglycopeptide (SGP) as components of hen's egg yolk were found and their chemical structures were determined. SGP and FSGs were isolated from fresh egg yolk by treatment with phenol, gel filtration and successive chromatographies on columns of anion- and cation-exchangers. They were localized in the yolk plasma. The glycan moiety of SGP, which was liberated by PNGase digestion, was studied for the chemical structure by HPLC mapping with p-aminobenzoic ethylester-derivatization, sugar composition analysis, 1H nuclear magnetic resonance and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and the glycomoiety was found to be an N-linked disialyl-biantennary glycan. The amino acid sequence of the peptide moiety of SGP was determined to consist of Lys-Val-Ala-Asn-Lys-Thr, the Asn of which is modified with the disialylglycan moiety. FSGs were determined to be two free disialyl-biantennary glycans whose reducing end was either Man beta1-4GlcNAc (FSG-I) or Man beta1-4GlcNAc beta1-4GlcNAc (FSG-II). Since the molar value of SGP present in one egg yolk (2.8 micromol) is comparable to those of well-known major yolk proteins, low density lipoprotein, lipovitellins and phosvitin, it can be considered that SGP is one of the major components in hen's egg yolk.

MRI study of atherosclerotic plaque progression using ultrasmall superparamagnetic iron oxide in Watanabe heritable hyperlipidemic rabbits.

OBJECTIVE: The purpose of this study was to evaluate plaque progression by using MRI with ultrasmall superparamagnetic iron oxide (USPIO) and by histopathological studies. METHODS: We divided 12 Watanabe heritable hyperlipidemic (WHHL) rabbits into 4 groups based on their age (3, 9, 14 and 26 months) and injected them intravenously with 0.8 mmol (Fe) kg(-1) of USPIO (size, 32 nm; concentration, 15 mg dl(-1)). On the fifth post-injection day, they were again given an intravenous injection with 40 µmol kg(-1) of the same USPIO, and MR angiography (MRA) was performed. The signal-to-noise ratio (SNR) in regions of interest in the wall of the upper abdominal aorta was calculated on coronal images. Specimens from the same level of the aorta were subjected to iron staining and RAM-11 immunostaining and used for histopathological study. For statistical analysis of the MRA and histopathological findings, we used analysis of variance [Tukey's honest significant difference (HSD) test]. RESULTS: In 9-month-old rabbits, the SNR was significantly lower than in rabbits of the other ages (p < 0.01), and the area of RAM-11 (DAKO Corporation, Glostrup, Denmark) and iron uptake in the aortic wall was significantly larger (RAM-11, p < 0.01; iron, p < 0.05). These areas were the smallest in 3-month-old rabbits. CONCLUSION: Histopathologically, the number of macrophages was the greatest in 9-month-old rabbits. Our findings indicate that the SNR on MRI scans reflects the number of macrophages in the aortic wall of WHHL rabbits. ADVANCES IN KNOWLEDGE: USPIO-enhanced MRI visualized the accumulation of macrophages in early atherosclerotic plaques of WHHL rabbits in the course of natural progression.

Identification and characterization of N-acetylglucosamine-6-O-sulfate-specific beta1,4-galactosyltransferase in human colorectal mucosa.

6-Sulfo-sialyl Lewis X structure is attributable to recognition between lymphocytes and high endothelial venules. However, the biosynthetic pathway still remains unclear. We found that a beta-galactosyltransferase (betaGalT) in human colorectal mucosa preferentially acts on GlcNAc-6-O-sulfate (6S-GN). 6S-GN:beta4GalT was partially purified by UDP-hexanolamine-Sepharose and asialo-agalacto-ovomucin-Sepharose chromatographies. The optimum pH of this enzyme was found to be 6.5-7.5 and the Michaelis constants for 6S-GN and UDP-Gal were 0.43 mM and 16 microM, respectively. The enzymatic activity was dependent on divalent cations and the substrate specificity was not affected by alpha-lactalbumin. This is the first demonstration of the occurrence of 6S-GN:beta4GalT.

The level of erythrocyte aldose reductase is associated with the severity of diabetic retinopathy.

To examine whether the level of erythrocyte aldose reductase is a risk factor for the severity of diabetic retinopathy, the enzyme level in 97 non-insulin-dependent diabetes mellitus (NIDDM) patients was measured by the two-site enzyme-linked immunosorbent assay. Based on the results of fundus photography and biomicroscopy, the severity of retinopathy was classified among NIDDM patients of more than 10 years. The level of erythrocyte aldose reductase was significantly higher in the patients with active proliferative retinopathy than in those with nonproliferative or quiescent proliferative retinopathy. Multivariate logistic regression analysis demonstrated that the level of erythrocyte aldose reductase was an independent risk factor for active proliferative retinopathy (odds ratio, 1.28; 95% confidence interval, 1.01-1.61). The results suggest that a high level of erythrocyte aldose reductase in NIDDM patients may affect the prognosis of diabetic retinopathy. Patients with high enzyme levels would need to be closely followed up in the management of the retinal complication.

Ulcerative colitis in female siblings with an identical human leukocyte antigen haplotype (A24, Bw52, DR52 and DQw1).

Two female siblings with ulcerative colitis are reported. Both the 30-year-old elder sister and the 28-year-old younger sister developed bloody stools and diarrhea; ulcerative colitis was diagnosed by endoscopic and histologic examination of the colon. Their maternal aunt also suffered from the disease. Human leukocyte antigen analysis revealed an identical haplotype for the sisters (A24, Bw52, DR2 and DQw1), while the aunt had A24, Bw52 and DR2. The haplotypes of Japanese patients with familial ulcerative colitis are reviewed, and the role of human leukocyte antigens in this disease is discussed.

[A case of bilateral adrenal non-Hodgkin lymphoma].

A 47-year-old woman presented with the chief complaint of gait disturbance and pain of bilateral lower limbs. She was diagnosed as having necrotizing myelitis because myelography was normal and incidentally bilateral adrenal tumor was recognized by ultrasonography and computed tomography. Left adrenalectomy was performed under the diagnosis of bilateral non-functioning adrenal tumor or metastasis to the adrenal glands with unknown origin. The tumor was 10 x 6 x 3 cm in size and 175 g in weight. The pathological diagnosis was non-Hodgkin lymphoma (diffuse large cell type). After combination chemotherapy of vincristine, cyclophosphamide, prednisolone and adriamycin, residual right adrenal mass showed a remarkable reduction, but unfortunately she died 5 months later postoperatively because of complications of lung edema and pneumonitis.

Anti-tumour effects of transcatheter arterial embolisation administered in combination with thalidomide in a rabbit VX2 liver tumour model.

OBJECTIVE: Using a liver tumour model we investigated whether thalidomide enhances the anti-tumour effect of transcatheter arterial embolisation (TAE). METHOD: First, the viability of VX2 tumour cells co-cultured with thalidomide in a 21% and 1% O(2) atmosphere was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Second, we randomly assigned 20 rabbits bearing VX2 liver tumours to 4 groups: Group 1 (thalidomide plus TAE), Group 2 (TAE only), Group 3 (thalidomide only) and Group 4 (control). Thalidomide was orally administered for 5 days. The anti-tumour effects were assessed by the tumour proliferation rate using MRI and by immunohistochemical analysis of the area of intratumoural vessels. Analysis of variance and Tukey's honestly significant difference test were used for statistical analysis. RESULTS: The viability of cells grown under hypoxic and normal conditions was not significantly different, nor was there a difference among the four groups. The tumour size increased by 55.9±29.3% in Group 1, 250.6±73.3% in Group 2, 355.2±51.7% in Group 3 and 424.7±110.7% in Group 4; the difference between Group 1 and the other three groups was significant. The area of intratumour vessels in specimens was 0.22±0.28% in Group 1, 0.42±0.29% in Group 2, 1.44±1.00% in Group 3 and 6.00±2.17% in Group 4; the difference between Group 1 and the other groups was statistically significant, as was the difference between Groups 3 and 4. CONCLUSION: Thalidomide used in combination with TAE enhanced anti-tumour effects in rabbits bearing VX2 liver tumours.

Does the concomitant intra-arterial injection of asialoerythropoietin and edaravone mitigate ischaemic mucosal damage after acute superior mesenteric artery thromboembolism in a rabbit autologous fibrin clot model?

To increase the survival rate of patients with acute superior mesenteric artery thromboembolism (ASMAT) treated by catheter thrombolysis, we examined the effects of delivering edaravone and asialoerythropoietin, agents with tissue-protective activities, using a rabbit autologous fibrin clot ASMAT model. Japanese white rabbits (n=32) were randomly separated into four equal groups. 45 min after introducing autologous fibrin clot, Group U received urokinase and heparin; Group E received urokinase and heparin plus edaravone; Group A received urokinase and heparin plus asialoerythropoietin; and Group EA received urokinase, heparin and edaravone plus asialoerythropoietin via a catheter. The intestines were removed 6 h later and intestinal mucosal damage was scored using the Park's injury score. Survival time was assessed. Average mucosal injury was 5.78+/-1.52 (Group U), 2.88+/-0.72 (Group E), 1.90+/-1.23 (Group A) and 1.18+/-1.25 (Group EA). The degree of mucosal injury was significantly lower in Group EA than in Groups U and E (p<0.05). Conversely, there was no significant difference between Group A and Group EA, or between Group A and Group E. The survival times were 31.50+/-13.30 h (Group U), 51.00+/-24.74 h (Group E), 48.00+/-16.97 h (Group A) and 82+/-51.07 h (Group EA); the difference among the four groups was not significant. In conclusion, the concomitant administration of asialoerythropoietin and edaravone reduced mucosal membrane injury significantly compared with edaravone alone. However, to improve the survival of ASMAT rabbit models, the delivery of an appropriate dose of asialoerythropoietin is required, together with the development of methods to assess peripheral recanalisation.

A combination of cisplatin-eluting gelatin microspheres and flavopiridol enhances anti-tumour effects in a rabbit VX2 liver tumour model.

The aim of this study was to investigate whether the combination of cisplatin-eluting gelatin microspheres (GMSs) and flavopiridol enhances anti-tumour effects in a rabbit VX2 liver tumour model. Tumour-bearing rabbits (n = 21) were divided into five groups and infused from the proper hepatic artery. Group 1 (n = 5) received cisplatin-eluting GMSs (1 mg kg(-1)) and flavopiridol (3 mg kg(-1)), group 2 (n = 5) cisplatin-eluting GMSs alone (1 mg kg(-1)), Group 3 (n = 5) flavopiridol (3 mg kg(-1)), Group 4 (n = 3) GMSs alone (1 mg kg(-1)), and Group 5 (n = 3) was the control group receiving physiological saline (1 ml kg(-1)). On days 0 and 7 after procedures the liver tumour volume was measured using a horizontal open MRI system and the relative tumour volume growth rates for 7 days after treatment were calculated. On T(1) weighted images, the tumours were visualised as circular, low-intensity areas just below the liver surface. After treatment, the signals remained similar. The relative tumour volume growth rate for 7 days after treatment was 54.2+/-22.4% in Group 1, 134.1+/-40.1% in Group 2,166.7+/-48.1% in Group 3, 341.8+/-8.6% in Group 4 and 583.1+/-46.9% in Group 5; the growth rate was significantly lower in Group 1 than the other groups (p<0.05). We concluded that in our rabbit model of liver tumours the combination of cisplatin-eluting GMSs and flavopiridol was effective.

Cisplatin-conjugated degradable gelatin microspheres: fundamental study in vitro.

The object of this study was to generate cisplatin-conjugated gelatin microspheres (GMSs) and to confirm the subsequent release of cisplatin in vitro. The GMSs (1 mg) were immersed in 50 microl of a cisplatin solution (0.06, 0.15, 0.27, 0.30 or 0.54 mg ml(-1)) at 38 degrees C to allow conjugation. The cisplatin-conjugated GMSs were then extensively washed in double-distilled water and freeze-dried. The platinum concentration in the GMSs samples was investigated as a function of the concentration of cisplatin solution used in their preparation, the number of immersions in cisplatin (1, 2, 3, 4 or 5) and the period of immersion (1, 6 or 11 h). In vitro release tests were performed at different time intervals (1, 3, 6, 12 or 24 h) to allow the rate of cisplatin release to be calculated. The platinum concentration of the GMSs increased in proportion to the concentration of cisplatin solution and the length or number of immersions in cisplatin. In vitro release tests demonstrate that the release rate (%) from GMSs after 1, 3, 6, 12 or 24 h was 4.8, 5.5, 7.6, 10.0 and 12.4, respectively. We demonstrated the ability of GMSs to bind cisplatin forming cisplatin-conjugated GMSs. Moreover, we showed that cisplatin continued to bind GMSs strongly during the in vitro release test.