α(1,6)Fucosyltransferase expression is an independent prognostic factor for disease-free survival in colorectal carcinoma.

We previously reported that α(1,6)fucosyltransferase (Enzyme class 2.4.1.68) activity and expression are increased in colorectal cancer, suggesting a role for this enzyme in tumor development and progression. However, the possible impact of α(1,6)fucosyltransferase activity or expression on clinical outcomes in colorectal cancer patients has never been studied. Thus, the present study was conducted to determine the value of α(1,6)fucosyltransferase as a prognostic factor for colorectal cancer. α(1,6)Fucosyltransferase expression was analyzed using immunohistochemistry in 141 colorectal tumors, and α(1,6)fucosyltransferase activity was determined in 39 tumors. A complete standardized follow-up of patients was documented until the end of the observation period of 5 years or patient death. Univariate analysis demonstrated the absence of a correlation between enzyme activity and disease evolution. However, in patients with moderate or strong α(1,6)fucosyltransferase expression, a significant decrease in the overall (P = .04) and disease-free (P = .03) survival rates was observed. In addition, when local and distant disease recurrence were considered separately, enzyme expression was found to correlate with local tumor recurrences (P = .01). Furthermore, multivariate analysis showed that α(1,6)fucosyltransferase expression has independent value for predicting tumor recurrences and, specifically, local recurrences. These findings suggest that α(1,6)fucosyltransferase expression may be a good indicator of poor prognosis in colorectal cancer and, therefore, a helpful tool to choose the most effective treatment.

Effect of human colorectal carcinogenesis on the neural cell adhesion molecule expression and polysialylation.

OBJECTIVE: Although downregulation of neural cell adhesion molecule (NCAM) has been correlated with poor prognosis in colorectal cancer (CRC), it is also possible that colon cancer spreading comes from reducing tumor cell adhesion through NCAM polysialylation, as occurs in lung carcinoma or Wilms' tumor. METHODS: To prove this hypothesis, we have performed a prospective study on tumor and control specimens from 39 CRC patients, which were immunostained for NCAM and PSA (polysialic acid) expression. RESULTS: Tumor versus control expression of NCAM and PSA epitopes in tissue specimens, as well as correlation between tumor expression and clinicopathological features, were statistically analyzed. Results showed a low constitutive expression of NCAM and PSA (PSA-NCAM) in control tissue, which reached a statistically significant increase in the tumor tissue. Likewise, the presence and number of lymph node metastases at surgery were correlated with NCAM expression and PSA/NCAM coexpression. CONCLUSIONS: These data highlight the importance of taking into account PSA-associated epitopes when dealing with NCAM cell expression studies in tumor development and progression. The analysis of PSA and NCAM expression in CRC suggests a new way, other than downregulation of NCAM, in order to escape contact inhibition and promote cell tumor spreading in colorectal cancer.

α(1,2)fucosylation in human colorectal carcinoma.

Lewis(b) and Lewis(y) (Le) antigens are known to be elevated in colorectal tumours. Alterations in the catalytic behaviour of GDP-L-fucose:ß-D-galactoside α(1,2)fucosyltransferase [α(1,2)FT, EC: 2.4.1.69], the key enzyme in their synthesis, have been suggested as being responsible for these changes. In particular, an aberrant tumour-specific α(1,2)FT activity that converts Le(a) and Le(x) to Le(b) and Le(y) determinants, respectively, has been reported in colorectal cancer tissues. To clarify the catalytic function of this enzyme during colorectal tumorigenesis, we analyzed α(1,2)FT activity levels in healthy and tumour colon specimens using different acceptor substrates and determined the kinetic properties of the enzyme. To complete the study, the aberrant Le(a)/Le(x) α(1,2)fucosylation was determined in healthy and tumour colorectal tissues. A correlation analysis between the activity levels and various standard clinicopathological features, such as tumour stage, was also carried out to elucidate the role of these activities in tumour progression. The results obtained confirm the enhanced α(1,2)fucosylation in colorectal neoplastic tissues and the importance of the aberrant Le(a)/Le(x) α(1,2)FT activity in this increase. However, taking into account the high levels of Le(a)/Le(x) fucosylation observed in healthy control tissues, we must rule out the idea of a colorectal tumour-specific α(1,2)FT. On the other hand, no significant association was observed between α(1,2)FT activity levels and the clinicopathological characteristics. Overall, our results suggest that α(1,2)FT activity plays a critical role in the accumulation of Le(b) and Le(y) antigens in human colorectal carcinoma.

Correlation analysis between tumor-associated antigen sialyl-Tn expression and ST6GalNAc I activity in human colon adenocarcinoma.

OBJECTIVES: Sialyl-Tn (sTn) is a mucin carbohydrate-associated antigen that is strongly expressed in a large number of colorectal carcinomas. In this study, we combined immunohistochemical and enzymatic techniques in order to find the correlation between sTn tissue expression and the sialyltransferase activity (ST6GalNAc I) responsible for its synthesis in colorectal cancer (CRC) patients. METHODS: We compared sTn expression in healthy (n = 46), tumorous (n = 60) and transitional tissue (n = 46) from CRC patients, and correlated sTn altered expression with clinicopathologic variables of the patient. Furthermore, we determined ST6GalNAc I tissue activity employing asialo-ovine submaxillary mucin (asialo-OSM) as glycoprotein acceptor (n = 27). RESULTS: The rates of sTn positive expression obtained for healthy, tumorous and transitional tissues were 15, 67 and 63%, respectively. These rates led to statistically significant differences between healthy and tumorous or transitional tissue (p = 0.001); sTn expression was related to the first stages of the tumor invasion in transitional tissue. As regards ST6GalNAc I activity, we found an enhancement in transitional tissue. Statistical correlation analysis did not reveal association between sTn expression and ST6GalNAc I activity. CONCLUSIONS: Our findings indicated that sTn antigen tissue expression and ST6GalNAc I activity levels were not correlated in CRC, in spite of the overexpression of the antigen in tumorous and transitional tissue.

Alterations of CMP-NeuAc:asialofetuin sialyltransferase activities in human colorectal adenocarcinoma.

It has been reported that surface glycoconjugates in tumour cells have more complex and more heavily sialylated sugar chains in glycoproteins. Here, we analysed CMP-NeuAc:asialofetuin sialyltransferase activities in total cell membranes from colorectal cancer tissue with respect to normal adjacent tissue, finding enhanced sialyltransferase activities. Determination of the kinetic parameters for the donor substrate, CMP-NeuAc, revealed that the apparent K(m) in tumour tissue was decreased as regards to the normal value. Furthermore, we failed to find any correlation between this activity and the characteristics of the samples such as the age or sex of the patient, or Dukes' stage of the tumour. In order to know which sialyltransferase activity was altered, we studied the incorporation of NeuAc into N- and O-linked chains from asialofetuin. The results allow us to conclude that it is on N-linked chains where the activity is enhanced. With respect to alpha(2,3)- and alpha(2,6)-NeuAc linkages, we observed that enhanced activity in tumour tissues affects both linkages equally.

N-acetyl-beta-hexosaminidase activity and isoenzymes in human gastric adenocarcinoma.

The enhancement of lysosomal beta-hexosaminidase degradative activity in different human cancer tissues is fairly well documented. Gastric tumors have attracted considerable attention on the basis of their social incidence and clinical recurrence. Here we report a comparative study of beta-hexosaminidase activity and of its isoenzymes beta-hexosaminidase A (HA) and beta-hexosaminidase B (HB) from gastric adenocarcinoma and normal mucosa. Tumor beta-hexosaminidase activity from crude extracts and chromatographically resolved HA and HB forms were analyzed as regards their physicochemical and enzymatic properties and were compared to similar samples obtained from control tissue. The existence of one active site in the beta-hexosaminidase enzyme responsible for both N-acetyl-beta-D-glucosaminidase and N-acetyl-beta-D- galactosaminidase activities was determined. Apart from their relative contributions to beta-hexosaminidase activities, two major differences appeared in tumor HA and HB forms with respect to the corresponding controls: (1) the presence of an atypical heat-stable HB isoenzyme in gastric adenocarcinoma, and (2) a significantly increased Vmax of the HA form acting on both p-nitrophenyl-N-acetyl-beta-D-glucosaminide and p-nitrophenyl-N-acetyl-beta-D-galactosaminide substrates. The results show that the beta-hexosaminidase HA and HB isoenzymes from gastric adenocarcinoma display different patterns of response from the same forms from other human tumors.

Chronic alcoholization in rats by free-choice ingestion of a hydroalcoholic solution.

The present work addresses a schedule designed to render rats alcoholic by offering them free access to a 20% (v/v) ethanol solution over 6 weeks. A solid diet and water (for controls) were fully available ad lib. in the animals' cages. The treatment was observed to achieve some effects suitable for the chronic alcoholization of rats: (1) Ethanol furnished the 36% of the calories of the diet of ethanol-treated rats; (2) The daily ethanol intake achieved was 12.05 +/- 1.18 g ethanol/kg weight; (3) The morning blood ethanol level was 0.45 g/litre; (4) Ethanol-treated rats deprived of ethanol showed several withdrawal symptoms. These results agree with those obtained by standard liquid diets, suggesting that the current protocol can be used to achieve a status of chronic alcoholization in rats.

Inactivating peptide of the Shaker B potassium channel: conformational preferences inferred from studies on simple model systems.

Previous studies on the interaction between the inactivating peptide of the Shaker B K+ channel (ShB peptide, H2N-MAAVAGLYGLGEDRQHRKKQ) and anionic phospholipid vesicles, used as model targets, have shown that the ShB peptide: (i) binds to the vesicle surface with high affinity; (ii) readily adopts a strongly hydrogen-bonded beta-structure; and (iii) becomes inserted into the hydrophobic bilayer. We now report fluorescence studies showing that the vesicle-inserted ShB peptide is in a monomeric form and, therefore, the observed beta-structure must be intramolecularly hydrogen-bonded to produce a beta-hairpin conformation. Also, additional freeze-fracture and accessibility-to-trypsin studies, which aimed to estimate how deeply and in which orientation the folded monomeric peptide inserts into the model target, have allowed us to build structural models for the target-inserted peptide. In such models, the peptide has been folded near G6 to configure a long beta-hairpin modelled to produce an internal cancellation of net charges in the stretch comprising amino acids 1-16. As to the positively charged C-terminal portion of the ShB peptide (RKKQ), this has been modelled to be in parallel with the anionic membrane surface to facilitate electrostatic interactions. Since the negatively charged surface and the hydrophobic domains in the model vesicle target may partly imitate those present at the inactivation 'entrance' in the channel protein [Kukuljan, M., Labarca, P. and Latorre, R. (1995) Am. J. Physiol. Cell Physiol. 268, C535-C556], we believe that the structural models postulated here for the vesicle-inserted peptide could help to understand how the ShB peptide associates with the channel during inactivation and why mutations at specific sites in the ShB peptide sequence, such as that in the ShB-L7E peptide, result in non-inactivating peptide variants.

Alterations of glycosidases in human colonic adenocarcinoma.

OBJECTIVES: We have carried out a detailed study of some glycosidases in an attempt to explain the differential profile of enzyme activity between human colonic adenocarcinoma and normal mucosa. DESIGN AND METHODS: Several glycosidase activities associated with human colonic adenocarcinoma and control tissues were submitted to a detailed structural and functional characterization. RESULTS: Tumoral and control samples were assayed for beta-D-galactosidase, beta-D-glucuronidase, alpha-D-mannosidase, beta-NAc-D-glucosaminidase and beta-NAc-D-galactosaminidase activities. Tumoral tissue showed higher beta-D-galactosidase, beta-NAc-D-glucosaminidase, and beta-NAc-D-galactosaminidase activities than control tissue. Glycosidases from tumoral and control tissues demonstrated no differences in optimum pH, subcellular distribution, pH and thermal stability. However, the kinetic analysis showed a statistically significant increased Vmax in tumoral colon with respect to the control for beta-D-galactosidase, beta-NAc-D-glucosaminidase, and beta-NAc-D-galactosaminidase activities. The Km remained unaltered. CONCLUSIONS: The increased Vmax detected for some glycosidase activities in human colonic adenocarcinoma could correspond with a greater presence of enzyme proteins in the tumoral cells, and not to changes in protein and/or active site structure.

Elevation of acid glycosidase activities in thyroid and gastric tumors.

Numerous investigators have suggested that cell glycoconjugates are modified by the development of cancer and the progression of this to a malignant form. Accordingly, in the present work, beta-D-galactosidase, alpha-L-fucosidase, beta-N-acetyl-D-glucosaminidase and beta-N-acetyl-D-galactosaminidase activities were studied in human thyroid and gastric tumours. Samples were obtained from human gastric mucosa and thyroid gland tumours together with a part of the surrounding normal tissue (control). Enzyme activity was determined spectrophotometrically based on the release of p-nitrophenol from suitable p-nitrophenyl-derivative substrates. Results showed that beta-D-galactosidase, alpha-L-fucosidase, beta-N-acetyl-D-glucosaminidase and beta-N-acetyl-D-galactosaminidase activities were detected in tumour and control samples from thyroid and gastric tissues. The gastric mucosa also showed alpha-L-mannosidase activity. The specific activities of these glycosidases were higher (two- or three-fold) in tumour tissues as compared with their controls. beta-D-galactosidase, beta-N-acetyl-D-glucosaminidase and beta-N-acetyl-D-galactosaminidase activities from thyroid and gastric tumours showed a significant increase in V(max) as compared with their respective controls (P < 0.05 or P < 0.001). Thyroid alpha-L-fucosidase activity showed a statistically and significantly increased affinity (lower K(m)) in tumour samples as compared to normal tissue. In conclusion both gastric and thyroid tumours showed enhanced glycosidase activity as compared with enzyme activity observed in normal tissue. These results are in agreement with the notion of a markedly raised degradation within lysosomes of tumour cells.

Effects of an acute dose of ethanol on dopaminergic and serotonergic systems from rat cerebral cortex and striatum.

Intraperitoneal injection 10 min before sacrifice of 1.5 g ethanol/kg weight produced an increase in rat striatal levels of homovanillic acid (HVA) (p < 0.05) but did not affect the striatal concentrations of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA). A similar ethanol treatment led to decreases in 5-HT (p < 0.05) and 5-HIAA (p < 0.05) from cerebral cortex (prefrontal and anterior cingulate areas). The results point to several ethanol-linked alterations in central serotonergic and dopaminergic systems.

Effect of chronic treatment with ethanol and withdrawal of ethanol on binding of [3H]SCH23390 to D1 dopamine receptor in rat visual cortex and hippocampus. An autoradiographic study.

Male Wistar rats, treated with ethanol for 8 weeks and pair-control animals, were used to study the effects of chronic treatment with ethanol, and withdrawal of ethanol for 24 and 48 hr on [3H]SCH23390 binding. The visual cortex (Laminae III-IV and Lamina VI), the superficial grey layer of the superior colliculus, and the molecular layer of the dentate gyrus of the hippocampus were the cerebral areas analysed. Non significant changes were observed in hippocampus and Laminae III-IV of the visual cortex after treatments with alcohol. More interesting results were obtained from Lamina VI, where the chronic treatment with ethanol did not modify the binding of [3H]SCH23390, whereas the withdrawal of ethanol produced a statistically significant increase in binding values. In addition, superficial grey layer of the superior colliculus showed a significant increase in binding values between 48 hr withdrawal and ethanol treated groups. The results herein reported suggest that some structures involved in visual functions are related to responses of adaptation to ethanol.

Effects of glycosylation enzymes from membrane fractions, induced by chronic ethanol administration.

To study the effect of chronic ethanol administration on the enzyme activities involved in the glycosylation processes (glycosidases and glycosyltransferases), we used 6-week ethanol-treated female Wistar rats and pair-control rats. Biological material was membrane fractions of microsomes and plasma membrane obtained by subcellular fractionation technique. Ethanol treatment increased with statistical significance the Vmax of N-acetyl-beta-D-glucosaminidase (P less than 0.10), beta-D-glucuronidase and alpha-D-mannosidase (P less than 0.001) activities from microsomal fractions and likewise it increased the Km value of beta-D-glucuronidase (P less than 0.001). In vitro doses of ethanol (0.1-1.7 M) were added to delucidate hypothetical membrane tolerance responses. However in vitro addition of ethanol provoked no differential effects in treated and untreated rats. Glycosyltransferase assays were carried out using [14C]sugar derivatives. We detected several glycosyltransferase activities in both microsome and plasma membrane fractions. Chronic ethanol exposure appeared to affect N-acetyl-neuraminyltransferase activity from both membrane fractions, producing a greatly increased incorporation in the presence of asialofetuin. Glucosyl and mannosyltransferase activities from plasma membrane fractions were also altered by ethanol treatment, producing an increased enzyme activity when reaction was performed in the presence of phosphatidylcholine + dolichol-phosphate liposomes.