Serum sCD26 for colorectal cancer screening in family-risk individuals: comparison with faecal immunochemical test.

BACKGROUND: The development of specific screening programs for individuals with a family history of colorectal cancer (CRC) is a priority. This study evaluates the diagnostic performance of serum soluble CD26 (sCD26) in family-risk individuals and compares this marker with the faecal immunochemical test for the detection of advanced neoplasia (AN) (CRC or advanced adenomas; AA). METHODS: Five hundred and sixteen asymptomatic individuals with at least one first-degree relative with CRC were included. Serum sCD26 was measured in all the individuals who also underwent a colonoscopy (53 AA and four cancer cases were found) and a faecal immunochemical test. RESULTS: Setting specificity to 90% and 95%, respectively, sCD26 showed a sensitivity of 39.6% and 28.3% for AA, and of 42.1% and 28.1% for AN. The combination of sCD26 and the faecal test detected AA and AN with a 52.8% and 56.1% sensitivity, corresponding to 93.5% specificity. CONCLUSIONS: The combination of serum sCD26 and the faecal blood test could result a valuable strategy for detecting AN in familial-risk CRC screening.

Calprotectin: a novel biomarker for the diagnosis of pleural effusion.

BACKGROUND: Novel non-invasive biomarkers for the precise diagnosis of malignancy in pleural effusion (PE) are needed. The aim of this study was to determine the diagnostic accuracy of calprotectin for predicting malignancy in patients with exudative PE. METHODS: Calprotectin concentration was measured in 156 individuals diagnosed with exudative PE (67 malignant and 89 benign). Calprotectin accuracy for discriminating between malignant and benign PE was evaluated using receiver operating characteristic (ROC) curves. Univariate and multivariate logistic regression were performed to test the association between calprotectin levels and malignant PE. RESULTS: Calprotectin levels were significantly lower in malignant pleural fluid (257.2 ng ml(-1), range: 90.7-736.4) than benign effusions (2627.1 ng ml(-1), range: 21-9530.1). The area under the curve was 0.963. A cutoff point of ≤ 736.4 ng ml(-1) rendered a sensitivity of 100%, with a specificity of 83.15%, which could prove useful to delimit those patients with negative cytology tests that should be referred for more invasive diagnostic procedures. Logistic regression demonstrated a strong association between calprotectin and malignancy (adjusted OR 663.14). CONCLUSION: Calprotectin predicts malignancy in pleural fluid with high accuracy and could be a good complement to cytological methods.

Proteomic evidence of a paedomorphic evolutionary process within a marine snail species: a strategy for adapting to extreme ecological conditions?

The exposed and sheltered ecotypes of the marine snail Littorina saxatilis from European rocky shores are considered a key model system to study adaptation and ecological speciation. Previous studies showed that two ecotypes (RB and SU) of this species in NW Spain have adapted differently to different shore levels and microhabitats. In order to understand how this divergent adaptive process has been accomplished, we followed a quantitative proteomic approach to investigate the proteome variation in a number of different biological factors, that is, ecotype, ontogeny and their interactions. This approach allowed testing the hypothesis that one of the ecotypes has evolved by paedomorphosis, and also whether or not the molecular mechanisms related to ecotype differentiation are set up in early developmental stages. Additionally, the identification of some candidate proteins using mass spectrometry provides some functional insights into these evolutionary processes. Results from this study provided evidence of higher ontogenetic differentiation at proteome level in the RB (metamorphic) than in SU (paedomorphic) ecotype that point to the possibility of juvenile stage retention in this latter ecotype. The level of protein expression (proteome) differences between ecotypes maintained nearly constant from late embryonic stages to adulthood, although some proteins involved in these changes considerably differed in embryonic compared to other ontogenetic stages. Paedomorphosis may be the evolutionary response of the SU ecotype of solving the trade-off during sexually immaturity that is caused by the evolution of small size arising from adaptation to the wave-exposed habitat. Some potential candidate genes of adaptation related to energetic metabolism have been identified, providing a promising baseline for future functional analyses.

The level of aryl acylamidase activity displayed by human butyrylcholinesterase depends on its molecular distribution.

Butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) display both esterase and aryl acylamidase (AAA) activities. Their AAA activity can be measured using o-nitroacetanilide (ONA). In human samples depleted of acetylcholinesterase, we noticed that the ratio of amidase to esterase activities varied depending on the source, despite both activities being due to BuChE. Searching for an explanation, we compared the activities of BuChE molecular forms in samples of human colon, kidney and serum, and observed that BuChE monomers (G(1)) hydrolyzed o-nitroacetanilide much faster than tetramers (G(4)). This fact suggested that association might cause differences in the AAA site between single and polymerized subunits. This and other post-translational modifications in BuChE subunits probably determine their level of AAA activity. The higher amidase activity of monomers could justify the presence of single BuChE subunits in cells as a way to preserve the AAA activity of BuChE, which could be lost by oligomerization.

Absence of activating mutations in the EGFR kinase domain in Spanish head and neck cancer patients.

The discovery of kinase domain mutations in the epidermal growth factor receptor gene (EGFR) in never-smoker patients, associated with an increased sensitivity to tyrosine kinase inhibitors (TKIs) such as gefitinib or erlotinib, has been one of the most relevant findings ever in non-small cell lung carcinomas (NSCLC). Since treatment with TKIs has furthermore shown a clinical benefit in head and neck squamous cell carcinoma (HNSCC) patients, we hypothesized that these mutations could also be present in this neoplasia. Current studies looking for EGFR mutations in HNSCC are limited and results are still controversial. In this work, we screened for EGFR tyrosine kinase mutations in tumour DNA obtained from 31 Spanish patients with HNSCC by PCR-single-strand conformational polymorphism analysis. None of the patients displayed a somatic EGFR mutation, previously described in NSCLC, but other DNA sequence variations were found in 9 of 31 HNSCC patients. Accordingly, activating EGFR mutations in HNSCC patients seem to be a rare event in Spanish patients, suggesting that there is little room for the administration of TKIs in HNSCC based on the presence of these mutations. Additional investigations about EGFR amplification are indicated to establish a potential relationship between EGFR overexpression and the response to anti-EGFR therapies.

Properties and kinetics of a neutral beta-galactosidase from rabbit kidney.

A neutral beta-galactosidase has been purified by concanavalin A-Sepharose affinity chromatography, DEAE-cellulose chromatography, Sephadex G-200 gel filtration and hydroxylapatite chromatography. The enzyme was purified 126-fold with a yield of about 21%. This form has a neutral optimal pH (7.5) and it is located in the cytosolic fraction. It shows a wide pH stability from pH 4.5 to 8.0, but it is very unstable at low pH values. Its isoelectric point is 4.9 and this value does not change on neuraminidase treatment. The estimated molecular weight was 47 000. The neutral form shows beta-D-galactosidase, beta-D-fucosidase and beta-D-glucosidase activities, all of them associated in a single peak in all the purification steps. p-Nitrophenyl beta-D-galactosides, p-nitrophenyl beta-D-fucosides and p-nitrophenyl beta-D-glucosides competed fully for a common active site in mixed-substrate experiments. Using gamma-D-galactonolactone as competitive inhibitor the Ki values were always coincident for the three activities. The effect of NaCl, methyl mannoside and some sugars (fucose, galactose and glucose) was studied.

Fucose levels in sera and in tumours of colorectal adenocarcinoma patients.

Fucose levels were determined in normal and tumour-derived tissues from patients with colorectal adenocarcinoma. Total, free and bound fucose were significantly higher (P < 0.001) in the tumoral tissue. Comparison of serum fucose levels between patients with colorectal cancer and control subjects indicated no statistically significant differences in total, free and bound fucose expressed as nmol/ml serum. However, when total and bound fucose were normalized to total protein (nmol/mg protein), an elevation in both parameters was found in colorectal cancer patients, although only that for total fucose was statistically significant (P < 0.001). A clear association between fucose content and clinical stage of patients according to the Dukes' classification can not be established, although patients at stage C showed higher levels of fucose both in serum and tissue. The current investigation provides direct evidence of a relationship between the elevation of fucose and the presence of tumour in colorectal cancer patients.

Sialic acid levels in serum and tissue from colorectal cancer patients.

Sialic acid levels were determined in serum and in both normal and tumour-derived tissues from 30 patients with colorectal cancer. Total sialic acid (TSA), bound sialic acid (BSA),TSA normalised to total protein (TSA/TP) and BSA normalised to total protein (BSA/TP) were significantly higher (P < 0.0001) in sera from patients than in normal subjects. We found a trend of increasing serum sialic acid levels (TSA/TP and BSA/TP) as the malignancy became more severe (i.e. Dukes' stages A to C). Comparison of sialic acid levels between normal and tumour-derived colorectal tissues indicated no statistically significant differences in TSA, BSA or FSA (free sialic acid) levels between both tissues; however, TSA/TP and BSA/TP values were significantly decreased (P < 0.05) in the tumoral tissue. In this study, the possible relation between serum and tumour sialic acid levels in colorectal cancer patients was investigated. Our results showed that in these patients there was no correlation between serum BSA and tumour BSA levels.

Purification and characterization of acid beta-D-galactosidase from rabbit spleen.

beta-D-Galactosidase has been purified to apparent homogeneity from rabbit spleen. The purification steps involved ammonium sulphate precipitation, DEAE-cellulose, concanavalin A-Sepharose, Sephadex G-200, and Sepharose 4B-(epsilon-aminocaproyl)-2-deoxy-beta-D-glucosylamine affinity chromatographies. In the DEAE-cellulose step, the beta-D-galactosidase was separated into two molecular forms, designated I and II, with similar pH optimum, Km, substrate specificity, and sensitivity to substrate analogues and other substances. Form I was purified 1,800-fold with a yield of about 2% of the total activity. This form is heat-labile, it has an acid optimal pH (4.0), an isoelectric point of 6.7 and a molecular weight of 75,000 daltons. Form II has an optimal pH of 3.6 and three different pI values (5.3, 5.7, and 6.7) whose relative proportions can be modified by treatment with neuraminidase. Form II appeared to be a multimeric form (IIA) of about 600,000 daltons at pH 4.0, which was reversibly dissociated to an oligomeric form (IIB) with an apparent molecular weight of 120,000 at neutral pH values. Both IIA and IIB were purified separately and showed an acid pH optimum and an heterogeneous pI (from 4.6 to 7.2). The dissociation of IIA into IIB can be generated spontaneously, but is increased by the presence of urea in the elution buffer, suggesting that both are aggregates of a common subunit.

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.

Clinical significance of preoperative serum sialic acid levels in colorectal cancer: utility in the detection of patients at high risk of tumor recurrence.

This study was conducted to evaluate the significance of preoperative serum sialic acid levels in the diagnosis and prognosis of colorectal cancer (CRC). Total sialic acid (TSA) was determined by the thiobarbituric acid method and normalized to total protein (TP). A postoperative follow-up of CRC patients classified as Dukes' stages A, B or C was performed and survival analysis was carried out to evaluate the impact of sialic acid levels on tumor recurrence. Our diagnostic studies indicate that TSA/TP is a better marker than either TSA or carcinoembryonic antigen (CEA), especially for the detection of CRC patients at an early stage. At a cutoff of 30.90 nmol/mg of protein, TSA/TP showed a sensitivity of 85% with a specificity of 97% to discriminate CRC patients from healthy donors. In survival analysis, both TSA and TSA/TP were found to be significant prognostic factors for tumor recurrence in CRC. Furthermore, TSA/TP could distinguish patients at high risk of recurrence within Dukes' stage B and in multivariate analysis it was identified as the best independent prognostic factor. According to our results, preoperative serum TSA/TP content could supply additional information to that provided by Dukes' stage about the prognosis of CRC patients.

Malabsorption syndrome observed in the common octopus Octopus vulgaris infected with Aggregata octopiana (Protista: Apicomplexa).

Octopus vulgaris infected with Aggregata octopiana were collected from an open-water culture system in the Ría of Aldán (NW Spain). Digestive tract infection values were determined with the use of a Neubauer chamber by counting the number of A. octopiana sporocysts. After determining enzyme activity values by the colorimetric Api-Zym system Biomerieux, one representative enzyme of glycosidases, peptid hydrolases and phosphoric hydrolases showing high activity was spectrophotometrically analysed. The enzymes were maltase and leucine-aminopeptidase (LAP) involved in the absorption process, and acid phosphatase, a lysosomic enzyme, respectively. Enzymatic activity of maltase and LAP decreased significantly, with increased sporocyst counts. However, acid phosphatase activity increased with severity of infection, indicating the presence of degradative enzymes from phagocytic cells in the infected area. A detrimental effect on gastrointestinal function may result from a decrease or malfunction of absorption enzymes. The results suggest a malabsorption syndrome resulting from parasitic infection.

Alteration of the serum levels of the epidermal growth factor receptor and its ligands in patients with non-small cell lung cancer and head and neck carcinoma.

Serum levels of the soluble epidermal growth factor receptor (sEGFR) and its ligands epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha) and amphiregulin (AR) were measured in healthy donors and patients with non-small cell lung cancer (NSCLC) and head and neck carcinoma (HNC). In NSCLC, we found sEGFR and EGF levels significantly lowered in patients with respect to healthy donors. In HNC patients, significantly diminished levels were found in the case of sEGFR, EGF and also AR. In both malignancies, no significant association was found between the serum levels of the molecules and the patients' gender, age or smoking habit. Only a significant association was found between the decrease of sEGFR and the absence of distant metastasis in NSCLC and the tumour stage in HNC. The most interesting result was that combining sEGFR and EGF, sensitivities of 88% in NSCLC and 100% in HNC were reached without losing specificity (97.8% in both cases). The use of discriminant analysis and logistic regression improved the sensitivity for NSCLC and the specificity for HNC. These data demonstrate a potentially interesting value of the serum levels of sEGFR and EGF, especially when combined, as markers for NSCLC and HNC.

Heterogeneity of acid beta-galactosidase from rabbit kidney.

1. Rabbit kidney acid beta-galactosidase can be resolved into three peaks (named A3, A2 and A1) by gel-filtration chromatography. Their estimated molecular weights were: more than 250,000, 150,000 and 17,000 respectively. 2. The purified acid form appeared as a single band of protein (Mr = 28,000) on electrophoresis in the presence of sodium dodecyl sulphate, suggesting that forms A3 and A2 are multimeric forms of beta-galactosidase A1. 3. Treatment with neuraminidase from Clostridium perfringens converts form A3 into a more basic form. This phenomenon occurs also when this form is stored for a week at 4 degrees C and parallels its disaggregation. 4. The data suggest that the sialic acids present in the multimeric forms are involved in the aggregation of the acidic form of beta-galactosidase.

Comparative studies of two acid beta-galactosidases from rabbit and bovine kidney.

Comparative studies of two acid beta-galactosidases from rabbit and bovine kidney have been made. The enzyme from rabbit (enzyme I) was purified 450-fold, and the activity of the bovine enzyme (enzyme II) was enriched 250-fold using conventional purification methods. Both purified enzymes were characterized and their properties were compared. Enzyme I showed a lower optimal temperature and was less pH stable. Enzyme II appeared to be homogeneous in charge, in contrast to the heterogeneity observed for enzyme I. Studies on their specificity using natural substrates showed that enzyme I hydrolyzed GM1, asialofetuin and lactose. However, enzyme II was only able to cleave galactose from the disaccharide. Some of the carbohydrates tested acted as activators for enzyme II, suggesting a mechanism of transglycosilation. Using lactose as substrate we confirmed the ability of enzyme II to transfer galactose residues to D-maltose and N-acetylgalactosamine.

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.

Value of the serum alpha-L-fucosidase activity in the diagnosis of colorectal cancer.

OBJECTIVES: The purpose of this study was to assess the value of the serum levels of alpha-L-fucosidase activity in the diagnosis of patients with colorectal cancer. METHODS: Using a fluorometric method we analyzed the alpha-L-fucosidase activity in preoperative sera from 137 colorectal cancer patients and in sera from 232 donors. RESULTS: The enzymatic activity of alpha-L-fucosidase was significantly lower (p <0.001) in patients (4.8+/-3.09 U/ml) than in donors (10.5+/-5.46 U/ml). Using the ROC curve, the ideal cut-off for the diagnostic value of alpha-L-fucosidase activity was determined to be 5.6 U/ml. The diagnostic efficiency for colorectal cancer of alpha-L-fucosidase activity was higher than that observed for carcinoembryonic antigen (cut-off 5.0 ng/ml), especially for tumors at an early stage. CONCLUSIONS: Our results suggest that preoperative serum alpha-L-fucosidase activity may be used as a cheap and easy complementary test, in addition to standard clinical procedures routinely used for the diagnosis of colorectal cancer.

Human colon sialidase: characterization and activity levels in normal mucosa and colonic adenocarcinoma.

Human colon sialidase has been characterized, and its activity levels in normal mucosa and colonic adenocarcinoma have been determined. Sialidase activity was maximal at pH 5.5, and was unstable with storage at 4 and -20 degrees C. The bulk of activity was pellet-associated, and could not be released with triton X-100 or 3-([3-cholamidopropyl]- dimethylammonio)-1-propanesulfonate. Using 2'-(4-methylumbelliferyl)alpha-D-N-acetylneuraminic acid as substrate, the Km and Vmax values were estimated to be 0.140 mmol/l and 63 mU/g, respectively. Furthermore, an inhibition by substrate concentrations above 1.5 mmol/l was detected. Neuraminic acid caused a competitive inhibition with a Ki of 3.5 mmol/l. A statistically significant increase (p < 0.001) in the sialidase specific activity was found in primary colonic adenocarcinoma (104.20 +/- 8.00 mU/g) compared to that of the normal mucosa (72.50 +/- 7.67 mU/g).

Immunohistochemical analysis of sialic acid and fucose composition in human colorectal adenocarcinoma.

The expression of different sialoglycoconjugates and fucoglycoconjugates in normal mucosa and adenocarcinoma samples from 43 colorectal cancer patients was investigated by using specific lectins and applying a semiquantitative analysis. A pronounced decrease in the intracellular binding of the Maackia amurensis lectin, which recognizes alpha(2,3)-linked sialic acid residues, was found in the tumoral tissue. In contrast, a significant increase in the staining with the Sambucus nigra lectin (SNA I), which binds to alpha(2,6)-linked sialic acid residues, was detected in the epithelial cells as well as in the mucins from tumors. No significant differences in the reactivity with the Aleuria aurantia lectin, which recognizes the sequence Fuc(alpha1,6)GlcNAc, between normal and malignant colorectal tissues were detected. Furthermore, the correlation between lectin-binding profiles and the prognosis of colorectal cancer patients was examined. After an average postoperative follow-up period of 31 months, patients with tumors showing a strong SNA I staining presented a greater probability of disease recurrence. This result suggests that the intensity of staining with SNA I could be a valid parameter for predicting recurrence in colorectal cancer.