Aberrant expression of MUC5AC and MUC6 gastric mucin genes in colorectal polyps

Altered mucin glycosylation and the de novo appearance of gastric mucin antigens have been described in colonic adenomas. The purpose of our study was to determine if expression of the gastric mucin genes MUC5AC and MUC6 occurs in colorectal adenomas and whether this correlates with histopathologic criteria of malignant potential. Immunohistochemical staining using antibodies against MUC5AC and MUC6 tandem repeat synthetic peptides was performed on specimens of normal colon mucosa (n = 26), hyperplastic polyps (n = 9) and adenomatous polyps (n = 111). Mucin mRNA levels were determined using RNase protection assays using riboprobes corresponding to unique non-repetitive sequences. MUC5AC and MUC6 staining were rarely detected and of low intensity in normal colon and hyperplastic polyps. The number of immunoreactive polyps and intensity of MUC5AC and MUC6 staining were greatest in larger adenomas of moderate villous histology and dysplasia. MUC5AC and MUC6 staining tended to decrease in highly villous polyps with severe dysplasia. Increased MUC5AC mRNA levels were found in 26/45 of adenomas tested compared with 0/9 normal colon specimens. MUC6 mRNA levels were found in 20/45 of adenomas compared with 1/9 normal colon specimens. MUC5AC and MUC6 mRNA were present more frequently and at higher levels in polyps with intermediate stages of size, villous histology and dysplasia. We conclude that aberrant expression of MUC5AC and MUC6 mucin genes is likely responsible for an expanded repertoire of mucin antigen expression in colorectal neoplasia.     

Progesterone stimulates production and secretion of MUC1 epithelial mucin in steroid-responsive breast cancer cell lines

The effects of oestrogen and progesterone on expression of the MUC1 epithelial mucin were examined in MCF7 and ZR-75-1 breast cancer cells grown in steroid-free medium. Oestrogen caused an increase in cell growth and both cellular and secreted MUC1 in both cell lines. However, after correcting for increases in cellular protein, there was no clear changes. Progesterone, in combination with oestrogen, caused significant increases (over 2-fold, P<0.01) in cellular and secreted MUC1 when compared with oestrogen alone in both cell lines despite no or small increases in cellular protein. Growth of ZR-75-1 cells in a competitive inhibitor of O-glycosylation demonstrated that the increased detection by ELISA was not due to altered glycosylation. Progesterone may modulate expression of MUC1 in steroid-responsive breast cancer cells.     

The MUC2 gene product: a human intestinal mucin

Sera from 82 cases of leptospirosis (confirmed by micro-agglutination tests or IFAT) and 108 patients with other diseases were investigated using the microcapsule agglutination test (MCAT) for leptospirosis. The overall sensitivity (90.2%), specificity (96.3%), positive predictive value (94.9%), negative predictive value (92.9%), and accuracy of the MCAT (93.7%) were encouraging. MCAT is simple, can be performed by unskilled personnel with minimum laboratory facilities, and produces results in 3 h. MCAT would be a reliable serodiagnostic test for rapidly screening individuals for leptospirosis, in various geographical areas of Thailand.     

Significance of p53 and VEGF expression in liver metastasis from colorectal cancer

BACKGROUND: Children with large anterior mediastinal masses frequently present with severe respiratory compromise and often pose a difficult diagnostic dilemma. A biopsy is preferred for diagnosis before treatment can begin; however, many of these children are at risk of acute clinical deterioration and cardiovascular arrest with the induction of anesthesia. The authors noted a correlation between pleural effusions and lymphoblastic lymphoma and recently diagnosed three cases of lymphoblastic lymphoma in children with a large anterior mediastinal mass and pleural effusion through cytological and flow cytometric examination of the pleural fluid. METHODS: To focus on this problem, 101 pediatric patients presenting with an anterior mediastinal mass between January 1980 and September 1994 were reviewed to determine if pleural effusions occur more frequently at initial presentation with lymphoblastic lymphoma than with Hodgkin's disease, thus offering a means of diagnosis in children with severe respiratory compromise. The patients' chest radiographs and/or computed tomograms for the 88 cases in which they were available were reviewed retrospectively in a blinded fashion to identify those children with pleural effusions at the time of presentation. RESULTS: In this study, 71% of patients with lymphoblastic lymphoma (10 of 14) had a pleural effusion at presentation, whereas only 11.7% of patients with Hodgkin's disease (7 of 60) had a pleural effusion on initial presentation. (P < .002 Fisher's Exact test). CONCLUSION: This retrospective review suggests that there is a significantly greater association of pleural effusions in patients with lymphoblastic lymphoma than with Hodgkin's disease. Our experience supports the conclusion that thoracentesis may provide a means of diagnosis in children presenting in severe respiratory compromise obviating the need for anesthesia and open biopsy.     

Effects of glycosylation on fragments of tumour associated human epithelial mucin MUC1

The glycodecapeptide AcPAPGS(alpha GalNAc)T(alpha GalNAc)APPA and the C-terminal glycohexapeptide AcS(alpha GalNAc)T(alpha GalNAc)APPA have been synthesized by applying the N-terminal Fmoc group in combination with the heptyl ester cleavable by lipase-catalyzed hydrolysis at pH 7. The solution conformation of these MUC1-related synthetic glycopeptides and the control, non-glycosylated decapeptide AcPAPGSTAPPA have been investigated using NMR spectroscopy. The structural studies indicate that the glycohexapeptide has a folded structure in solution. For this molecule, unrestrained molecular dynamics has been used to confirm the presence of the observed solution through-space connections. The results indicate that the non-globular nature of MUC1 is due to both protein core sequence and the effect of carbohydrate.     

Differential expression of human high-molecular-weight salivary mucin (MG1) and low-molecular-weight salivary mucin (MG2)

Two distinct mucin components of saliva, MG1 and MG2, have been identified based on chemical composition and molecular weights (high and low, respectively) in saliva. With the aim of characterizing the expression pattern of salivary mucins, we have prepared monoclonal antibodies (MAbs) directed against the peptide core of MG1 and against a synthetic peptide derived from the MG2 (MUC7) sequence. MAb PANH2 raised against partially deglycosylated MG1 stained a high-molecular-weight smear in Western blots of partially purified MG1. PANH2 binding was increased by deglycosylation with trifluoromethanesulfonic acid as well as with subsequent periodate treatment, and was eliminated by pronase treatment, strongly suggesting that MAb PANH2 was directed to a peptide epitope of MG1. MAb PANH3 raised against a synthetic peptide derived from the MG2 (MUC7) sequence reacted with the native molecule and stained a narrow smear of ca. 200,000 to 210,000 in Western blots of concentrated saliva and a lower-molecular-weight smear of trifluoromethanesulfonic-acid-treated MG2. Immunohistology on frozen sections of human salivary glands showed that MAb PANH2 selectively labeled mucous cells, whereas MAb PANH3 labeled subpopulations of serous cells. Double-direct immunofluorescence staining with PANH2 and PANH3 demonstrated that the staining patterns were non-overlapping. The development of these antibody probes will facilitate studies of mucin expression in diseases of salivary glands.     

Decreased MUC1 expression induces E-cadherin-mediated cell adhesion of breast cancer cell lines

Two breast cancer cell lines, YMB-S and ZR-75-1S, were established in our laboratory. They proliferated in suspension culture without aggregation in a complete liquid medium. We found that sodium butyrate (NaB) arrested the cells in the G0-G1 phase of the cell cycle, inhibited their proliferation, and induced cell-cell and cell-surface adhesion. In this study, we explored the mechanism of this adhesion. Adhesion was inhibited by an anti-E-cadherin antibody, suggesting a role for E-cadherin. However, there were no changes in the expression of E-cadherin, alpha-catenin, and beta-catenin. Northern blot analysis and cytofluorometry revealed that NaB-treated cells showed a lower expression of MUC1 than did untreated cells. To examine the possibility that the adhesion of these cells might be induced by decreased MUC1 expression, the level of MUCI expression was directly reduced using an antisense oligonucleotide. The MUC1 antisense oligonucleotide induced cell-cell and cell-surface adhesion of these breast cancer cells, just as NaB did. Our observations indicate that E-cadherin can be functionally suppressed by overexpression of MUC1 but resumes its activity after suppression of MUC1 expression. Thus, regulation of MUC1 might be a new strategy for cancer therapy.     

Identification of a major human high molecular weight salivary mucin (MG1) as tracheobronchial mucin MUC5B

The strain G89HT of Clostridium argentinense obtained by culture selection of the prototype G89 strain producing high titers of type G botulinal toxin was studied. Its cultural, biochemical and toxigenic characteristics and the presence of plasmids were tested. Both strains showed similar physiological features and carried a 83 MDa plasmid. A 170 MDa plasmid was also recognized in the G89HT strain. Notably, this strain was better sporulating and showed a higher toxigenicity than the prototype G89 C. argentinense strain. These two characteristics might permit a long term storage and perhaps yield high antitoxin titres.     

Differential expression of galectin 3 and galectin 1 in colorectal cancer progression

BACKGROUND & AIMS: Galectins are beta-galactoside-binding proteins possibly involved in tumor progression. The aim of this study was to determine the pattern of galectin 3 and galectin 1 expression and involvement in colorectal cancer progression. METHODS: Galectin 3 expression was examined immunohistochemically in 39 samples of normal mucosae, 25 adenomas, 87 carcinomas, and 39 lymph node metastases. Galectin 1 was analyzed in 25 samples of mucosae, 15 adenomas, 25 carcinomas, and 11 metastases. Western blot analysis was also performed. RESULTS: All normal mucosae showed strong nuclear galectin 3 expression, which was down-regulated in the neoplastic progression, because only 60% of adenomas, 48% of carcinomas, and 44% of metastases were strongly positive (P < 0.0001). Cytoplasmic expression was down-regulated in adenomas (16%) but increased again in carcinomas (64%) (P < 0.0001). Galectin 1 expression was mainly detected in stromal cells and correlated with tumor progression from normal mucosae to adenomas and carcinomas (P < 0.0001). CONCLUSIONS: Galectin 3 expression is down-regulated in the initial stages of neoplastic progression, whereas a dissociated cytoplasmic expression increases in later phases of tumor progression. Galectin 1 in colorectal mucosa is predominantly a stromal product whose overexpression is associated with the neoplastic progression of colorectal cancer.     

Structural features of the human salivary mucin, MUC7

Specific mgi mutations in the alpha, beta or gamma subunits of the mitochondrial F1-ATPase have previously been found to suppress rho0 lethality in the petite-negative yeast Kluyveromyces lactis. To determine whether the suppressive activity of the altered F1 is dependent on the F0 sector of ATP synthase, we isolated and disrupted the genes KlATP4, 5 and 7, the three nuclear genes encoding subunits b, OSCP and d. Strains disrupted for any one, or all three of these genes are respiration deficient and have reduced viability. However a strain devoid of the three nuclear genes is still unable to lose mitochondrial DNA, whereas a mgi mutant with the three genes inactivated remains petite-positive. In the latter case, rho0 mutants can be isolated, upon treatment with ethidium bromide, that lack six major F0 subunits, namely the nucleus-encoded subunits b, OSCP and d, and the mitochondrially encoded Atp6, 8 and 9p. Production of rho0 mutants indicates that an F1-complex carrying a mgi mutation can assemble in the absence of F0 subunits and that suppression of rho0 lethality is an intrinsic property of the altered F1 particle.     

A novel role for murine IL-4 in vivo: induction of MUC5AC gene expression and mucin hypersecretion

Mucus hypersecretion and plugging of lower respiratory tract airways contributes to the morbidity and mortality associated with asthma. Interleukin (IL)-4 plays a putative role in some forms of asthma. Thus, transgenic mice that overexpress murine IL-4 selectively within the lung were used to study the effect of IL-4 on mucus glycoprotein gene expression and mucin release. Histologic examination of lung sections from IL-4 mice revealed that nonciliated epithelial cells from conducting airways were hypertrophic, due at least in part to the accumulation of mucus glycoprotein. The cytoplasm of these cells stained positively for glycoproteins using mucicarmine, alcian blue (AB), and periodic acid-Schiff (PAS). Ciliated cells were also enlarged but did not show any mucin-specific staining. Inclusion granules typically found in nonciliated (Clara) cells of control mice were absent in the IL-4 transgenic mice. Northern blot analysis of total RNA from lung tissue revealed that the expression of the MUC5AC, but not MUC2, mucin gene was distinctly upgraded in IL-4 transgenic mice compared to transgene-negative controls. In addition, a 5- to 10-fold increase in AB- and PAS-positive material was found in lavage fluid from IL-4 overexpressing mice compared to transgene-negative controls. Thus, the overexpression of IL-4 locally within the lung enhances mucus glycoprotein synthesis by altering gene expression, results in the accumulation of mucus glycoprotein in nonciliated epithelial cells, and induces the release of mucus into the airway lumen. We therefore hypothesize that the overproduction of mucus seen in some patients with asthma may be a direct result of the action of IL-4 within the inflamed lung.     

Loss of Bcl-2 expression correlates with tumour recurrence in colorectal cancer

OBJECTIVE: To analyse the efficacy, correlations and adverse-event profile of placebo therapy from the initial placebo run-in period to beyond 2 years of treatment. PATIENTS AND METHODS: The effects of placebo therapy on prostate size, maximum urinary flow rate (Qmax) and symptoms were analysed, and adverse drug experiences documented, for a period of 25 months in 303 patients randomized to the placebo arm of a controlled trial evaluating finasteride in the treatment of BPH (the Canadian PROSPECT study). RESULTS: For all variables, the values during follow-up were significantly different from baseline (P < or = 0.001). Transrectal ultrasonography confirmed a progressive increase in prostate volume over 25 months (+8.4%) but Qmax improved for the first 5 months (to 1.4 mL/s over baseline) and remained 1.0 mL/s more than baseline at 25 months. The total symptom score improved by -2.9 points in the first 2 months on placebo and was ultimately 2.3 points below baseline at 25 months. The extent of the placebo response for symptoms (r=0.08, P=.180) and Qmax (r=0.04, P=0.550) was independent of age, but the response correlated with the initial severity of symptoms (r= -0.394, P < or = 0.001) and initial Qmax (r= -0.134, P=0.023). Patients with a prostate of < or = 40 mL had a clinically more important placebo response than those with larger prostates. In all, 246 patients (81.2%) reported adverse events thought to be secondary to placebo therapy. The most common complaint was urogenital (40.3%), specifically impotence (6.3%) and decreased libido (6.3%); 13.2% of patients discontinued placebo therapy because of significant adverse reactions. CONCLUSIONS: Placebo therapy rapidly produces a significant improvement in Qmax and symptoms of BPH but also causes clinically important adverse effects. The beneficial effect fades but remains after 2 years.     

Induction of HLA-A2-restricted CTLs to the mucin 1 human breast cancer antigen

HLA-A*0201/Kb transgenic mice were immunized with oxidized mannan-mucin 1 (MUC1) as a fusion protein (containing five repeats of the 20-amino-acid MUC1 VNTR (variable number of tandem repeats) that generated highly active CD8+ CTLs to MUC1 peptides. In a direct CTL assay, the MUC1 peptides could be presented specifically by both the transgenic murine HLA-A*0201/Kb and human HLA-A*0201 molecules. The 9-mer MUC1 peptide sequences (APDTRPA and STAPPAHGV) were presented by HLA-A*0201, although they did not contain L at P2 and L/V at P9, the preferred motifs; as a consequence, the binding was of relatively low affinity when compared with a high affinity-binding HIV peptide (ILKEPVHGV). In addition, when mice were immunized separately with the HLA-A*0201-binding peptides (STAPPAHGV or APDTRPAP-containing peptides-keyhole limpet hemocyanin-mannan), direct lysis of MCF-7 (HLA-A*0201+, MUC1+) also occurred. The findings are of interest for tumor immunotherapy, particularly as the CTLs generated to low affinity-binding peptides were highly active and could specifically lyse an HLA-A*0201+ human breast cancer cell line without further in vitro stimulation. The findings demonstrate that the range of peptides that can generate CTLs is broader than formerly considered.     

MUC4 is a major component of salivary mucin MG1 secreted by the human submandibular gland

High molecular weight salivary mucin (MG1) is an important component of saliva, contributing to the lubricative and tissue-protective functions of this biological fluid. We have shown previously that the human mucin gene MUC5B is expressed at high levels in sublingual gland and is a significant constituent of MG1. Since many epithelia express multiple mucin genes, it seemed likely that MG1 in salivary secretions is also a heterogeneous mixture of mucin gene products. The aim of this study was to determine whether MUC4, a mucin shown in Northern blotting experiments to be expressed in salivary glands, was a significant protein component of MG1 in salivary secretions. Two cDNA clones containing MUC4 tandem repeats were isolated from a human submandibular gland cDNA library. In addition, recombinant MUC4 produced in a bacterial expression system cross-reacted with an antibody directed against deglycosylated MG1. This shows conclusively that human salivary mucin MG1 contains both MUC5B and MUC4 gene products suggesting that each mucin may perform distinct functions in the oral cavity.     

Bcl-2 protein expression: association with p53 and prognosis in colorectal cancer

In myocardial SPECT perfusion imaging, reorientation algorithms from transaxial image planes are used to generate short- and long-axis views of myocardial tracer uptake. We performed phantom experiments with 201Tl to delineate how image reorientation affects the results of quantitative image analysis. METHODS: Thirty consecutive patient studies were analyzed to characterize the distribution of the angle of reorientation in a clinical setting. Short-axis SPECT images of a cardiac phantom with and without a 180 degrees cold-spot insert were reconstructed with three different backprojection filters (ramp, Metz and Butterworth) and reoriented through different angles ranging from 45 degrees to 89 degrees. Four interpolation algorithms were used to calculate from the transaxial images the pixel values of the reoriented images: (a) a simple interpolator that averages the pixel values of the eight neighboring pixels of the transaxial image; (b) a three-dimensional linear interpolator; (c) a hybrid interpolator that combines a two-dimensional linear in-plane with a one-dimensional cubic across-plane interpolation; and (d) a three-dimensional cubic convolution interpolator. Images were reoriented twice with opposite angles so that the original and the reoriented images could be directly compared. Circumferential profile analysis was applied to determine the root mean square error of corresponding profiles and the difference of the extent and the severity of perfusion defects. Single and multivariate analyses of variance (ANOVA) were used to compare the effects of the reorientation angle, the backprojection filter and the interpolation algorithm. RESULTS: In the clinical studies, the angle between the transaxial and reoriented images was 75 degrees +/- 10 degrees (s.d.). In 48 phantom experiments, multivariate ANOVA demonstrated that the backprojection filter and the interpolation algorithm significantly affect the circumferential profiles and the extent and severity of a perfusion defect (p < 0.05). In contrast, the angle of reorientation was not a significant factor (p = ns). By univariate analysis, the three-dimensional cubic interpolator was associated with significantly (p < 0.05) less error than the simple and three-dimensional linear algorithms. Relative computation times (simple interpolator = 100%) were 119% for the three-dimensional linear, 136% for the hybrid and 243% for the three-dimensional cubic interpolator. CONCLUSION: For quantitative analysis of myocardial SPECT perfusion images, a Metz filter for filtered backprojection in combination with a three-dimensional cubic convolution interpolation for image reorientation appears to offer improved accuracy.     

Germline mutation analysis in hMLH 1 and hMSH 2 genes in hereditary nonpolyposis colorectal cancer and colorectal cancer patients with familial history

Workplace violence has become a problem in modern American society. Health-care workers are particularly vulnerable because of the nature of their jobs dealing with clients, many of whom are emotionally disturbed. A brief review of the Occupational and Safety Health Administration (OSHA) "Guidelines for Preventing Workplace Violence Among Health Care and Social Workers" that was published in 1996 is presented. Some sensible ways to implement the OSHA guidelines are also discussed.     

MLH1 and MSH2 constitutional mutations in colorectal cancer families not meeting the standard criteria for hereditary nonpolyposis colorectal cancer

Genetic diagnosis of hereditary nonpolyposis colorectal cancer (HNPCC) may have a significant impact on the clinical management of patients and their at-risk relatives. At present, clinical criteria represent the simplest and most useful method for the identification of HNPCC families and for the selection of candidates for genetic testing. However, reports of mismatch repair (MMR) gene mutations in families not fulfilling the minimal diagnostic criteria point out the necessity to identify additional clinical parameters suggestive of genetic predisposition to colorectal cancer (CRC) related to MMR defects. We thus investigated a series of 32 Italian putative HNPCC individuals selected on the basis of one of the following criteria: 1) family history of CRC and/or other extracolonic tumors; 2) early-onset CRC; and 3) presence of multiple primary malignancies in the same individual. These patients were investigated for the presence of MLH1 and MSH2 mutations by single-strand conformation polymorphism analysis. Pathogenetic truncating mutations were identified in 4 (12.5%) cases, 3 of them involving MSH2 and 1 MLH1. In addition, 2 missense MLH1 variants of uncertain significance were observed. All pathogenetic mutations were associated with early age (<40 years) at onset and proximal CRC location. Our results support the contention that constitutional MMR mutations can also occur in individuals without the classical HNPCC pattern. Moreover, evaluation of the clinical parameters associated with MMR mutations indicates that early onset combined with CRC location in the proximal colon can be definitely considered suggestive of MMR-related hereditary CRC and should be included among the guidelines for referring patients for genetic testing.