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.     

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.     

The high-molecular-weight human mucin is the primary salivary carrier of ABH, Le(a), and Le(b) blood group antigens

Because many bacteria interact with the carbohydrate portions of receptor molecules, factors controlling glycosylation probably influence the ability of salivary components to mediate bacterial adherence/clearance. Important sources of diversity in glycosylation are the ABO, secretor, and Lewis genes, which code for glycosyltransferases that add specific sugar sequences to the termini of carbohydrate chains of glycolipids and glycoproteins. We identified, by Western blotting, salivary glycoproteins carrying the ABH and Le(a) or Le(b) antigens. Samples of whole, unstimulated saliva were obtained from 19 subjects whose blood group was determined by agglutination of red blood cells with specific antisera. After centrifugation, the samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose. Glycoproteins carrying blood group antigens were identified by staining the blot with monoclonal antisera specific for the A, B, H, Le(a), or Le(b) antigens. The most intensely staining component from all the samples migrated at the same position as the high-molecular-weight mucin. Saliva samples from the nonsecretors contained only the Le(a) antigen. Samples from the secretors contained one or more of the ABH antigens and, variably, the Le(b) antigen. In all cases, the salivary blood group antigens corresponded to those found on the red blood cells of the same subject. The functional consequences of the expression of blood group antigens on the high-molecular-weight mucin are not known, but their presence could modulate the adherence of certain oral microorganisms that interact preferentially with this molecule.     

Human low-molecular-weight salivary mucin expresses the sialyl lewisx determinant and has L-selectin ligand activity

Previously we showed that the low-molecular-weight mucin (MG2, encoded by MUC7), a major component of human submandibular/sublingual saliva, is a bacterial receptor that coats the tooth surface. Here we tested the hypothesis that the structure of its carbohydrate residues contains important information about its function. Purified MG2 (Mr 120 000) was digested with trypsin, and the resulting Mr 90 000 fragment, which carried primarily O-linked oligosaccharides, was subjected to reductive beta-elimination. The released oligosaccharides were characterized by using nuclear magnetic resonance spectroscopy and mass spectrometry. Of the 41 different structures we detected, the most prominent included NeuAcalpha2-->3Galbeta1-->3GalNAc-ol (sialyl-T antigen), Galbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-->6(Galbeta1 -->3)GalNAc-ol [type 2 core with Lewisx (Lex) determinant], and NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->6(Galbeta1--> 3) GalNAc-ol [type 2 core with sialyl Lex (sLex) determinant]. We also detected di-, tri-, and pentasaccharides with one sulfate group. Lex, sLex, and related sulfated structures are ligands for selectins, adhesion molecules that mediate leukocyte trafficking. Therefore, we investigated whether MG2 was a selectin ligand. In an enzyme-linked immunosorbent assay, L-selectin chimeras interacted with immobilized MG2 in a Ca2+-dependent manner. L-Selectin chimeras also bound to MG2 immobilized on nitrocellulose. Together, these results suggest that the saccharides that MG2 carries could specify some of its important functions, which may include mediating leukocyte interactions in the oral cavity.     

Molecular cloning, sequence, and specificity of expression of the gene encoding the low molecular weight human salivary mucin (MUC7)

Previous biochemical studies have determined that human saliva contains high and low molecular weight mucin glycoproteins (MG1 and MG2, respectively) that are structurally distinct. In this study, we describe the isolation and characterization of overlapping cDNA clones which code for the MG2 protein core. DNA sequencing revealed a translated region of 1131 nucleotides encoding a protein of 377 amino acid residues with a molecular mass of 39 kDa. The first 20 N-terminal residues were very hydrophobic and probably comprise the MG2 leader peptide. The region encoding the secreted protein can be divided into three distinct domains; unique 5'- and 3'-translated regions containing 4 and 1 potential N-glycosylation sites, respectively, and a central region of six almost perfect tandem repeats of 23 amino acid residues with a high number of Thr and Ser. No sequence homology with any other human or animal mucins, and no significant homology to any other proteins was found. MG2 mRNA is about 2.5 kilobases long, and its expression appears to be species-, tissue-, and cell-specific. We propose to name this gene MUC7 in accordance with the mucin genes cloned to date named MUC1-MUC6.     

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.     

Molecular cloning of cDNAs for mouse low-molecular-weight and high-molecular-weight prekininogens

5% Amlexanox oral paste (Aphthasol) was studied in four vehicle-controlled, randomized, double-blind, parallel group, multicenter, clinical studies involving 1335 subjects who had 1 to 3 aphthous ulcers less than 48 hours old at enrollment. Subjects applied study pastes directly to ulcers four times a day until ulcers healed or for the duration of the study, whichever occurred first. Ulcer size was measured by the investigator and pain was evaluated by the subject; the primary determinant of efficacy was the percentage of subjects with complete healing of ulcers and complete resolution of ulcer pain. The vehicle had marginal beneficial effects as would be expected from a covering material, but statistical significance over no treatment was inconsistent. However, these studies, both individually and collectively, clearly demonstrated in a highly significant and consistent manner that in comparison to both Vehicle and No Treatment 5% Amlexanox oral paste accelerates the resolution of pain and healing of aphthous ulcers.     

Differential expression of functional protease-activated receptor-2 (PAR-2) in human vascular smooth muscle cells

The protease-activated family of G protein-coupled receptors includes PAR-1 and PAR-3, which are activated by thrombin, and PAR-2, which is activated by trypsin and tryptase. PAR-2 has recently been shown to be expressed in human endothelial cells. In the present studies, we have examined the expression of PAR-2 in other cells, particularly vascular smooth muscle, and tested whether the receptors are functional. The results show that PAR-2 is present in human aorta and coronary artery smooth muscle cells, as well as in arteries traversing the walls of the small intestine. It was also detected in human keratinocytes, sweat glands, intestinal smooth muscle, and intestinal epithelium, but not at all in myocardial smooth muscle and only inconsistently in intestinal veins and venules. Activation of aortic smooth muscle cells in culture with PAR-2 peptide agonists caused a transient increase in the cytosolic Ca2+ concentration. In contrast, PAR-2 mRNA could not be detected in saphenous vein smooth muscle cells, and the same cells placed in culture showed little, if any, response to the PAR-2 agonist peptides. These observations show that PAR-2 is widely distributed in human vascular smooth muscle, particularly in arteries. However, this is not a universal finding and at least some venous smooth muscle cells, including those in saphenous veins, apparently do not express the receptor in detectable amounts.     

Differential expression and origin of membrane-type 1 and 2 matrix metalloproteinases (MT-MMPs) in association with MMP2 activation in injured human livers

Matrix metalloproteinase-2 (MMP2) activation is associated with basement membrane remodeling that occurs in injured tissues and during tumor invasion. The newly described membrane-type MMPs (MT-MMPs) form a family of potential MMP2 activators. We investigated the localization and steady-state levels of MT1-MMP and MT2-MMP mRNA, compared with those of MMP2 and tissue inhibitor of MMP-2 in 22 hepatocellular carcinomas, 12 liver metastases from colonic adenocarcinomas, 13 nontumoral samples from livers with metastases, 10 benign tumors, and 6 normal livers. MMP2 activation was analyzed by zymography in the same series. The expression of MT1-MMP mRNA and the activation of MMP-2 were increased in hepatocellular carcinomas, metastases, and cholestatic nontumoral samples. MT2-MMP mRNA was rather stable in the different groups. MT1-MMP mRNA levels, but not MT2-MMP mRNA, correlated with MMP-2 and tissue inhibitor of MMP-2 mRNA levels and with MMP2 activation. In situ hybridization showed that MT1-MMP mRNA was expressed in stromal cells, and MT2-MMP mRNA was principally located in both hepatocytes and biliary epithelial cells. Consistently, freshly isolated hepatocytes expressed only MT2-MMP mRNA, and culture-activated hepatic stellate cells showed high levels of MT1-MMP mRNA. These results indicate that in injured livers, MMP2 activation is related to a coordinated high expression of MMP2, tissue inhibitor of MMP-2, and MT1-MMP. Furthermore, the finding of a preferential expression of MT2-MMP in hepatocytes, together with our previous demonstration that the activation of stellate cell-derived MMP2 in co-culture requires interactions with hepatocytes (Am J Pathol 1997, 150:51-58), suggests that parenchymal cells might play a pivotal role in the MMP2 activation process.     

Significance of MUC1 and MUC2 mucin expression in colorectal cancer

AIMS: To compare the lineage specific distribution of MUC1 and MUC2 mucins in normal colorectal mucosa and adenocarcinoma and to identify pathological correlations. METHODS: Paraffin wax sections from 51 colorectal cancers were examined for the expression of MUC1 and MUC2, non-O-acetyl sialic acid and the carbohydrate epitopes Lex, Ley, sialosyl-Lex, sialosyl-Tn, and Tn using standard histochemical methods. RESULTS: MUC1, Lex and Ley co-localised with columnar cell secretions, whereas MUC2, mild periodic acid Schiff and sialosyl-Tn co-localised with goblet cell mucin in both normal and malignant tissues. Sialosyl-Lex and Tn were associated with both lineages. In normal tissues MUC1, Lex and Ley showed only trace expression by crypt base columnar cells. Cancers could be classified into four phenotypes (MUC2+/MUC1-, MUC2+/MUC1+, MUC2-/MUC1+, MUC2-/MUC1-). Particular phenotypes showed significant correlations with cancer type, lymph node spread and peritumoral lymphocytic infiltration and trends falling short of significance in relation to grade of differentiation and contiguous adenoma. CONCLUSIONS: Classification of colorectal cancer by means of lineage specific function may be relevant to both pathogenesis and prognosis.     

Differential expression of vasoactive intestinal peptide receptors 1 and 2 (VIP-R1 and VIP-R2) mRNA in murine lymphocytes

Vasoactive intestinal peptide (VIP), a neuropeptide present in the lymphoid microenvironment, modulates cytokine expression and affects T cell proliferation. Recent molecular studies identified two VIP receptors. VIP-R1 and VIP-R2, primarily in nonlymphoid cells. In this study, we investigate the expression of VIP-R1 and VIP-R2 mRNA in unstimulated and stimulated lymphocytes and thymocytes, and in various lymphocyte subpopulations. In contrast to VIP-R1 which is constitutively expressed, the expression of VIP-R2 is induced only following stimulation through the TCR-associated CD3 complex. Both CD4+ and CD8+ T cells express VIP-R1 and VIP-R2. Two T cell lines, EL-4.IL-2 and D10.G4.1 express exclusively VIP-R2. VIP induces the expression of the VIP-R2 gene in the absence of additional stimuli. Differential expression and regulation of the two VIP receptors in T lymphocytes suggests different physiological roles in mediating the immunomodulatory activities of VIP and related neuropeptides.     

Differential expression of FMR1, FXR1 and FXR2 proteins in human brain and testis

Lack of expression of the fragile X mental retardation protein (FMRP) results in mental retardation and macroorchidism, seen as the major pathological symptoms in fragile X patients. FMRP is a cytoplasmic RNA-binding protein which cosediments with the 60S ribosomal subunit. Recently, two proteins homologous to FMRP were discovered: FXR1 and FXR2. These novel proteins interact with FMRP and with each other and they are also associated with the 60S ribosomal subunit. Here, we studied the expression pattern of the three proteins in brain and testis by immunohistochemistry. In adult brain, FMR1, FXR1 and FXR2 proteins are coexpressed in the cytoplasm of specific differentiated neurons only. However, we observed a different expression pattern in fetal brain as well as in adult and fetal testis, suggesting independent functions for the three proteins in those tissues during embryonic development and adult life.     

Control of mucin molecular forms expression by salivary protease: differences with caries

1. A protease activity capable of degradation of the high mol. wt salivary mucus glycoprotein to a low mol. wt glycoprotein form was identified in human submandibular gland secretion. 2. The protease exhibited optimum activity at pH 7.0-7.4, and gave on SDS-PAGE under reducing conditions two major protein bands of 48 and 53 kDa. The enzyme showed susceptibility to PMSF, alpha 1antitrypsin, and egg white and soybean inhibitors, a characteristic typical to serine proteases. 3. The activity of the protease towards the high mol. wt mucus glycoprotein was found to be 3.8-fold higher in submandibular gland secretion of caries-resistant individuals than that of caries-susceptible. Furthermore, the enzyme from both groups displayed greater activity against the mucus glycoprotein of caries-resistant subjects. 4. Since the low mol. wt salivary mucus glycoprotein form is more efficient in bacterial clearance than the high mol. wt mucin, the enhanced expression of this indigenous salivary protease activity towards mucin may be the determining factor in the resistance to caries.     

Differential induction of peroxisomal enzymes by hypolipidaemics in human (HepG2) and rat (MH1C1) hepatoma cell lines

Human (HepG2) and rat (MH1C1) hepatoblastoma cells were incubated with different concentrations of the hypolipidaemics cetaben, clofibrate and thyroxine. The enzymatic activities of catalase, peroxisomal bifunctional enzyme, succinate dehydrogenase, and 3-oxoacyl-CoA thiolase were measured. In order to determine the point of regulation of the enzymatic activities Northern and Slot blot experiments with probes for peroxisomal bifunctional enzyme, catalase and fatty acyl CoA oxidase were performed on total RNA. Catalase activity was enhanced in HepG2 cells treated with 3 mmol/l clofibric acid to 135% of control and the mRNA value to 2.6 fold, whereas in cetaben treated cells the enhancement (up to 119% of control) was less pronounced. In MH1C1 cells catalase activity was not changed by any of the drugs. The activity of the peroxisomal bifunctional enzyme was not affected in HepG2 cells by clofibric acid and cetaben, whereas the mRNA level was elevated to 2.3 fold by 10 micromol/l cetaben. At high concentrations of cetaben all enzyme activities were decreased in both cell lines due to its high cytotoxicity. Our data show that, due to the differences in the genomic organisation, the regulation of the enzyme activities is different in human and rat, but the results from the human and rat hepatoblastoma cells correlate with the findings in whole man and rat, so that a human in vitro system is more suitable for pharmacological tests. These results suggest that the human hepatoma cell line HepG2 may be a useful model system for studies of the influence of hypolipidaemics on the peroxisomal enzyme system.     

Binding of the pili of Pseudomonas aeruginosa to a low-molecular-weight mucin and neutral cystatin of human submandibular-sublingual saliva

The HLA-B*27 group of alleles has been extensively studied due to the association of particular B*27 alleles with ankylosing spondylitis (AS). We describe here an HLA-B*27 allele (B*2712) encoding an antigen that lacks reactivity with B27 monoclonal antibodies (moabs) and alloantisera but reacts with some B40/B60 moabs and alloantisera and expresses the Bw6 public epitope. This allele was discovered by the segregation of an HLA-B allele undetectable by PCR-SSP within a Caucasian family from the British population referred for routine bone marrow transplant HLA typing and found in the haplotype A*29; B*2712; Cw*1203; DRB1*13; DQB1*0603. Serological typing showed a lack of reactivity with four B27 moabs and four alloantisera but positive reactivity with moabs and alloantisera specific for B40/B60 and Bw6 public epitopes. Subsequent sequencing showed the closest homology was with B*2708 with three mismatches in exon 2 at positions 204, 209 and 210. The intron 2 sequence was identical with other B*27 lineage alleles including a 2 base pair deletion at positions 95 and 96. The relationship between HLA-B*2712 and reported B60 associations with susceptibility to AS remains to be determined.     

Differential expression of cytochrome P4502E1 (CYP2E1) in Morris hepatomas and livers of tumor bearing rats

PURPOSE: Osmotic dilators (laminarias) have been used for gradual nontraumatic dilation of the cervical canal for various intrauterine procedures; however, this technique has not been well accepted in gynecological brachytherapy. The purpose of this study was to evaluate the efficacy of osmotic cervical dilation for brachytherapy in gynecologic cancer patients, without the use of general/regional anesthesia, and to assess patient tolerance, complications, and outcome. METHODS AND MATERIALS: Thirteen brachytherapy procedures were performed in 6 patients with clinical Stages I and II endometrial (5) and Stage IB cervical cancer (1), who were unable to tolerate general/regional anesthesia because of severe medical problems. An osmotic dilator (synthetic laminaria) was inserted into the cervical os 10-12 h before each brachytherapy procedure and removed just before the procedure. Standard Fletcher-Suit-Delclos tandem insertions with vaginal colpostats or cylinders were then performed. Degree of cervical dilation, patient discomfort, procedure time, intra- and postoperative complications were recorded, and local control and survival were assessed. Median follow-up was 31 months (range: 8-35 months). RESULTS: The diameter of the dilated cervical os after laminaria removal was adequate (> or = 5 mm) for tandem insertion, and no additional mechanical dilation was required in all but one procedure (1 of 13). All procedures were performed without general/regional anesthesia. The mean duration of the procedures was 44 min (range, 20-60 min). Discomfort was minimal in all cases. There were no intra- or postoperative complications. All patients maintained local control until death (1 of metastatic disease, 2 of intercurrent disease) or last follow-up (2 with no evidence of disease, 1 alive with metastatic disease). CONCLUSION: This preliminary study suggests that osmotic cervical dilation with a synthetic laminaria is a useful technique to facilitate intrauterine tandem insertion in patients who cannot tolerate general/regional anesthesia. This technique may reduce treatment-associated morbidity, shorten procedure time, and allow the delivery of adequate radiation therapy in this uncommon but challenging patient population.