Immunohistochemical staining patterns of MUC1, MUC2, MUC4, and MUC5AC mucins in hyperplastic polyps, serrated adenomas, and traditional adenomas of the colorectum.

We studied the distribution of the four human apomucins MUC1, MUC2, MUC4, and MUC5AC in hyperplastic polyps, serrated adenomas, and traditional adenomas of the colorectum using immunohistochemical techniques, with the aim of comparing and contrasting their patterns of expression. A series of 12 hyperplastic polyps, 27 serrated adenomas, and 20 traditional adenomas was studied. No significant change in apomucin expression was observed in traditional adenomas compared with normal colorectal epithelium, except for MUC5AC, which was present in 12 of the adenomas (60%) and only 20% of the normal samples. In both hyperplastic polyps and serrated adenomas, MUC2 and MUC5AC mucin expression was consistently and markedly increased. In 50% of the hyperplastic polyps, MUC4 was reduced but in the remaining cases was similar to normal. Loss of MUC4 expression was observed in all serrated adenomas. MUC1 was not increased in the hyperplastic polyps but increased expression was seen in 17 of the serrated adenomas (63%). Similar altered distribution patterns of MUC2, MUC4, and MUC5AC were seen in hyperplastic polyps and serrated adenomas, whereas traditional adenomas showed little change from normal patterns of expression. Although hyperplastic polyps are commonly defined as benign lesions without neoplastic potential, the similar phenotypes of hyperplastic and serrated adenomas and the existence of mixed polyps suggest that these lesions may represent a histogenetic continuum.     

Expression profiles of MUC1, MUC2, and MUC4 mucins in human neoplasms and their relationship with biological behavior

Mucins are high molecular weight glycoproteins that play important roles in carcinogenesis or tumor invasion. To clarify the relationship of the expression patterns of mucins in human neoplasms with their biological behavior, we examined the expression profiles of MUC1, MUC2, and MUC4 mucins in various human neoplasms using immunohistochemistry and in situ hybridization, and compared them with clinicopathologic factors including outcome of the patients. MUC1 or MUC4 expression is related with the aggressive behavior of human neoplasms and a poor outcome of the patients. In contrast, MUC2 expression tends to be related with the indolent behavior of human neoplasms and a favorable outcome of the patients, although indolent pancreatobiliary neoplasms sometimes show invasive growth with MUC1 expression in the invasive areas. The expression of MUC2 mucin in indolent pancreatobiliary neoplasms coincided with expression of MUC2 mRNA. Our recent studies to clarify the MUC2 gene regulation mechanism disclosed that DNA methylation and histone modification in the 5' flanking region of the MUC2 promoter may play an important role. Further studies of the epigenetics also in MUC1 and MUC4 gene expression may be needed to understand the relationship between the expression of mucins in human neoplasms with their biological behavior.     

MUC1 mediates Pneumocystis murina binding to airway epithelial cells

Previous studies have shown that Pneumocystis binds to pneumocytes, but the proteins responsible for binding have not been well defined. Mucins are the major glycoproteins present in mucus, which serves as the first line of defence during airway infection. MUC1 is the best characterised membrane‐tethered mucin and is expressed on the surface of most airway epithelial cells. Although by electron microscopy Pneumocystis primarily binds to type I pneumocytes, it can also bind to type II pneumocytes. We hypothesized that Pneumocystis organisms can bind to MUC1 expressed by type II pneumocytes. Overexpression of MUC1 in human embryonic kidney HEK293 cells increased Pneumocystis binding, while knockdown of MUC1 expression by siRNA in A549 cells, a human adenocarcinoma‐derived alveolar type II epithelial cell line, decreased Pneumocystis binding. Immunofluorescence labelling indicated that MUC1 and Pneumocystis were co‐localised in infected mouse lung tissue. Incubation of A549 cells with Pneumocystis led to phosphorylation of ERK1/2 that increased with knockdown of MUC1 expression by siRNA. Pneumocystis caused increased IL‐6 and IL‐8 secretion by A549 cells, and knockdown of MUC1 further increased their secretion in A549 cells. Taken together, these results suggest that binding of Pneumocystis to MUC1 expressed by airway epithelial cells may facilitate establishment of productive infection.     

MUC5AC,but not MUC2, is a prominent mucin in respiratory secretions

Airway mucus was collected from healthy and chronic bronchitic subjects. The chronic bronchitic sputum was separated into gel and sol phase by centrifugation and mucins were isolated using isopycnic density-gradient centrfugation in CsCl. The presence of the MUC5AC and MUC2 mucins was investigated with antisera raised against synthetic peptides with sequences from the respective apoproteins. The gel and sol phase of chronic bronchitic sputum as well as healthy respiratory secretions were shown to contain MUC5AC whereas the MUC2 mucin could not be detected. Rate-zonal centrifugation showed that the MUC5AC mucin was large, polydisperse in size and that reduction yielded subunits. Ion-exchange HPLC revealed the presence of two subunit populations in all secretions, the MUC5AC subunits always being the more acidic. MUC5AC is thus the first large, subunit-based, gel-forming respiratory mucin identified and this glycoprotein is biochemically distinct from at least one other population of large, gel-forming mucins also composed of subunits but lacking a genetic identity.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate - CF cystic fibrosis - DFP diisopropylphosphofluoridate - DTT dithiothreitol - EDTA ethylenedinitrilotetraacetic acid - NEM N-ethylmaleimide - PAS periodic acid/Schiffs - PMSF phenylmethylsulphonyl fluoride - Tris Tris(hydroxymethyl)aminomethane - VNTR variable number of tandem repeats     

MUC1蛋白的结构、功能及应用

MUC1粘蛋白是一种大分子量的糖蛋白,主要存在于乳腺、胰腺、卵巢等上皮性组织和器官中。它在癌变上皮细胞表面高度异常表达,结构发生相应改变,从而成为免疫应答的靶点,并在乳腺癌的发展、转移、预后及免疫治疗中起到重要作用。     

Degradation of MUC7 and MUC5B in Human Saliva

Background

Two types of mucins, MUC7 and MUC5B constitute the major salivary glycoproteins, however their metabolic turnover has not been elucidated in detail to date. This study was conducted to examine turnover of MUC7 and MUC5B in saliva, by focusing on the relationship between their deglycosylation and proteolysis.

Methodology/Principal Findings

Whole saliva samples were collected from healthy individuals and incubated at 37°C in the presence of various protease inhibitors, sialidase, or a sialidase inhibitor. General degradation patterns of salivary proteins and glycoproteins were examined by SDS-polyacrylamide-gel-electrophoresis. Furthermore, changes of molecular sizes of MUC7 and MUC5B were examined by Western blot analysis. A protein band was identified as MUC7 by Western blot analysis using an antibody recognizing an N-terminal epitope. The MUC7 signal disappeared rapidly after 20-minutes of incubation. In contrast, the band of MUC7 stained for its carbohydrate components remained visible near its original position for a longer time indicating that the rapid loss of Western blot signal was due to the specific removal of the N-termimal epitope. Pretreatment of saliva with sialidase facilitated MUC7 protein degradation when compared with samples without treatment. Furthermore, addition of sialidase inhibitor to saliva prevented proteolysis of N-terminus of MUC7, suggesting that the desialylation is a prerequisite for the degradation of the N-terminal region of MUC7. The protein band corresponding to MUC5B detected in both Western blotting and glycoprotein staining showed little sign of significant degradation upon incubation in saliva up to 9 hours.

Conclusions/Significance

MUC7 was highly susceptible to specific proteolysis in saliva, though major part of MUC5B was more resistant to degradation. The N-terminal region of MUC7, particularly sensitive to proteolytic degradation, has also been proposed to have distinct biological function such as antibacterial activities. Quick removal of this region may have biologically important implication.     

C-Mannosylation of MUC5AC and MUC5B Cys subdomains

We expressed recombinant Cys subdomains in COS-7 cells to examine the role of this highly conserved protein domain in mucin biosynthesis. The entire Cys1 and Cys5 and Cys1 and Cys3 subdomains in MUC5AC and MUC5B, respectively, each with six carboxyl terminal histidine residues, were pulse-labeled with [(35)S]cysteine/methionine, and the labeled proteins were examined in the culture medium. Under nonreducing conditions, secreted Cys subdomains were monomers, indicating the absence of interchain disulfide bonds. Cross-linking studies suggested the domains are able to interact through very weak noncovalent interactions. Though the domains had apparent M(r) consistent with the absence of N- and O-glycans, they could be purified with mannose-specific lectins. Lectin binding was prevented by mutation of the first tryptophan residue in the putative C-mannosylation acceptor motif WXXW, indicating that C-mannosylation is responsible for lectin binding. As judged by pulse-chase experiments, C-mannosylation occurred very early during the domain biosynthesis, likely in the endoplasmic reticulum (ER). Mutation of the WXXW motif or expression of the unmutated domain in CHO-Lec35.1 cells, a C-mannosylation-defective cell line, resulted in reduced secretion of the corresponding Cys subdomains. Live cell imaging of green fluorescent protein fused to the Cys subdomains clearly revealed increased presence of Cys subdomains in the ER of CHO-Lec35.1 cells when compared to the same domains expressed in CHO-K1 cells. Considered together, these studies suggest that the Cys subdomains of MUC5AC and MUC5B are C-mannosylated in their respective WXXW motifs. C-mannosylation is likely required for proper folding of the Cys subdomains and/or for some aspect of ER export during mucin biosynthesis.     

Quantification of Alternaria,Cladosporium, Fusarium and Penicillium verrucosum in Conventional and Organic Grains by qPCR

We analysed the levels of Alternaria, Cladosporium, Fusarium and Penicillium verrucosum in grain samples harvested in 2011 and 2012 from conventional and organic farms using qPCR. In general, both Alternaria and Cladosporium occurred in all cereal grains in the highest quantities, followed by P. verrucosum and Fusarium. Alternaria, Cladosporium and P. verrucosum had the highest levels in crop mixtures, barley and rye and lower levels in wheat, while Fusarium levels were the highest in crop mixtures and wheat. The levels of Alternaria and P. verrucosum were higher in organic rye and wheat than conventional grains. Although the level of Fusarium was higher in conventional than organic rye, opposite results were obtained for crop mixtures. A positive correlation was found between Alternaria, Cladosporium and P. verrucosum, indicating that similar factors might affect the distribution of these fungi in grains.     

Altered expression of MUC2 and MUC5AC in response to Shigella infection, an in vivo study

Infection of mucosal epithelial cells by Shigella species leads to an intense and acute inflammatory bowel disease that is characterized by watery diarrhea and purulent discharge. Mucin production is a common defense mechanism to protect the underlying mucosa against pathogens. The molecular mechanism(s) underlying mucin induction is unknown in Shigellosis. In this study, we have evaluated the relationship between Shigella infection, the expression of MUC2 and MUC5AC and the participation of signaling molecules TNF-alpha, PKC and ERK1/2. Shigella infection up-regulated MUC2 and MUC5AC expression in 6-8 h, through activation of TNF-alpha, PKC and ERK1/2. These results confirm that, in response to Shigella infection, the normal expression pattern of MUC-2 and MUC-5AC is altered. This in vivo study brings new insights into the molecular pathogenesis of Shigellosis and new potential therapeutic targets for Shigellosis.     

MUC17, a novel membrane-tethered mucin

Membrane mucins have several functions in epithelial cells including cytoprotection, extravasation during metastases, maintenance of luminal structure, and signal transduction. In this paper we describe a large membrane mucin expressed in the normal intestine. This novel mucin, designated MUC17, contains an extended, repetitive extracellular glycosylation domain and a carboxyl terminus with two EGF-like domains, a SEA module domain, a transmembrane domain, and a cytoplasmic domain with potential serine and tyrosine phosphorylation sites. RNA blot analysis and in situ hybridization indicates that MUC17 is expressed in select pancreatic and colon cancer cell lines and in intestinal absorptive cells. Radiation hybrid mapping localized MUC17 to chromosome 7q22 where it resides in close proximity with three other membrane mucin genes, MUC3A, MUC3B, and MUC12. Thus, these membrane mucins reside together in a gene cluster, but are expressed in different tissues and are likely to have different functions as well.     

Effects of cyanobacteria,fish kairomones,and the presence of ostracods on the demography of Simocephalus vetulus (Cladocera)

Simocephalus vetulus is a large (2.0–4.0 mm at maturity) cladoceran often found in the littoral region of lakes and ponds, and capable of moderate growth rates even on poor‐quality cyanobacterial diets. It frequently co‐occurs with fishes and similar sized ostracods such as Heterocypris incongruens, but little is known of its response to fish kairomones or its interactions with potential competitors. We studied the demographic responses of S. vetulus fed the green alga Scenedesmus acutus, Microcystis cf. aeruginosa strain A, Microcystis cf. aeruginosa strain B, or Limnothrix sp. Experiments were conducted separately and together in the presence of Heterocypris incongruens and cichlid fish (Oreochromis) kairomones. A diet of Limnothrix sp. resulted in the lowest population growth rate (0.21±0.023 d^?1), while on diets of S. acutus or Microcystis, population growth was higher (0.30±0.009 d^?1). The presence of ostracods resulted in significantly higher growth rates of S. vetulus fed Limnothrix (0.33±0.01 d^?1), but not Microcystis or S. acutus. Regardless of the diet, the presence of fish kairomones resulted in significantly higher growth rates as compared with controls, particularly when ostracods were also present. Coexistence with ostracods may be beneficial to S. vetulus, particularly when food quality is poor.     

Classification of crucigenoid algae: phylogenetic position of the reinstated genus Lemmermannia,Tetrastrum spp. Crucigenia tetrapedia,and C. lauterbornii (Trebouxiophyceae,Chlorophyta)1

The subfamily Crucigenioideae was traditionally classified within the well‐characterized family Scenedesmaceae (Chlorophyceae). Several morpho‐logical revisions and questionable taxonomic changes hampered the correct classification of crucigenoid species resulting in a high number of synonymous genera. We used a molecular approach to determine the phylogenetic position of several Tetrastrum and Crucigenia species. The molecular results were correlated with morphological and ontogenetic characters. Phylogenetic analyses of the SSU rDNA gene resolved the position of Tetrastrum heteracanthum and T. staurogeniaeforme as a new lineage within the Oocystis clade of the Trebouxiophyceae. Crucigenia tetrapedia, T. triangulare, T. punctatum, and T. komarekii were shown to be closely related to Botryococcus (Trebouxiophyceae) and were transferred to Lemmer‐mannia. Crucigenia lauterbornii was not closely related to the other Crucigenia strains, but was recovered within the Chlorella clade of the Trebouxiophyceae.     

A combined morphological,ultrastructural, molecular,and biochemical study of the peculiar family Gomontiellaceae (Oscillatoriales) reveals a new cylindrospermopsin‐producing clade of cyanobacteria

Members of the morphologically unusual cyanobacterial family Gomontiellaceae were studied using a polyphasic approach. Cultured strains of Hormoscilla pringsheimii, Starria zimbabweënsis, Crinalium magnum, and Crinalium epipsammum were thoroughly examined, and the type specimen of the family, Gomontiella subtubulosa, was investigated. The results of morphological observations using both light microscopy and transmission electron microscopy were consistent with previous reports and provided evidence for the unique morphological and ultrastructural traits of this family. Analysis of the 16S rRNA gene confirmed the monophyletic origin of non‐marine repre‐sentatives of genera traditionally classified into this family. The family was phylogenetically placed among other groups of filamentous cyanobacterial taxa. The presence of cellulose in the cell wall was analyzed and confirmed in all cultured Gomontiellaceae members using Fourier transform infrared spectroscopy and fluorescence microscopy. Evaluation of toxins produced by the studied strains revealed the hepatotoxin cylindrospermopsin (CYN) in available strains of the genus Hormoscilla. Production of this compound in both Hormoscilla strains was detected using high‐performance liquid chromatography in tandem with high resolution mass spectrometry and confirmed by positive PCR amplification of the cyrJ gene from the CYN biosynthetic cluster. To our knowledge, this is the first report of CYN production by soil cyanobacteria, establishing a previously unreported CYN‐producing lineage. This study indicates that cyanobacteria of the family Gomontiellaceae form a separate but coherent cluster defined by numerous intriguing morphological, ultrastructural, and biochemical features, and exhibiting a toxic potential worthy of further investigation.     

MUC1 and cancer

The MUC1 membrane mucin was first identified as the molecule recognised by mouse monoclonal antibodies directed to epithelial cells, and the cancers which develop from them. Cloning the gene showed that the extracellular domain is made up of highly conserved repeats of 20 amino acids, the actual number varying between 25 and 100 depending on the allele. Each tandem repeat contains five potential glycosylation sites, and between doublets of threonines and serines lies an immunodominant region which contains the epitopes recognised by most of the mouse monoclonal antibodies. The O-glycans added to the mucin produced by the normal breast are core 2 based and can be complex, while the O-glycans added to the breast cancer mucin are mainly core 1 based. This means that some core protein epitopes in the tandem repeat which are masked in the normal mucin are exposed in the cancer associated mucin. Since novel carbohydrate epitopes are also carried on the breast cancer mucin, the molecule is antigenically distinct from the normal breast mucin. (Changes in glycosylation in other epithelial cancers have been observed but are not so well documented.) Immune responses to MUC1 have been seen in breast and ovarian cancer patients and clinical studies have been initiated to evaluate the use of antibodies to MUC1 and of immunogens based on MUC1 for immunotherapy of these patients. The role of the carbohydrates in the immune response and in other interactions with the effector cells of the immune system is of particular interest and is discussed.