Complete C-terminal sequence and genomic organization of MUC5B?
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Amino Acid Sequence↗
Biomedical subjects
A Laine
Publications and source records attributed to A Laine.
Human mucin genes MUC2, MUC3, MUC4, MUC5AC, MUC5B, and MUC6 express stable and extremely large mRNAs and exhibit a variable length polymorphism. An improved method to analyze large mRNAs.
Of the nine mucin genes that have been characterized, only MUC1 and MUC7 have been fully sequenced, and their transcripts can be detected as distinct bands of predicted size by Northern blot analysis. In contrast, the RNA patterns observed for each of the other MUC genes have usually shown a very high degree of polydispersity. This polydispersity has been believed to be one of the typical features of the mucin mRNAs, but until now, its origin has remained unexplained. In the work described in the present paper, we investigated two possible kinds of explanation for this phenomenon: namely that the extensive polydispersity results from a biological mechanism or that it is artifactual in origin. The data obtained, as a result of improving the purification and blotting methods, allowed us to show that in all of the tissues analyzed, each of the genes, MUC2-6, expresses mRNAs that are stable and are of an unusually large size to be found in eukaryotes (14-24 kilobases). Moreover, allelic variations in length of these mucin transcripts were observed. We demonstrate that these variations are directly related to the variable number of tandem repeat polymorphisms seen at the DNA level.
Apolipoprotein B-100↗
Evolutionary history of the 11p15 human mucin gene family.
The four human mucin genes MUC6, MUC2, MUC5AC, and MUC5B are located at chromosome 11p15.5. It has been demonstrated that the three mucins MUC2, MUC5AC, and MUC5B contain several Cys-subdomains of 108 amino acid residues. In contrast, little information is available concerning MUC6. These Cys-subdomains contain 10 cysteine residues that have a highly conserved position. We present here a coherent probable evolutionary history of this human gene family after comparison of the nucleotide sequences of these Cys-subdomains. The three MUC loci MUC2, MUC5AC, and MUC5B may have evolved from a common ancestral gene by two successive duplications. Moreover, we can postulate that MUC5AC and MUC5B have evolved in a concerted manner, while MUC2 has evolved separately.
Base Sequence↗
Human ornithine decarboxylase-overproducing NIH3T3 cells induce rapidly growing, highly vascularized tumors in nude mice.
Overexpression of human ornithine decarboxylase (ODC) under the control of strong promoters induces morphological transformation of immortalized NIH3T3 and Rat-1 fibroblasts [M. Auvinen et al., Nature (Lond.), 360: 355-358, 1992]. We demonstrate here that ODC-overproducing NIH3T3 cells are tumorigenic in nude mice, giving rise to rapidly growing, large fibrosarcomas at the site of inoculation. The tumors are capable of invading host fat and muscle tissues and are vascularized abundantly. To disclose the molecular mechanism(s) driving the tumorigenic, invasive, and angiogenic phenotype of the tumors, the ODC-overproducing cell lines and tumor tissues were analyzed for the expression of various potential regulators and mediators of cell proliferation, matrix degradation, and angiogenesis. The tumorigenicity of ODC transformants was associated with elevated polyamine levels and down-regulated growth factor receptors. The invasiveness of the ODC-induced tumors could not be attributed to overexpression of various known extracellular matrix-degrading proteases or matrix metalloproteinases. The induction of the tumor neovascularization proved not to be elicited by vascular endothelial growth factor or basic fibroblast growth factor. Instead, the ODC-overexpressing cells appeared to secrete a novel angiogenic factor(s) that was able to promote migration of bovine capillary endothelial cells in collagen gels and increase the proliferation of human endothelial cells in vitro. In parallel, ODC-transformed cells displayed down-regulation of thrombospondin-1 and -2, the negative regulators of angiogenesis. Thus, the induction of the angiogenic phenotype of the ODC transformants is likely due both to increased expression and secretion of the new angiogenesis-stimulating factor(s) and decreased production and release of the antiangiogenic thrombospondins.
3T3 Cells↗
Genomic organization of the 3' region of the human mucin gene MUC5B.
MUC5B, mapped clustered with MUC6, MUC2, and MUC5AC to chromosome 11p15.5, is a human mucin gene of which the genomic organization is being elucidated. We have recently published the sequence and the peptide organization of its huge central exon, 10,713 base pairs (bp) in length. We present here the genomic organization of its 3' region, which encompasses 10,690 bp. The genomic sequence has been completely determined. The 3' region of MUC5B is composed of 18 exons ranging in size from 32 to 781 bp, contrasting thus with the very large central exon. The sizes of the 18 introns range from 114 to 1118 bp. Some repetitive sequences were identified in four introns. The peptide deduced from the sequence of the 18 exons consists of an 808-amino acid peptide. This carboxyl-terminal region exhibits extensive sequence similarity to MUC2, MUC5AC, and von Willebrand factor, particularly the number and the positions of the cysteine residues, suggesting that this domain may be derived from a common ancestral gene. The presence in these components of a cystine knot also found in growth factors such as transforming growth factor-beta is of particular interest. Moreover, one part of this peptide is identical to the 196-amino acid sequence deduced from the cDNA clone pSM2-1, which codes for a part of the high molecular weight mucin MG1 isolated from human sublingual gland. Considering the expression pattern of MUC5B and the origin of MG1, we can thus conclude that MUC5B encodes MG1.
Amino Acid Sequence↗
Human mucin gene MUC5B, the 10.7-kb large central exon encodes various alternate subdomains resulting in a super-repeat. Structural evidence for a 11p15.5 gene family.
Human mucin gene MUC5B is mapped clustered with MUC6, MUC2, and MUC5AC on chromosome 11p15.5. We report here the isolation of three overlapping genomic clones of human MUC5B spanning approximately 40 kilobases. We have determined their partial restriction maps and the intron-exon boundaries of the central region encoding a single open reading frame. This coding region has been completely sequenced. Its length is 10,713 base pairs, and it encodes a 3570-amino acid peptide. Nineteen subdomains have been individualized. Some subdomains show similarity to each other, creating larger composite repeat units that we have called super-repeats. Four super-repeats of 528 amino acid residues are thus observed within the central exon. Each comprises (i) a subdomain composed of 11 repeats of the irregular repeat of 29 amino acid residues, (ii) a unique conserved subdomain with no typical repeat, and (iii) a cysteine-rich subdomain. This latter subdomain has high sequence similarity to the cysteine-rich domains described in MUC2 and MUC5AC. Sequence data of these three genes, together with their clustered organization, lead us to suggest that they may be a part of a multigene family. The super-repeat present in MUC5B is the largest ever determined in mucin genes and the central exon of this gene is, by far, the largest reported for a vertebrate gene.
Amino Acid Sequence↗
Prescreening entire mammograms for masses with artificial neural networks: preliminary results.
RATIONALE AND OBJECTIVES: The authors evaluated the feasibility of combining wavelet transform and artificial neural network (ANN) technologies to prescreen mammograms for masses. METHODS AND MATERIALS: Fifty-five mammograms (29 with masses and 26 without) were digitized to 100-mm resolution and processed by using wavelet transformation. These wavelets were subjected to a linear output sequential recursive auto-associative memory ANN and cluster analysis with feature vector formation. These vectors were used in two separate experiments-one with 13 cases and another with seven cases held out in a test set-to train feed-forward ANNs to detect the mammograms with a mass. The experiments were repeated with rerandomization of the data, four and six times, respectively. RESULTS: There was a statistically significant correlation (P < .01) between the network's prediction of a mass and the presence of a mass. With majority voting, the feed-forward ANNs detected masses with 79% sensitivity and 50% specificity. CONCLUSION: Although preliminary, the combination of wavelet transform and ANN is promising and may provide a viable method to prescreen mammograms for masses with high sensitivity and reasonable specificity.
Breast Neoplasms↗
Human mucin genes assigned to 11p15.5: identification and organization of a cluster of genes.
Four distinct genes that encode mucins have previously been mapped to chromosome 11p15.5. Three of these genes (MUC2, MUC5AC, and MUC6) show a high level of genetically determined polymorphism and were analyzed in the CEPH families. Linkage analysis placed all three genes on the genetic map in a cluster between HRAS and INS, and more detailed analysis of recombinant breakpoints revealed that MUC6 is telomeric to MUC2. Using these recombinants D11S150 was mapped close to MUC2. Ten of the 11 recombinant chromosomes studied in detail were paternal, and the recombinant events were distributed throughout the 11p15 region, suggesting that the high level of recombination observed in 11p15.5 is not due to a particular recombinational hot spot. Pulsed-field gel electrophoresis was used to make a detailed physical map of the MUC cluster and to integrate the physical and genetical maps. The gene order was determined to be HRAS-MUC6-MUC2-MUC5AC-MUC5B-IGF2. The MUC genes span a region of some 400 kb and the map extends 770 kb and contains 15 putative CpG islands. The order of the MUC genes on the map corresponds to the relative order of their expression along the anterior-posterior axis of the body, suggesting a possible functional significance to the gene order.
Adult↗
[Glycoforms of alpha-1 antichymotrypsin in infectious process. Diagnostic value and follow-up].
OBJECTIVES: Three alpha 1-antichymotrypsin (alpha 1-ACT) glycans have been identified. We followed their levels during the septic processes in 24 patients in order to determine their diagnostic value in correlation with C-reactive peptide in patients with infection. METHODS: Sera were collected for assay on days 0, 3 and 6 after starting antibiotics. Erythrocyte sedimentation rate, alpha 1-ACT and C-reactive protein were determined. RESULTS: Erythrocyte sedimentation rate changed little while C-reactive protein fell sharply as in other inflammatory processes. Crossed immunoaffinoelectrophoresis showed a decreased formation of the Con-A non reactive fraction which disappeared rapidly after initiating antibiotics. Glycan microheterogeneity returned to normal at 6 days while C-reactive protein and alpha 1-ACT were still elevated. CONCLUSION: Synthesis and glycosylation of alpha 1-ACT are independent. Study of alpha 1-ACT glycan microheterogeneity may provide a useful test in the diagnosis of inflammatory processes of unknown origin and may be helpful in following-up patients with sepsis.
Adult↗
Identification of a 42-kDa nuclear factor (NF1-MUC5B) from HT-29 MTX cells that binds to the 3' region of human mucin gene MUC5B.
MUC5B gene is one of the four human mucin genes mapped to chromosome 11p15. The identification of three potential Sp1 binding sites located between the tandem repeat and the 3' end of MUC5B suggests a possible regulatory role for this region. In this report we show by electrophoretic mobility shift assay that only one potential Sp1 binding site (NAU62) leads to a specific interaction with a nuclear factor from HT-29 MTX cells which does not exist in parental HT-29 cells. By using mutated versions of NAU62, an 18 mer sequence within this later was shown to be directly involved in the interaction. The nuclear factor called NF1-MUC5B which binds to this element has a Mr of 42000 and is not Sp1. These results suggest that MUC5B contains a sequence in its 3' region that might act as a cis-element. This report opens the field of transcriptional regulation of human mucin genes encoding secreted mucins.
Base Sequence↗
Acute effects of 1,1,1-trichloroethane inhalation on the human central nervous system.
The object of this study was to examine the immediate nervous effects of variable 1,1,1-trichloroethane (TCE) exposure combined with physical exercise. The effects on the quantitative electroencephalography (EEG), visual evoked potentials (VEP) and body sway were analyzed. Nine male volunteers were exposed to either a stable or a fluctuating exposure pattern with the same time-weighted average concentration of 200 ppm (8.1 mumol/l). In both cases, the subjects engaged in physical exercise during the exposures. Exercise alone induced an increase in the dominant alpha frequency in the EEG and, after an initial drop, an increase in the alpha percentage with a concomitant decrease in theta, whereas delta and beta bands remained unaffected. By contrast, exposure to TCI and exercise did not affect the alpha, theta or delta activities but induced changes in beta during the morning recordings at peak exposure to TCE. The body sway tended to decrease slightly during the fluctuating TCE exposure, and the later peaks in VEPs showed slight prolongations. Overall, no deleterious effects of exposure were noted.
Air Pollutants, Occupational↗
Integrin alpha 2 beta 1 in tumorigenic human osteosarcoma cell lines regulates cell adhesion, migration, and invasion by interaction with type I collagen.
Human osteosarcomas are aggressive bone tumors. Here we propose that their progression requires altered cell interaction with extracellular matrix. Since type I collagen is the main matrix molecule found in bone and thus obligated to interact with tumor cells, we analyzed the expression and function of different integrin-type collagen receptors in tumor cell-collagen interaction by using eight human osteogenic sarcoma (HOS) cell lines. Virally (Kirsten sarcoma virus) transformed derivatives of HOS cells (KHOS-NP) and chemically [N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)] transformed tumorigenic subclones of human osteogenic sarcoma cells (HOS-MNNG) expressed alpha 2 beta 1 integrin in remarkably larger amounts than the six other nontumorigenic cell lines (HOS, MG-63, Saos-2, KHOS-240, KHOS-312, and G292). Concomitantly, Mg(2+)-dependent adhesion of tumorigenic cells to type I collagen was increased. We also show that the migration of tumorigenic cells on and invasion through type I collagen is faster than that of HOS cells. HOS cells forced to express alpha 2 integrin by cDNA transfections showed increased Mg(2+)-dependent cell adhesion to type I collagen and also accelerated migration and invasion rate, indicating that the overexpression of alpha 2 beta 1 integrin in tumorigenic cells alone explains the altered cell-collagen interaction. Finally, HOS cells forced to express alpha 2 integrin subunit did not grow s.c. in athymic mice, suggesting that overexpression of alpha 2 integrin is not efficient to make these cells tumorigenic.
Animals↗
Identification of Epstein-Barr virus nuclear antigen 1 protein domains that direct interactions at a distance between DNA-bound proteins.
The EBNA1 protein of Epstein-Barr virus (EBV) binds to and activates DNA replication from the EBV latent origin of replication, oriP, via a direct interaction with the two noncontiguous subelements of oriP. The EBNA1 molecules bound to the oriP subelements interact efficiently with each other by a DNA looping mechanism. We have previously mapped a region of EBNA1 (termed the looping region) that is required to mediate the interaction of the EBNA1 molecules bound to the oriP subelements. We now demonstrate that two fragments of this region of EBNA1, which consist largely of an eight amino acid repeat, can mediate homotypic interactions when transferred to another DNA-binding protein. Protein interactions mediated by the EBNA1 looping region appear to be dependent on DNA binding since these interactions were detected between DNA-bound forms of the proteins only.
Amino Acid Sequence↗
Characterization of the human mucin gene MUC5AC: a consensus cysteine-rich domain for 11p15 mucin genes?
To date five human mucin cDNAs (MUC2, 5A, 5B, 5C and 6) mapped to 11p15.3-15.5, so it appears that this chromosome region might contain several distinct gene loci for mucins. Three of these cDNAs, MUC5A, B and C, were cloned in our laboratory and previously published. A common number, 5, was recommended by the Human Gene Mapping Nomenclature Committee to designate them because of their common provenance from human tracheobronchial mucosa. In order to define whether they are products of the same gene locus or distinct loci, we describe in this paper physical mapping of these cDNAs using the strategy of analysis of CpG islands by pulse-field gel electrophoresis. The data suggest that MUC5A and MUC5C are part of the same gene (called MUC5AC) which is distinct from MUC5B. In the second part of this work, complete sequences of the inserts corresponding to previously described (JER47, JER58) and novel (JER62, JUL32, MAR2, MAR10 and MAR11) cDNAs of the so-called MUC5AC gene are presented and analysed. The data show that in this mucin gene, the tandem repeat domain is interrupted several times with a subdomain encoding a 130 amino acid cysteine-rich peptide in which the TR3A and TR3B peptides previously isolated by Rose et al. [Rose, Kaufman and Martin (1989) J. Biol. Chem., 264, 8193-8199] from airway mucins are found. A consensus peptide sequence for these subdomains involving invariant positions of most of the cysteines is proposed. The consensus nucleotide sequence of this subdomain is also found in the MUC2 gene and in the MUC5B gene, two other mucin genes mapped to 11p15. The functional significance for secreted mucins of these cysteine-rich subdomains and the modular organization of mucin peptides are discussed.
Amino Acid Sequence↗
The MUC5AC gene: RFLP analysis with the Jer58 probe.
We have recently obtained evidence that the locus corresponding to three groups of partial tracheobronchial cDNAs (A = Jer47, B = Jer57, C = Jer58) which mapped to chromosome 11p15 and was given the symbol MUC5 corresponds to two distinct genes which we have provisionally called MUC5B and MUC5AC. Here we describe the detection, using the Jer58 probe, which contains a 24-bp tandem repeat, of polymorphism in the MUC5AC gene with seven different restriction enzymes.
Bronchi↗
Human mucin genes: genomic organization and expression of MUC4, MUC5AC and MUC5B.
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DNA, Complementary↗
Plasma antiproteinase screen and neutrophil-mediated platelet activation. A major role played by alpha 1 antitrypsin.
Upon activation, human polymorphonuclear neutrophils (PMN) release two serine proteinases, cathepsin G (Cat.G) and elastase (HLE), which in turn synergize to activate nearby platelets. We looked for the inhibitory effect of plasma and the involvement of alpha 1 antichymotrypsin (alpha 1 ACT) and alpha 1 antitrypsin (alpha 1 AT), on this cell-to-cell cooperation. It was observed that inhibition by plasma of PMN-mediated platelet activation was rather correlated with an effect on HLE (r = 0.95) than on Cat.G (r = 0.65) enzymatic activity. Purified alpha 1 AT suppressed in a concentration-dependent manner HLE activity present in the supernatant of activated PMN. When HLE was fully blocked, alpha 1 AT started to inhibit Cat.G activity. By contrast and as expected, purified alpha 1 ACT inhibited only Cat.G activity. Using specific blocking polyclonal antibodies against alpha 1 AT and alpha 1 ACT, it was demonstrated that the inhibitory effect of plasma vs. HLE was entirely mediated by alpha 1 AT. By contrast, blockade of Cat.G activity was only partly due to plasma alpha 1 ACT and around 50% was attributable to alpha 1 AT. When plasma from patients with an acute inflammatory state was used in place of plasma from normal subjects, the inhibitory effect was more pronounced, while plasma depleted in alpha 1 AT and alpha 1 ACT was less effective. These data indicate a predominant role of alpha 1 AT in the inhibition by plasma of the PMN-mediated platelet activation.
Amino Acid Sequence↗
Heparin protects cathepsin G against inhibition by protein proteinase inhibitors.
Cathepsin G, a cationic serine proteinase present in neutrophils and monocytes, is able to cleave biologically important proteins and may thus participate in tissue destruction during inflammation. Its activity is physiologically controlled by the fast-acting serpins, alpha 1-anti-chymotrypsin (ka = 5 x 10(7) M-1 S-1) and alpha 1-proteinase inhibitor (ka = 2.7 x 10(5) M-1 S-1). We have shown that cathepsin G forms a tightly bound 1:1 complex with a 5-kDa heparin fragment (Kd = 1.9 x 10-8 M). The partial enzymatic activity retained by this complex is inhibited extremely slowly by the above 68- and 53-kDa serpins. The activity of the complex is also virtually resistant to inhibition by eglin c, and 8-kDa non-serpin inhibitor. A detailed kinetic investigation showed that the inhibition of heparin-bound cathepsin G by the three proteins proceeded via a two-step mechanism. [formula: see text] The three inhibitors have widely different Ki* values (0.18-13 microM) (Ki* = k-1/k1). Their isomerization constants k2 are, however, all in the same range and their extremely low values (0.7-3 ms-1) account for the very low rate of cathepsin G inhibition. The second-order inhibition rate constants k2/Ki* were 4300, 700, and 52 M-1 S-1 for alpha 1-antichymotrypsin, alpha 1-antitrypsin, and eglin c, respectively, indicating that, if heparin is present in vivo, the two former physiological inhibitors will be unable to prevent cathepsin G-mediated proteolysis. Neutrophil elastase binds the 5-kDa heparin fragment with an affinity identical to that of cathepsin G. alpha 1-Proteinase inhibitor reacts, however, much faster with heparin-elastase (ka = 1.8 x 10(6) M-1 S-1) than with heparin-cathepsin G (k2/Ki* = 700 M-1 S-1).
Cathepsin G↗