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Research Interests: Cancer, Biology, Immunohistochemistry, Enzyme Inhibitors, Cancer Research, and 14 moreMedicine, Western blotting, Signal Transduction, Humans, Calcium Signaling, Immunoprecipitation, Fluorescent Antibody Technique, Epidermal Growth Factor, Colon cancer, Cancer Du Sein, Cell Proliferation, reverse transcriptase polymerase chain reaction, Src family kinases, and Colonic Neoplasms
Research Interests: Chemistry, Flow Cytometry, Biology, Cell Biology, Cytoskeleton, and 15 moreCell line, Humans, Medical Physiology, Epithelial cells, Cancer Cell, Matrix Metalloproteinases, Cell Proliferation, Imidazoles, MAP Kinase, Enzyme Linked Immunosorbent Assay, FASEB J, Maleimides, Filamin, Biochemistry and cell biology, and Chlorides
Vasoactive intestinal peptide (VIP) receptors were characterized in epithelial plasma membranes from human small intestine. Native VIP inhibited the binding of 125I-labeled VIP to jejunal membranes, and Scatchard analysis of these data... more
Vasoactive intestinal peptide (VIP) receptors were characterized in epithelial plasma membranes from human small intestine. Native VIP inhibited the binding of 125I-labeled VIP to jejunal membranes, and Scatchard analysis of these data was consistent with the existence of one class of receptor with a dissociation constant of 42 pM and a maximal binding of 256 fmol/mg membrane protein. VIP stimulated adenylyl cyclase activity in human jejunal membranes in the 0.01 nM-1 microM range [half-maximal effective dose = 0.7 nM]. Coupling of VIP receptors with a Gs protein was further assessed by the ability of GTP (10(-8) to 10(-3) M) to inhibit 125I-VIP binding to membranes. 125I-VIP binding was seven to eight times higher in villus cells than in crypt cells. Finally, 125I-VIP binding was detectable throughout the small and large intestines with the highest binding in jejunum. Among the natural peptides structurally related to VIP, some inhibited 125I-VIP binding with the following order of potency: VIP = pituitary adenylate cyclase-activating peptide (PACAP)-27 = PACAP-38 > helodermin >> peptide histidine methionineamide = human growth hormone-releasing factor > secretin. The same order of potency of peptides for inhibiting 125I-VIP or 125I-labeled PACAP was observed, supporting that the two tracers bound to a common VIP-PACAP receptor site. This order of potency was also observed for the stimulation of adenylyl cyclase activity by these peptides. 125I-VIP was cross-linked to membranes using disuccinimidyl suberate. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, one single band of 70,000 mol wt was observed.(ABSTRACT TRUNCATED AT 250 WORDS)
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We describe here two human VIP receptor cDNA clones isolated from a jejunal epithelial cell cDNA library. The hIVR8 cDNA encodes a human VIP receptor consisting of 460 amino acids and has seven putative transmembrane domains like other G... more
We describe here two human VIP receptor cDNA clones isolated from a jejunal epithelial cell cDNA library. The hIVR8 cDNA encodes a human VIP receptor consisting of 460 amino acids and has seven putative transmembrane domains like other G protein-coupled receptors. When expressed in COS-7 cells, hIVR8 conferred specific [125I]VIP binding sites (dissociation constant = 0.6 nM) with displacement patterns characteristic of the human common VIP/PACAP receptor, i.e., VIP = PACAP-27 > PACAP-38 > helodermin > growth hormone-releasing factor = peptide methionineamide > secretin. It also conferred stimulation of cAMP production by VIP (half-maximal stimulation for 0.5 nM peptide). Another clone, hIVR5, encodes a 495 amino acid VIP receptor-related protein exhibiting 100% homology with the functional VIP receptor (hIVR8) over the 428 amino acids at the C-terminus but a completely divergent 67 amino acid N-terminal domain. When expressed in COS-7 cells, this VIP receptor-related protein does not bind 125I-VIP, although it is normally addressed at the plasma membrane as assessed by immunofluorescence studies.
Research Interests: Molecular Biology, Kinetics, Biology, Medicine, Gene expression, and 15 moreCell line, Humans, cyclic AMP, Animals, Biochemical, Jejunum, Epithelium, Vasoactive Intestinal Peptide, Receptor, Transfection, Amino Acid Sequence, Base Sequence, Biochemistry and cell biology, Molecular Sequence Data, and Medical biochemistry and metabolomics
Research Interests: Kinetics, Biology, Biological Chemistry, Medicine, Colorectal cancer, and 15 moreCell Division, Biological Sciences, Humans, Protease, Biological, Phosphorylation, Mitogen Activated Protein Kinase, CHEMICAL SCIENCES, Epidermal Growth Factor Receptor, Colon cancer, Cell Proliferation, Protein Tyrosine Phosphatases, Quinazolines, Medical and Health Sciences, and Colonic Neoplasms(Cell Division, Biological Sciences, Humans, Protease, Biological, Phosphorylation, Mitogen Activated Protein Kinase, CHEMICAL SCIENCES, Epidermal Growth Factor Receptor, Colon cancer, Cell Proliferation, Protein Tyrosine Phosphatases, Quinazolines, Medical and Health Sciences, and Colonic Neoplasms)
(Cell Division, Biological Sciences, Humans, Protease, Biological, Phosphorylation, Mitogen Activated Protein Kinase, CHEMICAL SCIENCES, Epidermal Growth Factor Receptor, Colon cancer, Cell Proliferation, Protein Tyrosine Phosphatases, Quinazolines, Medical and Health Sciences, and Colonic Neoplasms)
Vasoactive intestinal peptide (VIP) receptors were characterized in epithelial plasma membranes from human small intestine. Native VIP inhibited the binding of 125I-labeled VIP to jejunal membranes, and Scatchard analysis of these data... more
Vasoactive intestinal peptide (VIP) receptors were characterized in epithelial plasma membranes from human small intestine. Native VIP inhibited the binding of 125I-labeled VIP to jejunal membranes, and Scatchard analysis of these data was consistent with the existence of one class of receptor with a dissociation constant of 42 pM and a maximal binding of 256 fmol/mg membrane protein. VIP stimulated adenylyl cyclase activity in human jejunal membranes in the 0.01 nM-1 microM range [half-maximal effective dose = 0.7 nM]. Coupling of VIP receptors with a Gs protein was further assessed by the ability of GTP (10(-8) to 10(-3) M) to inhibit 125I-VIP binding to membranes. 125I-VIP binding was seven to eight times higher in villus cells than in crypt cells. Finally, 125I-VIP binding was detectable throughout the small and large intestines with the highest binding in jejunum. Among the natural peptides structurally related to VIP, some inhibited 125I-VIP binding with the following order of potency: VIP = pituitary adenylate cyclase-activating peptide (PACAP)-27 = PACAP-38 > helodermin > peptide histidine methionineamide = human growth hormone-releasing factor > secretin. The same order of potency of peptides for inhibiting 125I-VIP or 125I-labeled PACAP was observed, supporting that the two tracers bound to a common VIP-PACAP receptor site. This order of potency was also observed for the stimulation of adenylyl cyclase activity by these peptides. 125I-VIP was cross-linked to membranes using disuccinimidyl suberate. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, one single band of 70,000 mol wt was observed.(ABSTRACT TRUNCATED AT 250 WORDS)
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Cryptdins are antimicrobial peptides of the defensin family that are expressed specifically by Paneth cells in small intestinal crypts (M.E. Selsted, S.I. Miller, A.H. Henschen, and A.J. Ouellette. J. Cell Biol. 118: 929-936, 1992), and... more
Cryptdins are antimicrobial peptides of the defensin family that are expressed specifically by Paneth cells in small intestinal crypts (M.E. Selsted, S.I. Miller, A.H. Henschen, and A.J. Ouellette. J. Cell Biol. 118: 929-936, 1992), and at least 17 cryptdin isoforms have been reported in mouse small intestine (A.J. Ouellette, M.M. Hsieh, M.T. Nosek, D.F. Cano-Gauci, K.M. Huttner, R.N. Buick, and M.E. Selsted. Infect. Immun. 62: 5040-5047, 1994). Analysis of cryptdin gene expression in adult mouse small bowel revealed that the cryptdin-4 isoform is differentially expressed along the proximal-to-distal intestinal axis. By peptide-specific reverse transcriptase-polymerase chain reaction-based assays, cryptdin-4 mRNA was found to be absent from the proximal small bowel, increasing to maximal levels in the ileum. In contrast, intestinal content of cryptdin-1 and -5 mRNAs was equivalent in duodenum, jejunum, and ileum, and Northern blot hybridization experiments were consistent with both sets of data. Similarly, individual crypts isolated from duodenum contain cryptdin-1 mRNA but not cryptdin-4 mRNA. Taken together, the results show that Paneth cells are heterogeneous, depending on their position along the longitudinal axis of the small bowel. The positional specificity of defensin gene expression suggests that cryptdins may be useful markers for investigating the establishment and maintenance of this epithelial lineage in the mouse small intestine.
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Research Interests: Endocrinology, Biology, Immunohistochemistry, Fetal development, DNA, and 15 moreHumans, Internal Medicine, Clinical, Energy Balance, Amniotic Fluid, Food intake, Clinical Sciences, Intestines, Digestive Tract, Esophagus, Gastrointestinal Tract, Fetus, Dna Synthesis, culture medium, and Gastric mucosa
Research Interests: Pathology, Molecular Biology, Kinetics, Biology, Calcium, and 15 moreImmunohistochemistry, Enzyme Inhibitors, Flavonoids, Medicine, Cell Culture, Cell Division, Motility, In Vitro, Humans, Colon cancer, Cell Proliferation, The American, reverse transcriptase polymerase chain reaction, Medical and Health Sciences, and Colonic Neoplasms
76 Serine proteases elicit cellular effects by activating G protein-coupled protease-activated receptors (PARs). The PAR family includes four members: PAR1, PAR2, PAR3 and PAR4. PARs are activated by an unusual irreversible proteolytic... more
76 Serine proteases elicit cellular effects by activating G protein-coupled protease-activated receptors (PARs). The PAR family includes four members: PAR1, PAR2, PAR3 and PAR4. PARs are activated by an unusual irreversible proteolytic event in which the protease binds to and cleaves the amino-terminus of the receptor generating a new amino terminus that functions as a tethered ligand. This activation initiates signal transduction resulting in stimulation of phosphoinositide breakdown and cytosolic calcium mobilization leading to the activation of MEK-ERK1/2. Our recent studies have shown the implication of proteinase-activated receptors (PARs) in colon cancer (Darmoul et al. Br. J. Cancer 2001; Am J Pathol., 2003) .Using the HT29 colon cancer cell line, we identified a cross-talk between proteinase activated receptors and epidermal growth factor receptor (Darmoul et al. J. Biol. Chem. 2004) . This cross talk explains the activation of ERK signal transduction pathway and the proliferative effects induced by serine proteases in colon cancer cells. In this study we show that : 1) the mechanism of EGFR transactivation is not a general mechanism since it is found only in 50% of the colon cancer cell lines analyzed. Indeed, AG1478, the EGFR tyrosine kinase inhibitor abrogated the PAR induced ERK phosphorylation in HT29, T84 and Caco2 cells. In contrast, this inhibitor has no effect on the PARs-induced ERKs phosphorylation in other colon cancer cells such as HCT116, SW480 and LoVo cells; 2) we did not find any correlation between the expression level of the EGFR receptor on the cell membrane and the involvement of the EGFR as a transducer molecule; 3) PKC is not involved in ERKs activation. Indeed, the PKC inhibitor GF10903X has no effect on ERKs activation induced by PARs in some colon cancer cells. We conclude that PARs induce ERKs activation and subsequent colon cancer cell proliferation through both EGFR-dependent and-independent pathways. Thus PARs stimulate a non described pathway to activate ERKs in some colon cancer cells.
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AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 4379 The thrombin receptor, Proteinase-activated receptor-4 (PAR4, belongs to a new G protein-coupled receptor subfamily activated by serine proteinases. The presence and role of PAR4... more
AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 4379 The thrombin receptor, Proteinase-activated receptor-4 (PAR4, belongs to a new G protein-coupled receptor subfamily activated by serine proteinases. The presence and role of PAR4 in human gastrointestinal system functions have not been fully investigated. The aim of this study is to investigate the expression and the potential role of the thrombin receptor PAR4 in human colon cancer. The presence of PAR4 in colon cancer cell lines was assessed by immunofluorescence detection and RT-PCR, and its presence in resected human colonic tumors and normal colon was assessed by immunohistochemistry. By using the serine protease thrombin and the specific receptor-activating peptide (AYPGKF-NH) AP4, we tested the functionality of PAR-4 on calcium mobilization and cell proliferation in human colon cancer cells HT29. The following results were obtained: 1) immunofluorescence studies using polyclonal anti-PAR4 antibodies revealed expression of PAR-4 in HT29 cells . Immunohistochemical studies on paraffin embedded section of human colonic cancer showed a strong staining with the PAR4 antibody in some dysplastic areas and cancers. In contrast no staining was observed in normal human colonic epithelium; 2) RT-PCR analysis showed expression of PAR-4 mRNA in most human colon cancer cell lines (10 out of 14). In contrast, no mRNA was detected in normal epithelial cells from human colon; 3) microspectrofluorimetry using Fura 2-loaded cells demonstrated a prompt increase in intracellular calcium ([Ca2+]i) transients in HT29 in response to thrombin (10 nM) or AP4 (10-200 µM) challenges; 4) HT-29 cells exhibited dramatic mitogenic responses when grown in serum-deprived media supplemented with thrombin or AP4. This stimulatory effect on cell proliferation was observed also in several other human colon cancer cell lines; 5) we also show here that the PAR4-dependent increase in HT29 cell proliferation isdependent on src activity and transactivation of epidermal growth factorreceptor (EGFR). Indeed, PAR4-mediated cell proliferation wasblocked by the src inhibitor PP2, the metalloproteinase inhibitor batimastat and the specificEGFR tyrosine kinase inhibitors AG1478 and PD 168393. In conclusion thrombin exerts proliferative responses in colon cancer cells through aberrant expression of PAR4.
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The expression of dipeptidyl peptidase IV (DPP IV), an intestinal brush-border hydrolase, has been studied in the rat jejunum crypt-villus axis. DPP IV enzyme activity is lower in crypt cells than in villus cells. Indirect... more
The expression of dipeptidyl peptidase IV (DPP IV), an intestinal brush-border hydrolase, has been studied in the rat jejunum crypt-villus axis. DPP IV enzyme activity is lower in crypt cells than in villus cells. Indirect immunofluorescence studies, using a polyclonal antibody raised against purified DPP IV, show a gradient of immunoreactivity from the crypts to the villi that was quantified using confocal laser scanning microscopy. Accordingly, Western blot analysis demonstrates that the steady-state amount of DPP IV is much lower in crypt cells than in villus cells. Northern blot analysis was performed using our cDNA probe for human DPP IV that presents more than 94% homology with rat DPP IV cDNA. Results clearly show that there is approximately seven times less DPP IV mRNA in crypt cells than in villus cells. We conclude that the differentiation-dependent expression of DPP IV in rat jejunum is primarily controlled at the mRNA level.
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In rodents, the four intestinal epithelial cell lineages differentiate and become morphologically distinct during the first 2-3 postnatal wk. In studies reported here, reverse transcriptase-polymerase chain reaction (RT-PCR)-based assays... more
In rodents, the four intestinal epithelial cell lineages differentiate and become morphologically distinct during the first 2-3 postnatal wk. In studies reported here, reverse transcriptase-polymerase chain reaction (RT-PCR)-based assays detected Paneth cell defensin mRNAs in intestinal RNA from 1-day-old (P1) mice before crypt formation and maturation of the epithelium. Analysis of these defensin-coding RT-PCR products from P1 mice showed that 69% of clones sequenced coded for cryptdin-6, suggesting that it is the most abundant enteric defensin mRNA in the newborn. Paneth cell mRNAs, including cryptdins-4 and -5, lysozyme, matrilysin, and defensin-related sequences, also were detected in RNA from P1 mouse intestine. Unlike adult mice, where only Paneth cells are immunopositive for cryptdin, cryptdin-containing cells were distributed throughout the newborn intestinal epithelium and not in association with rudimentary crypts. Cryptdin immunoreactivity in the P1 mouse intestine was sp...
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Accumulating evidence indicates that serine proteinase family of the kallikrein-related-peptidases (KLK1-KLK15) are deregulated in cancer and their up-regulation is often associated with poor patient prognosis. Although primarily known as... more
Accumulating evidence indicates that serine proteinase family of the kallikrein-related-peptidases (KLK1-KLK15) are deregulated in cancer and their up-regulation is often associated with poor patient prognosis. Although primarily known as cancer biomarkers, recent evidence implicates KLKs in many cancer-related processes, including cell-growth, angiogenesis, invasion and metastasis. In addition, KLKs have emerged as signalling molecules by selectively targeting members of a unique subfamily of cell surface G-protein coupled receptors termed, proteinase-activated receptors (PARs). PARs are now considered important players in the development of human colon cancer. Indeed, we have previously demonstrated that in colonic tumours, upregulated PAR-2 and aberrantly expressed PAR-1 and PAR-4 control human colon cancer cell migration and proliferation. Recently, we showed that some members of the aberrantly expressed KLKs mediate these responses by signalling of through PARs. We hypothesized...
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Kallikrein-related peptidases (KLKs) are implicated in many cancer-related processes. KLK6, one of the 15 KLK family members, is a promising biomarker for diagnosis of many cancers and has been associated with poor prognosis of colorectal... more
Kallikrein-related peptidases (KLKs) are implicated in many cancer-related processes. KLK6, one of the 15 KLK family members, is a promising biomarker for diagnosis of many cancers and has been associated with poor prognosis of colorectal cancer (CRC) patients. Herein, we evaluated the expression and cellular functions of KLK6 in colon cancer-derived cell lines and in clinical samples from CRC patients. We showed that, although many KLKs transcripts are upregulated in colon cancer-derived cell lines, KLK6, KLK10, and KLK11 are the most highly secreted proteins. KLK6 induced calcium flux in HT29 cells by activation and internalization of protease-activated receptor 2 (PAR2). Furthermore, KLK6 induced extracellular signal–regulated kinases 1 and 2 (ERK1/2) phosphorylation. KLK6 suppression in HCT-116 colon cancer cells decreased the colony formation, increased cell adhesion to extracellular matrix proteins, and reduced spheroid formation and compaction. Immunohistochemistry (IHC) anal...
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AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 4379 The thrombin receptor, Proteinase-activated receptor-4 (PAR4, belongs to a new G protein-coupled receptor subfamily activated by serine proteinases. The presence and role of PAR4... more
AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 4379 The thrombin receptor, Proteinase-activated receptor-4 (PAR4, belongs to a new G protein-coupled receptor subfamily activated by serine proteinases. The presence and role of PAR4 in human gastrointestinal system functions have not been fully investigated. The aim of this study is to investigate the expression and the potential role of the thrombin receptor PAR4 in human colon cancer. The presence of PAR4 in colon cancer cell lines was assessed by immunofluorescence detection and RT-PCR, and its presence in resected human colonic tumors and normal colon was assessed by immunohistochemistry. By using the serine protease thrombin and the specific receptor-activating peptide (AYPGKF-NH) AP4, we tested the functionality of PAR-4 on calcium mobilization and cell proliferation in human colon cancer cells HT29. The following results were obtained: 1) immunofluorescence studies using polyclonal anti-PAR4 antibodies revealed expressi...
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Maharani, Mayzestika. 2010. Pengaruh Pertumbuhan Ekonomi, Pendapatan Asli Daerah (PAD), dan Dana Alokasi Umum (DAU) terhadap Belanja Modal. Skripsi. Jurusan Akuntansi. Fakultas Ekonomi, Universitas Negeri Semarang. Pembimbing I: Dra. Sri... more
Maharani, Mayzestika. 2010. Pengaruh Pertumbuhan Ekonomi, Pendapatan Asli Daerah (PAD), dan Dana Alokasi Umum (DAU) terhadap Belanja Modal. Skripsi. Jurusan Akuntansi. Fakultas Ekonomi, Universitas Negeri Semarang. Pembimbing I: Dra. Sri Kustini, Pembimbing II: Amir Mahmud, S.Pd, M.Si. Kata Kunci: Pertumbuhan ekonomi, Laporan APBD, Agency Theory Pergeseran komposisi belanja merupakan upaya logis yang dilakukan pemerintah daerah setempat dalam rangka meningkatkan tingkat kepercayaan publik. Pergeseran ini ditunjukkan untuk peningkatan investasi modal dalam bentuk aset tetap, yakni peralatan, bangunan, infrastruktur, dan harta tetap lainnya. Alokasi belanja modal ini didasarkan pada kebutuhan daerah akan sarana dan prasarana baik untuk kelancaran pelaksanaan tugas pemerintah maupun untuk fasilitas publik. Oleh karena itu, dalam upaya meningkatkan kualitas pelayanan publik, pemerintah daerah seharusnya mengubah komposisi belanjanya. Selama ini belanja daerah lebih banyak digunakan untu...
Research Interests: Biochemistry, RNA, Biology, Biological Chemistry, Medicine, and 15 moreSignal Transduction, Biological Sciences, DNA, Sequence alignment, Mice, Animals, Biological, CHEMICAL SCIENCES, T lymphocytes, Amino Acid Sequence, Base Sequence, Molecular Sequence Data, Restriction Mapping, Thymus Gland, and Medical and Health Sciences
A cDNA (DPCR1) specific for human intestinal dipeptidyl peptidase IV (DPP IV) has been isolated. This 1.7-kilobase cDNA, together with a previously published partial sequence, covers the entire open reading frame of human DPP IV plus 67... more
A cDNA (DPCR1) specific for human intestinal dipeptidyl peptidase IV (DPP IV) has been isolated. This 1.7-kilobase cDNA, together with a previously published partial sequence, covers the entire open reading frame of human DPP IV plus 67 base pairs of the 3'-untranslated end. Human DPP IV is a 766-amino acid polypeptide with a high degree of homology with the rat liver protein. The characterization of this molecular probe allowed us to definitively confirm the identity of DPP IV with CD 26, a mouse thymocyte activation antigen, a conclusion strengthened by the fact that we observed identical patterns on Southern blot of human genomic DNA hybridized either with human DPP IV or mouse CD 26 cDNA probe. Using this new tool, we have investigated the expression of DPP IV during the onset of enterocytic differentiation of two cultured human colon cancer cell lines, HT-29 and Caco-2. Whatever the cell line and the culture conditions, DPP IV expression strictly correlates with the presence of a differentiated phenotype, as shown by enzyme activity and the steady state amount of the protein measured by indirect immunofluorescence and Western blot. Accordingly, DPP IV biosynthesis exclusively increases in cells that display an enterocytic differentiation. Neither the glycosylation nor the stability of the protein appear to be dependent on the state of enterocytic differentiation. The DPP IV mRNA level remains very low in undifferentiated cell populations and specifically increases in cells that undergo an enterocytic differentiation. These results strongly suggest that DPP IV gene expression is controlled at the transcriptional or posttranscriptional level during intestinal differentiation.
Research Interests: Molecular Biology, Biology, Biological Chemistry, Medicine, Gene expression, and 15 moreWestern blotting, Biological Sciences, DNA, Cell Differentiation, Humans, Biological, Jejunum, CHEMICAL SCIENCES, Molecular cloning, Fluorescent Antibody Technique, Amino Acid Sequence, Base Sequence, Molecular Sequence Data, Medical and Health Sciences, and Colonic Neoplasms
Paneth cells in crypts of the small intestine express antimicrobial peptides, including α-defensins, termed cryptdins in mice. Of the known Paneth cell α-defensins, the cryptdin 4 gene is unique, because it is inactive in the duodenum and... more
Paneth cells in crypts of the small intestine express antimicrobial peptides, including α-defensins, termed cryptdins in mice. Of the known Paneth cell α-defensins, the cryptdin 4 gene is unique, because it is inactive in the duodenum and expressed at maximal levels in the distal small bowel (D. Darmoul and A. J. Ouellette, Am. J. Physiol. 271:G68–G74, 1996). With a cryptdin 4-specific antibody, immunohistochemical staining of ileal Paneth cells was strong and specific for cytoplasmic granules, demonstrating that this microbicidal peptide is a secretory product of Paneth cells in the distal small intestine. Consistent with the pattern of cryptdin 4 mRNA distribution along the length of the gut, the cryptdin 4 peptide was not detected in duodenum. Structurally, the cryptdin 4 gene resembles other Paneth cell α-defensin genes. Its two exons, transcriptional start site, intron, splice sites, and 3′ flanking sequences are characteristic of the highly conserved mouse α-defensin genes. Ho...
Research Interests: Immunohistochemistry, Innate immunity, Gene expression, Biological Sciences, Intestinal Mucosa, and 15 moreMice, Animals, Infection, Antimicrobial Peptide, Gene Structure, Amino Acid Sequence, Base Sequence, Gene Family, Transcription Start Site, Small Intestine, DNA sequence, Differential expression, Molecular Sequence Data, small bowel, and Medical and Health Sciences
The human colon carcinoma cell line Caco-2 was used as an enterocyte model to study the expression of the facilitative glucose transporters GLUT-1 and GLUT-2, and of the putative hexose transporter GLUT-5, which are expressed specifically... more
The human colon carcinoma cell line Caco-2 was used as an enterocyte model to study the expression of the facilitative glucose transporters GLUT-1 and GLUT-2, and of the putative hexose transporter GLUT-5, which are expressed specifically in the gut. Northern blots indicate that Caco-2 cells express GLUT-1 and GLUT-5 mRNAs but not the mRNA coding for the basolateral glucose transporter GLUT-2. The level of GLUT-5 mRNA is growth dependent, being detectable only in postconfluent differentiated cells. In addition, the expression of GLUT-5 increases with the number of cell passages and is approximately 10 times higher in later passages (passage 184) than in early ones (passage 26). With the use of polyclonal antibodies directed against the COOH-terminus of GLUT-5, indirect immunofluorescence and Western blotting indicate that GLUT-5 is mainly localized to the brush border of Caco-2 cells. GLUT-5 is also found to be associated with the brush border of epithelial cells from fetal and norm...
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The expression of dipeptidyl peptidase IV (DPP IV), an intestinal brush-border hydrolase, has been studied in the rat jejunum crypt-villus axis. DPP IV enzyme activity is lower in crypt cells than in villus cells. Indirect... more
The expression of dipeptidyl peptidase IV (DPP IV), an intestinal brush-border hydrolase, has been studied in the rat jejunum crypt-villus axis. DPP IV enzyme activity is lower in crypt cells than in villus cells. Indirect immunofluorescence studies, using a polyclonal antibody raised against purified DPP IV, show a gradient of immunoreactivity from the crypts to the villi that was quantified using confocal laser scanning microscopy. Accordingly, Western blot analysis demonstrates that the steady-state amount of DPP IV is much lower in crypt cells than in villus cells. Northern blot analysis was performed using our cDNA probe for human DPP IV that presents more than 94% homology with rat DPP IV cDNA. Results clearly show that there is approximately seven times less DPP IV mRNA in crypt cells than in villus cells. We conclude that the differentiation-dependent expression of DPP IV in rat jejunum is primarily controlled at the mRNA level.
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The expression of dipeptidyl peptidase IV (DPP IV), an intestinal brush-border hydrolase, has been studied in the rat jejunum crypt-villus axis. DPP IV enzyme activity is lower in crypt cells than in villus cells. Indirect... more
The expression of dipeptidyl peptidase IV (DPP IV), an intestinal brush-border hydrolase, has been studied in the rat jejunum crypt-villus axis. DPP IV enzyme activity is lower in crypt cells than in villus cells. Indirect immunofluorescence studies, using a polyclonal antibody raised against purified DPP IV, show a gradient of immunoreactivity from the crypts to the villi that was quantified using confocal laser scanning microscopy. Accordingly, Western blot analysis demonstrates that the steady-state amount of DPP IV is much lower in crypt cells than in villus cells. Northern blot analysis was performed using our cDNA probe for human DPP IV that presents more than 94% homology with rat DPP IV cDNA. Results clearly show that there is approximately seven times less DPP IV mRNA in crypt cells than in villus cells. We conclude that the differentiation-dependent expression of DPP IV in rat jejunum is primarily controlled at the mRNA level.
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The expression of subunits of the guanine nucleotide regulatory protein that mediates hormonal stimulation of adenylyl cyclase (Gs) and of the guanine nucleotide regulatory protein that mediates hormonal inhibition of adenylyl cyclase... more
The expression of subunits of the guanine nucleotide regulatory protein that mediates hormonal stimulation of adenylyl cyclase (Gs) and of the guanine nucleotide regulatory protein that mediates hormonal inhibition of adenylyl cyclase (Gi) was studied during cell migration and differentiation in the rat small intestine crypt-villus axis. Proliferative crypt cells were separated from nonproliferative mature villus cells and the following data were obtained: 1) alpha s subunits were more abundant in crypt cells than in villus cells as evidenced by cholera toxin-catalyzed [32P]NAD ribosylation and Western blotting of this relative molecular weight (M(r)) 42,000 protein; 2) alpha i2- and alpha i3-subunits (M(r) 40,000 and M(r) 41,000, respectively) were preferentially expressed in villus cells as evidenced by pertussis toxin-catalyzed [32P]NAD ribosylation and Western blotting (alpha i1-subunit was not detectable in intestinal epithelium by using these techniques); 3) Western blotting s...
Research Interests: Physiology, RNA, Membrane Proteins, Cell Biology, Cell Differentiation, and 15 moreIntestinal Mucosa, Animals, Male, Jejunum, Medical Physiology, Alkaline phosphatase, Rats, Epithelium, Epithelial cells, Molecular weight, NAD, Gtp Binding Proteins, Crypt, Biochemistry and cell biology, and Gene Expression Regulation
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Research Interests: Cancer, Biology, Immunohistochemistry, Enzyme Inhibitors, Cancer Research, and 13 moreMedicine, Western blotting, Signal Transduction, Humans, Calcium Signaling, Immunoprecipitation, Fluorescent Antibody Technique, Colon cancer, Cancer Du Sein, Cell Proliferation, reverse transcriptase polymerase chain reaction, Src family kinases, and Colonic Neoplasms
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Serine proteinases signal through proteinase-activated receptors (PARs). Our studies have implicated PAR 1 and PAR 4 (thrombin receptors) and PAR 2 (trypsin receptor) in human colon cancer growth. However, endogenous activators of PARs in... more
Serine proteinases signal through proteinase-activated receptors (PARs). Our studies have implicated PAR 1 and PAR 4 (thrombin receptors) and PAR 2 (trypsin receptor) in human colon cancer growth. However, endogenous activators of PARs in colon tumors are still unknown. Recently, members of the kallikrein-related peptidase (KLK) family have been shown to activate PARs in many cell systems. Thus, our aim was to determine if KLK14, a KLK family member, is expressed in colonic tumors and to see if this KLK can activate PARs in human colon cancer cells. The presence of KLK14 in colon cancer cell lines was assessed by RT-PCR, ELISA and immunofluorescence, and its expression in human colonic tissue was assessed by immunohistochemistry. The effects of recombinant KLK14 in parallel with PAR 1/2 agonists (SFLLRN-NH 2 , an activating peptide for both PAR 1 and PAR 2 ; TFLLR-NH 2, a selective activating peptide for PAR 1 and the selective PAR 2 agonists, 2-furoyl-LIGRLO-NH 2 ; SLIGRL-NH 2 ) were assessed for calcium flux, receptor internalization and ERK1/2 phosphorylation in a human colon cancer-derived HT29 cell line that expresses PAR 1 and PAR 2 . We found that: a) KLK14 mRNA (RT-PCR) was present in 16 out of 16 human colon cancer cell lines, b) KLK14 protein is present (ELISA) in HT29 conditioned media and other colon cancer cell lines; c) there was strong staining of KLK14 in the cytoplasmic compartment of HT29 cells and of human colonic tumors (immunofluorescence and immunohistochemistry; paraffin sections) d) KLK14 (0.1 µM) caused prompt increases in intracellular calcium ([Ca2+] i ) in HT29 cells (Microspectrofluorimetry with Fura 2/fluo-3). The KLK14-induced Ca 2+ -flux was abrogated after an initial challenge of the cells with SFLLRN-NH 2 (100 µM), which desensitizes PAR 1 and PAR 2 simultaneously. Desensitization with 2-furoyl-LIGRLO-NH 2 (10 µM) a potent PAR 2 agonist, attenuated most of the Ca 2+ flux induced by KLK14, whereas responses to TFLLR-NH 2, the PAR 1 agonist, were minimally affected. Consistently, a first challenge with thrombin did not affect KLK14-induced Ca 2+ flux. These results strongly suggest that KLK14 activates preferentially PAR 2 in HT29 cells. e) KLK14 initiates a dramatic loss of cell surface PAR 2 (microscopic analysis) due to its internalization and f) KLK14 induces a rapid (within 5-10 min) and significant ERK1/2 phosphorylation in HT29 cells. This KLK14-PAR signaling pathway may represent a novel therapeutic target for colon tumorigenesis. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4104.
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We hypothesized that kallikrein-related peptidase 14 (KLK14) is produced by colonic tumors and can promote tumorigenesis by activating proteinase-activated receptors (PARs). We found that KLK14 is expressed in human colon adenocarcinoma... more
We hypothesized that kallikrein-related peptidase 14 (KLK14) is produced by colonic tumors and can promote tumorigenesis by activating proteinase-activated receptors (PARs). We found that KLK14 is expressed in human colon adenocarcinoma cells but not in adjacent cancer-free tissue; KLK14 mRNA, present in colon cancer, leads to KLK14 protein expression and secretion; and KLK14 signals viaPAR-2 in HT-29 cells to cause (1) receptor activation/internalization, (2) increases in intracellular calcium, (3) stimulation of ERK1/2/MAP kinase phosphorylation, and (4) cell proliferation. We suggest that KLK14, acting via PAR-2, represents an autocrine/paracrine regulator of colon tumorigenesis.
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Research Interests:
We report the nucleotide sequence and derived amino-acid sequence of a cDNA clone encoding the 3' end of human intestinal dipeptidylpeptidase IV (DPP-IV). This cDNA probe identifies a 4 kb mRNA in the human colon cancer cell line... more
We report the nucleotide sequence and derived amino-acid sequence of a cDNA clone encoding the 3' end of human intestinal dipeptidylpeptidase IV (DPP-IV). This cDNA probe identifies a 4 kb mRNA in the human colon cancer cell line Caco-2. We demonstrate here an extensive homology between this human DPP-IV cDNA and the recently published rat liver DPP-IV cDNA. Using the human DPP-IV cDNA to probe genomic DNA from a panel of somatic cell hybrids we have assigned the gene encoding human DPP-IV to chromosome 2.
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Research Interests: Pathology, Molecular Biology, Kinetics, Calcium, Immunohistochemistry, and 14 moreEnzyme Inhibitors, Flavonoids, Cell Culture, Cell Division, Motility, In Vitro, Thrombin, Humans, Colon cancer, Cell Proliferation, The American, reverse transcriptase polymerase chain reaction, Medical and Health Sciences, and Colonic Neoplasms
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Research Interests:
Research Interests:
Serine proteases are now considered as crucial contributors to the development of human colon cancer. We have shown recently that thrombin is a potent growth factor for colon cancer cells through activation of the aberrantly expressed... more
Serine proteases are now considered as crucial contributors to the development of human colon cancer. We have shown recently that thrombin is a potent growth factor for colon cancer cells through activation of the aberrantly expressed protease-activated receptor 1 (PAR1). Here, we analyzed the signaling pathways downstream of PAR1 activation, which lead to colon cancer cell proliferation in HT-29 cells. Our data are consistent with the following cascade of events on activation of PAR1 by thrombin or specific activating peptide: (a) a matrix metalloproteinase-dependent release of transforming growth factor-alpha (TGF-alpha) as shown with TGF-alpha blocking antibodies and measurement of TGF-alpha in culture medium; (b) TGF-alpha-mediated activation of epidermal growth factor receptor (EGFR) and subsequent EGFR phosphorylation; and (c) activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and subsequent cell proliferation. The links between these events are shown by ...
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76 Serine proteases elicit cellular effects by activating G protein-coupled protease-activated receptors (PARs). The PAR family includes four members: PAR1, PAR2, PAR3 and PAR4. PARs are activated by an unusual irreversible proteolytic... more
76 Serine proteases elicit cellular effects by activating G protein-coupled protease-activated receptors (PARs). The PAR family includes four members: PAR1, PAR2, PAR3 and PAR4. PARs are activated by an unusual irreversible proteolytic event in which the protease binds to and cleaves the amino-terminus of the receptor generating a new amino terminus that functions as a tethered ligand. This activation initiates signal transduction resulting in stimulation of phosphoinositide breakdown and cytosolic calcium mobilization leading to the activation of MEK-ERK1/2. Our recent studies have shown the implication of proteinase-activated receptors (PARs) in colon cancer (Darmoul et al. Br. J. Cancer 2001; Am J Pathol., 2003) .Using the HT29 colon cancer cell line, we identified a cross-talk between proteinase activated receptors and epidermal growth factor receptor (Darmoul et al. J. Biol. Chem. 2004) . This cross talk explains the activation of ERK signal transduction pathway and the prolife...
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Research Interests: Biology()
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Research Interests: Biology()
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Oral epithelial cells in vivo show marked changes in cytokeratin expression during embryonic and fetal development (1). In the present study we used cell culture techniques which permit identifying cy.tokeratins in early stages of... more
Oral epithelial cells in vivo show marked changes in cytokeratin expression during embryonic and fetal development (1). In the present study we used cell culture techniques which permit identifying cy.tokeratins in early stages of differentiation and present data which show similar changes of cytokeratin expression in culture. Our immunocytochemical results on human gingival cells show that during the stages of epithelial migration/proliferation, and the stage of coloni formation, the cells express the same set of cytokeratins made of; basal cells markers CK 5 and 14, hyperproliferation markers CK 6 and 16, and simple epithelial markers CK 8, 18 and 19. At the stage of stratification, the markers of nonkeratinised stratified epithelia CK 13 and 4 are expressed. Cytokeratin 13 was expressed once individual cells started to overlap the basal cells of the colonies then CK 4 appeared in the well-spread squames. In older cultures, the simple epithelial cytokeratins became less prominent in the stratified epithelium. Coloni non-forming cells in older cultures (10 days) showed morphological evidence of maturation while attached to glass and expressed cyt.)keratin 4, not 13. These changes are consistent with those obtained in vivo and suggest that the cells pass a programmed differentiation. The difference in the expression of CK 4 and 13 suggests that CK 13 is a marker for stratification whereas CK 4 is a marker for postmitotic cell differentiation in gingival epithelial cells.