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Biomedical subjects

M C Rio

Publications and source records attributed to M C Rio.

At least 19 recordsLinked to original sources
PubMed2002

Ghrelin/motilin-related peptide is a potent prokinetic to reverse gastric postoperative ileus in rat.

A novel peptide called ghrelin or motilin-related-peptide (MTLRP) was found in the stomach of various mammals. We studied its effect on the motor function of the rat gastrointestinal tract. In normal, conscious unoperated animals, ghrelin/MTLRP (5 or 20 microg/kg iv) significantly accelerated the gastric emptying of a methylcellulose liquid solution (gastric residue after 15 min: 57 +/- 7, 42 +/- 11, 17 +/- 4, and 9 +/- 3% of the ingested meal with doses of 0, 1, 5, and 20 microg/kg iv, respectively) Transit of the methylcellulose liquid solution was also accelerated by ghrelin/MTLRP in the small intestine but not in the colon. Des-[Gln(14)]ghrelin, also found in the mammalian stomach, was as potent as ghrelin in emptying the stomach (gastric residue after 15 min: 12 +/- 3% at a dose of 20 microg/kg iv). In rats in which postoperative gastrointestinal ileus had been experimentally induced, ghrelin/MTLRP (20 microg/kg iv) reversed the delayed gastric evacuation (gastric residue after 15 min: 28 +/- 7% of the ingested meal vs. 82 +/- 9% with saline). In comparison, the gastric ileus was not modified by high doses of motilin (77 +/- 7%) or erythromycin (82 +/- 6%) and was only partially improved by calcitonin gene-related peptide (CGRP) 8-37 antagonist (59 +/- 7%). Ghrelin/MTLRP, therefore, accelerates the gastric emptying and small intestinal transit of a liquid meal and is a strong prokinetic agent capable of reversing the postoperative gastric ileus in rat.

Animals↗
PubMed2002

Adipocyte hypertrophy in stromelysin-3 deficient mice with nutritionally induced obesity.

Several matrix metalloproteinases (MMPs), including the stromelysins MMP-3 and MMP-11, are expressed in adipose tissue. To investigate a potential role of MMP-11 (stromelysin-3) in adipose tissue development, five-week-old male wild-type mice (MMP-11+/+) or mice with deficiency of MMP-11 (MMP-11-/-) were fed a high fat diet (HFD, 42% fat) for 15 weeks. Haematologic parameters, including white and red blood cells, platelets, haemoglobin and haematocrit, and metabolic parameters including glucose, triglycerides and total cholesterol were not different for both genotypes. At the time of sacrifice, the body weight of the MMP-11-/- mice was higher than that of the MMP-11+/+ mice (36+/-1.4 g versus 29+/-0.9 g, p = 0.0002). The weight of the isolated subcutaneous (SC) and gonadal (GON) fat deposits was also higher in MMP-11-/- mice (620+/-150 mg versus 280+/-28 mg for SC fat, and 970+/-180 mg versus 430+/-62 mg, p < 0.05, for GON fat). Adipocytes in MMP-11-/- adipose tissue were hypertrophic as compared to MMP-11+/+ adipocytes (volume of 57+/-12 x 10(3) microm3 versus 31+/-2.4 x 10(3) microm3 for SC fat, and 100+/-18 x 10(3) microm3 versus 57+/-7.6 x 10(3) microm3 for GON fat; both p < 0.06). In nutritionally induced obesity models in mice a potential role of the fibrinolytic system was suggested in adipocyte hypertrophy. The hypertrophy observed in this model is, however, not related to changes in fibrinolytic parameters, as suggested by our finding that levels of t-PA, u-PA and PAI-1 antigen as well as t-PA and u-PA activity were not different in SC or GON adipose tissue extracts of both genotypes. As the main biological function of MMP-11 remains unknown, it is not clear whether the adipocyte hypertrophy in MMP-11-/- adipose tissue is directly related to the deficiency or to other pathways affected by MMP-11.

Adipocytes↗
PubMed2001

Transcription regulation and protein subcellular localization of the truncated basic hair keratin hHb1-DeltaN in human breast cancer cells.

An aberrant truncated hHb1 hair keratin transcript, named hHb1-DeltaN, was previously identified in breast carcinomas. No normal tissue tested so far, including hairy skin, expressed hHb1-DeltaN, indicating that hHb1-DeltaN is related to carcinogenesis. In the present study, we investigated the mechanism by which such truncated transcript was generated in breast cancer cell lines. We found that hHb1-DeltaN transcription is initiated at an unusual cryptic promoter within the fourth intron of the hHb1 gene and is dependent on two proximal Sp1 binding sites for its baseline activity. Moreover, hHb1-DeltaN transcription is increased in response to DNA demethylation by the 5-aza-2'-deoxycytidine drug. This induction is dependent on protein neosynthesis, indicating that an additional factor is required. In addition, we showed that the hHb1-DeltaN transcript is translated in vivo as a truncated hHb1 protein that is missing the 270 amino-terminal residues. The hHb1-DeltaN protein exhibits a filament pattern throughout the cytoplasm and partially co-localizes with cytokeratin filaments, indicating its participation in the cytoskeleton network. hHb1-DeltaN might alter the adhesive properties of cancer cells.

Base Sequence↗
PubMed2001

Crystal structure of the stromelysin-3 (MMP-11) catalytic domain complexed with a phosphinic inhibitor mimicking the transition-state.

Stromelysin-3 (ST3) is a matrix metalloproteinase (MMP-11) whose proteolytic activity plays an important role in tumorigenicity enhancement. In breast cancer, ST3 is a bad prognosis marker: its expression is associated with a poor clinical outcome. This enzyme therefore represents an attractive therapeutic target. The topology of matrix metalloproteinases (MMPs) is remarkably well conserved, making the design of highly specific inhibitors difficult. The major difference between MMPs lies in the S(1)' subsite, a well-defined hydrophobic pocket of variable depth. The present crystal structure, the first 3D-structure of the ST3 catalytic domain in interaction with a phosphinic inhibitor mimicking a (d, l) peptide, clearly demonstrates that its S(1)' pocket corresponds to a tunnel running through the enzyme. This open channel is filled by the inhibitor P(1)' group which adopts a constrained conformation to fit this pocket, together with two water molecules interacting with the ST3-specific residue Gln215. These observations provide clues for the design of more specific inhibitors and show how ST3 can accommodate a phosphinic inhibitor mimicking a (d, l) peptide. The presence of a water molecule interacting with one oxygen atom of the inhibitor phosphinyl group and the proline residue of the Met-turn suggests how the intermediate formed during proteolysis may be stabilized. Furthermore, the hydrogen bond distance observed between the methyl of the phosphinic group and the carbonyl group of Ala182 mimics the interaction between this carbonyl group and the amide group of the cleaved peptidic bond. Our crystal structure provides a good model to study the MMPs mechanism of proteolysis.

Amino Acid Sequence↗
PubMed2001

Mouse Trefoil factor genes: genomic organization, sequences and methylation analyses.

The mammalian Trefoil Factors (TFFs), TFF1/pS2, TFF2/SP and TFF3/ITF, are expressed and secreted throughout the gastrointestinal tract with a specific and complementary pattern. These proteins exhibit common functions in the protection and repair process of the gastrointestinal epithelial barrier. Here, we report the clustered organization of the three mouse TFF genes in a 40 kb DNA segment, in a head to tail orientation in the following order: TFF1, TFF2, and TFF3. Computer comparison of the mouse TFF promoter sequences to their human counterparts revealed conserved boxes in both mouse and human genes. Promoter methylation analyses showed that, in tissues where these genes are normally expressed, the proximal promoters of TFF1 and TFF2 are specifically not methylated and that of TFF3 is partially demethylated. In contrast, in organs that do not express TFFs, the promoters of the three genes are methylated. These findings strongly argue for the involvement of epigenetic mechanisms in the regulation of TFF expression in normal and pathological conditions.

Animals↗
PubMed2001

High cancer cell death in syngeneic tumors developed in host mice deficient for the stromelysin-3 matrix metalloproteinase.

Matrix metalloproteinases (MMPs) are extracellular enzymes. Some of them are known to be involved in tumor development and/or progression. Several cellular functions have been proposed for MMPs during malignant processes. Notably, they may be involved in tissue-remodeling processes through their ability to digest matrix components or to participate in tumor neoangiogenesis and, subsequently, in cancer cell proliferation. One of these MMPs, stromelysin-3 (ST3/MMP11), although devoid of enzymatic activity against the matrix components, is associated with human tumor progression and poor patient clinical outcome. Using several in vivo experimental models, it has been demonstrated that ST3 expression by the fibroblastic cells surrounding malignant epithelial cells promotes tumorigenesis in a paracrine manner. The present study was devoted to the identification of the cellular function underlying this ST3-induced tumor promotion using a syngeneic tumorigenesis model in mice. Our results show that ST3 exhibits a new and unexpected role for a MMP, because ST3-increased tumorigenesis does not result from increased neoangiogenesis or cancer cell proliferation but from decreased cancer cell death through apoptosis and necrosis. Thus, during malignancy, the cellular function of ST3 is to favor cancer cell survival in the stromal environment.

Animals↗
PubMed2001

Identification of CGA as a novel estrogen receptor-responsive gene in breast cancer: an outstanding candidate marker to predict the response to endocrine therapy.

The estrogen receptor (ER) status of breast tumors is used to identify patients who may respond to endocrine agents such as tamoxifen. However, ER status alone is not perfectly predictive, and there is a pressing need for more reliable markers of endocrine responsiveness. Here, we identified the well-known CGA gene (coding for the alpha subunit of glycoprotein hormones) as a new ERalpha-responsive gene in human breast cancer cells. We used a real-time quantitative reverse transcription-PCR assay to quantify CGA mRNA copy numbers in a large series of breast tumors. CGA overexpression (> 10 SD above the mean for normal breast tissues) was observed in 44 of 131 (33.6%) breast tumor RNAs, ranging from 20 to 16,500 times the level in normal breast tissues; the highest levels of CGA gene expression were close to those observed in placenta. Significant links were observed between CGA gene overexpression and Scarff-Bloom-Richardson histopathological grade I+II (P = 0.015), and progesterone (P = 0.0009) and estrogen (P < 10(-7)) receptor positivity, which suggested that CGA is a marker of low tumor aggressiveness. We observed CGA mRNA overexpression in 44 of 90 (48.9%) ERalpha-positive tumors and in none of the 41 ERalpha-negative tumors. Immunohistochemical studies demonstrated that human chorionic gonadotropin alpha protein was strictly limited to ERalpha-positive tumor cells. Overexpression of the CGA gene was not accompanied by overexpression of the CGB gene. Our results also suggest that CGA could be a more reliable marker than PS2 and PR for ERalpha functionality and, thus, for endocrine responsiveness. Moreover, the CGA marker has the added value of dichotomizing ERalpha-positive patients into two subgroups of similar size. Specific antibodies directed to secreted human chorionic gonadotropin alpha protein are commercially available, thus facilitating the future application of this marker to the clinical management of breast cancer.

Adult↗
PubMed2001

Identification of cDNA encoding motilin related peptide/ghrelin precursor from dog fundus.

A novel protein expressed by entero-endocrine cells of the mouse stomach was named prepromotilin Related Peptide (ppMTLRP) since it shares sequence similarities with the prepromotilin (Tomasetto et al.). The mouse ppMTLRP was found identical to the rat precursor of ghrelin (ppghrelin), an endogenous ligand specific for the Growth Hormone Secretagogue receptor identified from rat stomach (Kojima et al.). In the present study the cDNA encoding the dog counterpart of ppMTLRP/Ghrelin has been isolated and sequenced. The dog ppMTLRP/Ghrelin cDNA showed scores of respectively 80% and 75% homology with its human and mouse counterparts. By translation of the dog ppMTLRP/Ghrelin cDNA sequences, two ORFs could be deduced encoding either a 117 amino acid ppMTLRP/Ghrelin or the deleted Gln14 ppMTLRP/Ghrelin, as it was also known in mouse, rat and man. The dog ppMTLRP/Ghrelin shared 91% similarity and 78% identity, and 89% similarity and 78% identity with the human and mouse ppMTLRP/Ghrelin proteins respectively. The best score of homology was found in the MTLRP/Ghrelin sequence itself. Indeed the dog MTLRP/Ghrelin peptide shared 100% similarity and 93% identity, and 96% identity and similarity, with the human and mouse MTLRP/Ghrelin. Using Northern blot analysis to study dog ppMTLRP/Ghrelin gene expression on dog adult gut tissues, maximal expression level was found in the stomach fundus and corpus, and no expression could be detected in the stomach antrum nor in the duodenum, jejunum, ileum, colon or liver. In conclusion, we have identified ppMTLRP/Ghrelin from dog, and found that it is highly conserved with man, mouse or rat. The expression pattern along the gastro-intestinal tract is similar to the expression pattern previously described in mouse.

Amino Acid Sequence↗
PubMed2001

In vivo and in vitro effects of ghrelin/motilin-related peptide on growth hormone secretion in the rat.

Ghrelin (Ghr), a 28 amino acid gastric peptide with an n-octanoylation on Ser 3, has recently been identified as an endogenous ligand of the growth hormone secretagogue (GHS) receptor. A cDNA was also isolated from a mouse stomach library encoding a protein named prepromotilin-related peptide (ppMTLRP) which shares sequence similarities with prepromotilin. Mouse and rat ppMTLRP sequences (rGhr) are identical and show 89% identity with human ghrelin (hGhr). By analogy with promotilin, cleavage of proMTLRP into an 18 amino acid endogenous processed peptide can be assumed on the basis of a conserved dibasic motif in position 9-10 of its sequence. In the present work, we compared the GH-releasing activity of rGhr28/MTLRP and of hGhr28/MTRLP with that of a shorter form of the peptide, hGhr18. A short peptide devoid of Ser-3 n-octanoylation hGhr18[-] was also tested. Addition of rGhr28, hGhr28 and hGhr18 stimulated GH release to the same extent from superfused pituitaries. The effect was dose dependent in a 10(-8) to 10(-6) M concentration range. In contrast, hGhr 18[-] was inactive. In freely moving animals, both rGhr28 and hGhr28 (10 microg, i.v.) stimulated GH release, whereas the same dose of hGhr18 or of hGhr18[-] was ineffective. After rGhr28, GH plasma levels increased as early as 5 min after injection and returned to basal values within 40-60 min. Expressed as percent stimulation, administration of rGhr28 was equally effective when injected during troughs or peaks of GH. Plasma concentrations of prolactin, adrenocorticotropin and leptin were not modified. Spontaneous GH secretory episodes were no longer observed within 3 h of rGhr28 treatment, but repeated administration of the secretagogue at 3- to 4-hour intervals resulted in a similar GH response. Activation of somatostatin (SRIH) release by ether stress did not blunt the GH response to rGhr28. This suggests that the secretagogue acts in part by inhibiting endogenous SRIH, as further substantiated by the ability of rGhr28 (10(-6) M), to decrease the amplitude of 25 mM K+-induced SRIH release from perifused hypothalami. In conclusion, (1) n-octanoylation of Ghrs and the shorter form hGhr18 is essential for the direct pituitary GH-releasing effect of this new family of endogenous GHSs; (2) only the longer forms are active in vivo and (3) inhibition of SRIH release appears involved in the mechanism of Ghr action.

Adrenocorticotropic Hormone↗
PubMed2000

The steroidogenic acute regulatory protein homolog MLN64, a late endosomal cholesterol-binding protein.

MLN64 is a transmembrane protein that shares homology with the cholesterol binding domain (START domain) of the steroidogenic acute regulatory protein. The steroidogenic acute regulatory protein is located in the inner membrane of mitochondria, where it facilitates cholesterol import into the mitochondria. Crystallographic analysis showed that the START domain of MLN64 is a cholesterol-binding domain. The present work was undertaken to determine which step of the intracellular cholesterol pathway MLN64 participates in. Using immunocytofluorescence, MLN64 colocalizes with LBPA, a lipid found specifically in late endosomes. Electron microscopy indicates that MLN64 is restricted to the limiting membrane of late endosomes. Microinjection or endocytosis of specific antibodies shows that the START domain of MLN64 is cytoplasmic. Deletion and mutagenesis experiments demonstrate that the amino-terminal part of MLN64 is responsible for its addressing. Although this domain does not contain conventional dileucine- or tyrosine-based targeting signals, we show that a dileucine motif (Leu(66)-Leu(67)) and a tyrosine residue (Tyr(89)) are critical for the targeting or the proper folding of the molecule. Finally, MLN64 colocalizes with cholesterol and Niemann Pick C1 protein in late endosomes. However, complementation assays show that MLN64 is not involved in the Niemann Pick C2 disease which, results in cholesterol lysosomal accumulation. Together, our results show that MLN64 plays a role at the surface of the late endosomes, where it might shuttle cholesterol from the limiting membrane to cytoplasmic acceptor(s).

Animals↗
PubMed2000

C2PA, a new protein expressed during mouse spermatogenesis.

C2PA is a novel protein that contains a C2 membrane binding domain, a PDZ protein/protein interaction domain, and an ATP/GTP binding domain. C2PA is expressed during embryogenesis from 8.5 days post-coitum (dpc) until birth. After birth, C2PA expression is mainly observed in the post-natal and adult testis. During spermatogenesis, C2PA transcripts are specifically observed in the spermatocytes, whereas spermatogonia and spermatids are negative. Taken together, these results suggest that C2PA might be involved in cell signaling pathways occurring during spermatogenesis.

Amino Acid Sequence↗
PubMed2000

Demonstration in vivo that stromelysin-3 functions through its proteolytic activity.

Stromelysin-3 (ST3), a matrix metalloproteinase (MMP) expressed in aggressive carcinomas, has been shown to promote tumor development in different in vivo experimental models. However, the inability of its mature form to degrade extracellular matrix components casts doubt on whether ST3 functions in vivo as a protease. In this study, we evaluated whether the ST3 tumor-promoting effect could be ascribed to its proteolytic activity and whether this putative protease could be targeted with MMP inhibitors. Catalytically inactive mutant cDNA of human (h) ST3 or mouse (m) ST3 were generated and transfected into MCF7 cells. When injected into nude mice in the presence of matrigel, the mutant-bearing cells did not exhibit the enhanced tumorigenicity elicited by MCF7 cells transfected with wild-type ST3 cDNA. In a second approach, TIMP2 overproduction in MCF7 cells expressing hST3 was induced by retroviral infection. The co-expression of ST3 and TIMP2 failed to enhance the tumorigenicity of MCF7 cells. Notably, matrigel depleted of low-molecular-weight proteins and growth factors failed to promote the tumorigenicity of ST3-expressing MCF7 cells. These findings provide the first in vivo evidence that ST3 is indeed a protease that can modulate cancer progression by remodeling extracellular matrix and probably by inducing it to release the necessary microenvironmental factors. Thus, ST3 represents an interesting target for specific MMP inhibition.

Adenocarcinoma↗
PubMed2000

pS2/TFF1 interacts directly with the VWFC cysteine-rich domains of mucins.

BACKGROUND & AIMS: Trefoil factors (TFFs) are secreted gastrointestinal proteins that have been shown to protect and promote healing of the gastrointestinal tract. Moreover, pS2/TFF1 is essential for normal differentiation of the gastric mucosa because deficient mice develop antropyloric adenomas. To date, it is unclear how TFFs mediate their functions. METHODS: Using the yeast 2-hybrid system, we attempted to identify murine TFF1 interacting proteins by screening a stomach and duodenum complementary DNA (cDNA) expression library. RESULTS: Four positive clones were isolated. Sequence and expression studies showed that they corresponded to the murine counterpart of human cDNA sequences encoding carboxy-terminal fragments of mMuc2 (489 residues) and mMuc5AC (427, 430, and 894 residues) mucin proteins. Mutagenesis experiments showed that TFF1 interacts with the 2 mucins through binding with their VWFC1 and VWFC2 (von Willebrand factor C) cysteine-rich domains. CONCLUSIONS: These results show that the gastrointestinal protective effect of TFF1, and presumably of the other TFFs, is caused at least partially by their participation, via mucin binding, in the correct organization of the mucous layer that protects the apical side of the mucosa from deleterious luminal agents.

Amino Acid Sequence↗
PubMed2000

Identification and characterization of a novel gastric peptide hormone: the motilin-related peptide.

BACKGROUND & AIMS: This study looked for new proteins with expression restricted to the gastric epithelium that may provide insight to the differentiation and function of the gastric unit. METHODS: A novel complementary DNA was isolated and sequenced, and its expression was examined in mouse tissues at both messenger RNA and protein levels. Subcellular localization was studied using immunoelectron microscopy. The posttraductional processing of the protein was analyzed in vitro by protein microsequencing and in vivo by Western blotting. RESULTS: We identified a novel protein that is mainly expressed by the secretory granules of the stomach enteroendocrine cells. This protein has sequence similarity with prepromotilin, the precursor of the motilin hormone and the motilin-associated peptide. As for the prepromotilin, a posttraductional maturation leads to a secreted peptide that is further cleaved at a dibasic site and gives rise to the motilin-related peptide (MTLRP) and MTLRP-associated peptide. CONCLUSIONS: We have identified and characterized a novel gene encoding the preproMTLRP protein. MTLRP presents similarity to motilin and is specifically expressed by enteroendocrine cells of the stomach and therefore represents a novel hormone.

Animals↗
PubMed1999

Presence of high levels of MT1-MMP protein in fibroblastic cells of human invasive carcinomas.

Matrix metalloproteinase 2 (MMP2) activity is associated with the aggressiveness of human cancers. Therefore, the mechanisms regulating its activation are of great interest for a better understanding of malignant invasive processes. MT1-MMP, a membrane-bound MMP, is involved in the conversion of the latent form of MMP2 to the active one. In the present study, we have raised 3 monoclonal antibodies (Mabs) directed against 3 different epitopes of human MT1-MMP, which we used to investigate the expression and cellular localization of MT1-MMP protein in human carcinomas. MT1-MMP protein was present in all invasive carcinomas tested, and it was almost exclusively located to the stromal cells and not to cancer cells as previously reported, suggesting that MMP2 activation might be a peri-fibroblastic event.

Animals↗
PubMed1999

Phosphinic pseudo-tripeptides as potent inhibitors of matrix metalloproteinases: a structure-activity study.

Several phosphinic pseudo-tripeptides of general formula R-XaaPsi(PO(2)-CH(2))Xaa'-Yaa'-NH(2) were synthesized and evaluated for their in vitro activities to inhibit stromelysin-3, gelatinases A and B, membrane type-1 matrix metalloproteinase, collagenases 1 and 2, and matrilysin. With the exception of collagenase-1 and matrilysin, phosphinic pseudo-tripeptides behave as highly potent inhibitors of matrix metalloproteinases, provided they contain in P(1)' position an unusual long aryl-alkyl substituent. Study of structure-activity relationships regarding the influence of the R and Xaa' substituents in this series may contribute to the design of inhibitors able to block only a few members of the matrix metalloproteinase family.

Kinetics↗
PubMed1999

Purification of active matrix metalloproteinase catalytic domains and its use for screening of specific stromelysin-3 inhibitors.

The matrix metalloproteinase (MMP) stromelysin-3 (ST3) has been shown to be involved in malignant tumor progression and therefore represents an attractive therapeutical target. In order to screen for ST3 synthetic inhibitors, we have produced and purified the catalytic domain of ST3, matrilysin, stromelysin-2, and membrane type-1 MMP from inclusion bodies in a bacterial system. Our strategy allowed the purification of MMPs directly in the active form, thereby avoiding in vitro activation. A total of 140,000 synthetic compounds from the Bristol-Myers Pharmaceutical Research Institute chemical deck were tested, using a substrate-based colorimetric enzymatic assay, in which ST3 activity was evaluated through its ability to cleave and inactivate alpha-1 proteinase inhibitor. One ST3 inhibitor belonging to the cephalosporin family of antibiotics was thereby identified.

Animals↗