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C Chakraborty

Publications and source records attributed to C Chakraborty.

At least 19 recordsLinked to original sources
PubMed2006

COX-2-mediated stimulation of the lymphangiogenic factor VEGF-C in human breast cancer.

Increased expression of COX-2 or VEGF-C has been correlated with progressive disease in certain cancers. Present study utilized several human breast cancer cell lines (MCF-7, T-47D, Hs578T and MDA-MB-231, varying in COX-2 expression) as well as 10 human breast cancer specimens to examine the roles of COX-2 and prostaglandin E (EP) receptors in VEGF-C expression or secretion, and the relationship of COX-2 or VEGF-C expression to lymphangiogenesis. We found a strong correlation between COX-2 mRNA expression and VEGF-C expression or secretion levels in breast cancer cell lines and VEGF-C expression in breast cancer tissues. Expression of LYVE-1, a selective marker for lymphatic endothelium, was also positively correlated with COX-2 or VEGF-C expression in breast cancer tissues. Inhibition of VEGF-C expression and secretion in the presence of COX-1/2 or COX-2 inhibitors or following downregulation of COX-2 with COX-2 siRNA established a stimulatory role COX-2 in VEGF-C synthesis by breast cancer cells. EP1 as well as EP4 receptor antagonists inhibited VEGF-C production indicating the roles of EP1 and EP4 in VEGF-C upregulation by endogenous PGE2. Finally, VEGF-C secretion by MDA-MB-231 cells was inhibited in the presence of kinase inhibitors for Her-2/neu, Src and p38 MAPK, indicating a requirement of these kinases for VEGF-C synthesis. These results, for the first time, demonstrate a regulatory role of COX-2 in VEGF-C synthesis (and thereby lymphangiogenesis) in human breast cancer, which is mediated at least in part by EP1/EP4 receptors.

Breast Neoplasms↗
PubMed2004

RNA interference: potential therapeutic targets.

One of the most exciting findings in recent years has been the discovery of RNA interference (RNAi). RNAi methodologies hold the promise to selectively inhibit gene expression in mammals. RNAi is an innate cellular process activated when a double-stranded RNA (dsRNA) molecule of greater than 19 duplex nucleotides enters the cell, causing the degradation of not only the invading dsRNA molecule, but also single-stranded (ssRNAs) RNAs of identical sequences, including endogenous mRNAs. The use of RNAi for genetic-based therapies has been widely studied, especially in viral infections, cancers, and inherited genetic disorders. As such, RNAi technology is a potentially useful method to develop highly specific dsRNA-based gene-silencing therapeutics.

Animals↗
PubMed2003

Factors regulating trophoblast migration and invasiveness: possible derangements contributing to pre-eclampsia and fetal injury.

Impaired trophoblast invasiveness and spiral arterial remodelling, which results in poor placental perfusion during early pregnancy, is believed to cause fetal injury and growth retardation, and also endothelial cell activation/dysfunction in a susceptible mother, leading to clinical manifestations of pre-eclampsia. This article briefly reviews the regulatory roles of certain locally active factors in trophoblast migration and invasiveness. This background is then used to discuss and debate whether derangements or dysfunction of some of these factors can manifest as early serum markers predictive of the disease, as opposed to the intermediate and late stage markers which may reflect manifestations and consequences of the disease. Of particular significance are the observed derangements in uPA/uPAR/PAI system, IGFBP-1, HGF, HB-EGF and TGFbeta, factors which are known to regulate trophoblast migration and invasiveness in situ. An emphasis is placed on the need for longitudinal studies in order to identify predictive serum markers which may help strategies for prevention or amelioration of fetal injury and pre-eclampsia.

Adult↗
PubMed2002

Human placental trophoblast as an in vitro model for tumor progression.

The human placenta is a highly invasive tumor-like structure in which a subpopulation of placental trophoblast cells known as the "extravillous trophoblast" (EVT) invades the uterine decidua and its vasculature to establish adequate fetal-maternal exchange of molecules. By utilizing in vitro-propagated short-lived EVT cell lines we found that molecular mechanisms responsible for their invasiveness are identical to those of cancer cells; however, unlike cancer cells, their proliferation, migration, and invasiveness in situ are stringently controlled by decidua-derived transforming growth factor (TGF)-beta. By SV40T antigen transfection of normal EVT cells followed by a forced crisis regimen in culture we produced an immortalized premalignant derivative that is hyperproliferative, hyperinvasive, and deficient in gap-junctional intercellular communication. Both premalignant and malignant EVT (JAR and JEG-3 choriocarcinoma) cell lines were found to be TGF-beta-resistant. Using these cell lines, we investigated genetic changes responsible for transition of the normal EVT cells to premalignant and malignant phenotype. Hyperinvasiveness in both cases resulted from a downregulation of tissue inhibitor of metalloprotease (TIMP)-1 and plasminogen activator inhibitor (PAI)-1 genes. In contrast to normal EVT cells, both cell types failed to upregulate these genes in response to TGF-beta. Loss of TGF-beta response in malignant EVT cells was explained by the loss of expression of Smad3 gene. Differential mRNA display of normal and premalignant EVT cells identified up- and down-regulation of numerous known or novel genes in premalignant EVT cells, with potential oncogenic and (or) tumor-suppressor functions, e.g., loss of fibronectin and insulin-like growth factor binding protein (IGFBP-5). Premalignant EVT cells also lost IGF receptor type 2 (IGFR-II). IGFBP-5 was shown to be a negative regulator of IGF-1-induced proliferation of premalignant EVT cells, so that loss of IGFBP-5 as well as IGFR-II permitted their unrestricted proliferation in an IGF-I-rich microenvironment of the fetal-maternal interface. The present model may be a good prototype for identifying genetic changes underlying epithelial tumor progression.

Cell Line↗
PubMed2001

Differential gene expression in premalignant human trophoblast: role of IGFBP-5.

Tumorigenesis results from genetic alterations that occur in a stepwise manner giving rise to cells with increasingly cancer-like characteristics. We used in vitro propagated first trimester human extravillous trophoblast (EVT) cells to identify genetic changes responsible for the transition of the EVT from a normal to premalignant stage. The model used consisted of a normal invasive EVT (HTR8) cell line and its premalignant derivative (RSVT2/C) generated by transfection with the SV40 Tag and selected using a forced crisis regimen. RSVT2/C display increased proliferative, migratory and invasive behavior, unresponsiveness to anti-proliferative and anti-invasive signals of TGFbeta and a deficiency in gap junctional intercellular communication. These cells, however, were unable to form colonies on soft agar or tumors in nude mice and are thus defined as premalignant. Differential display revealed 18 gene sequences, 7 with unknown and 11 with known identity, showing altered expression between the normal HTR8 and premalignant RSVT2/C cell lines. The known sequences include the potential tumor suppressors insulin-like growth factor binding protein (IGFBP)-5 and fibronectin (FN) and potential protooncogenes such as chromokinesin (KIF4), alternative splicing factor (SF2), dynein, DNA polymerase epsilon (DNApol epsilon) and NF-kappaB activating kinase (NAK). The role of the remaining 4 genes upregulated in the premalignant EVT is presently unknown and these are FK506 binding protein (FKBP) 25, histone protein (HP1Hs)-gamma, nucleoporin (Nup) 155 and an 82 kDa acidic human protein. The functional role of IGFBP-5 was examined in the control of proliferation, migration and invasiveness of RSVT2/C cells measured in vitro. IGFBP-5 alone had no effect on these properties of RSVT2/C cells. Furthermore, unlike normal EVT cells, RSVT2/C cells exhibited refractoriness to the migration stimulating signals of IGF-II, which was explained by the loss or downregulation of the IGF type 2 receptor (IGF-R2). RSVT2/C cells, however, expressed the IGF type 1 receptor (IGF-R1) and responded to IGF-I by increased proliferation. This response was blocked with increasing concentrations of IGFBP-5. These results suggest that the loss of IGFBP-5 and possibly IGF-R2, both of which can sequester IGF-I from IGF-R1, permits unhindered proliferation of the premalignant EVT in an IGF-I rich environment of the fetal-maternal interface. The functions of the other differentially expressed genes, some of which are essential for cell cycle progression or cell survival require further investigation.

Base Sequence↗
PubMed2001

Expression of TGF-beta signaling genes in the normal, premalignant, and malignant human trophoblast: loss of smad3 in choriocarcinoma cells.

We had earlier shown that TGF-beta controls proliferation, migration, and invasiveness of normal human trophoblast cells, whereas premalignant and malignant trophoblast cells are resistant to TGF-beta. To identify signaling defects responsible for TGF-beta resistance in premalignant and malignant trophoblasts, we have compared the expression of TGF-beta signaling molecules in a normal trophoblast cell line (HTR-8), its premalignant derivative (RSVT2/C), and two choriocarcinoma cell lines (JAR and JEG-3). RT-PCR analysis revealed that all these cell lines expressed the mRNA of TGF-beta1, -beta2, and -beta3, TGF-beta receptors type I, II, and III, and post-receptor signaling genes smad2, smad3, smad4, smad6, and smad7 with the exception that TGF-beta2 and smad3 were undetectable in JAR and JEG-3 cells. Immunoblot analysis confirmed the absence of smad3 protein in choriocarcinoma cells. Treatment with TGF-beta1 induced smad3 phosphorylation and smad3 translocation to the nucleus in the normal and premalignant trophoblast cells. These results suggest that loss of smad3 may account for a functional disruption in the TGF-beta signaling pathway in choriocarcinomas, but not in the premalignant trophoblast.

Choriocarcinoma↗
PubMed2001

Cyclooxygenase inhibitors retard murine mammary tumor progression by reducing tumor cell migration, invasiveness and angiogenesis.

Tumor-derived prostaglandins (PGs) have been implicated in the progression of murine and human breast cancer. Chronic treatment with a non-selective PG inhibitor indomethacin was shown in this laboratory to retard the development and metastasis of spontaneous mammary tumors in C3H/HeJ female retired breeder mice. The present study examined the role of endogenous prostaglandins in the proliferation/survival, the migratory and invasive behavior and angiogenic ability of a highly metastatic murine mammary tumor cell line, C3L5, originally derived from a C3H/HeJ spontaneous mammary tumor. This cell line was shown to express high levels of cyclooxygenase (COX) -2 mRNA and protein as detected by Northern and Western blotting as well as immunostaining. PGE(2) production by C3L5 cells was primarily owing to COX-2, since this was blocked similarly with non-selective COX inhibitor indomethacin and selective COX-2 inhibitor NS-398, but unaffected with the selective COX-1 inhibitor valeryl salicylate. C3L5 cell proliferation/survival in vitro was not influenced by PGs, since their cellularity remained unaffected in the presence of PGE(2) or NS-398 or PG-receptor (EP1/EP2) antagonist AH6809; a marginal decline was noted only at high doses of indomethacin, which was not abrogated by addition of exogenous PGE(2). Migratory and invasive abilities of C3L5 cells, as quantitated with in vitro transwell migration/invasion assays, were inhibited with indomethacin or NS-398 or AH6809 in a dose-dependent manner; the indomethacin and NS-398-mediated inhibition was partially reversed upon addition of exogenous PGE(2). An in vivo angiogenesis assay that used subcutaneous implants of growth factor-reduced matrigel inclusive of tumor cells showed a significant inhibition of blood vessel formation in these implants in animals treated with indomethacin compared with animals receiving vehicle alone. These studies show that selective and nonselective COX-2 inhibitors retarded tumor progression in this COX-2-expressing murine mammary tumor model by inhibiting tumor cell migration, invasiveness and tumor-induced angiogenesis. The inhibitory effects were not entirely PG dependent; some PG-independent effects were also noted.

Animals↗
PubMed2001

Role of nitric oxide in carcinogenesis and tumour progression.

Nitric oxide (NO) is a short-lived molecule required for many physiological functions, produced from L-arginine by NO synthases (NOS). It is a free radical, producing many reactive intermediates that account for its bioactivity. Sustained induction of the inducible form of NOS (iNOS) in chronic inflammation may be mutagenic, through NO-mediated DNA damage or hindrance to DNA repair, and thus potentially carcinogenic. Expression of iNOS is positively associated with P53 mutation in tumours of the colon, lung, and oropharynx. Progression of a large majority of human and experimental tumours seems to be stimulated by NO resulting from activation of iNOS or constitutive NOS, whereas inhibition is documented in others. This discrepancy is largely explained by differential sensitivity of tumour cells to NO-mediated cytostasis or apoptosis and clonal evolution of NO-resistant and NO-dependent cells. P53 mutation or loss is one of many events linked with NO resistance and dependence. NO can stimulate tumour growth and metastasis by promoting migratory, invasive, and angiogenic abilities of tumour cells, which may also be triggered by activation of cyclo-oxygenase (COX)-2. Thus, selective inhibitors of NOS, COX, or both may have a therapeutic role in certain cancers.

Apoptosis↗
PubMed2001

Insulin-like growth factor-binding protein 1 stimulates human trophoblast migration by signaling through alpha 5 beta 1 integrin via mitogen-activated protein Kinase pathway.

A highly migratory subpopulation of the human placental trophoblast, known as the extravillous trophoblast (EVT), invades the uterus and its vasculature, to establish adequate exchange of key molecules between the maternal and fetal circulations. During their formation, EVT cells selectively acquire alpha 5 beta 1 integrin. We had shown that alpha 5 beta 1 is required for their migratory function, and that EVT cell migration is stimulated by insulin-like growth factor-binding protein (IGFBP)-1 produced by the uterine decidua. The present study examined whether this stimulation is dependent on binding of the Arg-Gly-Asp (RGD) domain of IGFBP-1 to an RGD binding site on the alpha 5 beta 1 integrin, followed by activation of focal adhesion kinase (FAK) and stimulation of the mitogen-activated protein kinase (MAPK) pathway. IGFBP-1 treatment increased migration of EVT cells, whereas an anti-alpha 5 beta 1 integrin antibody blocked migration regardless of IGFBP-1 treatment. Migration stimulation by IGFBP-1 was abrogated by pretreatment with a Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), but not a Gly-Arg-Gly-Glu-Ser-Pro (GRGESP) hexapeptide, and by mutation of the RGD domain of IGFBP-1 to Trp-Gly-Asp (WGD). IGFBP-1 treatment caused a rapid localization of immunoreactive FAK to cellular lamellipodia, a rapid increase in phosphorylation of FAK and extracellular-signal regulated kinases 1 and 2. Preincubation of EVT cells with Herbimycin A, a tyrosine kinase inhibitor, abrogated IGFBP-1 effects; whereas an MAPK kinase inhibitor, PD 98059, reduced migration regardless of IGFBP-1 treatment. These results indicate that IGFBP-1 stimulation of EVT cell migration occurs by binding of its RGD domain to the alpha 5 beta 1 integrin, leading to activation of FAK and stimulation of MAPK pathway.

Animals↗
PubMed2001

Stimulation of human extravillous trophoblast migration by IGF-II is mediated by IGF type 2 receptor involving inhibitory G protein(s) and phosphorylation of MAPK.

We have earlier shown that migration and invasiveness of first trimester human extravillous trophoblast cells are stimulated by IGF-II, independently of IGF type 1 receptor and that migration stimulation is the primary reason for increased extravillous trophoblast cell invasiveness induced by IGF-II. In the present study we examined the functional role of IGF type II receptor in IGF-II stimulation of extravillous trophoblast cell migration and the underlying signal transduction pathways including the participation of inhibitory G protein(s) and MAPK. The migratory ability of a well characterized in vitro propagated human first trimester extravillous trophoblast cell line expressing the phenotype of extravillous trophoblast cells in situ was quantitated with a Transwell migration assay under different experimental conditions. We found that the extravillous trophoblast cells expressed an abundance of IGF type 2 receptor as detected by immunostaining and Western blots, and recombinant human IGF-II promoted their migration in a dose- and time-dependent manner. Both polyclonal and monoclonal IGF type 2 receptor-blocking antibodies blocked migration-stimulating effects of IGF-II. Two synthetic IGF-II analogs ([Leu27]IGF-II, which can bind to IGF type 2 receptor and IGF-binding proteins, but not IGF type 1 receptor, and [QAYL-Leu27]IGF-II, which can bind to IGFR-II, but neither IGFR-I nor IGF-binding proteins) both stimulated extravillous trophoblast cell migration to levels higher than those induced by wild-type IGF-II. These results reveal that IGF-II action was mediated by IGF type 2 receptor, independently of IGF type 1 receptor and IGF-binding proteins. Treatment of extravillous trophoblast cell membrane preparations with IGF-II decreased adenylyl cyclase activity in a concentration-dependant manner, indicating the participation of inhibitory G proteins in IGF-II action. This was substantiated further with the findings that increasing intracellular cAMP using forskolin or (Bu)2cAMP inhibited basal extravillous trophoblast cell migration and blocked IGF-II stimulation of migration. IGF-II treatment rapidly stimulated phosphorylation of MAPK (ERK-1 and -2), which was blocked by pretreatment of extravillous trophoblast cells with the MAPK kinase (MEK) inhibitor PD98059. Treatment with this inhibitor also blocked extravillous trophoblast cell migration in the presence or absence of IGF-II. These results, taken together, reveal that IGF-II stimulates extravillous trophoblast cell migration by signaling through IGF type 2 receptor, involving inhibitory G proteins and activating the MAPK pathway.

Adenylyl Cyclases↗
PubMed2000

Nitric oxide promotes murine mammary tumour growth and metastasis by stimulating tumour cell migration, invasiveness and angiogenesis.

The contributory role of nitric oxide (NO) on tumour growth and metastasis was evaluated in a murine mammary tumour model. NO synthase (NOS) protein expression levels were examined in spontaneously arising C3H/HeJ mammary adenocarcinomas and respective lung metastases. In addition, 2 clonal derivatives of a single spontaneous tumour differing in metastatic phenotype (C3L5 and C10; highly and weakly metastatic, respectively) were utilised to investigate (i) the relationship between NOS expression levels and the biological behaviour of tumour cells (e.g., in vitro migratory and invasive capacities, in vivo tumour growth rate and metastatic and angiogenic capacities) and (ii) whether tumour-derived NO stimulated the invasive, migratory and angiogenic capacities of tumour cells. A heterogeneous pattern of endothelial NOS (eNOS) expression was observed in tumour cells in spontaneous primary tumours, and eNOS expression was higher in undifferentiated relative to differentiated tumour zones. However, tumour cells in lung metastatic sites were always strongly eNOS-positive, suggesting that eNOS expression facilitated metastasis. Findings using clonal derivatives supported this notion; s.c. primary tumour growth rate, efficiency of spontaneous metastasis and eNOS expression were higher for C3L5 relative to C10 cell lines. Nevertheless, lung metastases derived from both tumour cell lines were always strongly and homogeneously eNOS-positive. C3L5 cells were more invasive than C10 cells in vitro, but the migratory capacities of the cell lines did not differ. However, migration and invasiveness of both cell lines were inhibited with L-NAME and restored with excess L-arginine. Tumour-associated angiogenesis, measured in Matrigel implants inclusive of tumour cells, was higher for C3L5 relative to C10 cells, and C3L5-induced angiogenesis was reduced with chronic L-NAME treatment of host animals. These findings suggest that tumour-derived eNOS promoted tumour growth and metastasis by multiple mechanisms: stimulation of tumour cell migration, invasiveness and angiogenesis.

Adenocarcinoma↗
PubMed1995

Expression of pituitary peptide 23 in the rat uterus: regulation by estradiol.

Peptide 23 was first identified in pituitary cell conditioned medium as a secreted protein which was regulated in a similar fashion to growth hormone. It was subsequently found to be a member of the C-type lectin gene superfamily and identical to pancreatis associated protein (PAP). It is widely expressed in the gastrointestinal tract. Our present study demonstrates that peptide 23 gene is also expressed in the uterus. Peptide 23/PAP mRNA was at highest levels during estrus and was not detectable in the immature rat uterus. A single injection of 17 beta-estradiol resulted in a transient induction of peptide 23/PAP mRNA in ovariectomized rats whereas a sustained induction was seen with diethylstilbestrol implants. In situ hybridization localized peptide 23/PAP mRNA to the luminal epithelial cells. During gestation, peptide 23/PAP mRNA was detected only in the uterine samples from day 12 to 18 of pregnancy with maximal expression on day 12. Peptide 23 expression was confined to the uterus itself and not expressed in either the decidua or the fetal tissues. PSP/reg, another closely related member of the C-lectin gene family was not expressed in any of these uterine tissues. These results indicate that estrogen may act as a physiological regulator of peptide 23 in the uterus and suggests that this protein may have some role in estrogen action.

Animals↗
PubMed1995

Molecular cloning and expression of peptide 23, a growth hormone-releasing hormone-inducible pituitary protein.

Peptide 23 is a newly identified protein secreted by rat pituitary cells in primary culture. Although the secretion of this protein is stimulated by GH-releasing hormone and inhibited by somatostatin, the N-terminal amino acid sequence of peptide 23 shows no homology to rat GH. Using the polymerase chain reaction technique, we cloned and sequenced the peptide 23 complementary DNA (cDNA). By means of the mixed oligonucleotide-primed amplification of cDNA technique, primers corresponding to the NH2-amino acid sequence of peptide 23 were used to amplify, clone, and sequence a 74-basepair cDNA of peptide 23. This polymerase chain reaction product was then used as a primer to amplify the complete peptide 23 cDNA by means of the rapid amplification of cDNA ends procedure. The cDNA of peptide 23 obtained by the rapid amplification of cDNA ends procedure contained 777 nucleotides and encoded a 175-amino acid protein with a 26-amino acid putative signal peptide. The calculated mol wt of the mature protein (16,613 daltons) was in good agreement with that estimated by polyacrylamide gel electrophoresis (16 kilodaltons). Northern blot analysis revealed a major messenger RNA species of about 0.9 kilobase and a minor species of about 1.7 kilobases in cultured rat anterior pituitary cells. In rats, peptide 23 was most abundant in the pancreas and gastrointestinal tract. A GenBank sequence search revealed complete sequence identity between peptide 23 cDNA and pancreatitis-associated protein cDNA, an approximately 73% homology with human hepatocellular carcinoma cDNA from human hepatocellular carcinoma, 64% homology with bovine pancreatic thread protein cDNA, and 55% homology with rat and human reg cDNAs, which have been reported to be expressed in regenerating pancreatic islets. Therefore, peptide 23 is identical to pancreatitis-associated protein and a member of the C-type lectin supergene family.

Amino Acid Sequence↗
PubMed1995

Age-related changes in peptide-23/pancreatitis-associated protein and pancreatic stone protein/reg gene expression in the rat and regulation by growth hormone-releasing hormone.

Peptide-23 is a 16-kilodalton protein secreted by rat pituitary cells that was first identified because it was regulated by GRF and somatostatin in a similar fashion to GH. Cloning of peptide-23 complementary DNA revealed that it is identical to pancreatitis-associated protein (PAP) and a member of the c-lectin gene family. We examined the expression of peptide-23/PAP and a structurally related protein, pancreatic stone protein (PSP/reg), in the rat gastrointestinal tract. Here we report age-related changes in the expression and GRF regulation of peptide-23. Both peptide-23/PAP messenger RNA (mRNA) and PSP/reg mRNA were virtually undetectable in the small intestine of newborn and 1- and 2-week-old rats. A dramatic increase in the expression of both genes was seen at the time of weaning in the third week postpartum. The abundance of both of these mRNA decreases after 3 and 6 months of age. Peptide-23/PAP mRNA is most abundant in the ileum, whereas PSP/reg is maximally expressed in the pancreas and duodenum. Human GRF analog pellets were implanted sc into adult male rats for 2 weeks to study the chronic effects of GRF on the expression of these genes. Both peptide-23/PAP and PSP/reg mRNA levels in duodenum and jejunum were increased in these rats compared with levels in control rats. However, no increase in peptide-23/PAP mRNA in response to GRF treatment was seen in the ileum, where the level of expression of this gene is very high, and GRF had no effect on peptide-23/PSP expression in the heart, pituitary, or hypothalamus, where expression is normally undetectable. In situ hybridization was used to localize peptide-23/PSP in the small intestine and pancreas of GRF-treated rats. An increase in peptide-23/PAP mRNA was restricted to acinar cells close to islets, whereas little expression was seen in acinar cells distant from islets, suggesting that either peptide-23/PAP may have some paracrine action on the islets, or alternatively, an islet-derived factor may function as a paracrine modulator of peptide-23/PAP expression. These data demonstrate that GRF modulates peptide-23/PAP expression in the gastrointestinal tract in a similar fashion to that previously reported for pituitary cells in primary culture.

Aging↗
PubMed1995

Plasma clearance, tissue uptake and expression of pituitary peptide 23/pancreatitis-associated protein in the rat.

The secretion of peptide 23 by rat pituitary cells is stimulated by growth hormone-releasing hormone and inhibited by somatostatin. Recent cloning of the cognate cDNA for peptide 23 revealed that it is identical to pancreatitis-associated protein (PAP). In the present study, the clearance and tissue uptake of recombinant peptide 23/PAP in normal adult male rats was assessed. The plasma half-life of recombinant peptide 23/PAP was 4.8 +/- 1.4 (S.D.) min. Maximal accumulation of radio-labelled peptide 23/PAP was observed in the kidney, stomach, small intestine and pancreas whereas negligible uptake was seen in the liver, lung or heart. Peptide 23/PAP was detected in a variety of tissue extracts using a radioimmunoassay. Extracts of ileum contained the highest concentrations of peptide 23/PAP. In situ hybridization analysis showed that peptide 23/PAP mRNA was highly expressed in the columnar epithelial cells of ileum, jejunum and duodenum. These observations demonstrate that peptide 23/PAP, a protein previously thought to be of pituitary origin, is widely expressed in the gastrointestinal tract and that it is rapidly removed from the circulation by the kidney and by tissues which express peptide 23/PAP.

Animals↗
PubMed1992

Expression of thromboxane A2 receptor gene and thromboxane A2 synthase in bovine corpora lutea.

Studies were undertaken to investigate the expression of thromboxane (TXA2) receptor gene, from mRNA to functional receptor protein in terms of ligand binding, along with the cellular and subcellular distribution of the enzyme that catalyzes the formation of the ligand for the receptors. Bovine corpora lutea contained a single TXA2 receptor mRNA transcript of 2.8 kb. All the cell types in bovine corpora lutea contained immunoreactive TXA2 synthase, TXB2, TXA2 receptor transcripts, and receptor protein that bound the TXA2 antagonist 9,11-dimethylmethano-11,12-methano-16 (3-iodo-4-hydroxyphenyl)-13-14-dihydro-13-aza-15 alpha beta-omega-tetranor TXA2. The large luteal cells (20-35 microns) contained more receptor transcripts, receptor protein, and immunoreactive TXA2 synthase than did the small luteal cells (12-19 microns), luteal blood vessels, and nonluteal cells (7-12 microns). After correction for the cellular area differences, small luteal cells were seen to contain more receptor protein than did large luteal cells and nonluteal cells. All the cells showed an increase of TXA2 receptors and catalytically active TXA2 synthase from mid-luteal phase to early pregnancy, suggesting the possibility that TXA2 could be a luteotropic eicosanoid. Bovine lung homogenates (a positive control), bovine luteal plasma membranes-mitochondria-lysosomes fraction, rough-smooth endoplasmic reticulum-Golgi fraction, and highly purified nuclei contained 65-kDa immunoreactive protein, presumably representing TXA2 synthase. In addition, the luteal fractions, but not bovine lung, contained other small and large molecular-size immunoreactive proteins. Immunogold electron microscopy showed that immunoreactive TXA2 synthase was present primarily in plasma membranes, rough endoplasmic reticulum, nuclear membranes, and chromatin; and immunoreactive TXB2 was present primarily in different-size vesicles and nuclear chromatin. In summary, the present studies demonstrate for the first time that primarily small and large luteal cells and secondarily blood vessels and nonluteal cells in bovine corpora lutea express TXA2 receptor gene along with the functional TXA2 synthase. The presence of functional enzyme in luteal cell nuclei suggests that the enzyme and/or its product may have previously unrecognized functions in nuclei.

Animals↗
PubMed1990

Catechol estrogen formation in the mouse uterus and its role in implantation.

Estradiol-2/4-hydroxylase (E-2/4-H) activity was determined in the mouse uterus during early pregnancy as well as in ovarian steroid hormone-treated ovariectomized uterus. Under the assay conditions used, E-4-H was the predominant catechol estrogen-forming monooxygenase enzyme. The inhibition of E-4-H activity by SKF-525A, metyrapone and alpha-naphthoflavone suggested involvement of cytochrome P450-dependent monooxygenases. A haloestrogen, 2-fluoroestradiol (2-FL-E2), also inhibited this activity. During the peri-implantation period, no change in uterine E-4-H activity was noted on the morning of days 2 through 5, but the activity significantly (P less than 0.01) increased in the afternoon of day 4 of pregnancy. A single injection of estradiol-17 beta (E2, 100 ng/mouse) to ovariectomized mice significantly (P less than 0.01) elevated the level of E-4-H activity at 24 h as did injections of progesterone (P4, 2 mg/mouse) for 2 days. When 2 days of P4 (2 mg/mouse) treatment was combined with a single injection of E2 (20 ng/mouse), E-4-H activity increased 1.3-fold (P less than 0.05) by 24 h above that of P4 treatment alone. Dexamethasone (200 micrograms/mouse) and cholesterol (2 mg/mouse) treatment for 2 days had no effect on E-4-H activity. Thus, the stimulatory effect of P4 and E2 on E-4-H activity appeared to be specific. The increased activity of uterine E-4-H prior to implantation on day 4 evening and the modulation of its activity by P4 and/or E2 suggest an involvement of 4-hydroxyestradiol in embryo implantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗