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PubMed2001

Platelet activation via the collagen receptor GPVI is not altered in platelets from chronic myeloid leukaemia patients despite the presence of the constitutively phosphorylated adapter protein CrkL.

In this study, we show that the adapter proteins CrkL and Cbl undergo increases in tyrosine phosphorylation and form an intracellular complex in platelets stimulated with the snake venom toxin convulxin, a selective agonist at the collagen receptor glycoprotein VI (GPVI). Constitutive tyrosine phosphorylation of CrkL has previously been reported in platelets from chronic myeloid leukaemia (CML) patients. This was confirmed in the present study, and shown to result in a weak constitutive association of CrkL with Cbl and a number of other unidentified tyrosine-phosphorylated proteins. There was no further increase in phosphorylation of CrkL in CML platelets in response to GPVI activation, whereas phosphorylation of Cbl and its association with CrkL were potentiated. In addition, this was accompanied by a small increase in p42/ 44 mapkinase (MAPK) activity in CML platelets. The functional consequence of the presence of constitutively phosphorylated proteins in CML platelets was investigated by measurement of aminophospholipid exposure and alpha-granule secretion. This revealed little alteration in the concentration-response curves for either in CML platelets stimulated via GPVI, although maximal levels of P-selectin were depressed. Despite the minimal effect on platelet activation in CML patients, we cannot exclude a role for CrkL or Cbl in signal transduction pathways stimulated via GPVI.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Constitutional delay in growth and puberty (CDGP) is associated with hypoleptinaemia.

OBJECTIVE: Serum leptin concentrations are higher in early adolescence compared with childhood and may play a facilitatory role in pubertal development. Constitutional delay in growth and puberty (CDGP) is a disorder of the tempo of physical maturation and may be associated with relative hypoleptinaemia. We have therefore compared serum leptin concentrations in normal boys with those in boys exhibiting constitutional delay of growth, controlling for pubertal status, age and body mass index (BMI). PATIENTS: 23 boys with constitutional delay in growth (n = 17, prepubertal) and puberty (n = 6, early pubertal) and 88 normal boys (n = 64 prepubertal, n = 24 Tanner stage 2). MEASUREMENTS: Serum leptin was measured in a single, non-fasted morning serum sample by radioimmunoassay. Using the data from normal boys, leptin standard deviation scores (sds) were calculated, to account for the independent influences of age and body mass index (BMI) sds. Both chronological age and bone age were used in the calculation of leptin sds in those with delay. RESULTS: BMIsds was significantly lower in prepubertal delays compared with controls but was not different in pubertal subjects. Leptin concentrations were higher in early puberty compared with prepuberty (P < 0.001) in normal boys but were not significantly elevated in pubertal boys with delay compared with prepubertal delays. Although leptin sds, for both chronological age and bone age, was similar in prepubertal controls and delays, it was significantly lower in the pubertal delay group (P < 0.05). In controls leptin sds did not correlate with age. However, in delays leptin sds was negatively correlated with both chronological age (r = - 0.43, P < 0.05) and bone age (r = - 0.68, P < 0.01), indicating that in the older delays leptin levels were lower than expected given their age and BMIsds. CONCLUSIONS: These data indicate that significantly higher leptin levels in Tanner stage 2 compared with prepuberty are not a prerequisite for progression into puberty. However, the absence of elevated leptin concentrations may be associated with delayed puberty in boys.

Biomarkers↗
PubMed1998

Specific NF-kappaB blockade selectively inhibits tumour necrosis factor-alpha-induced COX-2 but not constitutive COX-1 gene expression in HT-29 cells.

Cyclo-oxygenase (COX) is the key regulatory enzyme of the prostaglandin/eicosanoid pathway. While COX-1 is mostly constitutively expressed, the COX-2 isoform is inducible by proinflammatory cytokines. We used an adenoviral vector containing an NF-kappaB super-repressor (Ad5IkappaB) to investigate the role of NF-kappaB in tumour necrosis factor-alpha (TNF-alpha)-mediated COX-2 gene expression in a colonic epithelial cell line. COX-1 mRNA and protein were constitutively expressed in uninfected, control Ad5LacZ- or Ad5IkappaB-infected HT-29 cells with no apparent change following TNF-alpha exposure. COX-2 mRNA and protein expression was undetectable in unstimulated cells but was strongly up-regulated after TNF-alpha stimulation in uninfected and Ad5LacZ-infected HT-29 cells. This induction was prevented in Ad5IkappaB cells. TNF-alpha increased prostaglandin E2 production by 20-fold in Ad5LacZ-infected HT-29 cells compared with uninfected cells and was significantly inhibited in Ad5IkappaB-infected cells in agreement with the COX-2 mRNA findings. We conclude that NF-kappaB activation is critical in mediating COX-2, but not COX-1 gene expression in HT-29 cells. Selective inhibition of COX-2 expression with the NF-kappaB super-repressor may be useful in distinguishing the role of inducible versus constitutive prostaglandins in intestinal function and provides greater specificity than pharmacological inhibitors.

Adenoviridae↗
PubMed2001

Constitutive activation of Lck and Fyn tyrosine kinases in large granular lymphocytes infected with the gamma-herpesvirus agents of malignant catarrhal fever.

Large granular lymphocytes (LGL) with a T or natural killer (NK) lymphoblast morphology and indiscriminate (non-major histocompatibility complex-linked) cytotoxicity for a variety of target cells can be derived in culture from the tissues of animals infected with either alcelaphine herpesvirus-1 (AlHV-1) or ovine herpesvirus-2 (OvHV-2). In this study, LGL survival in the absence of exogenous interleukin-2 was inhibited by the protein kinase inhibitor genestein, but not the p70 s6 kinase inhibitor rapamycin. Constitutive activation of the src kinases Lck and Fyn was demonstrated in a bovine LGL line infected with OvHV-2 and in two rabbit LGL lines infected with AlHV-1. The p44 erk1 and p42 erk2 mitogen-activated protein kinases (MAPK) were also constitutively activated in the LGLs but not control T cells. Lck and Fyn kinase activity in the LGLs did not increase after mitogen (concanavalin A or concanavalin A plus phorbol ester) stimulation of the cells, in contrast to control T cells. Control T cells, but not the LGLs, proliferated after mitogen stimulation. An analysis of tyrosine phosphorylated proteins in the cells indicated that the LGLs exhibited some similarities and differences to activated control T cells. The results demonstrate that the activated phenotype of the LGLs, associated with malignant catarrhal fever virus infection and in the absence of exogenous interleukin-2, involves constitutively activated Lck and Fyn kinases. These are normally crucial for the initial activation of T cells via several cell-surface receptors (e.g. the T-cell receptor and CD2). The inability of the LGLs to proliferate in response to mitogen may be due to an inability of Lck and Fyn to become further activated after mitogen stimulation.

Animals↗
PubMed2000

Constitutively synthesized nitric oxide is a physiological negative regulator of mammalian angiogenesis mediated by basic fibroblast growth factor.

We recently reported that the systemically administered nitric oxide synthase (NOS) inhibitor Nw-nitro-L-arginine methyl ester, L-NAME, administered before, during and after the angiogenic treatment stimulated angiogenesis induced by basic fibroblast growth factor, bFGF, in the rat. This suggests that suppression of constitutively expressed NOS, cNOS, plus inducible NOS, iNOS, and thus reduced production of nitric oxide, NO, was the stimulating factor. In those studies, the rat mesenteric-window angiogenesis assay was used. Moreover, the systemic administration of a NO releaser inhibited bFGF-mediated angiogenesis. Using the same experimental system, we have now studied whether the inhibition of cNOS alone in adult animals under physiological conditions, i.e. prior to the administration of the angiogenic stimulation with bFGF, affected the subsequent angiogenic response. cNOS constitute endothelial cell NOS (ecNOS) and neuronal NOS (nNOS). L-NAME or its inactive enantiomer Nw-nitro-D-arginine methyl ester, D-NAME, were given continuously in the drinking water (1.0 g/L) during 14 days prior to the start of the treatment with bFGF. The treatment with L-NAME significantly enhanced the subsequent angiogenic response. NO synthesized under physiological conditions by ecNOS in endothelial cells and platelets or nNOS in platelets may thus act as a first constitutional angiostatic factor in bFGF-mediated mammalian angiogenesis.

Animals↗
PubMed1996

In vitro characterization of constitutive CysB proteins from Salmonella typhimurium.

Expression of the cysteine regulon in Salmonella typhimurium and Escherichia coli is controlled by the LysR-type transcriptional activator CysB and by the inducer N-acetyl-L-serine. Sulphide and thiosulphate are anti-inducers. Two highly purified constitutive CysB proteins, CysB(T149M) and CysB(T149P), were found to bind to the cysJIH, cysK and cysP promoters, to activate transcription from the cysJIH and cysK promoters in the absence of N-acetyl-L-serine, and to be insensitive to the effects of anti-inducers. At 10 mM MgCl2, the in vitro transcription activity of CysB(T149M) was maximal without N-acetyl-L-serine, but that of CysB(T149P) was increased by inducer. At 2 mM MgCl2, both proteins were fully active without inducer. A third mutant protein, CysB(W166R), was totally inactive at 10 mM MgCl2, but gave constitutive expression of the cysK and cysJIH promoters at 2 mM MgCl2. Surprisingly, wild-type CysB was also constitutive for the cysK promoter at 2 mM mgCl2 but not at 10 mM MgCl2; it required inducer for cysJIH promoter activation at both concentrations. Mutagenic studies indicated that this difference between promoters is due to the distance between activation site half-sites, which are separated by 1 bp in the cysJIH promoter and by 2 bp in the cysK promoter. We speculate that inducer acts to decrease the distance between the binding domains of two CysB subunits that interact with an activation site. In vitro activities of wild-type and mutant CysB proteins correlated much better with in vivo behaviour at 2 mM than at 10 mM MgCl2, suggesting that the former is the more physiological concentration.

Alleles↗
PubMed2003

A six amino acid deletion, partially overlapping the VanSB G2 ATP-binding motif, leads to constitutive glycopeptide resistance in VanB-type Enterococcus faecium.

Enterococcus faecium clinical isolate BM4524, resistant to vancomycin and susceptible to teicoplanin, harboured a chromosomal vanB cluster, including the vanSB/vanRB two-component system regulatory genes. Enterococcus faecium strain BM4525, isolated two weeks later from the same patient, was resistant to high levels of both glycopeptides. The ddl gene of BM4525 had a 2 bp insertion leading to an impaired d-alanine:d-alanine ligase. Sequencing of the vanB operon in BM4525 also revealed an 18 bp deletion in the vanSB gene designated vanSBDelta. The resulting six amino acid deletion partially overlapped the G2 ATP-binding domain of the VanSBDelta histidine kinase leading to constitutive expression of the resistance genes. Sequence analysis indicated that the deletion occurred between two tandemly arranged heptanucleotide direct repeats, separated by 11 base-pairs. The VanSB, VanSBDelta and VanRB proteins were overproduced in Escherichia coli and purified. In vitro autophosphorylation of the VanSB and VanSBDelta histidine kinases and phosphotransfer to the VanRB response regulator did not differ significantly. However, VanSBDelta was deficient in VanRB phosphatase activity leading to accumulation of phosphorylated VanRB. Increased glycopeptide resistance in E. faecium BM4525 was therefore a result of the lack of production of d-alanyl-d-alanine ending pentapeptide and to constitutive synthesis of d-alanyl-d-lactate terminating peptidoglycan precursors, following loss of d-alanine:d-alanine ligase and of VanSB phosphatase activity respectively. We suggest that the heptanucleotide direct repeat in vanSB may favour the appearance of high level constitutively expressed vancomycin resistance through a 'slippage' type of genetic rearrangement in VanB-type strains.

Bacterial Proteins↗
PubMed2002

Constitutive gain-of-function mutants in a nucleotide binding site-leucine rich repeat protein encoded at the Rx locus of potato.

Rx in potato encodes a protein with a nucleotide binding site (NBS) and leucine-rich repeats (LRR) that confers resistance against Potato virus X. The NBS and LRR domains in Rx are present in many disease resistance proteins in plants and in regulators of apoptosis in animals. To investigate structure-function relationships of NBS-LRR proteins we exploited the potential of Rx to mediate a cell death response. With wild-type Rx cell death is elicited only in the presence of the viral coat protein. However, following random mutagenesis of Rx, we identified mutants in which cell death is activated in the absence of viral coat protein. Out of 2500 Rx clones tested there were seven constitutive gain-of-function mutants carrying eight independent mutations. The mutations encoded changes in the LRR or in conserved RNBS-D and MHD motifs of the NBS. Based on these findings we propose that there are inhibitory domains in the NBS and LRR. The constitutive gain-of-function phenotypes would be due to deletion or modification of these inhibitory domains. However activation of Rx is not simply release of negative regulation by the LRR and adjacent sequence because deleted forms of Rx that lack constitutive gain of function mutations are not active unless the protein is overexpressed.

Agrobacterium tumefaciens↗
PubMed2000

Mouse brain and muscle tissues constitutively express high levels of Homer proteins.

In order to characterize expression of Homers in mouse brain and peripheral tissues we have developed a coupled reverse transcription (RT)-PCR/restriction digestion approach. This has allowed us to determine the molecular composition and relative levels of the constitutive expression of the Homer-1, -2 and -3 mRNAs across mouse tissues. We report here that mammalian brain constitutively expresses high levels of the Homer-1, -2 and -3 mRNAs. Expression of the Homer-1 mRNAs reaches 66% of the brain total Homer mRNAs expression, followed by Homer-3 mRNA (22%) and Homer-2 mRNAs (12%). Quantitative RT-PCR analysis and the Western blotting using pan-Homer antibody revealed that mouse heart, skeletal muscle and diaphragm constitutively express high levels of the Homer proteins and their mRNAs. We have shown that the molecular profile of expression of Homer-1, -2 and -3 mRNAs in muscle containing tissues resembles that obtained for mammalian brain.

Animals↗
PubMed2003

Constitutive expression of the low-affinity neurotrophin receptor and changes during axotomy-induced death of sensory neurones in the neonatal rat dorsal root ganglion.

Sensory neurones in the dorsal root ganglion (DRG) of the neonatal rat express the 75-kDa low-affinity neurotrophin receptor (p75NTR) and these neurones degenerate rapidly after axotomy. p75NTR belongs to the tumour necrosis factor superfamily, several members of which have a role in cell death and it is constitutively expressed within a subpopulation of DRG neurones. p75NTR has been implicated in mediating the degeneration of these neurones after axotomy. In this study, we characterize the expression of p75NTR in sensory neurones of the newborn rat DRG using immunohistochemistry. Furthermore, we investigate the change in constitutive expression pattern of p75NTR in these neurones following axotomy. In the C7 and C8 DRG of the newborn rat, p75NTR is expressed in approximately 70% of DRG neurones. Those expressing p75NTR can be classified into subpopulations with moderate or intense p75NTR expression, each present in approximately equal proportions. Whilst p75NTR expression is observed in neurones throughout the entire neuronal diameter range, a correlation exists between neuronal diameter and p75NTR expression intensity. We also found that the most vulnerable population following axotomy were those sensory neurones which constitutively express the highest levels of p75NTR, i.e. the large-diameter neurones.

Animals↗
PubMed1998

Membrane transport of neuronal nitric oxide synthase substrate L-arginine is constitutively expressed with CAT1 and 4F2hc, but not CAT2 or rBAT.

The potential induction of cationic and zwitterionic amino acid transport systems and mRNA transcripts was investigated in primary neuronal cultures from rat hypothalamus/brainstem. Cultures exposed to bacterial lipopolysaccharide (LPS) plus interferon-gamma (IFNgamma) were assessed with respect to northern blot analyses, L-leucine/L-arginine cross-inhibition uptake profiles in the presence and absence of Na+, and initial rate sodium-independent L-arginine transport kinetics. L-Arginine uptake activity was constitutively expressed along with uninduced steady-state levels of CAT1 and 4F2hc transcripts. However, neither the high-affinity nor the low-affinity alternatively spliced inducible isoforms of CAT2 or CAT2a transcripts (encoding system y+ in control astrocytes, lymphocytes, or liver) nor the rBAT transcripts (encoding system b(o,+) in control intestinal epithelial cells) were detected by northern analysis of neuronal mRNA. Cross-inhibition profiles were consistent with physiologic system y+ activity, but not system b(o,+) or system y+ L. Transport kinetics gave a single component with Vmax = 113 +/- 7 pmol/min/mg of protein and Km = 47 +/- 8 microM L-arginine; these kinetic parameters were not influenced by addition of LPS/IFNgamma at concentrations that up-regulated CAT2 mRNA and system y+ activity in control astroglia from the same area of the brain. The data are consistent with L-arginine membrane uptake occurring via only system y+ encoded by constitutive CAT1, with possible physiologic contribution by constitutive 4F2hc transcripts in primary neuronal cultures.

Amino Acid Transport Systems, Basic↗
PubMed1999

Involvement of the carboxyl terminus of the third intracellular loop of the cannabinoid CB1 receptor in constitutive activation of Gs.

The human cannabinoid receptor CB1 functionally couples primarily to Gi-, but also to Gs-mediated pathways to modulate intracellular cyclic AMP (cAMP) levels. To probe the features of the receptor that may be involved in promoting interactions with one G protein type over another, we generated the L341A/A342L mutant CB1 receptor. The double mutation involved the swap in position of two adjacent residues in the carboxyl-terminal segment of the third intracellular loop of CB1. This resulted in partial constitutive activation of the receptor and an agonist-independent enhancement in cAMP levels. Characterization following treatment with either pertussis or cholera toxin indicated that the constitutive activity is selective for a Gs- and not a Gi-mediated pathway. Treatment with the CB1-specific inverse agonist SR141716A inhibited the basal accumulation of cAMP in the presence of pertussis toxin, establishing that the effect is CB1 mediated. The binding of the agonist CP-55,940 to the L341A/A342L receptor was not markedly different from that for the wild-type receptor despite the constitutive Gs activity. This may reflect a preference of this ligand for an activated receptor state associated with the Gi coupling form and underscores the potential for developing therapeutics that selectively activate one pathway over another.

Animals↗
PubMed2001

Constitutive expression of functional GABA(B) receptors in mIL-tsA58 cells requires both GABA(B(1)) and GABA(B(2)) genes.

Studies of gamma-aminobutyric acid (GABA)(B) receptor function in heterologous cell systems have suggested that expression of two distinct seven transmembrane G-protein coupled receptor subunits is necessary for receptor activation and signal transduction. Some results suggest that both receptor proteins must be inserted into the plasma membrane to create heterodimers; however, it is possible that subunit monomers or homodimers are functional in cells which constitutively express GABA(B) receptors. A new pituitary intermediate lobe melanotrope cell clone (mIL tsA58) has been isolated which constitutively expresses GABA(B), D(2) and corticotrophin releasing factor receptors. Here, we report on characterization of the GABA(B) receptors. Solution hybridization-nuclease protection assays reveal the presence of GABA(B(1)) and GABA(B(2)) transcripts. Western blots show GABA(B(1a)) and one of two GABA(B(2)) proteins. Addition of the GABA(B) agonist baclofen to cultured mIL-tsA58 (mIL) cells inhibits high voltage activated Ca(2+) channels, as measured by agonist-induced inhibition of the K(+)-depolarization-stimulated increase in Ca(2+) influx. CGP55845, a GABA(B) antagonist, blocks the response to baclofen. Knockdown of either GABA(B(1)) or GABA(B(2)) subunits with selective antisense oligodeoxynucleotides reduced GABA(B) protein levels and completely abolished the GABA(B) receptor response in the mIL cells. Taken together, these results indicate that functionally active GABA(B) receptors in mIL cells require the constitutive expression of both GABA(B) genes. This is a physiologic validation of results from recombinant overexpression in naive cells and shows that the mIL cell line is a useful model for studying GABA(B) receptor expression, regulation and function.

Animals↗
PubMed2002

Constitutive activation of the opioid receptor-like (ORL1) receptor by mutation of Asn133 to tryptophan in the third transmembrane region.

We have introduced a series of point mutations into the human opioid receptor-like (ORL1) receptor and characterized them for their ability to constitutively activate G protein-coupled receptor signalling pathways. Among the 12 mutants generated, mutation at Asn133 (N133W) gave increased basal signalling through three separate pathways. N133W increased the basal activity of G14- and G16-dependent pathways by two- to three-fold. The constitutive activity of the mutant was confirmed by the finding that the enhanced activity is dependent on the level of receptor expression. In HEK-293 cells stably expressing N133W, signalling through Gi/o-dependent pathways was also observed. Radioligand binding studies revealed that the affinity for nociceptin of the wild-type ORL1 receptor and the N133W mutant do not differ significantly, suggesting that the ligand binding and signalling functions of constitutively active mutants of G protein-coupled receptors are not necessarily intrinsically linked. In conclusion, our results demonstrate that a mutation in the third transmembrane domain is able to increase the basal signalling activity of the human ORL1 receptor.

Amino Acid Substitution↗
PubMed2003

Constitutively and autonomously active protein kinase C associated with 14-3-3 zeta in the rodent brain.

Persistent activation of protein kinase C (PKC) is required for the expression of synaptic plasticity in the brain. There are several mechanisms proposed that can lead to the prolonged activation of PKC. These include long lasting production of lipid activators (diacylglycerol and fatty acid) through mitogen-activated protein (MAP) kinase pathway, and a modification of PKC by reactive oxygen species. In nerve growth factor (NGF)-differentiated PC12 cells, we found that constitutive and autonomous Ca2+-independent PKC activity is associated with 14-3-3 zeta. Because PKC and 14-3-3 zeta are both involved in synaptic plasticity and learning and memory, we examined whether PKC interacts with 14-3-3 zeta in the brain and whether the PKC/14-3-3 zeta complex has autonomous activity. Here we show that three subclasses of PKC, Ca2+-dependent classical PKC, Ca2+-independent novel PKC, and Ca2+-independent and diacylglycerol-insensitive atypical PKC, all interact with 14-3-3 zeta in the rodent brain. The pool size of 14-3-3 zeta bound form of PKC is small (1-4% of each PKC isoform), but they show constitutive and autonomous activity. Our study indicates that the binding of PKC with 14-3-3 zeta is at least in part independent of phosphorylation of PKC and that the C1 domain of PKC is involved in the binding. As both molecules are enriched in synaptic locus, the constitutive PKC activity and its interaction with 14-3-3 zeta could be a mechanism for the persistent PKC activation in the brain.

14-3-3 Proteins↗
PubMed2000

Indolinone derivatives inhibit constitutively activated KIT mutants and kill neoplastic mast cells.

Mastocytosis is a neoplastic disease caused at least in part by somatic mutations of the c-KIT proto-oncogene resulting in constitutive activation of its protein product, KIT, the receptor tyrosine kinase for stem cell factor. KIT stimulates mast cell proliferation and prevents apoptosis of neoplastic mast cells. To develop potential therapies for mastocytosis we used indolinones, small molecules that inhibit tyrosine kinases. Four indolinone derivatives (SU4984, SU6663, SU6577, and SU5614) inhibited wild-type KIT, but variably inhibited constitutively activated KIT mutants. SU4984, SU6577, and SU5614 were effective against KIT with juxtamembrane activating mutations, whereas only SU6577 could suppress KIT containing either juxtamembrane or kinase domain activating mutations. Furthermore, SU4984, SU6577, and SU5614 killed neoplastic mast cells expressing a juxtamembrane-mutated KIT, whereas SU4984 and SU6577 killed neoplastic mast cells expressing KIT bearing a kinase domain mutation. These data show a direct correlation between inhibition of constitutively activated KIT and the death of neoplastic mast cells, and point to specific tyrosine kinase inhibitors as a potential therapy aimed directly at a cause of mastocytosis.

Gene Expression Regulation↗
PubMed2003

Constitutive overexpression of human telomerase reverse transcriptase but not c-myc blocks terminal differentiation in human HaCaT skin keratinocytes.

Formation of a well structured epidermis strictly depends on a tight balance between proliferation and differentiation. Accordingly, telomerase, which is restricted to proliferating cells, is downregulated with differentiation. It is unclear, however, whether this inhibition is essential to or only a consequence of the differentiation process. By studying different variants of the HaCaT skin keratinocytes we now show that constitutive overexpression of human telomerase reverse transcriptase (hTERT) in HaCaT-TERT cells (lacking its own differentiation-sensitive promoter) and constitutive expression of the c-myc gene in HaCaT-myc cells caused increased proliferation in conventional cultures; however, this proliferative advantage was not maintained in tissue-like organotypic cocultures. Despite reduced stratification, HaCaT-myc cells were still able to develop a fully differentiated epithelium. HaCaT-TERT cultures, on the other hand, expressed all markers of early but not of terminal differentiation. The failure to differentiate terminally was observed in hTERT mass cultures and individual clones and correlated with an intense nuclear hTERT staining of the uppermost cells of the HaCaT-TERT epithelia. Thus, our data suggest that constitutive overexpression of hTERT does not interfere with epidermal differentiation per se but blocks the terminal stage of differentiation and therefore indicates that hTERT/telomerase plays an active part in the regulatory pathway of epidermal differentiation.

Biomarkers↗
PubMed2002

Constitutive expression and function of cyclooxygenase-2 in murine gastric muscles.

BACKGROUND & AIMS: Cyclooxygenase enzymes (COX) generate intermediates in the prostaglandin (PG) cascade. COX-1 is constitutively expressed in many cells, and COX-2 is typically thought to be an inducible isoform. METHODS: We evaluated constitutive expression and function of COX-2 in murine gastric muscles. RESULTS: Immunohistochemistry showed COX-2-like immunoreactivity (COX-2-LI) in myenteric neurons. Half the neurons with COX-2-LI expressed nitric oxide synthase (NOS). COX-2-LI was not observed in smooth muscle cells. Interstitial cells of Cajal within muscle layers (IC-IM) expressed COX-2-LI, suggesting a novel role for IC-IM. Molecular studies verified expression of COX-2 in gastric muscles. Quantitative polymerase chain reaction (PCR) showed equal expression of COX-1 and COX-2 in the antrum. COX-2 was more abundant in fundus. Indomethacin and GR253035X, a COX-2 inhibitor, increased antral phasic contractions and potentiated responses to ACh. Indomethacin, but not GR253035X, increased contractions and potentiated responses in tissues of COX-2 knockout mice. Indomethacin and GR253035X reduced tone in the fundus. CONCLUSIONS: COX-2 is constitutively expressed by IC-IM and neurons in the stomach and at levels similar to COX-1. Prostanoids produced by COX-2 regulate mechanical activities of fundus and antral muscles.

Acetylcholine↗