TP Activity Research Articles

Regulation of Thromboxane Receptor Trafficking Through the Prostacyclin Receptor in Vascular Smooth Muscle Cells

Prostacyclin (PGI2) and thromboxane (TxA2) effect disparate outcomes for atherogenesis and the response to vascular injury; PGI2, a vasodilator and inhibitor of platelet aggregation, limits the deleterious actions of TxA2, a vasoconstrictor and platelet activator. Dimerization of their G protein-coupled receptors, IP and TP, evokes a modified cellular response through which IP/TP counter-balance may be effected. We examined the consequence of IP/TP interaction for the regulatory pathways of both receptors. TPalpha overexpressed in HEK293 cells or expressed endogenously in aortic smooth muscle cells (ASMCs) was internalized after selective activation of either TP or IP. Homologous trafficking of TP was unaltered by coexpression of IP. Heterologous sequestration of TPalpha required the physical presence of activated IP, in transfected and native cells, but was independent of IP signaling to adenylyl cyclase. Reciprocal heterologous regulation of IP, via activated TP, was evident in both HEK293 cells and ASMCs. Homologous TP internalization led to receptor retention and degradation. In contrast, when internalization was IP-induced, TPalpha was recycled to the cell surface in coexpressing HEK293 cells, but not in ASMCs, in accord with the postendocytotic pathway of IP. IP/TPalpha interaction permits reciprocal regulation of receptor endocytosis via the trafficking pathway determined by the activated dimeric partner.

Influence of different condylar and incisal guidance ratios to the activity of anterior and posterior temporal muscle

Object Few studies quantify reactions of masticatory muscles during clenching at different occlusal positions in individuals with different anterior and posterior guidance relationship and different number of working-side occlusal contacts. The hypothesis that altered incisal guidance (IG) and a different number of working-side occlusal contacts in complete denture wearers (CDW) change a pattern of temporal muscle activity and loadings to the mandible during clenching in incisal and lateral positions was tested. Design EMG activity during clenching in incisal (IP) and left (LOP) and right lateral occlusal (ROP) positions was compared between dentate subjects (DS) with steeper IG than condylar guidance (PG) and canine or canine + first premolar guidance in laterotrusive movements with CDWs who had steeper PG than IG and group function. EMG values were expressed as percentages of maximum voluntary clenching in maximum intercuspation (ICP) in each individual. Results The CDWs exhibited significantly higher posterior temporal muscle (TP) activity in IP and during lateral biting on mediotrusive side than DSs. Their coronoid process had to be pulled backward by TP fibers to rotate condyle in a counter-clockwise direction (PG > IG); contrary DSs had to rotate condyle in a clockwise direction to compensate for vertical overlap (IG > PG). Group function allowed more working-side contacts in CDWs and significantly higher anterior temporalis (TA) activity. Conclusion Alteration of IG–PG ratio and a number of occlusal contacts during lateral clenching change a pattern of TA and TP activity in CDWs and a direction of mandibular loadings, although age related changes might also be responsible.

Alkyne hydrophosphinylation catalyzed by rhodium pyrazolylborate complexes

Despite numerous examples of stoichiometric bond activation reactions using rhodium pyrazolylborate complexes, the use of these complexes in catalytic reactions is still uncommon. We have evaluated the activity of Tp ∗Rh(PPh 3) 2 [Tp ∗ = hydrotris(3,5-dimethylpyrazolyl)borate] and Tp ∗Rh(cod) (cod = cyclooctadiene) in alkyne hydrophosphinylation relative to a series of known catalysts for this reaction. Both Tp ∗Rh(PPh 3) 2 and Tp ∗Rh(cod) are active catalysts for alkyne hydrophosphinylation, although neither is as active as Wilkinson’s catalyst. The ability of Tp ∗Rh(PPh 3) 2 to effect hydrophosphinylation of a series of alkyne substrates with diphenylphosphine oxide is also presented. In addition, we report the crystal structures of the products of the reactions between diphenylphosphine oxide and both [μ-ClRh(cod)] 2 and Tp ∗Rh(PPh 3) 2. The former is a possible catalytically relevant intermediate, while the latter is inactive.

Proteolytic activity and reduction of gliadin-like fractions by sourdough lactobacilli

To characterize the peptide hydrolase system of Lactobacillus plantarum CRL 759 and CRL 778 and evaluate their proteolytic activity in reducing gliadin-like fractions. The intracellular peptide hydrolase system of Lact. plantarum CRL 759 and CRL 778 involves amino-, di- (DP), tri- (TP) and endopeptidase activities. These peptidases are metalloenzymes inhibited by EDTA and 1,10-phenanthroline and stimulated by Co2+. DP and TP activities of Lact. plantarum CRL 759 and CRL 778, respectively, were completely inhibited by Cu2+. Lactobacillus plantarum CRL 778 showed the highest proteolytic activity and amino acids release in fermented dough. The synthetic 31-43 alpha-gliadin fragment was hydrolysed to 36% and 73% by Lact. plantarum CRL 778 and CRL 759 respectively. Lactobacillus plantarum CRL 759 and CRL 778 have an active proteolytic system, which is responsible for the high amino acid release during sourdough fermentation and the hydrolysis of the 31-43 alpha-gliadin-like fragment. This work provides new information of use when obtaining sourdough starters for bread making. Moreover, knowledge regarding lactobacilli capable of reducing the level of gliadin-like fractions, a toxic peptide for coeliac patients, has a beneficial health impact.

Paclitaxel and carboplatin (TP) for treatment of advanced or recurrent endometrial cancer: A retrospective study

5020 Background: For patients with advanced or recurrent endometrial cancer, a community standard treatment is TP, though there have been no large supportive studies. GOG trial #209 is comparing the control arm of paclitaxel, doxorubicin and cisplatin (TAP) to TP in the treatment of advanced or recurrent endometrial cancer but results of this trial are years away. Methods: A retrospective analysis was performed on patients with advanced or recurrent endometrial cancer who were treated with TP at our institution between 1/1/90 and 5/30/04. A total of 142 patients were identified, of which the first 68 consecutively treated patients are presented here. Forty patients who had received at least 1 cycle of therapy were eligible for analysis if they met the following conditions that correspond to GOG #209 entry criteria: advanced disease (defined as primary stage III or IV disease or stage III or IV patients with persistent, measurable disease after surgery), recurrent measurable disease, chemotherapy naïve. Twenty-eight patients were excluded (synchronous ovarian carcinoma, prior chemotherapy, carcinosarcoma pathology, incomplete records, no measurable disease at time of treatment). Data were extracted from electronic medical records. This study was approved by the IRB. Results: The median age at diagnosis was 59.9 years (range 47–80 years). Twenty-five percent of patients received treatment in the primary advanced setting, 75% at the time of disease recurrence. Prior radiation therapy had been delivered to 60% of patients. The median number of cycles of TP delivered was 4, with 35% of patients receiving 6 or more cycles of therapy. The overall response rate was 47%, with a complete response rate of 2% and a partial response rate of 45%. Median progression-free survival was 4.2 months (range 0.7–14.5 months). Median overall survival was 11 months (range 1.4–31.6 months). Conclusion: TP achieves high objective response rates in patients with advanced or recurrent endometrial cancer. All 142 patients will be analyzed to further characterize the activity of TP in patients with advanced or recurrent endometrial cancer. No significant financial relationships to disclose.

Hepatoprotective Activity of a Benzopyrone from Tephrosia purpurea Pers.

Objective: To study hepatoprotective activity of a new benzopyrone derivative (TP) isolated from the alcoholic extract of aerial parts of Tephrosia purpurea Pers. Materials and methods: Compound TP was isolated from alcoholic extract of T. purpurea by normal phase column chromatography using toluene: ethyl acetate (70:30) as mobile phase and structure was elucidated by spectroscopic methods. Hepatoprotective activity of TP and the alcoholic extract was evaluated using carbon tetrachloride, paracetamol and rifampicin as toxicants. The potency of TP (100 mg/kg) and the alcoholic extract of aerial parts (200 mg/kg), was compared with silymarin (100 mg/kg). The SGOT, SGPT, total and direct bilirubin levels of blood were measured spectrophotometrically. Results: Compound TP was found to be 3-hydroxy, 6-methoxy, 2-oxy (3-butanone), 7 (dioxolane-4-one), 2, 3,-dihydrobenzopyrone. The alcoholic extract and TP caused significant fall in the enzyme levels in serum in rats. Conclusion: Alcoholic extract of T. purpurea and TP have shown significant hepatoprotective activity in rats against all the toxicants that were studied.

Age-Related Differences in Vasoconstrictor Responses to Isoprostanes in Piglet Pulmonary and Mesenteric Vascular Smooth Muscle

Isoprostanes are prostaglandin (PG)-like compounds produced nonenzymatically by free radical-catalyzed peroxidation of arachidonic acid. Isoprostanes evoke potent vascular effects but their actions in the neonatal vasculature are poorly known. We aimed to study the effects of 8-iso-PGE(1), 8-iso-PGE(2), 8-iso-PGF(1alpha), 8-iso-PGF(1beta), 8-iso-PGF(2alpha), and 8-iso-PGF(2beta) in pulmonary arteries (PA), pulmonary veins (PV), and mesenteric arteries (MA) from newborn and 2-wk-old piglets. Isoprostanes produced concentration-dependent contractions of PA, PV, and MA (magnitudes up to 1.5- to 2-fold greater than the responses to 62.5 mM KCl) but they were markedly less potent vasoconstrictors than the thromboxane A(2) (TXA(2)) mimetic U46619. Neonatal PA were more sensitive to 8-iso-PGF(1alpha), 8-iso-PGF(1beta), and 8-iso-PGF(2beta) than 2-wk-old PA. Neonatal PV were more sensitive to 8-iso-PGE(2) and 8-iso-PGF(1alpha), and neonatal MA were more sensitive to 8-iso-PGE(2), 8-iso-PGF(1alpha), 8-iso-PGF(1beta), 8-iso-PGF(2alpha), and 8-iso-PGF(2beta) than the corresponding 2-wk-old vessels. The sensitivity to U46619 decreased with postnatal age in MA but did not change in PA and PV. The contractile responses to all the isoprostanes and to U46619 were reverted by the TXA(2) receptor (TP) antagonist SQ 29,548. Moreover, isoprostane-evoked contractions in 2-wk-old PA were reduced by inhibitors of tyrosine kinase (genistein) and Rho kinase (Y 27632 and hydroxyfasudil) but not by inhibitors of protein kinase C (chelerythrine), mitogen-activated protein kinase kinase (PD 98059) or p38-kinase (SB 203580). In conclusion, isoprostanes produced compound-, tissue-, and age-dependent constriction of neonatal porcine pulmonary and mesenteric vascular smooth muscle. Isoprostane-evoked PA vasoconstriction involved TP receptors and activation of tyrosine kinases and Rho kinases.

Dimerization of the Human Receptors for Prostacyclin and Thromboxane Facilitates Thromboxane Receptor-mediated cAMP Generation

Prostacyclin (PGI(2)) and thromboxane (TxA(2)) are biological opposites; PGI(2), a vasodilator and inhibitor of platelet aggregation, limits the deleterious actions of TxA(2), a vasoconstrictor and platelet activator. The molecular mechanisms involved in the counterregulation of PGI(2)/TxA(2) signaling are unclear. We examined the interaction of the receptors for PGI(2) (IP) and TxA(2) (TPalpha). IP-induced cAMP and TP-induced inositol phosphate generation were unaltered when the receptors were co-expressed in HEK 293 cells (IP/TPalpha-HEK). TP-cAMP generation, in response to TP agonists or a TP-dependent isoprostane, iPE(2)III, was evident in IP/TPalpha-HEK and in aortic smooth muscle cells, but not in cells expressing either receptor alone, or in IP-deficient aortic smooth muscle cells. Augmentation of TP-induced cAMP generation, with the IP agonist cicaprost, was ablated in IP-deficient cells and was independent of direct IP signaling. IP/TPalpha heterodimers were formed constitutively when the receptors were co-expressed, with no overt changes in ligand binding to the individual receptor sites. However, despite inefficient binding of iPE(2)III to either the IP or TPalpha, expressed alone or in combination, robust cAMP generation was evident in IP/TPalpha-HEK, suggesting the formation of an alternative receptor site. Thus, IP/TPalpha dimerization was coincident with TP-cAMP generation, promoting a "PGI(2)-like" cellular response to TP activation. This represents a previously unknown mechanism by which IP may limit the cellular effects of TP.

Human bronchial smooth muscle cell proliferation via thromboxane A 2 receptor

Thromboxane A 2 receptor (TP) mediates bronchial smooth muscle cell (BSMC) contraction, airway hyperresponsiveness, and airway inflammation in patients with asthma. In the present study, a pathogenic role of TP activation in airway remodeling was examined using primary cultures of human BSMC. A TP agonist, I-BOP, concentration-dependently enhanced not only bromodeoxyuridine (BrdU) uptake but also cell proliferation of BSMC. A TP-selective antagonist, AA-2414, blocked the effects of I-BOP on both BrdU uptake and cell proliferation. I-BOP-induced BrdU uptake was significantly blocked by two non-selective tyrosine kinase inhibitors, genistein and herbimycin A, or a Src family tyrosine kinase inhibitor, PP2, but not by an inhibitor of epidermal growth factor (EGF) receptor-associated tyrosine kinase, AG1478. In conclusion, TP receptor activation causes DNA synthesis and cell proliferation of human BSMC by activating tyrosine kinases including Src, but not by EGF receptor transactivation.

Selective activation of tibialis posterior: evaluation by magnetic resonance imaging.

To determine which exercise most selectively and effectively activates tibialis posterior. Five healthy adults (two men, three women; mean age 31 yr) with an Arch Index (AI) within 1 SD from norm performed three exercises, separated by 1-wk intervals. The exercises were: 1) closed chain resisted foot adduction (foot adduction), 2) unilateral heel raise (heel raise), and 3) open chain resisted foot supination (foot supination). Magnetic resonance transaxial images were obtained immediately before and after exercise using a 1.5-T MRI system. Changes in pre-to postexercise signal intensity were compared across five muscles: tibialis posterior, tibialis anterior, medial gastrocnemius, soleus, and peroneus longus. Postexercise signal intensity was normalized to baseline preexercise signal intensity. Tibialis posterior signal intensity increased after each exercise. The greatest TP increase (50 +/- 6%) occurred after foot adduction, whereas the mean increase in the other muscles was less than 5%. After the heel raise exercise, the signal intensity increase in TP was 27% (+/- 11%), soleus 39% (+/- 8%), peroneus longus 57% (+/- 14%), and medial gastrocnemius 99% (+/- 12%). The signal intensity of tibialis anterior decreased 4% (+/- 2%). After foot supination, the TP signal intensity increased 26% (+/- 7%), whereas the mean change in the other muscles was less than 10%. Multivariate analyses of variance revealed a significant difference in muscle activation between exercises. Posthoc analysis showed greater activation of TP during foot adduction than foot supination (P = 0.021). In individuals with a normal AI, TP was activated selectively and most effectively during foot adduction. Knowledge of selective activation of a muscle is necessary to provide an optimal environment for muscle strengthening and/or tendon rehabilitation.

Thymidine phosphorylase-mediated angiogenesis regulated by thymidine phosphorylase inhibitor in human ovarian cancer cells in vivo

Metastasis or progression of ovarian cancer cells is known to be due to the action of various angiogenic factors. We determined the expression of thymidine phosphorylase/platelet-derived endothelial cell growth factor (TP/PD-ECGF) and vascular endothelial growth factor (VEGF) in cell lines established from 3 serous adenocarcinomas, 3 clear cell carcinomas and 2 mucinous carcinomas of the human ovary. TP activity and the TP mRNA level were much higher in the serous adenocarcinoma cells than in the clear cells and mucinous carcinoma cells, and TP expression was extremely low in the clear cell carcinoma cells. Expression of VEGF mRNA was variable, but not significantly different between the 3 histological types of ovarian cancer. In vivo angiogenesis in the ovarian cancer cells was evaluated by the dorsal air sac assay and revealed that SHIN-3 and HRA serous adenocarcinoma cells, which have high levels of TP expression, induced angiogenesis, while KK clear cell carcinoma cells with low TP expression, did not. The degree of ovarian-cancer-induced angiogenesis seemed to be independent of expression of VEGF in the cells. To confirm that the serous adenocarcinoma-induced angiogenesis is dependent on TP levels, a potent and specific inhibitor of TP was administered orally to mice implanted with a chamber containing SHIN-3 or HRA cells. The TP inhibitor significantly inhibited the angiogenesis induced by the serous adenocarcinoma cells. These results suggest that the angiogenic potency of ovarian cancer cells differs with the histological type and is controlled by expression of TP/PD-ECGF, not by VEGF, and that TP-mediated angiogenesis may be the main factor responsible for progression or metastasis of ovarian serous adenocarcinomas.

Regulation of activator protein-1 by 8-iso-prostaglandin E2 in a thromboxane A2 receptor-dependent and -independent manner.

The thromboxane (TX) A(2) receptor (TP) encompasses two alternatively spliced forms, termed the platelet/placental (TP-P) and endothelial (TP-E) type receptors. Experimental evidence suggests that TP activity may be modulated by novel ligands, termed the isoprostanes, that paradoxically act as TP agonists in smooth muscle and TP antagonists in platelet preparations. Here we have investigated whether prototypical isoprostanes 8-iso-prostaglandin (PG)F(2 alpha) and 8-iso-PGE(2) regulate the activity of TP isoforms expressed in Chinese hamster ovary (CHO) cells using activator protein-1 (AP-1)-luciferase activity as a reporter. AP-1-luciferase activity was increased by a TP agonist [9,11-dideoxy-9 alpha,11 alpha-methanoepoxy PGF(2 alpha) (U46619)] in CHO cells transfected with the human TP-P and TP-E receptors, and this response was fully inhibited by TP antagonists [1S-[1 alpha,2 beta(Z),3 alpha,5 alpha]]-7-[3-[[4-iodophenyl)sulfonyl]amino]-6,6-dimethylbicyclo[3.1.1]hept-2-yl]-5-heptenoic acid (I-SAP) and [1S-[1 alpha,2 alpha(Z),3 alpha,4 alpha]]-7-[[2-[(phenylamino) carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1] hept-2-yl]-5-heptenoic acid (SQ 29,548)]. AP-1-luciferase activity was potently (nanomolar concentrations) increased by 8-iso-PGE(2) in CHO TP-P and TP-E cells, and this response was partially inhibited by cotreatment of cells with TP antagonists, whereas 8-iso-PGF(2 alpha) was without effect. Cyclooxygenase inhibitors did not abolish 8-iso-PGE(2) mediated AP-1-luciferase activity, indicating that this response is not dependent on de novo TXA(2) biosynthesis. Interestingly, 8-iso-PGE(2)-mediated AP-1-luciferase activity was near maximal in naive cells between 1 and 10 nM concentrations, and this response was not inhibited by TP antagonist or reproduced by agonists for TP or EP(1)/EP(3) receptors. These observations 1) support a role for novel ligands in the regulation of TP-dependent signaling, 2) indicate that TP-P and TP-E couple to AP-1, 3) provide further evidence that isoprostanes function as TP agonists in a cell-type specific fashion, and 4) indicate that additional targets regulated by 8-iso-PGE(2) couple to AP-1.

Soluble sugar permeases of the phosphotransferase system in Escherichia coli: evidence for two physically distinct forms of the proteins in vivo.

The bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) consists of a set of cytoplasmic energy-coupling proteins and various integral membrane permeases/sugar phosphotransferases, each specific for a different sugar. We have conducted biochemical analyses of three PTS permeases (enzymes II), the glucose permease (IIGlc), the mannitol permease (IIMtl) and the mannose permease (IIMan). These enzymes each catalyse two vectorial/chemical reactions, sugar phosphorylation using phosphoenolpyruvate (PEP) as the phosphoryl donor, dependent on enzyme I, HPr and IIA as well as IIBC (the PEP reaction), and transphosphorylation using a sugar phosphate (glucose-6-P for IIGlc and IIMan; mannitol-1-P for IIMtl) as the phosphoryl donor, dependent only on IIBC (the TP reaction). When crude extracts of French-pressed or osmotically shocked Escherichia coli cells are centrifuged in an ultracentrifuge at high speed, 5-20% of the enzyme II activity remains in the high-speed supernatant, and passage through a gel filtration column gives two activity peaks, one in the void volume exhibiting high PEP-dependent and TP activities, and a second included peak with high PEP-dependent activity and high (IIMan), moderate (IIGlc) or negligible (IIMtl) TP activities. Both log and stationary phase cells exhibit comparable relative amounts of pelletable and soluble enzyme II activities, but long-term exposure of cells to chloramphenicol results in selective loss of the soluble fraction with retention of much of the pelleted activity concomitant with extensive protein degradation. Short-term exposure of cells to chloramphenicol results in increased activities in both fractions, possibly because of increased lipid association, with more activation in the soluble fraction than in the pelleted fraction. Western blot analyses show that the soluble IIGlc exhibits a subunit size of about 45 kDa, and all three soluble enzymes II elute from the gel filtration column with apparent molecular weights of 40-50 kDa. We propose that enzymes II of the PTS exist in two physically distinct forms in the E. coli cell, one tightly integrated into the membrane and one either soluble or loosely associated with the membrane. We also propose that the membrane-integrated enzymes II are largely dimeric, whereas the soluble enzymes II, retarded during passage through a gel filtration column, are largely monomeric.

The Sp1 transcription factor contributes to the tumor necrosis factor-induced expression of the angiogenic factor thymidine phosphorylase in human colon carcinoma cells.

Thymidine phosphorylase (TP; also known as platelet-derived endothelial cell growth factor, PD-ECGF) is an angiogenic factor that is chemotactic for endothelial cells and has been found to induce neovascularization in vivo. TP is frequently overexpressed in human solid tumors, where its expression has been correlated with increased tumor microvessel density, invasion, and metastasis, and shorter patient survival. In this report, TP activity in the WiDr colon carcinoma cell line was found to be induced 100-fold by tumor necrosis factor (TNFalpha), a secretory product of activated macrophages that has indirect angiogenic activities. Increased TP activity was accompanied by increased TP mRNA levels and without an increase in mRNA stability. TNFalpha-induced TP mRNA levels were reduced by mithramycin, a DNA-binding transcription inhibitor specific for GC-rich sequences. Transcriptional regulation by TNFalpha was confirmed by transient transfection of WiDr with upstream TP sequences in a luciferase reporter construct. Deletion analysis of the reporter pinpointed two regions of the TP promoter with regulatory elements for both TNFalpha-inducible and basal expression, and they contained, respectively, three and one consensus binding sites for the Sp1-family of transcription factors. One additional region contributed only to basal TP expression, and it contained three Sp1 sites. TNFalpha-induced TP expression decreased when point mutations were made in three of the four Sp1 sites postulated to contribute to both basal and TNFalpha-inducible expression. Electrophoretic mobility shift assays further demonstrated binding of nuclear Sp1 to these three sites. Sp1-binding activity was also increased in cells treated with TNFalpha. These studies establish a role for Sp1 in the regulation of expression of the angiogenic factor TP in colon cancer WiDr cells.

Differential peristaltic motor effects of prostanoid (DP, EP, IP, TP) and leukotriene receptor agonists in the guinea-pig isolated small intestine.

1. Since the role of prostanoid receptors in intestinal peristalsis is largely unknown, the peristaltic motor effects of some prostaglandin (DP, EP, IP), thromboxane (TP) and leukotriene (LT) receptor agonists and antagonists were investigated. 2. Propulsive peristalsis in fluid-perfused segments from the guinea-pig small intestine was triggered by a rise of the intraluminal pressure and recorded via the intraluminal pressure changes associated with the peristaltic waves. Alterations of distension sensitivity were deduced from alterations of the peristaltic pressure threshold and modifications of peristaltic performance were reflected by modifications of the amplitude, maximal acceleration and residual baseline pressure of the peristaltic waves. 3. Four categories of peristaltic motor effects became apparent: a decrease in distension sensitivity and peristaltic performance as induced by the EP1/EP3 receptor agonist sulprostone and the TP receptor agonist U-46619 (1-1000 nM); a decrease in distension sensitivity without a major change in peristaltic performance as induced by PGD(2) (3-300 nM) and LTD(4) (10-100 nM); a decrease in peristaltic performance without a major change in distension sensitivity as induced by PGE(1), PGE(2) (1-1000 nM) and the EP1/IP receptor agonist iloprost (1-100 nM); and a decrease in peristaltic performance associated with an increase in distension sensitivity as induced by the EP2 receptor agonist butaprost (1-1000 nM). The DP receptor agonist BW-245 C (1-1000 nM) was without effect. 4. The peristaltic motor action of sulprostone remained unchanged by the EP1 receptor antagonist SC-51089 (1 micro M) and the DP/EP1/EP2 receptor antagonist AH-6809 (30 micro M), whereas that of U-46619 and LTD(4) was prevented by the TP receptor antagonist SQ-29548 (10 micro M) and the cysteinyl-leukotriene(1) (cysLT(1)) receptor antagonist tomelukast (10 micro M), respectively. 5. These observations and their pharmacological analysis indicate that activation of EP2, EP3, IP, TP and cysLT(1) receptors, but not DP receptors, modulate intestinal peristalsis in a receptor-selective manner, whereas activation of EP1 seems to be without influence on propulsive peristalsis. In a wider perspective it appears as if the effect of prostanoid receptor agonists to induce diarrhoea is due to their prosecretory but not peristaltic motor action.

Targeted deletion of both thymidine phosphorylase and uridine phosphorylase and consequent disorders in mice.

Thymidine phosphorylase (TP) regulates intracellular and plasma thymidine levels. TP deficiency is hypothesized to (i) increase levels of thymidine in plasma, (ii) lead to mitochondrial DNA alterations, and (iii) cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). In order to elucidate the physiological roles of TP, we generated mice deficient in the TP gene. Although TP activity in the liver was inhibited in these mice, it was fully maintained in the small intestine. Murine uridine phosphorylase (UP), unlike human UP, cleaves thymidine, as well as uridine. We therefore generated TP-UP double-knockout (TP(-/-) UP(-/-)) mice. TP activities were inhibited in TP(-/-) UP(-/-) mice, and the level of thymidine in the plasma of TP(-/-) UP(-/-) mice was higher than for TP(-/-) mice. Unexpectedly, we could not observe alterations of mitochondrial DNA or pathological changes in the muscles of the TP(-/-) UP(-/-) mice, even when these mice were fed thymidine for 7 months. However, we did find hyperintense lesions on magnetic resonance T(2) maps in the brain and axonal edema by electron microscopic study of the brain in TP(-/-) UP(-/-) mice. These findings suggested that the inhibition of TP activity caused the elevation of pyrimidine levels in plasma and consequent axonal swelling in the brains of mice. Since lesions in the brain do not appear to be due to mitochondrial alterations and pathological changes in the muscle were not found, this model will provide further insights into the causes of MNGIE.

Ethylene Polymerization Behavior of Tris(pyrazolyl)borate Titanium(IV) Complexes

A set of Tp‘TiCl3 and Tp‘TiCl2(OR) complexes containing tris(pyrazolyl)borate ligands with diverse steric properties has been evaluated for ethylene polymerization under MAO activation conditions (Tp‘ = HB(3-mesitylpyrazolyl)2(5-mesitylpyrazolyl)- (TpMs*), HB(3-mesitylpyrazolyl)3 - (TpMs), HB(3,5-Me2-pyrazolyl)3 - (Τp*), HB(pyrazolyl)3 - (Tp), BuB(pyrazolyl)3 - (BuTp)). The activity of Tp‘TiX3/MAO varies in the order TpMs*TiCl3 (10c) > TpMsTiCl3 ≫ Τp*TiCl3, TpTiCl3, BuTpTiCl3, Tp*TiCl2(OtBu), Tp*TiCl2(O-2-tBu-C6H4). The activity of 10c/MAO is similar to that of Cp2ZrCl2/MAO. High MAO levels or addition of AlMe3 decrease the activity of 10c/MAO, probably due to coordination of AlMe3 to the active Ti species. The predominant chain transfer mechanism for 10c/MAO is chain transfer to AlMe3, which results in broad molecular weight distributions at low Al/Ti ratios (Al/Ti = 200−1000). At very high Al levels (10c/5000 MAO or 10c/1000 MAO/4000 AlMe3) bimodal molecular weight distributions comprising a major low molecular weight fraction (M w/M n ca. 3) and a minor high molecular weight fraction are observed, which suggests that several active species are present, only one of which undergoes efficient chain transfer to Al.

Effect of slow wave and REM sleep on thyropharyngeus and stylopharyngeus activity during induced central apneas

The pharyngeal constrictors have been hypothesized to play an important role in the regulation of upper airway (UAW) patency in patients with sleep apnea. However, little research has focused on the activation and control of muscles that determine the lateral and posterior wall of the retropalatal airway dimensions. Our aim was to investigate the effects of slow wave sleep (SWS) and rapid eye movement (REM) sleep on the activation of pharyngeal constrictor (thyropharyngeus; TP) and dilator (stylopharyngeus; SP) muscles during eupneic breathing and induced central apneas. In nine goats, we found that eupneic TP and SP activity progressively decreased from awake to SWS (57 and 56%, respectively; P<0.01) and further in REM (25.6 and 19.9%, respectively; P<0.01). In contrast, diaphragm activity decreased equally during SWS and REM (89.3 and 87.7%, respectively; P<0.01) compared to awake. Following induced apneas while SP activity was eliminated in every state, maximal TP activity was highest in awake state (318.6% of control; P<0.02), less in SWS (157.6%; P<0.02), and nearly absent in REM (117.3%; P>0.02). During the recovery from an induced apnea when diaphragm activity was at 95% of its’ control, awake TP activity remained significantly elevated and SP reduced (P>0.02) while TP activity during SWS was elevated and SP had returned to control level. During REM, TP and SP activity were not different from their reduced controls (P>0.02). The data supports our hypotheses that SWS and REM sleep causes a reduction in the eupneic TP and SP activity, as well as a reduction in TP response to induced apneas. However, the relative imbalance in TP vs SP activity during the recovery from an apnea (awake and SWS) suggest that an imbalance of active neuromuscular forces may contribute to upper airway narrowing in mixed apneas, but not in central apnea during sleep.

The influence of elevated levels of platelet-derived endothelial cell growth factor/thymidine phosphorylase on tumourigenicity, tumour growth, and oxygenation

Purpose: Investigation of the effect of platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) on various aspects of tumour growth in a xenograft model, including growth rate, tumourigenicity and oxygenation levels. Methods and Materials: MDA 231 breast cancer cells overexpressing PD-ECGF/TP protein were made by retroviral transduction. These cells were grown in vitro and in vivo as xenografts. Direct measurement of tumours was used to record growth parameters, while the comet assay with the bioreductive drug RSU 1069 was used to assess tumour cell oxygenation. Results: We report that MDA 231 breast tumour cell lines expressing an increased range of levels of PD-ECGF/TP have increased tumourigenicity positively related to the level of PD-ECGF/TP when implanted in nude mice. As previously reported, tumours grown from these overexpressing cell lines grew faster than the parental line. These tumours expressed higher levels of TP activity and showed increased immunocytochemical staining for PD-ECGF. In addition, the rate of growth was found to be positively related to the level of PD-ECGF/TP expressed by the tumour cells. When the comet assay was used to compare the oxygenation status of cells between the parental and PD-ECGF/TP overexpressing tumours, the latter were found to have a larger proportion of well oxygenated cells. This is consistent with these tumours having an increased and functionally competent vascular supply in response to the expression of PD-ECGF/TP. Conclusion: PD-ECGF/TP appears to be capable of influencing tumourigenicity, angiogenesis and tumour growth in a proportional manner and can directly influence tumour oxygenation levels via its role in formation of functional vasculature.

Vinylic C-H Bond Activation and Hydrogenation Reactions of Tp'Ir(C(2)H(4))(L) Complexes.

The substitution of one of the ethylene ligands of the complexes Tp'Ir(C(2)H(4))(2) (Tp' = Tp(Me)()2, 1; Tp' = Tp, 1) by soft donors such as tertiary phosphines or carbon monoxide is a facile reaction that gives the corresponding Tp'Ir(C(2)H(4))(L) adducts. Spectroscopic studies support their formulation as five-coordinate, 18-electron species that possess a distorted trigonal bipyramidal geometry. This proposal has been confirmed by a single-crystal X-ray study carried out with the PMe(2)Ph complex Tp(Me)()2Ir(C(2)H(4))(PMe(2)Ph) (3b). Related hydride derivatives of Ir(III) can be obtained either by hydrogenation of the Ir(I) adducts (in general, this gives Tp'IrH(2)(L) compounds) or by thermal activation of one of the C-H bonds of the coordinated C(2)H(4) ligand of the Tp(Me)()2Ir(C(2)H(4))(L) compounds. All these reactions can be understood by invoking the participation of transient, 16-electron (eta(2)-Tp')Ir intermediates, but the thermodynamics of the [Ir](C(2)H(4)) to [Ir]H(CH=CH(2)) conversion does not require an overall change in the coordination mode of the Tp' ligand.