Microvascular effects of corticotropin-releasing hormone in human skin vary in relation to estrogen concentration during the menstrual cycle.

Females have a significantly greater life expectancy than males, which in part may be due to the cardio-protective effects of the female sex hormone, estrogen, on vascular function. However, the sex-specific mechanisms contributing to these differences are complex and not fully understood. Previously we have reported that corticotropin-releasing hormone (CRH) has potent dilator effects in the female skin circulation via mast cell degranulation. Furthermore the dilator response to CRH was more enhanced in females than in age-matched males, suggesting that estrogens may be involved. In this study we examined whether CRH-induced dilation and endothelial cell-dependent dilation in the skin circulation of pre-menopausal females were associated with changes in estrogen during the menstrual cycle. CRH-induced dilation (1 nM) was enhanced in the presence of high circulating concentrations of estrogen and a positive correlation was identified between CRH-induced dilation and plasma estrogen concentrations. Endothelial cell-dependent dilation was examined using acetylcholine. Acetylcholine-induced dilation (1 nM) was not correlated with circulating concentrations of estrogen. These data suggest the variation in CRH-induced dilation in the skin microvasculature during the menstrual cycle may be due to estrogenic effects on mast cell function and not due to direct changes in endothelial cell function.

Vasoactive effects of 8-epi-prostaglandin F(2alpha)in isolated human placental conduit and resistance blood vessels in vitro.

The effects of 8-epi-prostaglandin F(2alpha)(8-epi-PGF(2alpha)) and the thromboxane A(2)-mimetic U46619 were examined on isolated human fetal placental arteries obtained from normal pregnancies and from those complicated by pre-eclampsia. The effects of these agents were examined on both conduit and resistance arteries. 8-epi-PGF(2alpha)was found to be markedly less potent than U46619 in constricting both size vessels. Vasoconstrictor EC(50)s for 8-epi PGF(2alpha)were 4.10x10(-7) m (2.02-8.35x10(-7) m) (mean, 95 per cent CI and 2.05x10(-6) m (0.43-9.89 x10(-6) m) in conduit and resistance arteries, respectively. The maximum vasoconstriction produced by 8-epi-PGF(2alpha)(112+/-17 per cent), (relative to maximum KCl induced vasoconstriction) in conduit vessels was significantly less than that caused by U46619 (152+/-20 per cent). In resistance vessels the maximum vasoconstrictor effects to 8-epi-PGF(2alpha)(208+/-10 per cent) and U46619 (201+/-19 per cent) were similar, and in both cases significantly greater than the maximal effects seen in conduit vessels. U46619 displayed a similar vasoconstrictor potency in both conduit (EC(50)=1.21x10(-9) m, 0.58-2.51x10(-9) m) and resistance arteries [EC(50)=5.95x10(-9) m, (0.81-43.60x10(-9) m] as was found for 8-epi PGF(2alpha). 8-epi-PGF(2alpha)was equipotent in resistance arteries obtained from women with severely pre-eclamptic pregnancies (EC(50)=1.25x10(-6) m, 0.25-6.17x10(-6) m) compared with normotensive controls. However, the maximum vasoconstrictor effect induced by 8-epi-PGF(2alpha)in placental resistance arteries was significantly reduced (99+/-20 per cent) in vessels obtained from severely pre-eclamptic compared with normal pregnancies. These results indicate that 8-epi-PGF(2alpha)displays differential vasoconstrictor activity in the fetal-placental vasculature. Furthermore the vasoconstrictor effects of 8-epi-PGF(2alpha)are reduced in pre-eclampsia, the effect being selective to placental resistance vessels. This reduction may occur as a result of more serious disturbances in the placental microcirculation with the disease process in pre-eclampsia.

Dietary n-3 fatty acids alter the contractile response to thromboxane A(2) agonists of porcine coronary arteries.

Dietary supplementation with marine fish oils rich in n-3 fatty acids reduces circulating thromboxane A(2) (TxA(2)). However, the effects on thomboxane A(2) receptor mediated vascular reactivity are uncertain. The aim of this study was to test the hypothesis that dietary modification of TxA(2) levels alters vascular responsiveness to TxA(2) analogues. Juvenile female white pigs were fed a diet enriched in either 5% (w/w) fish oil or beef tallow for 6 weeks. Serum and myocardial tissue levels of eicosapentaenoic and docosahexaenoic acid reached a plateau during this period. Vascular responses were measured in isolated coronary arterial rings with intact endothelium by isometric tension measurement. Arteries from pigs fed fish oil produced a greater maximum vasoconstrictor tension to the TxA(2) analogue U46619 than did rings from pigs fed beef tallow (120 +/- 6% compared to 92 +/- 8%, values represented as a percentage relative to the maximum vasoconstrictor effect obtained to KCl, regression analysis, analysis of variance, P

Effects of nitrovasodilators on the human fetal-placental circulation in vitro.

This study examines the vasorelaxation of isolated human placental chorionic plate arteries and the perfused fetal-placental vasculature, in vitro, to a variety of nitrovasodilator compounds including glyceryl trinitrate (GTN) sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), S-nitroso-N-glutathione (SNG) and NaNO(2). The effects of these compounds were also examined under conditions of high (>450 mmHg) and low oxygen (<50 mmHg) tension. In a separate series of experiments the effects of GTN and NaNO(2)were further investigated with addition of the antioxidants cysteine (100 microm), glutathione (100 microm) or superoxide dismutase (SOD) (30 I.U./ml). The order of nitrovasodilator potency, when added directly to isolated fetal vessels was GTN=SNP>SNAP=SNG>NaNO(2). The order under low oxygen tension was similar, GTN=SNP>SNG= SNAP>or=NaNO(2). SNG ( approximately fourfold) and NaNO(2)( approximately 50-fold) were significantly more potent under low oxygen conditions. Cysteine, glutathione and SOD were without effect on GTN induced vasodilatation. However, all three agents significantly enhanced (six- to ninefold) the effects of NaNO(2)under similar conditions. When infused directly into the fetal-placental circulation during in vitro perfusion experiments the order of potency was GTN>SNP>or=SNG>or=SNAP>or=NaNO(2). When the nitrovasodilators were infused indirectly via the maternal intervillous space the order of potency was GTN>or=SNP>or=NaNO(2)>or=SNAP=SNG. Our observations suggest that there are important differences in the action of different classes of nitrovasodilator compounds on the fetal-placental circulation. The changes observed with SNG and NaNO(2)may be influenced by levels of tissue oxygenation.

Structural characterization of a guanine-quadruplex ligand complex.

The inhibition of telomerase by molecules such as disubstituted amidoanthraquinones is believed to be due to their stabilization of guanine-quadruplex complexes. The characterization is reported of a complex with the intermolecular parallel quadruplex formed from the sequence TGGGGT and a 1,4-bis-piperidino amidoanthraquinone. Crystals obtained did not give single-crystal diffraction; the fiber-like pattern has been interpreted in terms of a repeating unit with four guanine-quartets and two stacked/intercalated ligand molecules. The two categories of possible structures for the complex consistent with this interpretation have been examined by molecular dynamics simulations, with fully solvated environments and 1000 ps simulation times. The two central guanine-quartets in the intercalation model rapidly became highly distorted, whereas the two types of models with ligand stacked externally on the ends of the quadruplex remained very stable. It was concluded that the externally bound ligand complexes best represent the structure of this quadruplex complex, in agreement with earlier NMR results on related systems.

A novel inhibitor of human telomerase derived from 10H-indolo[3,2-b]quinoline.

The bis-dimethylaminoethyl derivative of quindoline (10H-indolo[3,2-b]quinoline), an alkaloid from the West African shrub Cryptolepis sanguinolenta, has been synthesised. This has been shown to have modest cytotoxicity, as well as inhibitory activity against the telomerase enzyme. It is hypothesised that the latter activity is due to stabilisation of an intermediate guanine-quadruplex complex, in accordance with computer modelling.

Fetal placental vascular responses to corticotropin-releasing hormone in vitro. Effects of variation in oxygen tension.

In this study, using the human placenta perfused in vitro with Krebs' bicarbonate solution, we have examined the effects of changes in oxygen tension on the vasoreactivity of fetal placental blood vessels to corticotropin releasing hormone (CRH). Vasodilatory responses to human synthetic CRH were measured during sub-maximal vasoconstriction of the fetal placental circulation with prostaglandin F(2alpha)(PGF(2alpha)) (1-100 micrometer). Decreases in fetal placental arterial perfusion pressure (FAP) were obtained with CRH under conditions of high oxygen or low oxygen tension, >/=450 mmHg and

Evidence for a hormonal tactic maximizing green turtle reproduction in response to a pervasive ecological stressor.

Mortality of breeding sea turtles due to excessive heat exposure after nesting activities is an unusual feature of the Raine Island green turtle rookery. Breeding turtles that fail to return to the ocean after oviposition can experience increasing body temperatures that exceed lethal limits (>39 degrees C) as ambient temperatures rise after sunrise. We investigated how acute increases in body temperature influenced plasma corticosterone (B) concentrations of individual turtles. Furthermore, interactions between progesterone (P) and testosterone (T) and increasing body temperature and the glucocorticoid corticosterone were examined for negative correlations. Breeding green turtles exhibited a 16-fold mean increase in plasma corticosterone concentration as body temperature (cloacal) rose from 28.2 to 40.7 degrees C in less than 6 h. However, the absolute increase in plasma B was small and much less than expected, despite the lethal stressor. Comparatively, the maximal B response to lethal heat stress was similar to plasma B concentrations obtained from breeding female turtles exposed to 8 h of capture stress. However, the maximal B response of breeding turtles exposed to heat and capture stressors was significantly less than the B response of nonbreeding adult female turtles subjected to an 8-h capture stressor. No negative correlations were observed between plasma T and plasma B, between plasma T and body temperature, between plasma P and plasma B, or between plasma P and body temperature. Our findings provide further evidence that reduced adrenocortical function operates in breeding green turtles in the presence of even the most pervasive of environmental stressors.

Structure-activity relationships among guanine-quadruplex telomerase inhibitors.

The ribonucleoprotein telomerase is responsible for maintaining the length of telomeric ends of chromosomes in tumour cells. It is activated in over 85% of the tumour cells, and is emerging as a major target for cancer chemotherapy. A range of molecules containing tricyclic and tetracyclic aromatic chromophores has been shown to inhibit the telomerase enzyme system at the micromolar level. There is evidence that they do so via stabilisation of a guanine-quadruplex structure, which provides a stop signal for further telomere elongation. The known structure-activity relationships for these compounds are summarised, and pointers for the development of future molecules with enhanced selectivity are described.

Nedd8 modification of cul-1 activates SCF(beta(TrCP))-dependent ubiquitination of IkappaBalpha.

Regulation of NF-kappaB occurs through phosphorylation-dependent ubiquitination of IkappaBalpha, which is degraded by the 26S proteasome. Recent studies have shown that ubiquitination of IkappaBalpha is carried out by a ubiquitin-ligase enzyme complex called SCF(beta(TrCP)). Here we show that Nedd8 modification of the Cul-1 component of SCF(beta(TrCP)) is important for function of SCF(beta(TrCP)) in ubiquitination of IkappaBalpha. In cells, Nedd8-conjugated Cul-1 was complexed with two substrates of SCF(beta(TrCP)), phosphorylated IkappaBalpha and beta-catenin, indicating that Nedd8-Cul-1 conjugates are part of SCF(beta(TrCP)) in vivo. Although only a minute fraction of total cellular Cul-1 is modified by Nedd8, the Cul-1 associated with ectopically expressed betaTrCP was highly enriched for the Nedd8-conjugated form. Moreover, optimal ubiquitination of IkappaBalpha required Nedd8 and the Nedd8-conjugating enzyme, Ubc12. The site of Nedd8 ligation to Cul-1 is essential, as SCF(beta(TrCP)) containing a K720R mutant of Cul-1 only weakly supported IkappaBalpha ubiquitination compared to SCF(beta(TrCP)) containing WT Cul-1, suggesting that the Nedd8 ligation of Cul-1 affects the ubiquitination activity of SCF(beta(TrCP)). These observations provide a functional link between the highly related ubiquitin and Nedd8 pathways of protein modification and show how they operate together to selectively target the signal-dependent degradation of IkappaBalpha.

G-quadruplexes as therapeutic targets.

The ends of chromosomes (telomeres) consist of tandem repeats of guanine-rich sequences. In eukaryotics, telomeric DNA is single stranded for the final few hundred bases. These single-stranded sequences can fold into a variety of four-stranded structures (quadruplexes) held together by quartets of hydrogen-bonded guanine bases. The reverse transcriptase enzyme telomerase is responsible for maintaining telomeric DNA length in over 85% of cancer cells by catalyzing the synthesis of further telomeric repeats. Its substrate is the single-stranded 3'-telomeric end. Inhibition of telomere maintenance can be achieved by stabilization of a quadruplex structure for the telomere end. A variety of small molecules have been devised to achieve this, ranging from anthraquinones to porphyrins, acridines, and complex polycyclic systems. Structural and mechanistic aspects of these quadruplex complexes are reviewed here, together with a discussion of the issues of selectivity/potency for quadruplex DNAs vs duplex DNA.

Molecular modeling studies on G-quadruplex complexes of telomerase inhibitors: structure-activity relationships.

Inhibition of the ability of the enzyme telomerase to add telomeric repeats to the end of chromosomes is a novel target for potential anticancer therapy. This paper examines the hypothesis that compounds possessing a planar aromatic chromophore inhibit telomerase via stabilization of, and binding to, a folded guanine quadruplex structure. Two series of telomerase inhibitors have been designed based on the 2,6-disubstituted amidoanthracene-9,10-dione and 3,6-disubstituted acridine chromophores in order to investigate structure-activity relationships between biological activity and substituent group size. The relative binding energies between these compounds and the folded human telomere DNA quadruplex were determined using molecular simulation methods, involving explicitly solvated structures. The results obtained are in excellent agreement with the biological activity as measured in vitro using a modified TRAP assay and in general agreement with the ranking order of binding enthalpies found in isothermal titration calorimetry studies. This broad agreement provides strong support for the hypothesis that guanine quadruplexes are the primary target for telomerase inhibitors with extended planar chromophores.

Crystal structure of human DT-diaphorase: a model for interaction with the cytotoxic prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954).

The crystal structure of human DT-diaphorase (NAD(P)H oxidoreductase (quinone); EC 1.6.99.2) has been determined to 2.3 A resolution. There are only minor differences in shape and volume between the active sites of the rat and human enzymes and in the hydrophobic environment in the vicinity of the substrate. The isoalloxazine ring of the bound FAD is more buried in the human structure. Molecular modeling was used to examine optimal positions for the antitumor prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) in both the human and rat enzyme active sites. This suggests that the position of CB1954 in the active site of the human enzyme is very similar to that in the rat, although there are detailed differences in the predicted patterns of hydrogen bonding between side chains and the drug. Some of the differences are a consequence of the shift in position for the FAD molecule and may contribute to the observed differences in rate of the two-electron reduction of CB1954.

Stabilization of DNA triple helices by a series of mono- and disubstituted amidoanthraquinones.

We have used quantitative DNase I footprinting to measure the relative affinities of four disubstituted and two monosubstituted amidoanthraquinone compounds for intermolecular DNA triplexes, and have examined how the position of the attached base-functionalized substituents affects their ability to stabilize DNA triplexes. All four isomeric disubstituted derivatives examined stabilize DNA triplexes at micromolar or lower concentrations. Of the compounds studied the 2,7-disubstituted amidoanthraquinone displayed the greatest triplex affinity. The order of triplex affinity for the other disubstituted ligands decreases in the order 2,7 > 1,8 = 1,5 > 2,6, with the equivalent monosubstituted compounds being at least an order of magnitude less efficient. The 1,5-disubstituted derivative also shows some interaction with duplex DNA. These results have been confirmed by molecular modelling studies, which provide a rational basis for the structure-activity relationships. These suggest that, although all of the compounds bind through an intercalative mode, the 2,6, 2,7 and 1,5 disubstituted isomers bind with their two side groups occupying adjacent triplex grooves, in contrast with the 1,8 isomer which is positioned with both side groups in the same triplex groove.

A new model of cancer cachexia: contribution of the ubiquitin-proteasome pathway.

A new model of cachexia is described in which muscle protein metabolism related to the ubiquitin-proteasome pathway was investigated. Cloning of the colon-26 tumor produced a cell line, termed R-1, which induced cytokine (noninterleukin-1beta, interleukin-6 and tumor necrosis factor-alpha)-independent cachexia. Implantation of R-1 cells in mice elicited significant (20-30%) weight loss and decreased blood glucose by 70%, and adipose tissue levels declined by 95% and muscle weights decreased by 20-25%. Food intake was unaffected. The decrease in muscle weight reflected a decline in insoluble, but not soluble, muscle protein that was associated with a significant increase in net protein degradation. The rate of ubiquitin conjugation of proteins was significantly elevated in muscles of cachectic mice. Furthermore, the proteasome inhibitor lactacystin blocked the increase in protein breakdown but had no significant effect on proteolysis. Several markers of the ubiquitin-proteasome pathway, E2(14k) mRNA and E2(14k) protein and ubiquitin-protein conjugates, were not elevated. Future investigations with this new model should gain further insights into the mechanisms of cachexia and provide a background to evaluate novel and more efficacious therapies.

2,7-Disubstituted amidofluorenone derivatives as inhibitors of human telomerase.

Telomerase is a major new target for the rational design of novel anticancer agents. We have previously identified anthraquinone-based molecules capable of inhibiting telomerase by stabilizing G-quadruplex structures formed by the folding of telomeric DNA. In the present study we describe the synthesis and biological evaluation of a series of analogous fluorenone-based compounds with the specific aims of, first, determining if the anthraquinone chromophore is a prerequisite for activity and, second, whether the conventional cytotoxicity inherent to anthraquinone-based molecules may be reduced by rational design. This fluorenone series of compounds exhibits a broad range of telomerase inhibitory activity, with the most potent inhibitors displaying levels of activity (8-12 microM) comparable with other classes of G-quadruplex-interactive agents. Comparisons with analogous anthraquinone-based compounds reveal a general reduction in the level of cellular cytotoxicity. Molecular modeling techniques have been used to compare the interaction of fluorenone- and analogous anthraquinone-based inhibitors with a human G-quadruplex structure and to rationalize their observed biological activities.

U46619-mediated vasoconstriction of the fetal placental vasculature in vitro in normal and hypertensive pregnancies.

OBJECTIVES: To measure in-vitro responses to the thromboxane A2 (TxA2) mimetic U46619 in the fetal placental vasculature of human placentae from normotensive women and those with pre-eclampsia. Furthermore, to compare fetal vascular responses to endothelin-1,5-hydroxytryptamine, potassium chloride (KCl) and prostacyclin (PGI2) in placentae from normal or pre-eclamptic pregnancies. METHODS: Single placental lobules of intact placentae were bilaterally perfused in situ (fetal and maternal) with constant flows of Krebs' solution. Changes in fetal arterial perfusion pressure during intra-arterial infusion of vasoactive agents were recorded. Fetal placental vasoconstrictor concentration response curves were obtained to U46619 (0.01-300 nmol/l), endothelin-1 (0.4-160 nmol/l), KCl (3-300 mmol/l) and 5-hydroxytryptamine (0.03-30 mumol/l). In addition, vasodilator concentration response curves were obtained for PGI2 (1.2-350 nmol/l) in the fetal placental circulation during submaximal increases in perfusion pressure with prostaglandin F2 alpha (PGF2 alpha; 0.7-2.0 mumol/l). RESULTS: The maximum increase in perfusion pressure caused by U46619 in placentae from normotensive women was 194 +/- 25 mmHg. The maximum response to U46619 was significantly reduced in the placentae from women with pre-eclampsia (104 +/- 21 mmHg). In contrast, there were no differences in constrictor responses to endothelin-1,5-hydroxytryptamine and KCl, or in dilator responses to PGI2 in placentae obtained from either normotensive women or those with pre-eclampsia. CONCLUSION: TxA2 receptor-mediated vasoconstriction is reduced in the fetal vasculature of placentae from women with pre-eclampsia, possibly to compensate for the increased levels of TxA2 seen in these conditions.

Human, rat and crocodile liver microsomal monooxygenase activities measured using diazepam and nifedipine: effects of CYP3A inhibitors and relationship to immunochemically detected CYP3A apoprotein.

Nifedipine oxidase and diazepam C3-hydroxylase were tested as activities for selectively measuring CYP3A enzymes using liver microsomes from male and female human organ donors, male and female Wistar rats and male and female estuarine crocodiles. The association between CYP3A enzymes and these monooxygenations was confirmed for the human samples. Male rat samples had lower specific contents of CYP3A apoprotein than the human samples but had equivalent (nifedipine) or higher (diazepam) monooxygenase specific activities. CYP3A apoprotein was undetectable in female rat samples which had very low activities towards both substrates. Enzyme inhibition studies showed that diazepam C3-hydroxylase of male rat liver was attributable to CYP3A but corresponding results for female rats suggested a contribution from non-CYP3A enzyme. Western blotting with immunochemical detection using anti-CYP3A4 IgG suggested the presence of putative CYP3A apoprotein in male and female crocodile liver samples and inhibition studies with diazepam as substrate suggested the presence of CYP3A subfamily monooxygenase activity in these enzyme preparations. Results for nifedipine oxidase with male and female rat liver and male crocodile liver suggested major contributions to catalysis from non-CYP3A enzymes. Inhibition studies suggested that a higher proportion of nifedipine oxidase in female crocodile liver may be attributable to the putative CYP3A enzyme(s) than in male crocodile liver. These results show the need for care in the assessment of CYP3A activity of fractionated tissues when using these substrates in cross-species studies and where gender is a variable.

Design, synthesis and evaluation of human telomerase inhibitors based upon a tetracyclic structural motif.

There is currently significant interest in the development of inhibitors of human telomerase for the treatment of cancer. We describe here the design and synthesis of a new class of mono-substituted small-molecule inhibitors of human telomerase based upon a tetracyclic structural motif. In contrast to the structurally related molecule 9-hydroxyellipticine, recently shown to inhibit telomerase activity in cell cultures but found to be inactive in a cell-free system, we demonstrate direct inhibition of the telomerase enzyme by the tetracyclic compounds in a modified cell-free TRAP assay. The most potent compounds exhibit activity in the low micromolar range and are thus comparable with some of the more active small-molecule telomerase inhibitors based on planar aromatic chromophores, previously described by ourselves and others. These compounds may represent useful leads for the development of more potent inhibitors of human telomerase.

Vasodilator actions of urocortin and related peptides in the human perfused placenta in vitro.

Urocortin, is a recently isolated peptide belonging to the CRH family that binds with high affinity to the CRH2 receptor. Like CRH, urocortin causes hypotension in the rat, but its vasoactive actions have not yet been studied in the human. We have compared the vasoactive properties of urocortin, CRH, and urotensin-1 in the human fetal placental vasculature in vitro. Single placental lobules were bilaterally perfused (maternal and fetal sides, 5 mL/min each; 95% O2-5% CO2; 37 C), and changes in fetal arterial perfusion pressure were recorded. Submaximal vasoconstriction was induced by PGF2alpha (4+/-0.7 micromol/L), which increased perfusion pressure from 19.6+/-1.4 to 100.7+/-3.1 mm Hg (n=38; P < 0.001). Subsequent fetal arterial infusion of urocortin (0.001-1 nmol/L) caused concentration-dependent vasodilatation. Urocortin was equipotent with urotensin-1 and 25 times more potent than CRH in causing vasodilatation. Nevertheless, the maximum vasodilator responses to each of the peptides were similar (P > 0.05). The CRH receptor antagonist, alpha-helical CRH-(9-41) (0.2 nmol/L) significantly attenuated the vasodilatation produced by urocortin, urotensin-1, and CRH (P < 0.05). These results indicate a possible physiological role for urocortin in the modulation of human fetal placental vascular tone by activation of CRH2-like receptors.