Getting the adrenaline going: Crystal structure of the adrenaline-synthesizing enzyme PNMT

Background: Adrenaline is localized to specific regions of the central nervous system (CNS), but its role therein is unclear because of a lack of suitable pharmacologic agents. Ideally, a chemical is required that crosses the blood-brain barrier, potently inhibits the adrenaline-synthesizing enzyme PNMT, and does not affect other catecholamine processes. Currently available PNMT inhibitors do not meet these criteria. We aim to produce potent, selective, and CNS-active PNMT inhibitors by structure-based design methods. The first step is the structure determination of PNMT. Results: We have solved the crystal structure of human PNMT complexed with a cofactor product and a submicromolar inhibitor at a resolution of 2.4 Angstrom. The structure reveals a highly decorated methyltransferase fold, with an active site protected from solvent by an extensive cover formed from several discrete structural motifs. The structure of PNMT shows that the inhibitor interacts with the enzyme in a different mode from the (modeled) substrate noradrenaline. Specifically, the position and orientation of the amines is not equivalent. Conclusions: An unexpected finding is that the structure of PNMT provides independent evidence of both backward evolution and fold recruitment in the evolution of a complex enzyme from a simple fold. The proposed evolutionary pathway implies that adrenaline, the product of PNMT catalysis, is a relative newcomer in the catecholamine family. The PNMT structure reported here enables the design of potent and selective inhibitors with which to characterize the role of adrenaline in the CNS. Such chemical probes could potentially be useful as novel therapeutics.

The urethral pressure profile and ultrasound imaging of the lower urinary tract

In a prospective study 105 patients with symptoms of stress incontinence underwent video-urodynamic testing, including resting urethral pressure profilometry and translabial ultrasound. The urethral pressure profile (UPP) included maximum urethral closure pressure (MUCP), functional length (FL) and area under the curve (AUC). Ultrasound parameters included urethral thickness, urethral rotation and bladder neck descent, as well as funneling/opening of the internal urethral meatus on Valsalva maneuver. Levator contraction strength was assessed measuring the cranioventral displacement of the internal meatus. Negative correlations between UPP data and age, parity and previous surgery were observed which were consistent with literature data. There was a positive correlation :between the urethral AP diameter on ultrasound and the MUCP, which agrees with reports showing reduced sphincter thickness or volume in stress-incontinent women. Hypermobility on ultrasound did not correlate with UPP data. However, a lower MUCP correlated with extensive opening of the bladder neck. Finally, there was a trend towards poorer pelvic floor function with lower MUCP measurements.

Reversal of cardiovascular remodelling with candesart

Cardiovascular remodelling, defined as ventricular and vascular hypertrophy together with fibrosis, characterises hypertension following inhibition of the production of the endogenous vasodilator, nitric oxide (NO). This study has determined whether the cardiovascular remodelling following chronic NO synthase inhibition can e reversed by administration of the selective angiotensin II AT(1)-receptor antagonist, candesartan. Male Wistar rats were treated with L-nitroarginine methyl ester (L-NAME, 400 mg/l in drinking water) for eight weeks and with candesartan cilexetil (2 mg/kg/day by oral gavage) for the last four weeks. L-NAME-treated rats became hypertensive with systolic blood pressure increasing from 110 +/- 4 mmHg (control) to 170 +/- 10 mmHg. Rats developed left ventricular hypertrophy (control 1.70 +/- 0.06; L-NAME 2.10 +/- 0.04 mg/kg body wt) with markedly increased deposition of perivascular and interstitial collagen. Candesartan returned blood pressure, left ventricular weights and collagen deposition to control values. Echo cardiographic assessment showed concentric hypertrophy with an increased fractional shortening; this was reversed by candesartan treatment. Heart failure was not evident. In the isolated Langendorff heart, diastolic stiffness increased in L-NAME-treated rats while the rate of increase in pressure (+dP/dt) increased after eight weeks only; candesartan reduced collagen deposition and normalised +dP/dt. In isolated left ventricular papillary muscles, the potency (negative log EC50) of noradrenaline as a positive inotropic compound was unchanged, (control 6.56 +/- 0.14); maximal increase in force before ectopic beats was reduced from 5.0 +/- 0.4 mN to 2.0 +/- 0.2 mN. Noradrenaline potency as a vasoconstrictor in thoracic aortic rings was unchanged, but maximal contraction was markedly reduced from 25.2 +/- 2.0 mN to 3.0 +/- 0.3 mN; this was partially reversed by candesartan treatment. Thus, chronic inhibition of NO production with L-NAME induces hypertension, hypertrophy and fibrosis with increased toxicity and significant decreases in vascular responses to noradrenaline. These changes were at least partially reversible by treatment with candesartan, implying a significant role of AT(1)-receptors in L-NAME-induced cardiovascular changes.

Development of response-selective agonists of human C5a anaphylatoxin: Conformational, biological, and therapeutic considerations

Numerous studies on the relationship between the structure and function of peptide agonists derived from the biologically active, C-terminal region of human C5a anaphylatoxin have been reported over the past decade. These studies have been performed with the objective of parlaying this structure-function information into the design of peptide/peptidomimetic modulators of C5a receptor (C5aR)-mediated function. In this review, we describe a rational approach for the development of conformationally biased, decapeptide agonists of C5a and described how these stabilized and specific conformational features relate to the expression of specific C5a-like activities in vitro and in vivo. The therapeutic potential of such response-selective C5a agonists is discussed and underscored by the results of one such response-selective C5a agonist that was used in vivo as an effective molecular adjuvant capable of generating antigen-specific humoral and cellular immune responses. Finally, we describe the synthesis of a new generation of highly response-selective, conformationally biased C5a agonist and discuss the in vitro and in vivo biologic results that so indicate this biologic selectivity.

Mango stem-end rot (Botryosphaeria dothidea) disease control by partial-pressure infiltration of fungicides

The in vitro efficacy of several fungicides against Botryosphaeria dothidea (syn. Dothiorella dominicana) and their in vivo efficacy in controlling mango cv. Kensington Pride stem-end rot on partial-pressure infiltration v. dip treatment of green mature fruit was evaluated. In vitro sensitivity of B. dothidea to Benlate (benomyl), Sportak (prochloraz) and Scala (pyrimethanil) at 10 dilutions of the manufacturer's recommended rate was first determined at typical cold (13degreesC) and shelf (23degreesC) storage temperatures. The effectiveness of partial-pressure infiltration and conventional hot (52degreesC) or cold (26degreesC) dipping of fruit after harvest was then evaluated using the commercially recommended rate for each fungicide. In vitro, Benlate and Sportak prevented the growth of B. dothidea at both storage temperatures and at all concentrations, while Scala partially controlled growth of the pathogen. Benlate was the most effective fungicide for stem-end rot control. Sportak and Scala resulted in stem-end rot control when applied by partial-pressure infiltration, but not as dips. Partial-pressure infiltration holds promise for enhancing the efficacy of otherwise less effective but alternative fungicides for control of stem-end rot diseases.

Repeated blood pressure measurements in a sample of Swedish twins: heritabilities and associations with polymorphisms in the renin-angiotensin-aldosterone system Iliadou, Anastasia, Lichtenstein, Paul, Morgenstern, Ralf, Forsberg, Lena, Svensson, Richard, de Faire, Ulf, Martin, Nicholas, Pedersen, Nancy

Background Twin and family studies have shown that genetic effects explain a relatively high amount of the phenotypic variation in blood pressure. However, many studies have not been able to replicate findings of association between specific polymorphisms and diastolic and systolic blood pressure. Methods In a structural equation-modelling framework the authors investigated longitudinal changes in repeated measures of blood pressures in a sample of 298 like-sexed twin pairs from the population-based Swedish Twin Registry. Also examined was the association between blood pressure and polymorphisms in the angiotensin-I converting enzyme and the angiotensin 11 receptor type 1 with the 'Fulker' test Both linkage and association were tested simultaneously revealing whether the polymorphism is a Quantitative Trait Locus (QTL) or in linkage disequilibrium with the QTL. Results Genetic influences explained up to 46% of the phenotypic variance in diastolic and 63% of the phenotypic variance in systolic blood pressure. Genetic influences were stable over time and contributed up to 78% of the phenotypic correlation in both diastolic and systolic blood pressure. Non-shared environmental effects were characterised by time specific influences and little transmission from one time point to the next. There was no significant linkage and association between the polymorphisms and blood pressure. Conclusions There is a considerable genetic stability in both diastolic and systolic blood pressure for a 6-year period of time in adult life. Non-shared environmental influences have a small long-term effect Although associations with the polymorphisms could not be replicated, results should be interpreted with caution due to power considerations. (C) 2002 Lippincott Williams Wilkins.

Selective loss of expression of glutamate GluR2/R3 receptor subunits in cerebellar tissue from a patient with olivopontocerebellar atrophy

Expression of the mRNAs encoding the astrocytic (EAAT1, EAAT2) and neuronal (EAAT3, EAAT4) excitatory amino acid transporters and the AMPA-type glutamate receptor subunits GluR2 and GluR3 was investigated in postmortem cerebellar extracts from a patient with olivopontocerebellar atrophy (OPCA) and in material from three age-matched controls. Decreased expression in the steady state level of EAAT4 mRNA in the OPCA sample was correlated with the selective loss of Purkinje cells. Neuropathological evaluation revealed reactive gliosis and concomitantly increased expression of the mRNA encoding astrocytic glial fibrillary acidic protein (GFAP). Expression of the mRNAs encoding the AMPA receptor subunits GluR2 and GluR3 subunits was found to be decreased in OPCA suggesting that excitotoxic mechanism could play a role in the pathogenesis of the selective neuronal cell death in this disorder.

Do female rainbowfish (Melanotaenia spp.) prefer to shoal with familiar individuals under predation pressure?

Shoaling with familiar individuals may have many benefits including enhanced escape responses or increased foraging efficiency. This study describes the results of two complimentary experiments. The first utilised a simple binary choice experiment to determine if rainbowfish (Melanotaenia spp.) preferred to shoal with familiar individuals or with strangers. The second experiment used a free range situation where familiar and unfamiliar individuals were free to intermingle and were then exposed to a predator threat. Like many other small species of fish, rainbowfish were capable of identifying and distinguishing between individuals and choose to preferentially associate with familiar individuals as opposed to strangers. Contrary to expectations. however. rainbowrish did not significantly increase their preference for familiar individuals in the presence of a stationary predator model. Griffiths [J Fish Biol (1997) 51:489-4951 conducted similar studies under semi-natural conditions examining, the shoaling preferences of European minnows and showed similar results. Both the current study and that of Griffiths were conducted using predator wary populations of fish. It is suggested that, in predator sympatric populations, the benefits of shoaling with familiar individuals are such that it always pays to stay close to familiar individuals even when the probability If predator attack is remote.

The effect of soft tissue deloading tape on thoracic spine pressure pain thresholds in asymptomatic subjects

The application of tape to deload soft tissue is used in the management of thoracic spine pain. A reported clinical feature of this treatment is reduced tenderness of the spine during postero-anterior mobilizations. A randomized, single blind, placebo controlled, repeated measures design study was employed to investigate the effects of deloading tape on pressure pain threshold measurements at the level of the T7 spinous process in an asymptomatic group of 24 subjects. Pressure pain thresholds were assessed prior to and following the application of deloading tape, placebo sham tape and no-tape control conditions. All subjects received all three conditions in a randomized order on three separate days. Differences between the pre- and post-measurements were used as indicators of change in a subject's pressure pain threshold. No significant change in pressure pain threshold measurements was found between conditions. In summary, this study demonstrated that deloading tape applied to the level of the T7 spinous process did not significantly change pressure pain threshold measurements in asymptomatic subjects, raising the possibility that any pain relieving effect may well be conditional upon pain being present. (C) 2002 Elsevier Science Ltd. All rights reserved.

Development of a selective photoactivatable antagonist for corticotropin-releasing factor receptor, type 2 (CRF2)

A novel photoactivatable analog of antisauvagine-30 (aSvg-30), a specific antagonist for corticotropin-releasing factor (CRF) receptor, type 2 (CRF2), has been synthesized and characterized. The N-terminal amino-acid D-Phe in aSvg-30 [D-Phe11,His12] Svg((11-40)) was replaced by a phenyldiazirine, the 4-(1-azi-2,2,2-trifluoroethyl) benzoyl (ATB) residue. The photoactivatable aSvg-30 analog ATB-[ His12] Svg was tested for its ability to displace [I-125-Tyr0] oCRF or [I-125-Tyr0]Svg from membrane homogenates of human embryonic kidney (HEK) 293 cells stably transfected with cDNA coding for rat CRF receptor, type 1 ( rCRF(1)) or mouse CRF receptor, type 2beta (mCRF(2beta)). Furthermore, the ability of ATB- [His12] Svg((12-40)) to inhibit oCRF- or Svg-stimulated cAMP production of transfected HEK 293 cells expressing either rCRF(1) (HEK-rCRF(1) cells) or mCRF(2beta) (HEK-mCRF(2beta) cells) was determined. Unlike astressin and photo astressin, ATB- [His12]Svg((12-40)) showed high selective binding to mCRF(2beta) (K-i = 3.1 +/- 0.2 nM) but not the rCRF(1) receptor (K-i = 142. 5 +/- 22.3 nM) and decreased Svg-stimulated cAMP activity in mCRF(2beta)-expressing cells in a similar fashion as aSvg-30. A66-kDa protein was identified by SDS/PAGE, when the radioactively iodinated analog of ATB- [His12]Svg((12-40)) was covalently linked to mCRF(2beta) receptor. The specificity of the photoactivatable I-125-labeled CRF2beta antagonist was demonstrated with SDS/PAGE by the finding that this analog could be displaced from the receptor by antisauvagine-30, but not other unrelated peptides such as vasoactive intestinal peptide (VIP).