Dp Geraghty

D-amino acid residue in a defensin-like peptide from platypus venom: effect on structure and chromatographic properties.

The recent discovery that the natriuretic peptide OvCNPb (Ornithorhynchus venom C-type natriuretic peptide B) from platypus (Ornithorynchus anatinus) venom contains a D-amino acid residue suggested that other D-amino-acid-containing peptides might be present in the venom. In the present study, we show that DLP-2 (defensin-like peptide-2), a 42-amino-acid residue polypeptide in the platypus venom, also contains a D-amino acid residue, D-methionine, at position 2, while DLP-4, which has an identical amino acid sequence, has all amino acids in the L-form. These findings were supported further by the detection of isomerase activity in the platypus gland venom extract that converts DLP-4 into DLP-2. In the light of this new information, the tertiary structure of DLP-2 was recalculated using a new structural template with D-Met2. The structure of DLP-4 was also determined in order to evaluate the effect of a D-amino acid at position 2 on the structure and possibly to explain the large retention time difference observed for the two molecules in reverse-phase HPLC. The solution structures of the DLP-2 and DLP-4 are very similar to each other and to the earlier reported structure of DLP-2, which assumed that all amino acids were in the L-form. Our results suggest that the incorporation of the D-amino acid at position 2 has minimal effect on the overall fold in solution.

Relationships between single nucleotide polymorphisms of antioxidant enzymes and disease

The presence and progression of numerous diseases have been linked to deficiencies in antioxidant systems. The relationships between single nucleotide polymorphisms (SNPs) arising from specific antioxidant enzymes and diseases associated with elevated oxidative stress have been studied with the rationale that they may be useful in screening for diseases. The purpose of this narrative review is to analyse evidence from these studies. The antioxidant enzyme SNPs selected for analysis are based on those most frequently investigated in relation to diseases in humans: superoxide dismutase (SOD2) Ala16Val (80 studies), glutathione peroxidise (GPx1) Pro197Leu (24 studies) and catalase C-262T (22 studies). Although the majority of evidence supports associations between the SOD2 Ala16Val SNP and diseases such as breast, prostate and lung cancers, diabetes and cardiovascular disease, the presence of the SOD2 Ala16Val SNP confers only a small, clinically insignificant reduction (if any) in the risk of these diseases. Other diseases such as bladder cancer, liver disease, nervous system pathologies and asthma have not been consistently related to this SOD SNP genotype. The GPx1 Pro197Leu and catalase C-262T SNP genotypes have been associated with breast cancer, but only in a small number of studies. Thus, currently available evidence suggests antioxidant enzyme SNP genotypes are not useful for screening for diseases in humans.

Binding characteristics of [125I]Bolton-Hunter [Sar9, Met(O2)11]substance P, a new selective radioligand for the NK1 receptor

The selective tachykinin agonist [Sar9,Met(O2)11]substance P (Sar-SP) was radioiodinated with [125I]Bolton-Hunter reagent and the product [125I]Bolton-Hunter-[Sar9,Met(O)2)11]SP (BHSar-SP) purified using reverse phase HPLC. Autoradiographic studies showed dense specific binding of BHSar-SP over the rat submandibular gland and over several regions in rat brain, with very low nonspecific binding, identical with the pattern of binding sites seen in a parallel study with [125I]Bolton-Hunter SP (BHSP). In homogenate binding experiments, BHSar-SP bound with high affinity to a single site in membranes from rat brain (KD 261 pM) and rat submandibular gland (KD 105 pM). Comparative values for BHSP were 495 and 456 pM, i.e. of two and four fold lower affinity than BHSar-SP. Association of BHSar-SP to membranes from brain (k+1 3.7 x 10(9) M-1 min-1) was faster than to membranes from salivary gland (k+1 5.6 x 10(8) M-1 min-1). In competition studies, BHSar-SP was displaced from salivary gland membranes by substance P (SP) approximately physalaemin greater than or equal to Sar-SP approximately SP-(3-11) greater than SP-(5-11) much greater than neurokinin A (NKA) approximately eledoisin = kassinin = SP-methyl ester greater than or equal to neurokinin B (NKB) much greater than [Nle10]NKA-(4-10) greater than [MePhe7]NKB-(4-10). In brain membranes, the rank potency order was SP greater than Sar-SP greater than or equal to physalaemin greater than SP-(3-11) greater than SP-(5-11) greater than NKA greater than or equal to eledoisin much greater than NKB greater than kassinin greater than SP-methyl ester: however [MePhe7]NKB-(4-10) and [Nle10]NKA-(4-10) were ineffective competitors at concentrations up to 1 microM. Both binding patterns are consistent with BHSar-SP binding to an NK1 site. With the exception of SP, Sar-SP, SP-(3-11) and physalaemin, all competitors were 5 to 54 times less potent at BHSar-SP binding sites in brain than in salivary gland. These data reveal some differences in characteristics of NK1 binding sites in brain and submandibular gland. Although of higher affinity, BHSar-SP does not appear greatly more selective than BHSP in its ability to define NK1 binding sites.

Respiratory action of capsaicin microinjected into the nucleus of the solitary tract: involvement of vanilloid and tachykinin receptors

The respiratory response to microinjection of capsaicin into the commissural nucleus of the solitary tract (cNTS) of urethane‐anaesthetized rats was investigated in the absence and presence of the competitive vanilloid (capsaicin) antagonist, capsazepine, and selective tachykinin NK1, NK2 and NK3 antagonists (RP 67580, SR 48968 and SR 142801, respectively). Microinjection of capsaicin reduced respiratory frequency but not tidal volume (VT), leading to an overall reduction in minute ventilation (V{dot above}E). The effect was dose‐dependent between 0.5 and 2 nmol capsaicin. Doses greater than 2 nmol produced apnoea. Tachyphylaxis was observed following repeated injection of capsaicin (1 nmol, 30 min apart). Capsazepine (1 nmol) had no effect on frequency or VT when injected alone but completely blocked the respiratory response to capsaicin (1 nmol). RP 67580 (1 but not 5 nmol) alone depressed frequency and VT slightly. Moreover, RP 67580 appeared to potentiate the bradypnoeic effect of capsaicin. In contrast, SR 48968 and SR 142801 (1 and 5 nmol) alone had no significant effect on respiration. However, both agents significantly attenuated the reduction in frequency produced by capsaicin. In conclusion, microinjection of capsaicin into the cNTS decreases overall ventilation, primarily by reducing frequency. The action of capsaicin appears from the data to be mediated by vanilloid receptors since it is blocked by the competitive vanilloid antagonist capsazepine and is subject to tachyphylaxis. However, since NK2 (SR 48968) and NK3 (SR 142801) receptor antagonists block the actions of capsaicin, we propose that capsaicin acts also by releasing tachykinins from central afferent terminals in the cNTS.

Respiratory actions of tachykinins in the nucleus of the solitary tract: characterization of receptors using selective agonists and antagonists

The respiratory response to microinjection of tachykinins and analogues into the commissural nucleus of the solitary tract (cNTS) of urethane‐anaesthetized rats was investigated in the presence and absence of selective tachykinin NK1, NK2 and NK3 antagonists (RP 67580, SR 48968 and SR 142801, respectively). All tachykinins, except for the selective NK2 agonist, [Nle10]‐NKA(4‐10), increased tidal volume (VT). The rank potency order of naturally‐occurring tachykinins was neurokinin A (NKA)substance P (SP)>>NKB, whereas the rank order for selective analogues was senktideseptide>> [Sar9,Met(O2)11]‐SP>>[Nle10]‐NKA(4‐10). Septide (NK1‐selective) and senktide (NK3‐selective) were 22 fold more potent (pD2∼12) at stimulating VT than SP (pD2∼10.5). Tachykinin agonists produced varying degrees of respiratory slowing, independent of changes in VT. At doses producing maximum stimulation of VT, agonists induced either a mild (<10 breaths min−1 decrease; SP and septide), moderate (10–25 breaths min−1 decrease; NKA, NKB and [Sar9,Met(O2]‐SP) or severe (∼40 breaths min−1 decrease; senktide) bradypnoea. [Nle10]‐NKA(4‐10) produced a dose‐dependent bradypnoea without affecting VT. RP 67580 significantly attenuated the VT response to SP (33 pmol) and NKA (10 pmol) but not NKB (100 pmol). In the presence of RP 67580, the mild bradypnoeic response to NKB was significantly enhanced whereas SP and NKA induced a bradypnoea which was not observed in the absence of RP 67580. SR 48968 had no effect on the VT response to SP or NKB, markedly enhanced the VT response to NKA and completely blocked the bradypnoeic response to [Nle10]‐NKA(4‐10). Only SR142801 attenuated the VT response to NKB. The present data suggest that all three tachykinin receptors (NK1, NK2 and NK3) are present in the cNTS and are involved in the central control of respiration.

Substrate Specificity of Platypus Venom L-to-D-Peptide Isomerase

The l-to-d-peptide isomerase from the venom of the platypus (Ornithorhyncus anatinus) is the first such enzyme to be reported for a mammal. In delineating its catalytic mechanism and broader roles in the animal, its substrate specificity was explored. We used N-terminal segments of defensin-like peptides DLP-2 and DLP-4 and natriuretic peptide OvCNP from the venom as substrates. The DLP analogues IMFsrs and ImFsrs (srs is a solubilizing chain; lowercase letters denote d-amino acid) were effective substrates for the isomerase; it appears to recognize the N-terminal tripeptide sequence Ile-Xaa-Phe-. A suite of 26 mutants of these hexapeptides was synthesized by replacing the second residue (Met) with another amino acid, viz. Ala, α-aminobutyric acid, Ile, Leu, Lys, norleucine, Phe, Tyr, and Val. It was shown that mutant peptides incorporating norleucine and Phe are substrates and exhibit l- or d-amino acid isomerization, but mutant peptides that contain residues with shorter, β-branched or long side chains with polar terminal groups, viz. Ala, α-aminobutyric acid, Ile, Val, Leu, Lys, and Tyr, respectively, are not substrates. It was demonstrated that at least three N-terminal amino acid residues are absolutely essential for l- to d-isomerization; furthermore, the third amino acid must be a Phe residue. None of the hexapeptides based on LLH, the first three residues of OvCNP, were substrates. A consistent 2-base mechanism is proposed for the isomerization; abstraction of a proton by 1 base is concomitant with delivery of a proton by the conjugate acid of a second base.

Effect of atorvastatin on kidney function in chronic kidney disease: A randomised double-blind placebo-controlled trial

BACKGROUND The effect of atorvastatin on kidney function was assessed in patients with stages 2-4 chronic kidney disease. METHODS We conducted a randomised, double-blind, placebo-controlled trial in chronic kidney disease clinics in Northern Tasmania and enrolled 132 patients with serum creatinine levels >120 μmol/l, not taking lipid-lowering therapy and at all levels of proteinuria and serum cholesterol. Patients were randomly assigned to receive either 10 mg of atorvastatin/day (64) or placebo (68) and were followed with trial visits 3-monthly for a mean of 2.5 yrs. The primary outcome was the rate of both MDRD eGFR and Cockcroft-Gault creatinine clearance (C-G CrCl) decline. Analysis was based on intention to treat and included all patients that had at least one follow-up visit. RESULTS The rate of MDRD eGFR decline was 29% lower; 1.04 ± 3.84 vs. 1.47 ± 3.74 ml/min/1.73 m(2)/yr (P=0.53), and the C-G CrCl was 20% lower; 1.88 ± 5.07 vs. 2.36 ± 4.61 ml/min/1.73 m(2)/yr (P=0.58) in atorvastatin-treated, compared with placebo-treated patients. Although blood pressure decreased in both atorvastatin and placebo-treated groups there were no differences between groups. In addition, there was no difference in concomitant medication intake including angiotensin converting enzyme inhibitors and angiotensin receptor blockers between groups. CONCLUSIONS There was a trend toward a slower eGFR decline in the atorvastatin-treated group that did not reach statistical significance. This may have been due to the lack of power of the study. However, atorvastatin may have a renoprotective effect in those patients with chronic kidney disease and cardiovascular disease. This needs to be assessed in further studies.

Mammalian l-to-d-amino-acid-residue isomerase from platypus venom

The presence of d‐amino‐acid‐containing polypeptides, defensin‐like peptide (DLP)‐2 and Ornithorhyncus venom C‐type natriuretic peptide (OvCNP)b, in platypus venom suggested the existence of a mammalian d‐amino‐acid‐residue isomerase(s) responsible for the modification of the all‐l‐amino acid precursors. We show here that this enzyme(s) is present in the venom gland extract and is responsible for the creation of DLP‐2 from DLP‐4 and OvCNPb from OvCNPa. The isomerisation reaction is freely reversible and under well defined laboratory conditions catalyses the interconversion of the DLPs to full equilibration. The isomerase is ∼50–60 kDa and is inhibited by methanol and the peptidase inhibitor amastatin. This is the first known l‐to‐d‐amino‐acid‐residue isomerase in a mammal.

The effect of 4-week chilli supplementation on metabolic and arterial function in humans

Objective:To investigate the effects of regular chilli ingestion on some indicators of metabolic and vascular function.Design:A randomized cross-over dietary intervention study.Setting:Launceston, Australia.Subjects:Healthy free-living individuals.Intervention:Thirty-six participants (22 women and 14 men), aged 46±12 (mean±s.d.) years; BMI 26.4±4.8 kg/m2, consumed 30 g/day of a chilli blend (55% cayenne chilli) with their normal diet (chilli diet), and a bland diet (chilli-free) for 4 weeks each. Metabolic and vascular parameters, including plasma glucose, serum lipids and lipoproteins, insulin, basal metabolic rate, blood pressure, heart rate, augmentation index (AIx; an indicator of arterial stiffness), and subendocardial-viability ratio (SEVR; a measure of myocardial perfusion), were measured at the end of each diet. In a sub-study, during week 3 of each dietary period, the vascular responses of 15 subjects to glyceryl-trinitrate (GTN) and salbutamol were also studied.Results:For the whole group, there were no significant differences between any of the measured parameters when compared at the end of the two dietary periods. When analysed separately, men had a lower resting heart rate (P=0.02) and higher SEVR (P=0.05) at the end of the chilli diet than the bland diet. In the sub-study, baseline AIx on the chilli diet was lower (P<0.001) than on the bland diet, but there was no difference in the effects of GTN and salbutamol between the two diets.Conclusion:Four weeks of regular chilli consumption has no obvious beneficial or harmful effects on metabolic parameters but may reduce resting heart rate and increase effective myocardial perfusion pressure time in men.

Effect of capsaicin and dihydrocapsaicin on in vitro blood coagulation and platelet aggregation

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in developed countries. The growing clinical and financial burden of CVD is, at least in part, being driven by aging populations, and thus represents a significant challenge to health care systemswith regard to diagnosis, prevention and treatment. Novel cost-effective approaches that slow or prevent the onset and/or reduce the incidence of CVD warrant investigation. Capsaicinoids, including capsaicin and dihydrocapsaicin, are the major pungent constituents of ‘hot chilli peppers’ of the Capsicum genus. These spice principles have been documented to increase carbohydrate metabolism [1,2], energy expenditure [3] and lipid metabolism [4–7], in rats and/or humans, and have been successfully used in the treatment of painful conditions such as rheumatoid arthritis, osteoarthritis and peripheral neuropathies [8]. Furthermore, we have recently shown that regular intake of chilli delays copperinduced oxidation of serum lipids in vitro [4] and lowers and improves post-prandial insulin and glucose profiles [9], actions that may help in reducing CVD risk. Their effect(s) however, on haemostasis have not been extensively investigated, and their potential “anti-haemostatic” properties may also reduce CVD risk. Limited reports have demonstrated that capsaicin inhibits platelet aggregation, although these studies investigated platelets from non-human species [10,11]. Furthermore, it was reported that capsaicin had no effect on blood coagulation [12], although the potential effects on individual clotting factors were not determined. The aim of this study was to therefore investigate and compare the effects of capsaicin and dihydrocapsaicin on markers of in vitro haemostasis, viz, blood coagulation and platelet aggregation. This studywas approved by the Human Research Ethics Committee (Tasmanian) Network (Ref No: H0009477). Informed consent was obtained from each subject in accordance with the Declaration of Helsinki of the World Medical Association. For blood coagulation experiments, normal reference plasma (NRP; Helena Laboratories, Beaumont, USA) was spiked with increasing concentrations (3.125100 μmol/L) of capsaicin (Sigma Chemical Company, St Louis, USA) or dihydrocapsaicin (Tocris Bioscience, Ellisville, USA). Each capsaicinoid was initially dissolved in 100% ethanol to make a stock solution of 1 mol/L and then diluted for further experiments in normal buffered saline, pH 7.1. After incubation for 30 minutes at 37 °C, prothrombin time (PT), activated partial thromboplastin time (aPTT) and the activities of clotting factors II, V, VII, VIII:C, IX, X, XI and XII were determined using a STarT4 coagulation analyzer (Diagnostica Stago, Parsippany, USA). For platelet aggregation experiments, venous whole blood was collected using 21 gauge syringes from six healthy subjects