Calcium(II)3 (3,5-Diisopropylsalicylate)6(H2O)6 Activates Nitric Oxide Synthase: An Accounting for its Action in Decreasing Platelet Aggregation

Purposes of these studies were first; to determine whether or not Calcium(II)3 (3,5- diisopropylsalicylate)6(H2O)6 [Ca(II)3(3,5-DIPS)6], a lipophilic calcium complex, could decrease activated-platelet aggregation, and second; to determine whether or not it is plausible that Ca(II)3(3,5-DIPS)6 decreases activated-platelet aggregation by facilitating the synthesis of Nitric Oxide (NO) by Nitric Oxide Synthase (NOS). The influence of Ca(II)3(3,5-DIPS)6 on the initial rate of activated-platelet aggregation was determined by measuring the decrease in rate of increase in transmission at 550 nm for a suspension of Thrombin-CaCl2 activated platelets following the addition of 0, 50, 100, 250, or 500 μM Ca(II)3(3,5-DIPS)6. To establish that the Ca(lI)3(3,5- DIPS)6-mediated decrease in aggregation was due to activation of NOS, the effect of ʟ-NMMA, an inhibitor of NOS, on the inhibition of platelet aggregation by Ca(II)3(3,5-DIPS)6 was determined using a suspension of activated platelets contaimng 0 or 250 μM Ca(II)3(3,5-DIPS)6 without or with 1 mM ʟ-NMMA. An in vitro Bovine Brain NOS reaction mixture, containing CaCl2 for the activation of Phosphodiesterase-3' ,5'-Cyclic Nucleotide Activator required for the activation of NOS, was used to determine whether or not Ca(II)3(3,5-DIPS)6 could be used as a substitute for the addition of Ca. The decrease in absorbance at 340 nm, lambda maximum for NADPH, was measured to determine NOS activity following the addition of NOS to the complete reaction mixture containing either CaCl2, Ca(II)3(3,5-DIPS)6, or neither Ca compound. Increasing the concentration of Ca(II)3(3,5-DIPS)6 caused a concentration related decrease in activated platelet aggregation. The addition of ʟ-NMMA to activated platelets, in the absence of Ca(II)3(3,5-DIPS)6, caused a 129% increase in initial rate of platelet aggregation. The initial rate of platelet aggregation decreased 74% with the addition of 250 μM Ca(II)3(3,5-DIPS)6 and the addition of ʟ-NMMA plus 250 μM Ca(II)3(3,5-DIPS)6 caused a 197% decrease in initial rate of aggregation compared to the initial rate observed width the presence of 1 mM ʟ-NMMA alone. There was only a small, 27%, increase in initial rate of 0.4 mM NADPH oxidation when 0.9 mM CaCl2 was added to the NOS reaction mixture in comparison to the initial rate of NADPH oxidation with no addition of CaCI2. Addition of an equivalent amount of Ca in the form of Ca(II)3(3,5-DIPS)6, 333 μM, caused a 37% increase in initial rate of NADPH oxidation compared to the addition of 0.9 mM CaCl2. Addition of increasing concentrations of ʟ-NMMA plus 0.9 mM CaCl2 or 333 μM Ca(II)3(3,5-DIPS)6 to the NOS reaction mixture caused a concentration related increase in initial rate of NADPH oxidation. Addition of ʟ-NMMA while expected to decrease NADPH oxidation actually increased the rate of NADPH oxidation. Additions of 133 μM or 267 μM Ca(II)3(3,5- DIPS)6 also caused concentration related increases in initial rate of NADPH oxidation in the presence of 113 μM ʟ-NMMA. However, the addition of 533 μM Ca(II)3(3,5-DIPS)6 caused a dramatic decrease in initial rate of NADPH oxidation by NOS. It is concluded that: 1) Ca(II)3(3,5- DIPS)6 activates platelet NOS in preventing platelet aggregation, 2) in vitro NOS activity can be observed spectrophotometrically by following the consumption of NADPH as a decrease in absorbance at 340 nm, 3) Ca(II)3(3,5-DIPS)6 plays a role in enhancing Bovine Brain NOS activity resulting in an increased rate of NADPH oxidation by NOS, 4) Ca(II)3(3,5-DIPS)6 is a useful form of Ca in activating NOS and superior to CaCl2 with regard to the facilitation of a NADPH oxidation, and 5) ʟ-NMMA stimulates Bovine Brain NOS activity rather than causing an inhibition of this enzyme and must serve as a reducible substrate for Bovine Brain NOS

Down-Regulation of Porcine Heart Diaphorase Reactivity by Trimanganese Hexakis(3,5-Diisopropylsalicylate), Mn3(3,5-DIPS)6, and Down-Regulation of Nitric Oxide Synthase Reactivity by Mn3(3,5-DIPS)6 and Cu(II)2(3,5-DIPS)4

Purposes of this work were to examine the plausible down-regulation of porcine heart diaphorase (PHD) enzyme reactivity and nitric oxide synthase (NOS) enzyme reactivity by trimanganese hexakis(3,5-diisopropylsalicylate), [Mn3(3,5-DIPS)6] as well as dicopper tetrakis(3,5- diisopropylsalicylate, [Cu(II)2(3,5-DIPS)4] as a mechanistic accounting for their pharmacological activities

Essential Metalloelement Chelates Facilitate Repair of Radiation Injury

Treatment with essential metalloelement (Cu, Fe, Mn, and Zn) chelates or combinations of them before and/or after radiation injury is a useful approach to overcoming radiation injury. No other agents are known to increase survival when they are used to treat after irradiation, in a radiorecovery treatment paradigm. These chelates may be useful in facilitating de novo syntheses of essential metalloelement-dependent enzymes required to repair radiation injury. Reports of radioprotection, which involves treatment before irradiation, with calcium-channel blockers, acyl Melatonin homologs, and substituted anilines, which may serve as chelating agents after biochemical modification in vivo, as well as Curcumin, which is a chelating agent, have been included in this review. These inclusions are intended to suggest additional approaches to combination treatments that may be useful in facilitating radiation recovery. These approaches to radioprotection and radiorecovery offer promise in facilitating recovery from radiation-induced injury experienced by patients undergoing radiotherapy for neoplastic disease and by individuals who experience environmental, occupational, or accidental exposure to ultraviolet, x-ray, or γ-ray radiation. Since there are no existing treatments of radiation-injury intended to facilitate tissue repair, studies of essential metalloelement chelates and combinations of them, as well as combinations of them with existing organic radioprotectants, seem worthwhile

Radioprotectant Activity of 5-Diethylsulfonamoylsalicylatocopper(II) in Gamma Irradiated Mice

Survival and changes in mean body mass of whole-body irradiated mice were determined to examine the radioprotectant activity of 5-diethylsulfonamoylsalicylatocopper(II) [Cu(II) (5-DESS)]. One of four groups of 25 female C57BL/6 mice were treated subcutaneously (sc)with 0, 10, 20, 40, 60, 80, 100, or 120 μmol Cu(II)(5- DESS)/kg of body mass 3 hours before exposure to 8.0 Gy, gamma irradiation. In this paradigm, doses of Cu(II)(5- DESS) increased survival up to 92% above vehicle-treated control mice (P = 0.008). Mean body mass determinations revealed that mice treated with 80 to 120 μmol Cu(II)(5-DESS)/kg of body mass exhibited a smaller decrease in body mass than other complex-treated groups. These results support the hypothesis that Cu(II)(5-DESS) is an effective radioprotectant

Phase II Open Label Study of Valproic Acid in Spinal Muscular Atrophy

UNLABELLED:Preliminary in vitro and in vivo studies with valproic acid (VPA) in cell lines and patients with spinal muscular atrophy (SMA) demonstrate increased expression of SMN, supporting the possibility of therapeutic benefit. We performed an open label trial of VPA in 42 subjects with SMA to assess safety and explore potential outcome measures to help guide design of future controlled clinical trials. Subjects included 2 SMA type I ages 2-3 years, 29 SMA type II ages 2-14 years and 11 type III ages 2-31 years, recruited from a natural history study. VPA was well-tolerated and without evident hepatotoxicity. Carnitine depletion was frequent and temporally associated with increased weakness in two subjects. Exploratory outcome measures included assessment of gross motor function via the modified Hammersmith Functional Motor Scale (MHFMS), electrophysiologic measures of innervation including maximum ulnar compound muscle action potential (CMAP) amplitudes and motor unit number estimation (MUNE), body composition and bone density via dual-energy X-ray absorptiometry (DEXA), and quantitative blood SMN mRNA levels. Clear decline in motor function occurred in several subjects in association with weight gain; mean fat mass increased without a corresponding increase in lean mass. We observed an increased mean score on the MHFMS scale in 27 subjects with SMA type II (p<or=0.001); however, significant improvement was almost entirely restricted to participants <5 years of age. Full length SMN levels were unchanged and Delta7SMN levels were significantly reduced for 2 of 3 treatment visits. In contrast, bone mineral density (p<or=0.0036) and maximum ulnar CMAP scores (p<or=0.0001) increased significantly. CONCLUSIONS:While VPA appears safe and well-tolerated in this initial pilot trial, these data suggest that weight gain and carnitine depletion are likely to be significant confounding factors in clinical trials. This study highlights potential strengths and limitations of various candidate outcome measures and underscores the need for additional controlled clinical trials with VPA targeting more restricted cohorts of subjects. TRIAL REGISTRATION:ClinicalTrials.gov

SMA CARNI-VAL Trial Part I: Double-Blind, Randomized, Placebo-Controlled Trial of L-Carnitine and Valproic Acid in Spinal Muscular Atrophy

Valproic acid (VPA) has demonstrated potential as a therapeutic candidate for spinal muscular atrophy (SMA) in vitro and in vivo.Two cohorts of subjects were enrolled in the SMA CARNIVAL TRIAL, a non-ambulatory group of "sitters" (cohort 1) and an ambulatory group of "walkers" (cohort 2). Here, we present results for cohort 1: a multicenter phase II randomized double-blind intention-to-treat protocol in non-ambulatory SMA subjects 2-8 years of age. Sixty-one subjects were randomized 1:1 to placebo or treatment for the first six months; all received active treatment the subsequent six months. The primary outcome was change in the modified Hammersmith Functional Motor Scale (MHFMS) score following six months of treatment. Secondary outcomes included safety and adverse event data, and change in MHFMS score for twelve versus six months of active treatment, body composition, quantitative SMN mRNA levels, maximum ulnar CMAP amplitudes, myometry and PFT measures.At 6 months, there was no difference in change from the baseline MHFMS score between treatment and placebo groups (difference = 0.643, 95% CI = -1.22-2.51). Adverse events occurred in >80% of subjects and were more common in the treatment group. Excessive weight gain was the most frequent drug-related adverse event, and increased fat mass was negatively related to change in MHFMS values (p = 0.0409). Post-hoc analysis found that children ages two to three years that received 12 months treatment, when adjusted for baseline weight, had significantly improved MHFMS scores (p = 0.03) compared to those who received placebo the first six months. A linear regression analysis limited to the influence of age demonstrates young age as a significant factor in improved MHFMS scores (p = 0.007).This study demonstrated no benefit from six months treatment with VPA and L-carnitine in a young non-ambulatory cohort of subjects with SMA. Weight gain, age and treatment duration were significant confounding variables that should be considered in the design of future trials.Clinicaltrials.gov NCT00227266

Phylogeny of snakes (Serpentes): combining morphological and molecular data in likelihood Bayesian and parsimony analyses

Copyright © 2007 The Natural history MuseumThe phylogeny of living and fossil snakes is assessed using likelihood and parsimony approaches and a dataset combining 263 morphological characters with mitochondrial (2693 bp) and nuclear (1092 bp) gene sequences. The ‘no common mechanism’ (NCMr) and ‘Markovian’ (Mkv) models were employed for the morphological partition in likelihood analyses; likelihood scores in the NCMr model were more closely correlated with parsimony tree lengths. Both models accorded relatively less weight to the molecular data than did parsimony, with the effect being milder in the NCMr model. Partitioned branch and likelihood support values indicate that the mtDNA and nuclear gene partitions agree more closely with each other than with morphology. Despite differences between data partitions in phylogenetic signal, analytic models, and relative weighting, the parsimony and likelihood analyses all retrieved the following widely accepted groups: scolecophidians, alethinophidians, cylindrophiines, macrostomatans (sensu lato) and caenophidians. Anilius alone emerged as the most basal alethinophidian; the combined analyses resulted in a novel and stable position of uropeltines and cylindrophiines as the second-most basal clade of alethinophidians. The limbed marine pachyophiids, along with Dinilysia and Wonambi, were always basal to all living snakes. Other results stable in all combined analyses include: Xenopeltis and Loxocemus were sister taxa (fide morphology) but clustered with pythonines (fide molecules), and Ungaliophis clustered with a boine-erycine clade (fide molecules). Tropidophis remains enigmatic; it emerges as a basal alethinophidian in the parsimony analyses (fide molecules) but a derived form in the likelihood analyses (fide morphology), largely due to the different relative weighting accorded to data partitions.Michael S. Y. Lee, Andrew F. Hugall, Robin Lawson & John D. Scanlo