Cytotoxic and mutagenic evaluation of extracts from plant species of the Miconia genus and their influence on doxorubicin-induced mutagenicity: An in vitro analysis

The Miconia genus, a plant widely used for medicine, occurs in tropical America and its extracts and isolated compounds have demonstrated antibiotic, antitumoral, analgesic and antimalarial activities. However, no study concerning its genotoxicity has been conducted and it is necessary to determine its potential mutagenic effects to develop products and chemicals from these extracts. This study assessed the cytotoxicity, mutagenicity and the protective effects of methanolic extracts from Miconia species on Chinese hamster lung fibroblast cell cultures (V79). The cytotoxicity was evaluated using a clonogenic assay. Cultures exposed to the extract of Miconia albicans up to a concentration of 30 mu g/mL, M. cabucu up to 40 mu g/mL, M. albicans up to 40 mu g/mL and M. stenostachya up to 60 mu g/mL exhibited a cytotoxic effect on the cells. The clonogenic assay used three non-cytotoxic concentrations (5, 10 and 20 mu g/mL) to evaluate mutagenicity and antimutagenicity of the extracts. Cultures were treated with these three extract concentrations (mutagenicity test) or the extract associated with doxorubicin (DXR) (antimutagenicity test) in three protocols (pre-, simultaneous and post-treatments). Distilled water and DXR were used as negative and positive controls, respectively. In the micronucleus (MN) test, a significant reduction was observed in MN frequency in cultures treated with DXR and extracts compared to those receiving only DXR; a significant reduction was also observed for the presence of mutagenicity in all treatments. This study confirmed the safe use of Miconia extracts at the concentrations tested and reinforced the therapeutic properties previously described for Miconia species by showing their protective effects on doxorubicin-induced mutagenicity. (C) 2010 Elsevier GmbH. All rights reserved.

[beta]-Hydroxy-F128b-Methylbutyrate (HMB) Supplementation and the Promotion of Muscle Growth and Strength

[beta]-Hydroxy [beta]-methylbutyrate (HMB), a metabolite of the essential amino acid leucine, is one of the latest dietary supplements promoted to enhance gains in strength and lean body mass associated with resistance training. Unlike anabolic hormones that induce muscle hypertrophy by increasing muscle protein synthesis, HMB is claimed to influence strength and lean body mass by acting as an anticatabolic agent, minimising protein breakdown and damage to cells that may occur with intense exercise. Research on HMB has recently tested this hypothesis, under the assumption that it may be the active compound associated with the anticatabolic effects of leucine and its metabolites. While much of the available literature is preliminary in nature and not without methodological concern, there is support for the claims made regarding HMB supplementation, at least in young, previously untrained individuals. A mechanism by which this may occur is unknown, but research undertaken to date suggests there may be a reduction in skeletal muscle damage, although this has not been assessed directly. The response of resistance trained and older individuals to HMB administration is less clear. While the results of research conducted to date appear encouraging, caution must be taken when interpreting outcomes as most manuscripts are presented in abstract form only, not having to withstand the rigors of peer review. Of the literature reviewed relating to HMB administration during resistance training, only 2 papers are full manuscripts appearing in peer reviewed journals. The remaining 8 papers are published as abstracts only, making it difficult to critically review the research. There is clearly a need for more tightly controlled, longer duration studies to verify if HMB enhances strength and muscular hypertrophy development associated with resistance training across a range of groups, including resistance trained individuals.

Time-reversal test for stochastic quantum dynamics

The calculation of quantum dynamics is currently a central issue in theoretical physics, with diverse applications ranging from ultracold atomic Bose-Einstein condensates to condensed matter, biology, and even astrophysics. Here we demonstrate a conceptually simple method of determining the regime of validity of stochastic simulations of unitary quantum dynamics by employing a time-reversal test. We apply this test to a simulation of the evolution of a quantum anharmonic oscillator with up to 6.022×1023 (Avogadro's number) of particles. This system is realizable as a Bose-Einstein condensate in an optical lattice, for which the time-reversal procedure could be implemented experimentally.

High-Frequency Turbulence and Sediment Flux Measurements in an Upper Estuarine Zone

In natural estuaries, the predictions of scalar dispersion are rarely predicted accurately because of a lack of fundamental understanding of the turbulence structure in estuaries. Herein detailed turbulence field measurements were conducted continuously at high frequency for 50 hours in the upper zone of a small subtropical estuary with semi-diurnal tides. Acoustic Doppler velocimetry was deemed the most appropriate measurement technique for such shallow water depths (less than 0.4 m at low tides), and a thorough post-processing technique was applied. In addition, some experiments were conducted in laboratory under controlled conditions using water and soil samples collected in the estuary to test the relationship between acoustic backscatter strength and suspended sediment load. A striking feature of the field data set was the large fluctuations in all turbulence characteristics during the tidal cycle, including the suspended sediment flux. This feature was rarely documented.

A PCR-based method of screening individuals of all ages, from neonates to the elderly, for familial hyperaldosteronism type I

Aim: Unless specifically treated (glucocorticoids in low doses), Familial Hyperaldosteronism Type I(FH-I) may result in early death from stroke. We report the successful application of a rapid, polymerase chain reaction (PCR)-based method of detecting the 'hybrid' 11 beta-hydroxylase (11 beta-OHase)/aldosterone synthase (AS) gene as a screening test for FH-I. Methods: 'Long-PCR' was used to amplify, concurrently, a 4 kb fragment of AS gene (both primers AS-specific) and a 4 kb fragment of the hybrid gene (5' primer 11 beta-OHase-specific, 3'primer AS-specific) from DNA extracted from blood either collected locally or transported from elsewhere. Sample collection and transport were straightforward. This 4 kb fragment contains all the currently recognised hybrid gene 'crossover' points. Results: Within a single family, long-PCR identified all 21 individuals known to have FH-I. Hypertension was corrected in all 11 treated with glucocorticoids. Nine with normal blood pressure are being closely followed for development of hypertension. Long-PCR cord blood analysis excluded FH-I in three neonates born to affected individuals. Long-PCR newly identified two other affected families: (1) a female (60 years) with a personal and family history of stroke and her normotensive daughter (40 years), and (2) a female (51 years) previously treated for primary aldosteronism with amiloride, her two hypertensive sons (14 and 16 years) and her hypertensive mother (78 years). No false negative or false positive results have yet been encountered. At least seven other centres have successfully performed this test. Conclusion: Long-PCR is a reliable method of screening individuals of all ages for FH-I.

Validity of (-)-[H-3]-CGP 12177A as a radioligand for the 'putative beta(4)-adrenoceptor' in rat atrium

1 We have recently suggested the existence in the heart of a 'putative beta(4)-adrenoceptor' based on the cardiostimulant effects of non-conventional partial agonists, compounds that cause cardiostimulant effects at greater concentrations than those required to block beta(1)- and Bz-adrenoceptors. We sought to obtain further evidence by establishing and validating a radioligand binding assay for this receptor with (-)-[H-3]-CGP 12177A ((-)-4-(3-tertiarybutylamino-2-hydroxypropoxy) benzimidazol-2-one) in rat atrium. We investigated (-)-[H-3]-CGP 12177A for this purpose for two reasons, because it is a nonconventional partial agonist and also because it is a hydrophilic radioligand. 2 Increasing concentrations of(-)-[H-3]-CGP 12177A, in the absence or presence of 20 mu M (-)-CGP 12177A to define non-specific binding, resulted in a biphasic saturation isotherm. Low concentrations bound to beta(1)- and beta(2)-adrenoceptors (pK(D) 9.4+/-0.1, B-max 26.9+/-3.1 fmol mg(-1) protein) and higher concentrations bound to the 'putative beta(4)-adrenoceptor' (pK(D) 7.5+/-0.1, B-max 47.7+/-4.9 fmol mg(-1) protein). In other experiments designed to exclude beta(1)- and beta(2)-adrenoceptors, (-)-[H-3]-CGP 12177A (1-200 nM) binding in the presence of 500 nM (-)-propranolol was also saturable (pK(D) 7.6+/-0.1, B-max 50.8+/-7.4 fmol mg(-1) protein). 3 The non-conventional partial agonists (-)-CGP 12177A (pK(i) 7.3+/-0.2), (+/-)-cyanopindolol (pK(i) 7.6+/-0.2), (-)-pindolol (pK(i) 6.6+/-0.1) and (+)-carazolol (pk(i), 7.2+/-0.2) and the antagonist (-)-bupranolol (pK(i) 6.6+/-0.2), all competed for (-)-[H-3]-CGP 12177A binding in the presence of 500 nM (-)-propranolol at the 'putative beta(4)-adrenoceptor', with affinities closely similar to potencies and affinities determined in organ bath studies. 4 The catecholamines competed with (-)-[H-3]-CGP 12177A at the 'putative beta(4)-adrenoceptor' in a stereoselective manner, (-)-noradrenaline (pK(iH) 6.3 +/- 0.3, pK(i), 3.5 +/- 0.1), (-)-adrenaline (pK(iH) 6.5 +/- 0.2, pK(iL) 2.9 +/- 0.1), (-)-isoprenaline (pK(iH) 6.2 +/- 0.5, pK(iL) 3.3 +/- 0.1), (+)-isoprenaline (pK(i) < 1.7), (-)-R0363 ((-)-(1-(3,4-dimethoxyphenethylamino)-3-(3,4-dihydroxyphenoxy)-2-propranol)oxalate, pK(i) 5.5 +/- 0.1). 5 The inclusion of guanosine 5-triphosphate (GTP 0.1 mM) had no effect on binding of (-)-CGP 12177A or (-)-isoprenaline to the 'putative beta(4)-adrenoceptor'. In competition binding studies, (-)-CGP 12177A competed with (-)-[H-3]-CGP 12177A for one receptor state in the absence (pK(i) 7.3 +/- 0.2) or presence of GTP (pK(i) 7.3 +/- 0.2). (-)-Isoprenaline competed with (-)-[H-3]-CGP 12177A for two states in the absence (pK(iH) 6.6 +/- 0.3, pK(iL) 3.5 +/- 0.1; % H 25 +/- 7) or presence of GTP (pK(iH) 6.2 +/- 0.5, pK(iL) 3.4 +/- 0.1; % H 37 +/- 6). In contrast, at beta(1)-adrenoceptors, GTP stabilized the low affinity state of the receptor for (-)-isoprenaline. 6 The specificity of binding to the 'putative beta(4)-adrenoceptor' was tested with compounds active at other receptors. High concentrations of the beta(4)-adrenoceptor agonists, BRL 37344 ((RR + SS)[4-[2-[[2-(3-chlorophenyl)-2-hydroxy -ethyl]amino]propyl]phenoxy]acetic acid, 6 mu M), SR 58611A (ethyl((7S)-7-[(2R)-2-(3-chlorophenyl)-2-hydroxyethylamino]-5,6,7,8-tetrahydronaphtyl-2-yloxy) acetate hydrochloride, 6 mu M), ZD 2079 ((+/-)-1-phenyl-2-(2-4-carboxymethylphenoxy)-ethylamino)ethan-1-ol, 60 mu M), CL 316243 (disodium (R,R)-5-[2-[2-(3-chlorophenyl)-2-hydroxyethyl-amino]propyl]- 1,3-benzodioxole-2,2-dicarboxylate, 60 mu M) and antagonist SR 59230A (3-(2-ethylphenoxy)-1-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-2S-2-propanol oxalate, 6 mu M) caused less than 22% inhibition of (-)-[H-3]-CGP 12177A binding in the presence of 500 nM (-)-propranolol. Histamine (1 mM), atropine (1 mu M), phentolamine (10 mu M), 5-HT(100 mu M) and the 5-HT4 receptor antagonist SE 207710 ((1-butyl-4-piperidinyl)-methyl 8-amino-7-iodo-1 ,4-benzodioxan-5-carboxylate, 10 nM) caused less than 26% inhibition of binding. 7 Non-conventional partial agonists, the antagonist (-)-bupranolol and catecholamines all competed for (-)-[H-3]-CGP 12177A binding in the absence of (-)-propranolol at beta(1)-adrenoceptors, with affinities (pK(i)) ranging from 1.6-3.6 log orders greater than at the 'putative beta(4)-adrenoceptor'. 8 We have established and validated a radioligand binding assay in rat atrium for the 'putative beta(4)-adrenoceptor' which is distinct from beta(1)-, beta(2)- and beta(3)-adrenoceptors. The stereoselective interaction with the catecholamines provides further support for the classification of the receptor as 'putative beta(4)-adrenoceptor'.

Synthesis, NMR studies and conformational analysis of oxazolidine derivatives of the beta-adrenoreceptor antagonists metoprolol, atenolol and timolol

Formaldehyde-derived oxazolidine derivatives 4-7 of the beta-adrenoreceptor antagonists metoprolol 1, atenolol 2 and timolol 3 have been synthesised. Conformational analysis of 1-3 and the oxazolidine derivatives 4-7 has been performed using H-1 NMR spectroscopy and computational methods. The H-1 NMR studies show that for the aryloxypropanolamine beta-adrenoreceptor antagonists there is a predominance of the conformer in which the amine group is approximately antiperiplanar or trans to the aryloxymethylene group. Both H-1 NMR data and theoretical studies indicate that the oxazolidine derivatives 4-7 and the aryloxypropanolamine beta-adrenoreceptor antagonists 1-3 adopt similar conformations around the beta-amino alcohol moiety. Thus, oxazolidine ring formation does not dramatically alter the preferred conformation adopted by the beta-amino alcohol moiety of 1-3. Oxazolidine derivatives of aryloxypropanolamine beta-adrenoreceptor antagonists may therefore be appropriate as prodrugs, or semi-rigid analogues, when greater lipophilicity is required for drug delivery.

Copper(II) complexes of mono- and di-nucleating hexaamines

The potentially hexadentate polyamines N,N,N',N'-tetrakis(2-aminoethyl)ethane-1,2-diamine (L-1) and the octamethylated analogue N,N,N',N'-tetrakis(2-dimethylaminoethyl)ethane 1,2-diamine (L-2) have been complexed with copper(II) and the crystal structures of their complexes determined. A trigonal-bipyramidal co-ordination geometry for [Cu(HL1)][ClO4](3) was found where one aminoethyl arm is not co-ordinated. By contrast, a dinuclear structure of formula [(H2O)Cu(L-2)Cu(OH)](3+) was determined for the N-methylated analogue, where the hexaamine acts as a bridging ligand between the two square-pyramidal metal centres. Electronic and EPR spectroscopy are both consistent with these structures being maintained in solution.

Mutational analysis of the cystathionine beta-synthase gene: A splicing mutation, two missense mutations and an insertion in patients with homocystinuria

RT-PCR and direct sequence analyses were used to define mutations in the cystathionine beta-synthase (CBS) gene in two unrelated male patients with vitamin B6 nonresponsive homocystinuria. Both patients were compound heterozygotes for CBS alleles containing point mutations. One patient had a maternally derived G-->A transition in the splice-donor site of intron 1, resulting in aberrant splicing of CBS mRNA. The other allele contained a missense mutation resulting in the previously reported E144K mutant CBS protein. The second patient had a maternally derived 4 bp insertion in exon 17, predicted to cause a CBS peptide of altered amino acid sequence. A 494G-->A transition was found in exon 4 of the other allele, predicting a C165Y substitution. Expression of recombinant CBS protein, containing the C165Y mutation, had no detectable catalytic activity. Each mutation was confirmed in genomic DNA. (C) 1998 Wiley-Liss, Inc.

(-)-CGP 12177 causes cardiostimulation and binds to cardiac putative beta(4)-adrenoceptors in both wild-type and beta(3)-adrenoceptor knockout mice

Some blockers of beta(1)- and beta(2)-adrenoceptors cause cardiostimulant effects through an atypical beta-adrenoceptor (putative beta(4)-adrenoceptor) that resembles the beta(3)-adrenoceptor. It is likely but not proven that the putative beta(4)-adrenoceptor is genetically distinct from the beta(3)-adrenoceptor. We therefore investigated whether or not the cardiac atypical beta-adrenoceptor could mediate agonist effects in mice lacking a functional beta(3)-adrenoceptor gene (beta(3)KO). (-)-CGP 12177, a beta(1)- and beta(2)-adrenoceptor blocker that causes agonist effects through both beta(3)-adrenoceptors and cardiac putative beta(4)-adrenoceptors, caused cardiostimulant effects that were not different in atria from wild-type (WT) mice and beta(3)KO mice. The effects of (-)-CGP 12177 were resistant to blockade by (-)-propranolol (200 nM) but were blocked by (-)-bupranolol (1 mu M) with an equilibrium dissociation constant of 15 nM in WT and 17 nM in beta(3)KO. (-)-[H-3]CGP 12177 labeled a similar density of the putative beta(4)-adrenoceptor in ventricular membranes from the hearts of both WT (B-max = 52 fmol/mg protein) and beta(3)KO (B-max = 53 fmol/mg protein) mice. The affinity of (-)-[H-3]CGP 12177 for the cardiac putative beta(4)-adrenoceptor was not different between WT (K-d = 46 nM) and beta(3)KO (K-d = 40 nM). These results provide definitive evidence that the cardiac putative beta(4)-adrenoceptor is distinct from the beta(3)-adrenoceptor.

Lemon oil to beta-cyclodextrin ratio effect on the inclusion efficiency of beta-cyclodextrin and the retention of oil volatiles in the complex

Microencapsulation of lemon oil was undertaken with beta-cyclodextrin using a precipitation method at the five lemon oil to beta-cyclodextrin ratios of 3:97, 6:94, 9:91, 12:88, and 15:85 (w/w) in order to determine the effect of the ratio of lemon oil to beta-cyclodextrin on the inclusion efficiency of beta-cyclodextrin for encapsulating oil volatiles. The retention of lemon oil volatiles reached a maximum at the lemon oil to beta-cyclodextrin ratio of 6:94; however, the maximum inclusion capacity of beta-cyclodextrin and a maximum powder recovery were achieved at the ratio of 12:88, in which the beta-cyclodextrin complex contained 9.68% (w/w) lemon oil. The profile and proportion of selected flavor compounds in the beta-cyclodextrin complex and the starting lemon oil were not significantly different.

The contribution of classical (beta(1/2)-) and atypical beta-adrenoceptors to the stimulation of human white adipocyte lipolysis and right atrial appendage contraction by novel beta(3)-adrenoceptor agonists of differing selectivities

The role of beta(3)- and other putative atypical beta-adrenaceptors in human white adipocytes and right atrial appendage has been investigated using CGP 12177 and novel phenylethanolamine and aryloxypropanolamine beta(3)-adrenoceptor (beta(3)AR) agonists with varying intrinsic activities and selectivities for human cloned PAR subtypes. The ability to demonstrate beta(1/2)AR antagonist-insensitive (beta(3) or other atypical beta AR-mediated) responses to CGP 12177 was critically dependent on the albumin batch used to prepare and incubate the adipocytes. Four aryloxypropanolamine selective beta(3)AR agonists (SB-226552, SB-229432, SB-236923, SB-246982) consistently elicited beta(1/2)AR antagonist-insensitive lipolysis. However, a phenylethanolamine (SB-220646) that was a selective full beta(3)AR agonist elicited full lipolytic and inotropic responses that were sensitive to beta(1/2)AR antagonism, despite it having very low efficacies at cloned beta(1)- and beta(2)ARs. A component of the response to another phenylethanolamine selective beta(3)AR agonist (SB-215691) was insensitive to beta(1/2)AR antagonism in some experiments. Because novel aryloxypropanolamine had a beta(1/2)AR antagonist-insensitive inotropic effect, these results establish more firmly that beta(3)ARs mediate lipolysis in human white adipocytes, and suggest that putative 'beta(4)ARs' mediate inotropic responses to CGP 12177. The results also illustrate the difficulty of predicting from studies on cloned beta ARs which beta ARs will mediate responses to agonists in tissues that have a high number of beta(1)- and beta(2)ARs or a low number of beta(3)ARs.

Comparative in vitro effects of closantel and selected beta-ketoamide anthelmintics on a gastrointestinal nematode and vertebrate liver cells

PNU-87407 and PrNU-88509, beta-ketoamide anthelmintics that are structurally related to each other and to the salicylanilide anthelmintic closantel, exhibit different anthelmintic spectra and apparent toxicity in mammals, The basis for this differential pharmacology was examined in experiments that measured motility and adenosine triphosphate (ATP) levels in larval and adult stages of the gastrointestinal nematode, Haemonchus contortus, and in a vertebrate liver cell line and mitochondria, PNU-87407 and PNU-88509 both exhibited functional cross-resistance with closantel in larval migration assays using closantel-resistant and -sensitive isolates of H, contortus. Each compound reduced motility and,ATP levels in cultured adult H. contortus in a concentration- and time-dependent manner: however, motility was reduced more rapidly by PNU-88509, and ATP levels were reduced by lower concentrations of closantel than the beta-ketoamides. Tension recordings from segments of adult H, contortus showed that PNU-88509 induces spastic paralysis, while PNU-87407 and closantel induce flaccid paralysis of the somatic musculature. Marked differences in the actions of these compounds were also observed in the mammalian preparations. In Chang liver cells, ATP levels were reduced after 3 h exposures to greater than or equal to 0.25 mu M PNU-87407 1 mu M closantel or 10 mu M PNU-88509, Reductions in ATP caused by PNU-88509 were completely reversible, while the effects of closantel and PNU-87407; were irreversible. PNU-87407, closantel and PNU-88509 uncoupled oxidative phosphorylation in isolated rat liver mitochondria, inhibiting the respiratory control index (with glutamate or succinate as substrate) by 50% at concentrations of 0.14, 0.9 and 7.6 mu M respectively.

Febrile seizures and generalized epilepsy associated with a mutation in the Na+-channel beta 1 subunit gene SCN1B

Febrile seizures affect approximately 3% of all children under six years of age and are by far the most common seizure disorder(1). A small proportion of children with febrile seizures later develop ongoing epilepsy with afebrile seizures(2). Segregation analysis suggests the majority of cases have complex inheritance(3) but rare families show apparent autosomal dominant: inheritance. Two putative loci have been mapped (FEB1 and FEB2), but specific genes have not yet been identified(4,5). We recently described a clinical subset, termed generalized epilepsy with febrile seizures plus (GEFS(+)), in which many family members have seizures with fever that may persist beyond six years of age or be associated with afebrile generalized seizures(6). We now report linkage, in another large GEFS(+) family, to chromosome region 19q13.1 and identification of a mutation in the voltage-gated sodium (Na+)-channel beta 1 subunit gene (SCN1B). The mutation changes a conserved cysteine residue disrupting a putative disulfide bridge which normally maintains an extracellular immunoglobulin-like fold. Go-expression of the mutant pr subunit with a brain Na+-channel alpha subunit in Xenopus laevis oocytes demonstrates that the mutation interferes with the ability of the subunit to modulate channel-gating kinetics consistent with a loss-of-function allele. This observation develops the theme that idiopathic epilepsies are a family of channelopathies and raises the possibility of involvement of other Na+-channel subunit genes in febrile seizures and generalized epilepsies with complex inheritance patterns.