Induction of apoptosis in murine leukemia by diarylheptanoids from Curcuma comosa Roxb.

Diarylheptanoids, isolated from the rhizome of Curcuma comosa Roxb., have several biological activities including anti-oxidant and anti-inflammation. The present study investigated the effect of five diarylheptanoids isolated from C. comosa rhizome on the proliferation of murine P388 leukemic cells. Compound-092, (3S)-1-(3,4-dihydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-ol, bearing a catechol moiety, was the most potent diarylheptanoid (IC₅₀ of 4 μM) in inhibiting P388 leukemic cell viability by causing DNA breakage and inducing apoptosis. Apoptotic cell death was characterized by the presence of chromatin condensation, formation of apoptotic bodies, DNA fragmentation, and externalization of plasma membrane phosphatidylserine. This compound increased caspase-3 activity about fivefold above the untreated control, decreased the intracellular reduced glutathione level, and impaired mitochondrial transmembrane potential. In the presence of Cu(II) ion, the compound exhibited a pro-oxidant activity causing DNA strand breakage and enhancing the anti-proliferative activity. The results provide evidence for the pro-oxidant activity of the diarylheptanoid bearing a catechol moiety in the induction of apoptosis in murine P388 leukemia.     

Identification of a novel voltage-driven organic anion transporter present at apical membrane of renal proximal tubule

A novel transport protein with the properties of voltage-driven organic anion transport was isolated from pig kidney cortex by expression cloning in Xenopus laevis oocytes. A cDNA library was constructed from size-fractionated poly(A)+ RNA and screened for p-aminohippurate (PAH) transport in high potassium medium. A 1856-base pair cDNA encoding a 467-amino acid peptide designated as OATV1 (voltage-driven organic anion transporter 1) was isolated. The predicted amino acid sequence of OATV1 exhibited 60-65% identity to those of human, rat, rabbit, and mouse sodium-dependent phosphate cotransporter type 1 (NPT1), although OATV1 did not transport phosphate. The homology of this transporter to known members of the organic anion transporter family (OAT family) was about 25-30%. OATV1-mediated PAH transport was affected by the changes in membrane potential. The transport was Na+-independent and enhanced at high concentrations of extracellular potassium and low concentrations of extracellular chloride. Under the voltage clamp condition, extracellularly applied PAH induced outward currents in oocytes expressing OATV1. The current showed steep voltage dependence, consistent with the voltage-driven transport of PAH by OATV1. The PAH transport was inhibited by various organic anions but not by organic cations, indicating the multispecific nature of OATV1 for anionic compounds. This transport protein is localized at the apical membrane of renal proximal tubule, consistent with the proposed localization of a voltage-driven organic anion transporter. Therefore, it is proposed that OATV1 plays an important role to excrete drugs, xenobiotics, and their metabolites driven by membrane voltage through the apical membrane of the tubular epithelial cells into the urine.     

Identification of a novel system L amino acid transporter structurally distinct from heterodimeric amino acid transporters

A cDNA that encodes a novel Na+-independent neutral amino acid transporter was isolated from FLC4 human hepatocarcinoma cells by expression cloning. When expressed in Xenopus oocytes, the encoded protein designated LAT3 (L-type amino acid transporter 3) transported neutral amino acids such as l-leucine, l-isoleucine, l-valine, and l-phenylalanine. The LAT3-mediated transport was Na+-independent and inhibited by 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, consistent with the properties of system L. Distinct from already known system L transporters LAT1 and LAT2, which form heterodimeric complex with 4F2 heavy chain, LAT3 was functional by itself in Xenopus oocytes. The deduced amino acid sequence of LAT3 was identical to the gene product of POV1 reported as a prostate cancer-up-regulated gene whose function was not determined, whereas it did not exhibit significant similarity to already identified transporters. The Eadie-Hofstee plots of LAT3-mediated transport were curvilinear, whereas the low affinity component is predominant at physiological plasma amino acid concentration. In addition to amino acid substrates, LAT3 recognized amino acid alcohols. The transport of l-leucine was electroneutral and mediated by a facilitated diffusion. In contrast, l-leucinol, l-valinol, and l-phenylalaninol, which have a net positive charge induced inward currents under voltage clamp, suggesting these compounds are transported by LAT3. LAT3-mediated transport was inhibited by the pretreatment with N-ethylmaleimide, consistent with the property of system L2 originally characterized in hepatocyte primary culture. Based on the substrate selectivity, affinity, and N-ethylmaleimide sensitivity, LAT3 is proposed to be a transporter subserving system L2. LAT3 should denote a new family of organic solute transporters.     

Identification of a novel Na+-independent acidic amino acid transporter with structural similarity to the member of a heterodimeric amino acid transporter family associated with unknown heavy chains

We identified a novel Na(+)-independent acidic amino acid transporter designated AGT1 (aspartate/glutamate transporter 1). AGT1 exhibits the highest sequence similarity (48% identity) to the Na(+)-independent small neutral amino acid transporter Asc (asc-type amino acid transporter)-2 a member of the heterodimeric amino acid transporter family presumed to be associated with unknown heavy chains (Chairoungdua, A., Kanai, Y., Matsuo, H., Inatomi, J., Kim, D. K., and Endou, H. (2001) J. Biol. Chem. 276, 49390-49399). The cysteine residue responsible for the disulfide bond formation between transporters (light chains) and heavy chain subunits of the heterodimeric amino acid transporter family is conserved for AGT1. Because AGT1 solely expressed or coexpressed with already known heavy chain 4F2hc (4F2 heavy chain) or rBAT (related to b(0,+)-amino acid transporter) did not induce functional activity, we generated fusion proteins in which AGT1 was connected with 4F2hc or rBAT. The fusion proteins were sorted to the plasma membrane and expressed the Na(+)-independent transport activity for acidic amino acids. Distinct from the Na(+)-independent cystine/glutamate transporter xCT structurally related to AGT1, AGT1 did not accept cystine, homocysteate, and l-alpha-aminoadipate and exhibited high affinity to aspartate as well as glutamate, suggesting that the negative charge recognition site in the side chain-binding site of AGT1 would be closer to the alpha-carbon binding site compared with that of xCT. The AGT1 message was predominantly expressed in kidney. In mouse kidney, AGT1 protein was present in the basolateral membrane of the proximal straight tubules and distal convoluted tubules. In the Western blot analysis, AGT1 was detected as a high molecular mass band in the nonreducing condition, whereas the band shifted to a 40-kDa band corresponding to the AGT1 monomer in the reducing condition, suggesting the association of AGT1 with other protein via a disulfide bond. The finding of AGT1 and Asc-2 has established a new subgroup of the heterodimeric amino acid transporter family whose members associate not with 4F2hc or rBAT but with other unknown heavy chains.     

Density estimates of two endangered nocturnal lemur species from northern Madagascar: new results and a comparison of commonly used methods

Very little information is known of the recently described Microcebus tavaratra and Lepilemur milanoii in the Daraina region, a restricted area in far northern Madagascar. Since their forest habitat is highly fragmented and expected to undergo significant changes in the future, rapid surveys are essential to determine conservation priorities. Using both distance sampling and capture-recapture methods, we estimated population densities in two forest fragments. Our results are the first known density and population size estimates for both nocturnal species. In parallel, we compare density results from four different approaches, which are widely used to estimate lemur densities and population sizes throughout Madagascar. Four approaches (King, Kelker, Muller and Buckland) are based on transect surveys and distance sampling, and they differ from each other by the way the effective strip width is estimated. The fifth method relies on a capture-mark-recapture (CMR) approach. Overall, we found that the King method produced density estimates that were significantly higher than other methods, suggesting that it generates overestimates and hence overly optimistic estimates of population sizes in endangered species. The other three distance sampling methods provided similar estimates. These estimates were similar to those obtained with the CMR approach when enough recapture data were available. Given that Microcebus species are often trapped for genetic or behavioral studies, our results suggest that existing data can be used to provide estimates of population density for that species across Madagascar.     

The contrasting genetic patterns of two sympatric flying fox species from the Comoros and the implications for conservation

Pteropus livingstonii and Pteropus seychellensis comorensis are endemic fruit bat species that are among the most threatened animals in the Comoros archipelago. Both species are pollinators and seed dispersers of native and cultivated plants and are thus of crucial importance for the regeneration of natural forests as well as for cultivated plantations. However, these species are subject to strong anthropogenic pressures and face one of the highest rates of natural habitat loss reported worldwide. Yet little is known about the population genetic structure of these two species, making it difficult to define relevant conservation strategies. In this study, we investigated for the two flying fox species (1) the level of genetic diversity within islands, as well as across the archipelago and (2) the genetic structure between the two islands (Anjouan and Mohéli) for P. livingstonii and between the four islands of the archipelago (Anjouan, Mohéli, Grande Comore and Mayotte) for P. s. comorensis using mitochondrial and microsatellite markers. The results revealed contrasting patterns of genetic structure, with P. s. comorensis showing low genetic structure between islands, whereas P. livingstonii exhibited high levels of inter-island genetic differentiation. Overall, the genetic analyses showed low genetic diversity for both species. These contrasting genetic patterns may be the result of different dispersal patterns and the populations’ evolutionary histories. Our findings lead us to suggest that in terms of conservation strategy, the two populations of P. livingstonii (on Anjouan and Mohéli islands) should be considered as two separate management units. We recommend focusing conservation efforts on the Anjouan population, which is the largest, exhibits the highest genetic diversity, and suffers the greatest anthropogenic pressure. As for P. s. comorensis, its four populations could be considered as a single unit for conservation management purposes. For this species, we recommend protecting roosting trees to reduce population disturbance.     

Relationship between African dust carried in the Atlantic trade winds and surges in pediatric asthma attendances in the Caribbean

Asthma is epidemic in developed and developing countries including those in the Caribbean where it is widely believed that African dust, transported in high concentrations in the Trade Winds every year, is a major causative factor. The link between asthma and dust in the Caribbean is based largely on anecdotal evidence that associates sharp increases in the occurrence of asthma symptoms with hazy conditions often caused by dust. Here we report on a 2-year study of the relationship between the daily concentrations of dust measured in on-shore Trade Winds at Barbados and pediatric asthma attendance rates at Queen Elizabeth Hospital (QEH). We looked for large increases in QEH daily attendances in relation to daily dust concentrations as previously suggested by anecdotal observations. We could not find any obvious relationship although there may be more subtle linkages between dust and asthma. Our measurements show, however, that the concentration of dust in the size range under 2.5 μm diameter is sufficiently high as to challenge United States Environmental Protection Agency air quality standards for respirable particles. Thus, African dust may constitute a health threat of a different nature, producing symptoms less obvious than those of asthma.     

Effects of organic nutrients and growth factors on biostimulation of tributyltin removal by sediment microorganisms and Enterobacter cloacae

Natural attenuation can reduce contamination of tributyltin (TBT), but persistence of the xenobiotic can cause long-term issues in the environment. Biostimulation is used to accelerate biodegradation. This study investigated the ability of individual organic nutrients and growth factors to enhance TBT biodegradation by sediment microorganisms (SED) and Enterobacter cloacae strain TISTR1971 (B3). The supplements that produced high biomass yield were selected for degradation enhancement. For TBT degradation at initial concentration of 0.1 mg/l, negative or limited degradation was observed in some selected supplements indicating that increasing the biomass did not necessarily promote degradation. Consequently, the addition of nutrients was expected to increase both dioxygenase activity and the degrader population. At different concentrations of supplements, a mixture of succinate/glycerol showed the highest removal for SED which reduced TBT by 77%, 75%, and 68% for 0.1×, 1×, and 10× supplement concentration, respectively. For B3, the addition of succinate showed degradation of 49% (0.1×), 75% (1×), and 77% (10×). Most nutrients and amino acids had an inhibitory effect at 1× or 10× levels. Excess amount of the nutrients added can inhibit the initial degradation of TBT. Therefore, TBT biostimulation requires supplements that increase the capability of TBT degraders at an appropriate amount.     

Molecular dynamics simulation,binding free energy calculation and molecular docking of human D-amino acid oxidase (DAAO) with its inhibitors

Schizophrenia is a mental illness; most affected people live in developing countries, and neither appropriate treatment nor commercial drugs are currently available. One possibility is to inhibit human-d-amino acid oxidase (h-DAAO). In this study, molecular dynamic simulations of the monomer, dimer and tetramer forms of h-DAAO complexed with the inhibitor 3-hydroxyquinolin-2(1H)-one(2) were performed. Seven residues, Leu51, Gln53, Leu215, Tyr228, Ile230, Arg283 and Gly313, were identified as essential for interacting with the inhibitor. Molecular docking of h-DAAO with pyrrole, quinoline and kojic acid derivatives, representing 69 known or potential h-DAAO inhibitors, was also performed. The results indicated that the activity of the inhibitor can be improved by modifying the compounds to have a substituent group capable of interacting with the side chain of Tyr228. Van der Waals interactions of the inhibitor with the hydrophobic pocket of h-DAAO and electrostatic interactions or H-bonds with Arg283 and Gly313 were important elements in determining the efficiency of the inhibitor. These results provide information on the interaction between h-DAAO and its inhibitors at the molecular level and can aid in the design of novel inhibitors against h-DAAO for new drug development in the treatment of schizophrenia.