Determination of Rhodamine 123 in cell lysate by HPLC with visible wavelength detection

Rhodamine 123 (R123) is widely used to quantify P-glycoprotein (P-GP) functional efflux activity in vitro. We developed a rapid and specific high-performance liquid chromatography (HPLC) method to quantify Rhodamine 123 for use in experimental cell culture studies. The R123 standards (2.5-250 ng/mL) and quality controls (QCs) (5, 75, 200 ng/mL) were prepared in cell lysis buffer consisting of 0.75% Triton 100X and 0.2% sodium chloride. The mobile phase consisted of acetonitrile, 1.5 mM tetrabutyl ammonium bromide in 20mM sodium acetate buffer (pH 4.0) (50:20:30) delivered at a rate of 1.0 mL/min. Samples (50 microl) were injected onto a C(18) reversed-phase HPLC column with detection at 500 nm. Analyte retention times were 1.4 and 4.3 min for R123 and internal standard (R6G), respectively. Intra- and inter-day coefficients of variation were < or = 4.2%. Samples were stable for at least three freeze-thaw cycles at room temperature for 24 and 48 h. This method was used to evaluate the functional activity of P-glycoprotein in renal tubule cell models including human kidney (HK-2), Madin-Darby canine kidney (MDCK) and multi-drug resistance gene-transfected MDCK cells (MDR1-MDCK).     

Octopamine receptors in the honeybee (Apis mellifera) brain and their disruption by RNA-mediated interference

Octopamine plays important neuromodulatory roles in the honeybee brain. Accordingly, mRNA from a recently identified honeybee octopamine receptor (AmOA1) is distributed throughout the brain. We have evaluated the occurrence of AmOA1 in the antennal lobe (AL) as well as rest of the brain (RB) by western blotting using an antiserum raised against a peptide selected from AmOA1 sequence. In addition to an expected band (78 kDa in the AL), one additional band (72 kDa) was identified from the AL and four bands (48, 60, 72 and 78 kDa) were observed in the RB. These bands were also recognized with antiserum against a different peptide segment from an octopamine receptor ortholog from the fruitfly (OAMB). Significant sequence identity with the peptide segment used to generate the antiserum was only found with OAMB and its splice variants in fruitfly; it was less conserved in other biogenic amine receptors from honeybee and other insects. Furthermore, western blot analysis performed on brains with dsRNA-treated antennal lobes showed a decrease in the intensity of all four bands. This suggests that AmOA1 antiserum specifically recognizes one or more types of AmOA1 receptors in the honeybee brain. We extend our earlier study of RNAi to quantify the rate of spread of dsRNA from a localized injection to other neuropils.     

Synthesis, crystal structures and, antibacterial and antiproliferative activities in vitro of palladium(II) complexes of triphenylphosphine and thioamides

Palladium(II) complexes with triphenylphosphine (PPh₃) and thioamides of the general formulae, [Pd(L)₂(PPh₃)₂]Cl₂ and [Pd(L)₂(PPh₃)₂] have been prepared and characterized by elemental analysis, IR and NMR (¹H, ¹³C and ³¹P) methods, and two of them (trans-[Pd(PPh₃)₂(Dmtu)₂]Cl₂·(H₂O)(CH₃OH)_0.5 (1) and trans-[Pd(PPh₃)₂(Mpy)₂] (2)) by X-ray crystallography; where L = thiourea (Tu), methylthiourea (Metu), N,N′-dimethylthiourea (Dmtu), tetramethylthiourea (Tmtu), 2-mercaptopyridine (Mpy), 2-mercaptopyrimidine (Mpm) and thionicotinamide (Tna). The spectral data of the complexes are consistent with the sulfur coordination of thioamides to palladium(II). The crystal structures of the complexes show that (1) has ionic character consisting of [Pd(PPh₃)₂(Dmtu)₂]⁺² cations and uncoordinated Cl⁻ ions, while (2) is a neutral complex with Mpy behaving as anionic thiolate ligand. The coordination environment around palladium in (2) is nearly regular square-planar, while in (1) the trans angles show significant distortions from 180°. The complexes were screened for antibacterial effects, brine shrimps lethality bioassay and antitumor activity. These complexes showed significant activities in most of the cases against the tested bacteria as compared to that of a standard drug. Their antitumor activity against prostate cancer cells (PC3) is comparable with doxorubicin, together with no cytotoxic effects in brine shrimps lethality bioassay study.     

Moderate reduction of Norrin signaling activity associated with the causative missense mutations identified in patients with familial exudative vitreoretinopathy

Mutations in Norrin signaling genes (NDP, FZD4 and LRP5) have been found in patients with familial exudative vitreoretinopathy (FEVR) and the altered signaling is suspected to play a critical role in its pathogenesis. To better understand this relationship, we systematically performed functional analyses on previously identified single nucleotide variants of LRP5, FZD4 and NDP, utilizing the Norrin dependent Topflash reporter assay. Cell surface binding assays and protein electrophoresis analysis of Norrin were also performed. Seven causative mutations and five possibly causative but indecisive variants were examined. We found: (1) a nonsense mutation in FZD4 completely abolished its signaling activity, while single missense mutations in LRP5 and FZD4 caused a moderate level of reduction (ranging from 26 to 48, 36% on average) and a double missense mutation in both genes caused a severe reduction in activity (71%). These observations correlated roughly with clinical phenotypes. (2) A mutational effect is suggested in four of five indecisive variants by signaling reductions comparable to those of missense mutations. (3) Norrin mutants demonstrated variable effects on signal transduction, and no apparent correlation with clinical phenotypes was observed. (4) The Norrin mutants examined demonstrated impaired cell surface binding, and some may have partially lost their ability to form a complex with unknown high molecular weight material(s). Our results illustrate the nature of FEVR in relation to Norrin signaling and further suggest the complexity of its disease causing mechanism. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evaluation of Haplotype Inference Using Definitive Haplotype Data Obtained from Complete Hydatidiform Moles, and Its Significance for the Analyses of Positively Selected Regions

The haplotype map constructed by the HapMap Project is a valuable resource in the genetic studies of disease genes, population structure, and evolution. In the Project, Caucasian and African haplotypes are fairly accurately inferred, based mainly on the rules of Mendelian inheritance using the genotypes of trios. However, the Asian haplotypes are inferred from the genotypes of unrelated individuals based on population genetics, and are less accurate. Thus, the effects of this inaccuracy on downstream analyses needs to be assessed. We determined true Japanese haplotypes by genotyping 100 complete hydatidiform moles (CHM), each carrying a genome derived from a single sperm, using Affymetrix 500 K Arrays. We then assessed how inferred haplotypes can differ from true haplotypes, by phasing pseudo-individualized true haplotypes using the programs PHASE, fastPHASE, and Beagle. We found that, at various genomic regions, especially the MHC locus, the expansion of extended haplotype homozygosity (EHH), which is a measure of positive selection, is obscured when inferred Asian haplotype data is used to detect the expansion. We then mapped the genome using a new statistic, XDiHH, which directly detects the difference between the true and inferred haplotypes, in the determination of EHH expansion. We also show that the true haplotype data presented here is useful to assess and improve the accuracy of phasing of Asian genotypes.     

A Unimodal Species Response Model Relating Traits to Environment with Application to Phytoplankton Communities

In this paper we attempt to explain observed niche differences among species (i.e. differences in their distribution along environmental gradients) by differences in trait values (e.g. volume) in phytoplankton communities. For this, we propose the trait-modulated Gaussian logistic model in which the niche parameters (optimum, tolerance and maximum) are made linearly dependent on species traits. The model is fitted to data in the Bayesian framework using OpenBUGS (Bayesian inference Using Gibbs Sampling) to identify according to which environmental variables there is niche differentiation among species and traits. We illustrate the method with phytoplankton community data of 203 lakes located within four climate zones and associated measurements on 11 environmental variables and six morphological species traits of 60 species. Temperature and chlorophyll-a (with opposite signs) described well the niche structure of all species. Results showed that about 25% of the variance in the niche centres with respect to chlorophyll-a were accounted for by traits, whereas niche width and maximum could not be predicted by traits. Volume, mucilage, flagella and siliceous exoskeleton are found to be the most important traits to explain the niche centres. Species were clustered in two groups with different niches structures, group 1 high temperature-low chlorophyll-a species and group 2 low temperature-high chlorophyll-a species. Compared to group 2, species in group 1 had larger volume but lower surface area, had more often flagella but neither mucilage nor siliceous exoskeleton. These results might help in understanding the effect of environmental changes on phytoplankton community. The proposed method, therefore, can also apply to other aquatic or terrestrial communities for which individual traits and environmental conditioning factors are available.     

Extracellular invertase is an essential component of cytokinin-mediated delay of senescence

Leaf senescence is the final stage of leaf development in which the nutrients invested in the leaf are remobilized to other parts of the plant. Whereas senescence is accompanied by a decline in leaf cytokinin content, exogenous application of cytokinins or an increase of the endogenous concentration delays senescence and causes nutrient mobilization. The finding that extracellular invertase and hexose transporters, as the functionally linked enzymes of an apolasmic phloem unloading pathway, are coinduced by cytokinins suggested that delay of senescence is mediated via an effect on source-sink relations. This hypothesis was further substantiated in this study by the finding that delay of senescence in transgenic tobacco (Nicotiana tabacum) plants with autoregulated cytokinin production correlates with an elevated extracellular invertase activity. The finding that the expression of an extracellular invertase under control of the senescence-induced SAG12 promoter results in a delay of senescence demonstrates that effect of cytokinins may be substituted by these metabolic enzymes. The observation that an increase in extracellular invertase is sufficient to delay leaf senescence was further verified by a complementing functional approach. Localized induction of an extracellular invertase under control of a chemically inducible promoter resulted in ectopic delay of senescence, resembling the naturally occurring green islands in autumn leaves. To establish a causal relationship between cytokinins and extracellular invertase for the delay of senescence, transgenic plants were generated that allowed inhibition of extracellular invertase in the presence of cytokinins. For this purpose, an invertase inhibitor was expressed under control of a cytokinin-inducible promoter. It has been shown that senescence is not any more delayed by cytokinin when the expression of the invertase inhibitor is elevated. This finding demonstrates that extracellular invertase is required for the delay of senescence by cytokinins and that it is a key element of the underlying molecular mechanism.     

Appearance of direct-acting mutagenicity of various foodstuffs produced in Japan and Southeast Asia on nitrite treatment

After nitrite treatment, various kinds of pickled vegetables and sun-dried fishes produced in Japan showed direct-acting mutagenicity on Salmonella typhimurium TA100, inducing 1900-18000 revertants/g. Kimchis, sun-dried fishes, sun-dried squid, soy sauces, fish sauces, bean pastes and shrimp paste produced in Korea, the Philippines and Thailand also showed direct-acting mutagenicity after nitrite treatment. All soy sauces and fish sauces tested contained as much tyramine as 17-1020 micrograms/ml, but very low or undetectable amounts of (-)-(1S,3S)- and (-)-(1R,3S)-1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acids.     

5-Aminolevulinic Acid-Induced Heavy Metal Stress Tolerance and Underlying Mechanisms in Plants

Journal of Plant Growth Regulation - Plants face different types of biotic and abiotic stresses during their life span. Heavy metal (HM) stress is considered as one of the most challenging and...