n-Butanol induces depolymerization of microtubules in vivo and in vitro

The effects of butanol on microtubules (MTs) were examined by immunofluorescence microscopy. Fragmentation of cortical MTs was induced by n-butanol, but not by s- and t-butanols, in cultured tobacco BY-2 cells. Taxol prevented n-butanol-induced MT fragmentation. Fragmented cortical MTs were still attached to the inner face of the plasma membrane when n-butanol-treated protoplasts were ruptured on the slide glass. Moreover, MTs were depolymerized in the presence of n-butanol in vitro. Therefore, n-butanol is not only an activator of phospholipase D but also an effective MT-depolymerizing agent.     

A comprehensive evaluation of imidazole‐zinc reverse stain for current proteomic researches

In this paper, we comprehensively evaluated the capability of imidazole‐zinc reverse stain (ZN) in comparative proteomics. Three commonly used protein gel staining methods, including silver (SN), SYPRO Ruby (SR), and CB stain were investigated alongside for comparison purpose. A transparency scanning procedure, which may deliver more even and contrasting gel images, was found best for documenting ZN stained gels. Our results showed that ZN was more sensitive than SN, SR, and CB. It may reveal as few as 1.8 ng of proteins in a gel. Moreover, ZN was found to provide a linear dynamic range of staining for revealing proteins up to 140 ng, and show an insignificant staining preference. To analyze a ZN stained 2‐D gel image that generally comprises an apparent but even background, the Melanie 4 software was found more suitable than others. Furthermore, ZN demonstrated an equivalent or better MS compatibility than the other three staining methods. Intense and comprehensive MS profiles were frequently observed for ZN stained gel spots. Approximate two‐third of ZN stained gel spots were successfully identified for protein identities. Taken together, our results suggest that the prompt, cost effective and versatile ZN is well suited for current proteomic researches.     

Red Fluorescent Protein pH Biosensor to Detect Concentrative Nucleoside Transport

Human concentrative nucleoside transporter, hCNT3, mediates Na⁺/nucleoside and H⁺/nucleoside co-transport. We describe a new approach to monitor H⁺/uridine co-transport in cultured mammalian cells, using a pH-sensitive monomeric red fluorescent protein variant, mNectarine, whose development and characterization are also reported here. A chimeric protein, mNectarine fused to the N terminus of hCNT3 (mNect.hCNT3), enabled measurement of pH at the intracellular surface of hCNT3. mNectarine fluorescence was monitored in HEK293 cells expressing mNect.hCNT3 or mNect.hCNT3-F563C, an inactive hCNT3 mutant. Free cytosolic mNect, mNect.hCNT3, and the traditional pH-sensitive dye, BCECF, reported cytosolic pH similarly in pH-clamped HEK293 cells. Cells were incubated at the permissive pH for H⁺-coupled nucleoside transport, pH 5.5, under both Na⁺-free and Na⁺-containing conditions. In mNect.hCNT3-expressing cells (but not under negative control conditions) the rate of acidification increased in media containing 0.5 mm uridine, providing the first direct evidence for H⁺-coupled uridine transport. At pH 5.5, there was no significant difference in uridine transport rates (coupled H⁺ flux) in the presence or absence of Na⁺ (1.09 ± 0.11 or 1.18 ± 0.32 mm min⁻¹, respectively). This suggests that in acidic Na⁺-containing conditions, 1 Na⁺ and 1 H⁺ are transported per uridine molecule, while in acidic Na⁺-free conditions, 1 H⁺ alone is transported/uridine. In acid environments, including renal proximal tubule, H⁺/nucleoside co-transport may drive nucleoside accumulation by hCNT3. Fusion of mNect to hCNT3 provided a simple, self-referencing, and effective way to monitor nucleoside transport, suggesting an approach that may have applications in assays of transport activity of other H⁺-coupled transport proteins.Nucleosides are hydrophilic molecules that require transport proteins to mediate their movement across the plasma membrane (1). Human (h)⁷ nucleoside transport (NT) proteins catalyze the vectorial transport of nucleosides, using either concentrative (C) or equilibrative (E) mechanisms (2). hCNTs use either a Na⁺ or H⁺ gradient to accumulate nucleosides against their concentration gradient, whereas hENTs mediate facilitated diffusion of nucleosides down their concentration gradient (3). Nucleoside transporters also transport anti-cancer and anti-viral drugs, and cellular expression of nucleoside transporters is important in cancer therapy as well as in the treatment of cardiovascular, parasitic, and viral diseases (4, 5).Members of the SLC28 family of concentrative nucleoside transporters (CNTs) divide into two phylogenetic subfamilies: hCNT1/2 belonging to one subfamily, and hCNT3 to the other (6–8). Cation substitution and charge/flux ratio studies suggest that hCNT1/2 couple the inward movement of nucleoside to the Na⁺ electrochemical gradient with a 1:1 stoichiometry, whereas hCNT3 can couple nucleoside transport to either the Na⁺ gradient (2 Na⁺:1 nucleoside) or a H⁺ gradient (1 H⁺:1 nucleoside) in the absence of Na⁺ (9, 10). The 2:1 coupling ratio of hCNT3 allows it to develop a trans-membrane nucleoside concentration gradient up to 10-fold higher than that of hCNT1 or hCNT2 (9, 11). At pH 5.5, hCNT3 also transports uridine in the presence of Na⁺ with a 2 cation:1 nucleoside stoichiometry, which raises the possibility that 1 H⁺ and 1 Na⁺ may be transported per nucleoside molecule in these conditions (9–12). Up to this point, however, there has been no direct demonstration that hCNT3 can transport H⁺.Concentrative nucleoside transport has previously been investigated using the Xenopus laevis oocyte expression system and both electrophysiology (two-microelectrode voltage clamp technique) and radioisotope flux measurements (6–9, 12). Electrophysiological experiments are advantageous in that they measure the current induced by addition of substrate in real-time, but they are time-consuming and require specialized equipment and skills. Radioisotope flux assays measure the accumulation of radiolabeled substrate. The need for radiolabeled substrate restricts the range of permeants able to be studied. In addition, radioisotope flux assays are not done in real-time and are labor-intensive, requiring large numbers of oocytes.An attractive alternative approach for the study of hCNT3 would be to measure pH in the immediate vicinity of its intracellular face during H⁺/nucleoside co-transport. These measurements could take advantage of the remarkable progress achieved in the development of genetically encoded fluorophores (13). Indeed, all members of the extended family of homologues and variants of the Aequorea victoria green fluorescent protein (avGFP) exhibit pH-dependent changes in their fluorescent intensity. The spectral changes that occur upon a change in pH can be intensiometric (14), excitation ratiometric (14), emission ratiometric (15), or both excitation and emission ratiometric (16). The apparent pK_a (pK_a′, equal to the pH at which the fluorescence is half-maximal in intensity) for a specific fluorescent protein (FP) is acutely dependent on specific amino acid substitutions in close proximity to the chromophore and can range from less than 3 (17, 18) to greater than 8 (19). Variants with pK_a′ values that are relatively close to intracellular pH values (i.e. ∼7.3 for the mammalian cytosol (20)) are particularly useful as genetically encoded biosensors for dynamic measurement of proton concentrations in living cells.A major development in the area of FP technology has been the identification (21) and subsequent optimization (22, 23) of red fluorescent protein (RFP) homologues of avGFP. The first (monomeric RFP 1 (mRFP1)) (23) and second (the mFruit series) (22) generation-optimized RFPs, derived from tetrameric Discosoma RFP (21), suffer from relatively low brightness relative to other common hues of FP. For example, of the three most red-shifted second generation mFruit variants (mTangerine, mStrawberry, and mCherry) (22), the brightest (mStrawberry) has only 44% of the intrinsic brightness (proportional to the product of extinction coefficient (ϵ) and quantum yield (Φ)) of the popular yellow FP (YFP) Citrine (24) and 76% of the brightness of enhanced avGFP. This limitation has been partially addressed by third generation mRFPs, specifically mApple and TagRFP-T, with fluorescent brightness values on par with, or better than, that of enhanced avGFP (25).Generally speaking, the most red-shifted RFPs derived from Discosoma RFP are relatively pH-insensitive, with the majority of variants having pK_a′ values < 5 (22, 25). A notable exception is the recently reported mApple variant with a pK_a′ of 6.5 (25). The more blue-shifted of the mFruit variants (i.e. mOrange) also have pK_a′ values of 6.5 (22). Several variants of mRFP1 with pK_a′ values >7.5 have been previously reported (26).Here we report the engineering of a pH-sensitive mFruit variant through multiple rounds of directed evolution by random mutagenesis. This RFP, called mNectarine, is appropriate to measure physiological pH changes in mammalian cells, because it has a pK_a′ of 6.9. We have developed a new method to measure H⁺/nucleoside co-transport in mammalian cells, which utilizes hCNT3''s H⁺ coupling characteristics and the pH sensitivity of mNectarine. We fused mNectarine to the cytosolic N terminus of hCNT3 to generate mNect.hCNT3. Fusion of the fluorescent H⁺ sensor to hCNT3 enabled measurement of pH at the intracellular surface of hCNT3, and provided insight into the mechanism of hCNT3 H⁺/uridine co-transport.     

GENOME SIZE IS NOT CORRELATED WITH EFFECTIVE POPULATION SIZE IN THE ORYZA SPECIES

Genome sizes vary widely across the tree of life and the evolutionary mechanism underlined remains largely unknown. Lynch and Conery (2003) proposed that evolution of genome complexity was driven mainly by nonadaptive stochastic forces and presented the observation that genome size was negatively correlated with effective population size (N_e) as a strong support for their hypothesis. Here, we analyzed the relation between N_e and genome size for 10 diploid Oryza species that showed about fourfold genome size variation. Using sequences of more than 20 nuclear genes, we estimated N_e for each species after correction for the effects of demography and heterogeneity of mutation rates among loci and species. Pairwise comparisons and correlation analyses did not detect a negative relationship between N_e and genome size despite about 6.5‐fold interspecies N_e variation. By calculating phylogenetically independent contrasts (PICs) for N_e, we repeated correlation analysis and did not find any correlation between N_e and genome size. These observations suggest that the genome size variation in the Oryza species cannot be explained simply by the effect of effective population size.     

Gene duplication of endothelin 3 is closely correlated with the hyperpigmentation of the internal organs (Fibromelanosis) in silky chickens

During early development in vertebrates, pluripotent cells are generated from the neural crest and migrate according to their presumptive fate. In birds and mammals, one of the progeny cells, melanoblasts, generally migrate through a dorsolateral route of the trunk region and differentiate to melanocytes. However, Silky is an exceptional chicken in which numerous melanoblasts travel via a ventral pathway and disperse into internal organs. Finally, these ectopic melanocytes induce heavy dermal and visceral melanization known as Fibromelanosis (Fm). To identify the genetic basis of this phenotype, we confirmed the mode of inheritance of Fm as autosomal dominant and then performed linkage analysis with microsatellite markers and sequence-tagged site markers. Using 85 backcross progeny from crossing Black Minorca chickens (BM-C) with F(1) individuals between White Silky (WS) and BM-C Fm was located on 10.2-11.7 Mb of chicken chromosome 20. In addition, we noticed a DNA marker that all Silky chickens and the F(1) individuals showed heterozygous genotyping patterns, suggesting gene duplication in the Fm region. By quantitative real-time PCR assay, Silky line-specific gene duplication was detected as an ~130-kb interval. It contained five genes including endothelin 3 (EDN3), which encoded a potent mitogen for melanoblasts/melanocytes. EDN3 with another three of these duplicated genes in Silky chickens expressed almost twofold of those in BM-C. Present results strongly suggest that the increase of the expression levels resulting from the gene duplication in the Fm region is the trigger of hypermelanization in internal organs of Silky chickens.     

Inhibitory activities of the heterotrimers formed from two α-type phospholipase A2 inhibitory proteins with different enzyme affinities and importance of the intersubunit electrostatic interaction in trimer formation

α-type phospholipase A₂ inhibitory protein (PLIα) isolated from the serum of the venomous snake Glyoidius brevicaudus, GbPLIα, is a homotrimer of subunits having a C-type lectin-like domain. The serum protein from nonvenomous snake Elaphe quadrivirgata, EqPLIα-LP, is homologous to GbPLIα, but it does not show any inhibitory activity against PLA₂s. When a mixture of denaturant-treated monomeric forms of GbPLIα and EqPLIα-LP was used to reconstitute their trimers, no significant amounts of heterotrimers composed of GbPLIα and EqPLIα-LP subunits could be formed. On the other hand, when a mixture of denaturant-treated monomeric forms of GbPLIα and the recombinant chimeric EqPLIα-LP, Eq13Gb37Eq, in which the residues 13–36 were replaced by those of GbPLIα, was used to reconstitute their trimers, significant amounts of their heterotrimers were observed. Furthermore, when a mixture of denaturant-treated monomeric forms of EqPLIα-LP and the recombinant chimeric GbPLIα, Gb13Eq37Gb, in which the residues 13–36 were replaced by those of EqPLIα-LP, was used, significant amounts of their heterotrimers were observed. By comparison of the respective inhibitory activities of the heterotrimeric subspecies, it was suggested that the inhibitory activity of the trimer was governed by one subunit with the highest activity, and not affected by the number of these subunits. The intermolecular electrostatic interactions between Glu23 and Lys28 of GbPLIα were also suggested to be important in stabilizing the trimeric structure. The importance of the electrostatic interaction was supported by the less stability of the homotrimeric structure of a mutant GbPLIα with a single amino acid substitution, GbPLIα(K28E).     

青海祁连地区四川绢蝶成虫习性的初步观察

为了了解四川绢蝶的生活习性,2009年6月至8月对青海省祁连地区的四川绢蝶Parnassius szechenyii Frivaldszky进行了实地调查研究。研究结果表明,该绢蝶喜欢在位于山谷到山顶间的中偏上部飞行活动,并对树林和人工破坏的区域有着很强的回避作用;雄性绢蝶的被捕捉机率要远大于雌性的;绢蝶一般喜欢在无风、阳光充足、气温较高的天气活动,其年活动最高峰大约在每年的7月中旬,日活动最高峰在每天的13：00左右。