Amino acids analysis using a monolithic silica column after derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F)

A fast HPLC method using a monolithic silica column was developed for the measurement of amino acids. The amino acids were pre-column derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and separated on a monolithic silica column (MonoClad C18-HS, 250 mm × 3 mm I.D.). The separation of 19 NBD-amino acids was achieved within 18 min, which was only one-fifth of the time taken by the methods using a conventional particle-packed column, with the gradient elution of a mobile phase at the flow rate of 1.4 mL/min. The sensitivity was good with a limit of detection for the individual amino acids ranging from 2.94 to 53.4 fmol. The calibration curves for all the amino acids were found to be linear in the range of 200 fmol to 20 pmol with correlation coefficients of 0.997 or better. The analytical method was successfully applied to determine the amino acids in a mouse plasma sample.     

Direct, simultaneous measurement of liposome-encapsulated and released drugs in plasma by on-line SPE-SPE-HPLC

A method for the simultaneous measurement of liposome-encapsulated and released drugs in mouse plasma by on-line solid phase extraction (SPE)-SPE-HPLC with direct plasma injection was developed using a doxorubicin (DXR)-containing liposome formulation as the model drug. During SPE, the released DXR was extracted on the 1st restricted-access media (RAM) SPE column, whereas the liposomes were eluted. The eluted liposomes were collapsed on-line, and the released DXR was delivered to the 2nd RAM SPE column for extraction. The retained DXR on the SPE columns was analyzed via HPLC-fluorescent detector by switching the valves. The method was validated and applied to the pharmacokinetic study of DXR in mice after intravenous injection of DXR-containing liposomes.     

Engineering the substrate specificity of Alcaligenes D-aminoacylase useful for the production of D-amino acids by optical resolution

D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (AxD-NAase) offers a novel biotechnological application, the production of D-amino acid from the racemic mixture of N-acyl-DL-amino acids. However, its substrate specificity is biased toward certain N-acyl-D-amino acids. To construct mutant AxD-NAases with substrate specificities different from those of wild-type enzyme, the substrate recognition site of the AxD-NAase was rationally manipulated based on computational structural analysis and comparison of its primary structure with other D-aminoacylases with distinct substrate specificities. Mutations of amino acid residues, Phe191, Leu298, Tyr344, and Met346, which interact with the side chain of the substrate, induced marked changes in activities toward each substrate. For example, the catalytic efficiency (k(cat)/K(m)) of mutant F191W toward N-acetyl-D-Trp and N-acetyl-D-Ala was enhanced by 15.6- and 1.5-folds, respectively, compared with that of the wild-type enzyme, and the catalytic efficiency (k(cat)/K(m)) of mutant L298A toward N-acetyl-D-Trp was enhanced by 4.4-folds compared with that of the wild-type enzyme. Other enzymatic properties of both mutants, such as pH and temperature dependence, were the same as those of the wild-type enzyme. The F191W mutant in particular is considered to be useful for the enzymatic production of D-Trp which is an important building block of some therapeutic drugs.     

Induction of apoptotic change in the rat hippocampus caused by ferric nitrilotriacetate

Iron, a source of oxidative stress, plays a major role in the pathology of neurodegenerative disease. In Alzheimer's disease, the hippocampus is vulnerable to oxidative stress, leading to impairment in memory formation. In our previous study, a brain oxidative reaction was induced after intraperitoneal injection of ferric nitrilotriacetate (Fe-NTA). However, since only a small amount of iron reached the brain in the previous study, Fe-NTA was administered into the hippocampus using an osmotic pump in this study. After continuous injection of Fe-NTA for 2 weeks, a high level of apoptotic change was induced in the hippocampus, in accordance with the iron localization. After injection for 4 weeks, the hippocampus was totally destroyed. A small amount of iron infiltrated into the cerebral cortex and the striatum, and deposition was observed at the choroid plexus and ependymal cells. However, no apoptotic reaction or clear tissue injury was observed in these areas. In addition, muscarinic acetylcholine receptors (M1, M2, and M4) were decreased in both the cortex and hippocampus while it increased in the striatum. Thus, the hippocampus is likely vulnerable to oxidative stress from Fe-NTA, and the oxidative stress is considered to bring the disturbance in the muscarinic acetylcholine receptors.     

The intron of tRNA-TrpCCA is dispensable for growth and translation of Saccharomyces cerevisiae

A part of eukaryotic tRNA genes harbor an intron at one nucleotide 3' to the anticodon, so that removal of the intron is an essential processing step for tRNA maturation. While some tRNA introns have important roles in modification of certain nucleotides, essentiality of the tRNA intron in eukaryotes has not been tested extensively. This is partly because most of the eukaryotic genomes have multiple genes encoding an isoacceptor tRNA. Here, we examined whether the intron of tRNA-Trp(CCA) genes, six copies of which are scattered on the genome of yeast, Saccharomyces cerevisiae, is essential for growth or translation of the yeast in vivo. We devised a procedure to remove all of the tRNA introns from the yeast genome iteratively with marker cassettes containing both positive and negative markers. Using this procedure, we removed all the introns from the six tRNA-Trp(CCA) genes, and found that the intronless strain grew normally and expressed tRNA-Trp(CCA) in an amount similar to that of the wild-type genes. Neither incorporation of (35)S-labeled amino acids into a TCA-insoluble fraction nor the major protein pattern on SDS-PAGE/2D gel were affected by complete removal of the intron, while expression levels of some proteins were marginally affected. Therefore, the tRNA-Trp(CCA) intron is dispensable for growth and bulk translation of the yeast. This raises the possibility that some mechanism other than selective pressure from translational efficiency maintains the tRNA intron on the yeast genome.     

Characterization of the human MATE2 proton-coupled polyspecific organic cation exporter

Human multidrug and toxic compound extrusion 2 (hMATE2) is a kidney-specific isoform of hMATE1, an exporter of toxic organic cations (OCs) of exogenous and endogenous origins at the final excretion step in the kidneys and liver (Otsuka et al., 2005), and contains a splicing variant, MATE2K, that has an exon of hMATE2 deleted (Masuda et al., 2006). In the present study, we characterized the degree of expression and the transport properties of hMATE2. Quantitative PCR analysis with probes specific for hMATE2 indicated the presence of hMATE2 mRNA in the kidneys, which corresponded to 39% of total mRNA encoding both hMATE2 and hMATE2K. hMATE2-specific antibodies immunostained the renal urinary tubules. Upon expression in HEK293 cells, hMATE2 was localized in intracellular vesicular structures, and thus transport activity of tetraethylammonium (TEA), a typical substrate for MATE transporters, by the cells was not detected. The hMATE2 protein was purified and reconstituted into liposomes. An artificially imposed pH gradient (ΔpH) across the proteoliposomal membrane drove the uptake of TEA. Dissipation of ΔpH by ammonium sulfate effectively inhibited the TEA uptake, while that of the membrane potential by valinomycin had little effect. The profiles of cis-inhibition of TEA transport by hMATE2 and hMATE2K are similar to each other. Thus, both hMATE2 and hMATE2K equally operate in the human kidneys to extrude OCs into the urine.     

Variation in seed production schedule among individual trees of a deciduous oak species <Emphasis Type="Italic">Quercus serrata</Emphasis>: its relation to seed characteristics

Intraspecific variation in seed production schedule and its effects on seed characteristics were examined in the deciduous oak species Quercus serrata in Iwate Prefecture, northern Japan. Seed production schedule differed markedly among 31 individual trees. The median dates of seed production differed by over 1 month between the earliest and latest individuals. Relationships between seed production schedule and seed characteristics were investigated at two different scales: the among-tree scale and the within-tree scale. At the within-tree scale, analyses revealed that the seeds dispersed by a tree later in the season tended to be larger in size and lower in tannin concentration. Similarly, at the among-tree scale, analysis revealed that trees with a later seed production schedule produced on average larger acorns. However, no clear tendency was observed between seed production schedule and mean tannin concentration. The observed variation in seed production schedule is likely to affect seed fate per se, and also via changes in seed characteristics.     

The putative endoglucanase PcGH61D from Phanerochaete chrysosporium is a metal-dependent oxidative enzyme that cleaves cellulose

Many fungi growing on plant biomass produce proteins currently classified as glycoside hydrolase family 61 (GH61), some of which are known to act synergistically with cellulases. In this study we show that PcGH61D, the gene product of an open reading frame in the genome of Phanerochaete chrysosporium, is an enzyme that cleaves cellulose using a metal-dependent oxidative mechanism that leads to generation of aldonic acids. The activity of this enzyme and its beneficial effect on the efficiency of classical cellulases are stimulated by the presence of electron donors. Experiments with reduced cellulose confirmed the oxidative nature of the reaction catalyzed by PcGH61D and indicated that the enzyme may be capable of penetrating into the substrate. Considering the abundance of GH61-encoding genes in fungi and genes encoding their functional bacterial homologues currently classified as carbohydrate binding modules family 33 (CBM33), this enzyme activity is likely to turn out as a major determinant of microbial biomass-degrading efficiency.     

Shal/K(v)4 channels are required for maintaining excitability during repetitive firing and normal locomotion in Drosophila

Background

Rhythmic behaviors, such as walking and breathing, involve the coordinated activity of central pattern generators in the CNS, sensory feedback from the PNS, to motoneuron output to muscles. Unraveling the intrinsic electrical properties of these cellular components is essential to understanding this coordinated activity. Here, we examine the significance of the transient A-type K⁺ current (I_A), encoded by the highly conserved Shal/K_v4 gene, in neuronal firing patterns and repetitive behaviors. While I_A is present in nearly all neurons across species, elimination of I_A has been complicated in mammals because of multiple genes underlying I_A, and/or electrical remodeling that occurs in response to affecting one gene.

Methodology/Principal Findings

In Drosophila, the single Shal/K_v4 gene encodes the predominant I_A current in many neuronal cell bodies. Using a transgenically expressed dominant-negative subunit (DNK_v4), we show that I_A is completely eliminated from cell bodies, with no effect on other currents. Most notably, DNK_v4 neurons display multiple defects during prolonged stimuli. DNK_v4 neurons display shortened latency to firing, a lower threshold for repetitive firing, and a progressive decrement in AP amplitude to an adapted state. We record from identified motoneurons and show that Shal/K_v4 channels are similarly required for maintaining excitability during repetitive firing. We then examine larval crawling, and adult climbing and grooming, all behaviors that rely on repetitive firing. We show that all are defective in the absence of Shal/K_v4 function. Further, knock-out of Shal/K_v4 function specifically in motoneurons significantly affects the locomotion behaviors tested.

Conclusions/Significance

Based on our results, Shal/K_v4 channels regulate the initiation of firing, enable neurons to continuously fire throughout a prolonged stimulus, and also influence firing frequency. This study shows that Shal/K_v4 channels play a key role in repetitively firing neurons during prolonged input/output, and suggests that their function and regulation are important for rhythmic behaviors.