Dendritic localization and a cis-acting dendritic targeting element of Kv4.2 mRNA

Kv4.2, a pore-forming α-subunit of voltage-gated A-type potassium channels, is expressed abundantly in the soma and dendrites of hippocampal neurons, and is responsible for somatodendritic I(A) current. Recent studies have suggested that changes in the surface levels of Kv4.2 potassium channels might be relevant to synaptic plasticity. Although the function and expression of Kv4.2 protein have been extensively studied, the dendritic localization of Kv4.2 mRNA is not well described. In this study, Kv4.2 mRNAs were shown to be localized in the dendrites near postsynaptic regions. The dendritic transport of Kv4.2 mRNAs were mediated by microtubule- based movement. The 500 nucleotides of specific regions within the 3'-untranslated region of Kv4.2 mRNA were found to be necessary and sufficient for its dendritic localization. Collectively, these results suggest that the dendritic localization of Kv4.2 mRNAs might regulate the dendritic surface level of Kv4.2 channels and synaptic plasticity.     

Informative SSR markers for commercial variety discrimination in watermelon (Citrullus lanatus)

SSR markers were used for variety discrimination and genetic assessment in watermelon varieties. Genetic characterization of 49 watermelon varieties was investigated using 30 SSR markers developed from melon and watermelon. A total of 121 polymorphic amplified fragments were obtained by using 30 SSR markers. The average polymorphism information content (PIC) was 0.502 ranging from 0.223 to 0.800. One hundred twenty one SSR loci were used to calculate Jaccard’s distance coefficients for unweighted pair group method using the arithmetic averages (UPGMA) cluster analysis. A clustering group of varieties, based on the results of SSR analysis, were categorized into 5 major groups corresponding to morphological traits. Inheritance mode of 2 SSR markers was investigated to F₁ plants and F₂ populations of 2 crosses. Parental alleles were transmitted from F1 plants and F2 populations. Therefore, these marker sets may prove to be effectively applicable to genetic assessment of germplasm, genome mapping, and fingerprinting of watermelon varieties.     

Population estimation of human embryonic stem cell cultures

Traditionally, the population of human embryonic stem cell (hESC) culture is estimated through haemacytometer counts, which include harvesting the cells and manually analyzing a fraction of an entire population. Obviously, through this highly invasive method, it is not possible to preserve any spatial information on the cell population. The goal of this study is to identify a fast and consistent method for in situ automated hESC population estimation to quantitatively estimate the cell growth. Therefore, cell cultures were fixed, stained, and their nuclei imaged through high‐resolution microscopy, and the images were processed with different image analysis techniques. The proposed method first identifies signal and background by computing an image specific threshold for image segmentation. By applying a morphological operator (watershed), we split most physically overlapping nuclei, leading to a pixel area distribution of isolated signal areas on the image. On the basis of this distribution, we derive a nucleus area model, describing the distribution of the area of cell debris, single nuclei, and small groups of connected nuclei. Through the model, we can give a quantitative estimation of the population. The focus of this study is on low‐density human embryonic stem cell populations; hence cultures were measured at days 2–3 after seeding. Compared with manual cell counts, the automatic method achieved higher accuracy with <6% error. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

3-Acyloxy-2-phenalkylpropyl amides and esters of homovanillic acid as novel vanilloid receptor agonists

A series of 3-acyloxy-2-phenalkylpropyl amides and esters of homovanillic acid were designed and synthesized as vanilloid receptor agonists containing the three principal pharmacophores of resiniferatoxin. Amide analogues 23, 5 and 11 were found to be potent agonists in vanilloid receptor assay both for ligand binding and for activation.     

Potential application of genetically identical somatic cell nuclear transfer-cloned dogs for gastrointestinal microbiota analysis

This study investigated the gastrointestinal microbiota in three genetically identical cloned dogs (A, B and C) by somatic cell nuclear transfer. We collected feces from three cloned dogs and their feed to investigate gastrointestinal microbiota using both culture-dependent and culture-independent methods. A total of 962 strains from the feces of cloned dogs were isolated using aerobic and anaerobic culture methods. The dominant microorganisms were Enterococcus faecalis and Enterococcus faecium in all fecal samples. In particular, the fecal sample from cloned dog C had similar proportions of three species (E. faecalis, E. faecium and Lactobacillus murinus). In all, 29 DNA fragments were identified by PCR-denaturing gradient gel electrophoresis (DGGE) analysis. The highest DGGE band intensities were for E. faecalis from cloned dogs A and C and for Clostridium sordellii from cloned dog B, with relative intensities of 15.2, 17.7 and 14.4%, respectively. The other strains identified from the cloned dogs were Chryseobacterium soldanellicola, Escherichia coli, L. murinus, Streptococcus alactolyticus, Weissella confusa and uncultured bacterium. Some microbes isolated from the fecal samples, including C. soldanellicola and W. confuse, were derived from the feed. Overall, gastrointestinal microbiota of all genetically identical cloned dogs, maintained under the same environmental and feeding conditions, showed similar profiles in terms of species diversity analyzed by PCR-DGGE, although there were proportional differences in the amounts of bacterial species. To our knowledge, this is the first report to investigate and compare gastrointestinal microbiota of three genetically identical dogs.     

Improved degradation of lignocellulosic biomass pretreated by Fenton-like reaction using Fe<Subscript>3</Subscript>O<Subscript>4</Subscript> magnetic nanoparticles

The ability of Fe₃O₄ Fenton-like reaction to produce glucose from lignocellulosic biomass was investigated. Fe₃O₄ magnetite nanoparticles were chemically synthesized from iron salts (a mixture of 1 M FeCl₂ and 2M FeCl₃) using an ammonia solution (30% NH₄OH). The synthesized Fe₃O₄ nanoparticles were further characterized by X-ray photoelectron spectroscopy, energy dispersive X-ray spectroscopy, scanning electron microscopy, and transmission electron microscopy. Reed stems and rice straw biomasses pretreated with optimized Fenton-like reagents (Fe₃O₄ and H₂O₂) increased glucose production by 177 and 87%, respectively, compared to the control without the catalysts.     

Enhancement of 2,3-butanediol production from Jerusalem artichoke tuber extract by a recombinant <Emphasis Type="Italic">Bacillus</Emphasis> sp. strain BRC1 with increased inulinase activity

A Bacillus sp. strain named BRC1 is capable of producing 2,3-butanediol (2,3-BD) using hydrolysates of the Jerusalem artichoke tuber (JAT), a rich source of the fructose polymer inulin. To enhance 2,3-BD production, we undertook an extensive analysis of the Bacillus sp. BRC1 genome, identifying a putative gene (sacC) encoding a fructan hydrolysis enzyme and characterizing the activity of the resulting recombinant protein expressed in and purified from Escherichia coli. Introduction of the sacC gene into Bacillus sp. BRC1 using an expression vector increased enzymatic activity more than twofold. Consistent with this increased enzyme expression, 2,3-BD production from JAT was also increased from 3.98 to 8.10 g L^?1. Fed-batch fermentation of the recombinant strain produced a maximal level of 2,3-BD production of 28.6 g L^?1, showing a high theoretical yield of 92.3%.     

Combination of newly developed SNP and InDel markers for genotyping the <Emphasis Type="Italic">Cf-9</Emphasis> locus conferring disease resistance to leaf mold disease in the tomato

The Cf-9 gene in the tomato is known to confer resistance against leaf mold disease caused by Cladosporium fulvum, and a gene-based marker targeted to the Cf-9 allele has been widely used as a crop protection approach. However, we found this marker to be misleading in genotyping. Therefore, we developed new single-nucleotide polymorphism (SNP) and insertion and deletion (InDel) markers targeted to the Cf-9 allele in order to increase genotyping accuracy and facilitate high-throughput screening. The DNA sequences of reported Cf-9, cf-9, Cf-0, and closely related Cf-4 alleles were compared, and two functional and non-synonymous SNPs were found to distinguish the Cf-9 resistance allele from the cf-9, Cf-0, and Cf-4 alleles. An SNP marker including these two SNPs was developed and applied to the genotyping of 33 tomato cultivars by high-resolution melting analysis. Our SNP marker was able to select all three Cf-9 genotypes (resistant, heterozygous, and susceptible alleles). Interestingly, two cultivars were grouped separately from these three genotypes. To further examine this outgroup, we preformed polymerase chain reaction (PCR) on two InDel regions identified by sequence comparison of the Cf-9 and Cf-4 genes. The band patterns revealed that these two cultivars carried Cf-4 rather than Cf-9 alleles and that three cultivars classified in the Cf-9 resistance group actually carried both Cf-9 and Cf-4 genes. To determine whether these genotyping results were consistent with disease resistance phenotypes, we examined the induction of a hypersensitive response by transiently expressing the corresponding effector genes, and found that the results matched perfectly with the genotyping results. These findings indicate that the combination of our SNP and InDel markers allows resistant Cf-9 alleles to be distinguished from cf-9 and Cf-4 alleles, which will be useful for marker-assisted selection of tomato cultivars resistant to C. fulvum.