Insertion of a novel transposable element in the tyrosinase gene is responsible for an albino mutation in the medaka fish, Oryzias latipes

In the medaka fish (Oryzias latipes) many mutants for body color have been isolated. A typical example is the recessive oculocutaneous albino mutant i, which has amelanotic skin and red-colored eyes with no tyrosinase activity. To cast light on the molecular basis of the albino mechanism, we performed Southern blot analysis of genomic DNA from the mutant with an authentic tyrosinase gene probe; the results demonstrate that an extra 1.9 kb fragment is present inside the first exon. The insertion is responsible for the oculocutaneous albinism. About 80 copies of this fragment are present in the genomes of albino-i and wild-type fish; these repeated sequences are here designated Tol1 elements and the particular element found in the tyrosinase gene of albino-i is denoted Tol1-tyr. The nucleotide sequence of Tol1-tyr shows that the fragment (i) carries terminal inverted repeats of 14 bp, and (ii) is flanked by duplicated 8 by segments of the host chromosome. These are properties of DNA-mediated transposable elements. Comparison of the nucleotide sequence of Tol1-tyr with other sequences in DNA databases, with special attention to sequences of transposable elements known to date, did not reveal any similarity. Thus, Tol1 constitutes a hitherto unknown family of DNA transposable elements.     

Cellular uptake of covalent conjugates of oligonucleotide with membrane-modifying peptide, peptaibol

Using the membrane-modifying peptide, trichorovin-XIIa (TV-XIIa), which is an 11-residual peptaibol isolated from the fungus Trichoderma viride, we synthesized covalent conjugates of 20-mer oligonucleotide with TV-XIIa to examine the potential use of TV-XIIa in cellular delivery. The results indicated that the conjugates were progressively taken up by human lung carcinoma A549 cells. Next, the antisense effects of the conjugates on p53 protein expression were examined. The p53 expression was significantly decreased by ca. 20-50% in the presence of the conjugates upon treatment with the transfection solution at the concentration of 5 μM.     

Regio- and Diastereo-Selectivity of Montmorillonite-Catalyzed Oligomerization of Racemic Adenosine 5′-Phosphorimidazolide

Clay is a possible candidate for an effective catalyst in prebiotic chemical evolution of biomolecules. Montmorillonite was reported to effectively catalyze oligomerization of racemic adenosine 5′-phosphorimidazolide (DL-ImpA). In the oligomerization reaction, considerable amounts of cyclic dimers as well as linear dimers were produced in the oligomerization reactions. To assess the regio- and diastereo-selectivities of the oligomerization reaction, the dimer products including cyclic dimers were completely identified by means of enzymatic degradation reactions of the products.     

Simultaneous fluorometric measurement of histamine and tele-methylhistamine levels in rodent brain by high-performance liquid chromatography

An improved high-performance liquid chromatography (HPLC) method was developed for simultaneous analysis of histamine (HA) and tele-methylhistamine (tele-MHA) levels in mouse and rat brain. The method consists of a solid-phase extraction (SPE) and subsequent HPLC with postcolumn derivatization of the amines with o-phthalaldehyde. The recovery rates of HA and tele-MHA during the SPE procedure were 82.8+/-3.4 and 86.0+/-1.7%, respectively. The detection limits for HA and tele-MHA were 8 and 12pg, respectively, with sufficient linearity up to 30pg. Using this newly developed system, we observed that the brain tele-MHA levels in H3 receptor knockout mice were significantly higher than those of wild-type mice by 2.1-fold. Furthermore, we also observed that the brain HA and tele-MHA levels in Zucker rats were significantly lower than those of lean rats by 76.6+/-5.3 and 77.8+/-5.0%, respectively. These observations coincided well with those of previous studies using radioimmunoassay or HPLC with precolumn OPA derivatization, confirming the utilization of the assay system.     

Identification of dicarboxylate carrier Slc25a10 as malate transporter in de novo fatty acid synthesis

Mitochondrial solute carrier family 25 member 10 (Slc25a10) transports dicarboxylates such as malate or succinate across the mitochondrial inner membrane. Although fatty acid synthesis in adipose tissue or the liver is initiated by citrate transport in exchange for malate across the mitochondrial membrane, the transporter responsible for supplying malate during citrate transport has not been identified. In the present study, we clarified the role of Slc25a10 in supplying malate for citrate transport and examined the effect of Slc25a10 suppression on the lipogenic pathway and lipid accumulation. We have reported an Slc25a10 increase in white adipose tissue in obese mouse models and a decrease in a fasted mouse model using expression profiles. Next, we examined the effect of Slc25a10 suppression by small interfering RNA on citrate transport in the lipogenic cell lines HepG2 and 3T3-L1. We observed that inhibition of malate transport by Slc25a10 suppression significantly reduced the citrate transport from the mitochondria to the cytosol. We also found that suppression of Slc25a10 down-regulated the lipogenic pathway, indicated by decreases in ACC1 expression and malonyl-CoA level. Furthermore, suppression of Slc25a10 decreased triglyceride lipid accumulation in adipose-differentiated 3T3-L1 cells. These results suggested that Slc25a10 plays an important role in supplying malate for citrate transport required for fatty acid synthesis and indicated that inhibition of Slc25a10 might effectively reduce lipid accumulation in adipose tissues.     

Spectroscopic Characterization of the Decadeoxynucleotide Duplex Modified with Dichloroethylenediamineplatinum(II)

Abstract

We investigated the structure of a decadeoxynucleotide duplex which was modified with Dichloroethylenediamineplatinum(II) (PtCl₂(en)) at the central GG site by using some spectroscopic techniques. The results suggest that the DNA structural changes induced by binding of PtCl₂(en) are quite similar to those of cisplatin.     

DNA cruciforms facilitate in vitro strand transfer on nucleosomal templates

A single, phased nucleosome assembled on a 240 by DNA duplex molecule blocked Escherichia coli RecA protein-promoted strand transfer of the complementary strand of the duplex onto a homologous single-stranded circle. However, when a four-armed cruciform structure was coupled to either end of the duplex the barrier to strand transfer was overcome and joint molecules were efficiently formed. Micrococcal nuclease digestion indicated that the nucleosome was dissociated by the juxtaposition of the cruciform. We interpret these results to mean that cruciform structures can act over a distance to destabilize adjacent nucleosomes and suggest that, as a consequence, the chromatin structure surrounding a crossed strand recombination intermediate might be disrupted, enabling other recombination events to initiate or the process of branch migration to proceed.     

The solution structure of the C-terminal domain of NfeD reveals a novel membrane-anchored OB-fold

Nodulation formation efficiency D (NfeD) is a member of a class of membrane-anchored ClpP-class proteases. There is a second class of NfeD homologs that lack the ClpP domain. The genes of both NfeD classes usually are part of an operon that also contains a gene for a prokaryotic homolog of stomatin. (Stomatin is a major integral-membrane protein of mammalian erythrocytes.) Such NfeD/stomatin homolog gene pairs are present in more than 290 bacterial and archaeal genomes, and their protein products may be part of the machinery used for quality control of membrane proteins. Herein, we report the structure of the isolated C-terminal domain of PH0471, a Pyrococcus horikoshii NfeD homolog, which lacks the ClpP domain. This C-terminal domain (termed NfeDC) contains a five-strand beta-barrel, which is structurally very similar to the OB-fold (oligosaccharide/oligonucleotide-binding fold) domain. However, there is little sequence similarity between it and previously characterized OB-fold domains. The NfeDC domain lacks the conserved surface residues that are necessary for the binding of an OB-fold domain to DNA/RNA, an ion. Instead, its surface is composed of residues that are uniquely conserved in NfeD homologs and that form the structurally conserved surface turns and beta-bulges. There is also a conserved tryptophan present on the surface. We propose that, in general, NfeDC domains may interact with other spatially proximal membrane proteins and thereby regulate their activities.     

Maturation of adult peripheral blood CD38(+)CD4(+) T cells demonstrated by cytokine production in response to a superantigen,TSST-1

This study looks at immunoincompetent CD4(+) T cells in adult peripheral blood (APB) using cytokine production in response to a superantigen as a measure of function. We compared the function of APB CD38(+)CD4(+) and CD38(-/low)CD4(+) T cells to that of cord blood (CB) CD4(+) T cells. APB CD4(+) T cell blasts produce substantial amounts of IL-2 in response to TSST-1 restimulation, while CB CD4(+) T cell blasts produce less. APB CD38(+)CD4(+) T cells produce low levels of IL-4 and IFN-gamma in response to TSST-1, even after activation, while APB CD38(-/low)CD4(+) T cells retain their ability to produce high levels of these cytokines despite high CD38 expression. These results suggest that the developmental stage of APB CD38(+)CD4(+) T cells lies between that of CB CD4(+) T cells and APB CD38(-/low)CD4(+) T cells and that APB CD38(+)CD45RO(-)CD4(+) T cells gradually cease to express CD38 as they acquire full function. We reconsider CD4(+) cell maturation and response to TSST-1 and discuss the implications of T cell maturity on infectious diseases.