Syntheses of 4-methylumbelliferyl-beta-D-xylobioside and 5-bromo-3-indolyl-beta-D-xylobioside for sensitive detection of xylanase activity on agar plates

4-Methylumbelliferyl-beta-D-xylobioside (MU-X2) and 5-bromo-3-indolyl-beta-D-xylobioside (BI-X2) were synthesized as substrates for the detection of xylanase activity on agar plates. A family F/10 xylanase from Streptomyces olivaceoviridis E-86 (FXYN) was able to be more sensitively detected than RBB-xylan by using MU-X2 as a substrate. A mutant xylanase E128H/FXYN having only 1/1000 of the activity of FXYN was also able to be detected on the MU-X2 plate but was not detected on the RBB-xylan plate. A family G/11 xylanase from Streptomyces lividans 66 (Xyn B) was not detected on the MU-X2 plate, but it was able to be detected on the RBB-xylan plate, suggesting that the MU-X2 substrate is specific to family F/10 xylanases. However, none of the xylanases were detected effectively by using BI-X2 as a substrate.     

Structural insight into activation of homoserine dehydrogenase from the archaeon Sulfolobus tokodaii via reduction

Homoserine dehydrogenase (HSD; 305 amino acid residues) catalyzes an NAD(P)-dependent reversible reaction between l-homoserine and aspartate 4-semialdehyde and is involved in the aspartate pathway. HSD from the hyperthermophilic archaeon Sulfolobus tokodaii was markedly activated (2.5-fold) by the addition of 0.8 mM dithiothreitol. The crystal structure of the homodimer indicated that the activation was caused by cleavage of the disulfide bond formed between two cysteine residues (C303) in the C-terminal regions of the two subunits.     

Perfusion of hearts with triglyceride-rich particles reproduces the metabolic abnormalities in lipotoxic cardiomyopathy

Hearts with overexpression of anchored lipoprotein lipase (LpL) by cardiomyocytes (hLpL(GPI) mice) develop a lipotoxic cardiomyopathy. To characterize cardiac fatty acid (FA) and triglyceride (TG) metabolism in these mice and to determine whether changes in lipid metabolism precede cardiac dysfunction, hearts from young mice were perfused in Langendorff mode with [14C]palmitate. In hLpL(GPI) hearts, FA uptake and oxidation were decreased by 59 and 82%, respectively. This suggests reliance on an alternative energy source, such as TG. Indeed, these hearts oxidized 88% more TG. Hearts from young hLpL(GPI) mice also had greater uptake of intravenously injected cholesteryl ester-labeled Intralipid and VLDL. To determine whether perfusion of normal hearts would mimic the metabolic alterations found in hLpL(GPI) mouse hearts, wild-type hearts were perfused with [14C]palmitate and either human VLDL or Intralipid (0.4 mM TG). Both sources of TG reduced [14C]palmitate uptake (48% with VLDL and 45% with Intralipid) and FA oxidation (71% with VLDL and 65% with Intralipid). Addition of either heparin or LpL inhibitor P407 to Intralipid-containing perfusate restored [14C]palmitate uptake and confirmed that Intralipid inhibition requires local LpL. Our data demonstrate that reduced FA uptake and oxidation occur before mechanical dysfunction in hLpL(GPI) lipotoxicity. This physiology is reproduced with perfusion of hearts with TG-containing particles. Together, the results demonstrate that cardiac uptake of TG-derived FA reduces utilization of albumin-FA.     

siRNA-mediated inhibition of endogenous Huntington disease gene expression induces an aberrant configuration of the ER network in vitro

Huntingtin is a ubiquitously expressed cytoplasmic protein encoded by the Huntington disease (HD) gene, in which a CAG expansion induces an autosomal dominant progressive neurodegenerative disorder; however, its biological function has not been completely elucidated. Here, we report for the first time that short interfering RNA (siRNA)-mediated inhibition of endogenous Hdh (a mouse homologue of huntingtin) gene expression induced an aberrant configuration of the endoplasmic reticulum (ER) network in vitro. Studies using immunofluorescence microscopy with several ER markers revealed that the ER network appeared to be congregated in various types of cell lines transfected with siRNA directed against Hdh, but not with other siRNAs so far tested. Other subcellular organelles and structures, including the nucleus, Golgi apparatus, mitochondria, lysosomes, microtubules, actin cytoskeletons, cytoplasm, lipid rafts, and plasma membrane, exhibited normal configurations. Western blot analysis of cellular prion protein (PrP(C)) revealed normal glycosylation, which is a simple marker of post-translational modification in the ER and Golgi compartments, and immunofluorescence microscopy detected no altered subcellular distribution of PrP(C) in the post-ER compartments. Further investigation is required to determine whether the distorted ER network, i.e., loss of the huntingtin function, participates in the development of HD.     

Design,synthesis, and biological evaluation of radioiodinated benzo[d]imidazole-quinoline derivatives for platelet-derived growth factor receptor β (PDGFRβ) imaging

Several malignant tumors and fibrotic diseases are associated with PDGFRβ overexpression and excessive signaling, making this receptor attractive for molecular targeting and imaging approaches. A series of benzo[d]imidazole-quinoline derivatives were designed and synthesized to develop radioiodinated compounds as PDGFRβ-specific imaging probes. The structure activity relationship (SAR) evaluation of the designed compounds was performed. Among them, 2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]-8-(piperazin-1-yl)quinoline (5a) and 4-{2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}morpholine (5d) exhibited a relatively high PDGFRβ-TK inhibitory potency, whereas iodinated 5a derivative 5-iodo-2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]-8-(piperazin-1-yl)quinoline (8) exhibited a superior inhibitory potency as PDGFRβ inhibitor than iodinated 5d derivative 4-{5-iodo-2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}morpholine (11). Furthermore, [¹²⁵I]8 and [¹²⁵I]11 were synthesized and evaluated for PDGFRβ radioligand ability, both in vitro and in vivo. Cellular uptake experiments showed that [¹²⁵I]8 had a higher uptake in BxPC3-luc cells as PDGFRβ-positive cells than [¹²⁵I]11. Incubation of [¹²⁵I]8 after pretreatment of PDGFRβ ligands significantly reduced the uptake of [¹²⁵I]8. In biodistribution experiments using tumor-bearing mice, [¹²⁵I]8 accumulation in the tumor 1?h postinjection was higher than that of the benzo[d]imidazol-quinoline derivative [¹²⁵I]IIQP, used in our previous research. These results indicate that [¹²⁵I]8 could be a promising PDGFRβ imaging agent. Although its clinical application requires further structural modifications, the results obtained in this research may be useful for the development of PDGFRβ-specific radioligands.     

Simultaneous quantification by HPLC of purines in umami soup stock and evaluation of their effects on extracellular and intracellular purine metabolism

Ribonucleotide flavor enhancers such as inosine monophosphate (IMP) and guanosine monophosphate (GMP) provide umami taste, similarly to glutamine. Japanese cuisine frequently uses soup stocks containing these nucleotides to enhance umami. We quantified 18 types of purines (nucleotides, nucleosides, and purine bases) in three soup stocks (chicken, consommé, and dried bonito soup). IMP was the most abundant purine in all umami soup stocks, followed by hypoxanthine, inosine, and GMP. The IMP content of dried bonito soup was the highest of the three soup stocks. We also evaluated the effects of these purines on extracellular and intracellular purine metabolism in HepG2 cells after adding each umami soup stock to the cells. An increase in inosine and hypoxanthine was evident 1 h and 4 h after soup stock addition, and a low amount of xanthine and guanosine was observed in the extracellular medium. The addition of chicken soup stock resulted in increased intracellular and extracellular levels of uric acid and guanosine. Purine metabolism may be affected by ingredients present in soups.     

Cloning of the blasticidin S deaminase gene (BSD) from Aspergillus terreus and its use as a selectable marker for Schizosaccharomyces pombe and Pyricularia oryzae

Aspergillus terreus produces a unique enzyme, blasticidin S deaminase, which catalyzes the deamination of blasticidin S (BS), and in consequence confers high resistance to the antibiotic. A cDNA clone derived from the structural gene for BS deaminase (BSD) was isolated by transforming Escherichia coli with an Aspergillus cDNA expression library and directly selecting for the ability to grow in the presence of the antibiotic. The complete nucleotide sequene of BSD was determined and proved to contain an open reading frame of 393 bp, encoding a polypeptide of 130 amino acids. Comparison of its nulceotide sequence with that of bsr, the BS deaminase gene isolated from Bacillus cereus, indicated no homology and a large difference in codon usage. The activity of BSD expressed in E. coli was easily quantified by an assay based on spectrophotometric recording. The BSD gene was placed in a shuttle vector for Schizosaccharomyces pombe, downstream of the SV40 early region promoter, and this allowed direct selection with BS at high frequency, following transformation into the yeast. The BSD gene was also employed as a selectable marker for Pyricularia oryzae, which could not be transformed to BS resistance by bsr. These results promise that the BSD gene will be useful as a new dominant selectable marker for eukaryotes.     

Cloning of rainbow trout SLC26A1: involvement in renal sulfate secretion

The kidney plays an important role in ion regulation in both freshwater and seawater fish. However, ion transport mechanisms in the teleost kidney are poorly understood, especially at the molecular level. We have cloned a kidney-specific SLC26 sulfate/anion exchanger from rainbow trout (Oncorhynchus mykiss) that is homologous to the mammalian SLC26A1 (Sat-1). Excretion of excess plasma sulfate concentration after Na2SO4 injection corresponded to significantly higher expression of the cloned SLC26A1 mRNA. Detailed morphological observation of rainbow trout renal tubules was also performed by light microscopy and transmission electron microscopy. According to the structure of brush border and tubular system in the cytoplasm, renal tubules of rainbow trout were classified into proximal tubule first and second (PI and PII) segments and distal tubules. In situ hybridization revealed that SLC26A1 anion exchanger mRNA is specifically localized in the PI segment of kidneys from both seawater- and freshwater-adapted rainbow trout. With immunocytochemistry, Na+-K+-ATPase and vacuolar-type H+-ATPase were colocalized to the same cells and distributed in the basolateral and the apical membranes, respectively, of the cells where the SLC26A1 mRNA expressed. These findings suggest that the cloned kidney-specific SLC26A1 is located in kidney proximal tubules and is involved in excretion of excess plasma sulfate in rainbow trout.