Determination of fecal shedding rates and genotypes of swine hepatitis E virus (HEV) in Korea.

Hepatitis E virus (HEV) infection induces an acute hepatitis or a subclinical disease in humans. It is known that HEV is a zoonotic agent and pigs are major reservoirs of HEV. This study was conducted to determine the fecal shedding rates of HEV in various age groups of pigs and identify the genotypes of swine HEV prevailing in Korea. A total of 565 fecal samples were collected from suckling piglets, post-weaning pigs, growing pigs, and sows at 12 swine farms. RT-PCR was used to detect the presence of swine HEV in the feces. Every swine farm examined in this study had HEV-infected pigs. The fecal shedding rates of the swine HEV at individual farms were in the range of 2.1-35.4%. The overall fecal shedding rate of HEV in individual pigs was 17.5%. The HEV shedding rates of suckling piglets, post-weaning pigs, growing pigs and sows in their feces were 6.3, 16.3, 38.0 and 9.3%, respectively. When the genotypes of swine HEVs identified in this study were determined, they were all grouped into genotype 3. They were further subdivided into subtype 3a together with human and swine HEVs isolated in the U.S.A.

Down-regulation of matrix metalloproteinase-9 expression by nitric oxide in lipopolysaccharide-stimulated rat primary astrocytes.

Immunologically activated astrocytes over-express matrix metalloproteinase-9 (MMP-9) and nitric oxide (NO). Because they have both beneficial and detrimental effects on the pathophyiological outcomes of several neurological diseases, their expression should be tightly regulated in the CNS. NO can modify the activity of other proteins either by directly modifying protein structure or regulating the expression of target proteins. In this study, we investigated the role of NO on the expression of MMPs in rat primary astrocytes. Rat primary astrocytes were stimulated with lipopolysaccharide (LPS), resulting in the over-expression of both MMP-9 and NO. Inhibition of NO production using nitric oxide synthase inhibitor, Nomega-nitro-l-arginine methyl ester (l-NAME), further increased MMP-9 expression, suggesting NO inhibits MMP-9 expression. In line with this observation, exogenous addition of NO donor, sodium nitroprusside (SNP) or S-nitroso-N-acetylpenicillamine (SNAP), inhibited MMP-9 expression in astrocytes. The inhibitory effect of NO was mediated by the down-regulation of mRNA and protein levels of MMP-9 but not by the direct modification of the enzymatic activity of MMP-9. The effect of NO on MMP-9 expression was mimicked by dibutyryl-cGMP and inhibited by PKG inhibitor KT5823, suggesting NO regulates MMP-9 expression via guanylate cyclase-PKG pathway. Finally, SNP or dibutyryl-cGMP inhibited the activation of ERK1/2 in LPS-stimulated astrocytes, which is an essential regulator of MMP-9 expression in astrocytes. The regulation of MMP-9 expression by NO may confer additional levels of fine-tuning of the level of MMP-9 during brain inflammatory conditions.

Surface modification for DNA and protein microarrays.

Microarrays of biomolecules are emerging as powerful tools for genomics, proteomics, and clinical assays, since they make it possible to screen biologically important binding events in a parallel and high throughput fashion. Because the microarrays are fabricated on a solid support, coating of the surface and immobilization strategy of the biomolecules are major issues for successful microarray fabrication. This review deals with both DNA microarrays and protein microarrays, and focuses on the various modification approaches for the two-dimensional surface materials and three-dimensional ones. In addition, the immobilization strategies including adsorption, covalent attachment, physical entrapment, and affinity attachment of the biomolecules are summarized, and advantage and limitation of representative efforts are discussed.

Nanoscale-controlled spacing provides DNA microarrays with the SNP discrimination efficiency in solution phase.

We have prepared solid substrates modified with a cone-shaped dendron that generates mesospacing (3.2 nm on average) on the surface. This nanoscale-controlled surface provided an ideal DNA microarray in which each probe DNA strand was given ample space for the incoming target DNA, resulting in selectivity as high as that in solution (100: < 1). In addition, high hybridization yield confirms that DNA probes on the mesospaced surface are sterically unhindered for the hybridization.

DNA microarrays on a dendron-modified surface improve significantly the detection of single nucleotide variations in the p53 gene.

Selectivity and sensitivity in the detection of single nucleotide polymorphisms (SNPs) are among most important attributes to determine the performance of DNA microarrays. We previously reported the generation of a novel mesospaced surface prepared by applying dendron molecules on the solid surface. DNA microarrays that were fabricated on the dendron-modified surface exhibited outstanding performance for the detection of single nucleotide variation in the synthetic oligonucleotide DNA. DNA microarrays on the dendron-modified surface were subjected to the detection of single nucleotide variations in the exons 5-8 of the p53 gene in genomic DNAs from cancer cell lines. DNA microarrays on the dendron-modified surface clearly discriminated single nucleotide variations in hotspot codons with high selectivity and sensitivity. The ratio between the fluorescence intensity of perfectly matched duplexes and that of single nucleotide mismatched duplexes was >5-100 without sacrificing signal intensity. Our results showed that the outstanding performance of DNA microarrays fabricated on the dendron-modified surface is strongly related to novel properties of the dendron molecule, which has the conical structure allowing mesospacing between the capture probes. Our microarrays on the dendron-modified surface can reduce the steric hindrance not only between the solid surface and target DNA, but also among immobilized capture probes enabling the hybridization process on the surface to be very effective. Our DNA microarrays on the dendron-modified surface could be applied to various analyses that require accurate detection of SNPs.

Catalytic Hydrolysis of Phosphate Diesters by Lanthanide(III) Cryptate (2.2.1) Complexes.

Lanthanide(III) Cryptate (2.2.1) chlorides (Ln(2.2.1)Cl(3); Ln = La (1a), Ce(1b), and Eu(1c); (2.2.1) = 4,7,13,16,21-pentaoxa-1,10-diazabicyclo[8.8.5]tricosane) are effective for the catalytic hydrolysis of bis(4-nitrophenyl) phosphate. Kinetic studies reveal that the europium(III) complex (1c) catalyzes the hydrolysis to produce 6 equiv of 4-nitrophenol with a significant rate (k(1) = 1.5 x 10(-)(4) s(-)(1) at 0.40 mM) at pH 8.5 and 50 degrees C. The catalytic activity of the complexes is increased with decreasing the ionic size, i.e, La < Ce < Eu. While the use of hydrogen peroxide further increase the activity of 1b (k(1) = 1.6 x 10(-)(3) s(-)(1) at 0.40 mM), the presence of molecular oxygen does not affect the activity at all. Crystal of 1a.CH(3)OH([La(2.2.1)(Cl)(2)](Cl)(CH(3)OH)) belongs to the space group Pnma with a = 17.072(3) Å, b = 19.037(3) Å, c = 14.725(2) Å, V = 4786(1) Å(3), Z = 8, D(x)() = 1.691 g cm(-)(3), &mgr; = 21.7 cm(-)(1). The encryptated metal ion is nine-coordinated, and all the heteroatoms of the cryptate (2.2.1) ligand coordinate the metal center to form a bowl-shaped structure. Two coordinating chloride anions are located on the open face with a cis geometry. The existence of coordinated water to the europium(III) complex 1c in the aqueous solution was confirmed by time-resolved Eu(III) luminescence spectroscopy. From the decay constants in H(2)O and D(2)O, the numbers of coordinated water molecules (q) are found to be 3.02 at pH of 5.0. The above kinetic and spectroscopic observation are supportive of mechanisms in which the metal complexes act as a center for binding and activation as well as a source of nucleophilic metal hydroxides.