Clinical Features of Drug-induced Liver Injury According to Etiology.

Drug-induced liver injury (DILI) is an increasingly common cause of acute hepatitis. We examined clinical features and types of liver injury of 65 affected patients who underwent liver biopsy according DILI etiology. The major causes of DILI were the use of herbal medications (43.2%), prescribed medications (21.6%), and traditional therapeutic preparations and dietary supplements (35%). DILI from herbal medications, traditional therapeutic preparations, and dietary supplements was associated with higher elevations in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels than was DILI from prescription medications. The types of liver injury based on the R ratio were hepatocellular (67.7%), mixed (10.8%), and cholestatic (21.5%). Herbal medications and traditional therapeutic preparations were more commonly associated with hepatocellular liver injury than were prescription medications (P = 0.002). Herbal medications and traditional therapeutic preparations induce more hepatocellular DILI and increased elevations in AST and ALT than prescribed medications.

Effect of modified glucose catabolism on xanthan production in Xanthomonas oryzae pv. oryzae.

In this study, the glucose 6-phosphate dehydrogenase gene (XOO2314) was inactivated in order to modulate the intracellular glucose 6-phosphate, and its effects on xanthan production in a wild-type strain of Xanthomonas oryzae were evaluated. The intracellular glucose 6-phosphate was increased from 17.6 to 99.4 µmol g⁻¹ (dry cell weight) in the gene-disrupted mutant strain. The concomitant increase in the glucose 6-phosphate was accompanied by an increase in xanthan production of up to 2.23 g l⁻¹ (culture medium). However, in defined medium supplemented with 0.4% glucose, the growth rate of the mutant strain was reduced to 52.9% of the wild-type level. Subsequently, when a family B ATP-dependent phosphofructokinase from Escherichia coli was overexpressed in the mutant strain, the growth rate was increased to 142.9%, whereas the yields of xanthan per mole of glucose remained approximately the same.

Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of the co-chaperonin XoGroES from Xanthomonas oryzae pv. oryzae.

Bacterial blight (BB), a devastating disease caused by Xanthomonas oryzae pv. oryzae (Xoo), causes serious production losses of rice in Asian countries. Protein misfolding may interfere with the function of proteins in all living cells and must be prevented to avoid cellular disaster. All cells naturally contain molecular chaperones that assist the unfolded proteins in folding into the native structure. One of the well characterized chaperone complexes is GroEL-GroES. GroEL, which consists of two chambers, captures misfolded proteins and refolds them. GroES is a co-chaperonin protein that assists the GroEL protein as a lid that temporarily closes the chamber during the folding process. Xoo4289, the GroES gene from Xoo, was cloned and expressed for X-ray crystallographic study. The purified protein (XoGroES) was crystallized using the hanging-drop vapour-diffusion method and a crystal diffracted to 2.0 Šresolution. The crystal belonged to the hexagonal space group P6(1), with unit-cell parameters a=64.4, c=36.5 Å. The crystal contains a single molecule in the asymmetric unit, with a corresponding VM of 2.05 Å3 Da(-1) and a solvent content of 39.9%.

Isolation and characterization of NgRLK1, a receptor-like kinase of Nicotiana glutinosa that interacts with the elicitin of Phytophthora capsici.

Elicitins, extracellular proteins from Phytophthora fungi, elicit a hypersensitivity response (HR), including systemic acquired resistance, in some plants. The elicitin capsicein (approximately 10 kDa) was purified by FPLC from culture filtrates of P. capsici. Purified native and recombinant capsicein induced a hypersensitive response in leaves of the non-host plants Nicotiana glutinosa and Brassica rapa subsp. pekinensis. To search for candidate capsicein-interacting proteins from N. glutinosa, a yeast two-hybrid assay was used. We identified a protein interactor that is homologous to a serine/threonine kinase of the plant receptor-like kinase (RLK) group and designated it NgRLK1. The ORF of NgRLK1 encodes a polypeptide of 832 amino acids (93,490 Da). A conserved domain analysis revealed that NgRLK1 has structural features typical of a plant RLK. NgRLK1 was autophosphorylated, with higher activity in the presence of Mn2+ than Mg2+.

Relationship between glucose catabolism and xanthan production in Xanthomonas oryzae pv. oryzae.

Two genes involved in central carbon metabolism were inactivated to modulate intracellular glucose 6-phosphate and to evaluate its effects on xanthan production in wild-type Xanthomonas oryzae pv. oryzae. Upon the inactivation of the phosphogluconate dehydratase gene (edd), intracellular glucose 6-phosphate increased from 0.05 to 1.17 mmol/g (dry cell wt). This was accompanied by increased xanthan production of up to 2.55 g/l (culture medium). In contrast, inactivation of 6-phosphogluconate dehydrogenase gene (gndA) did not influence intracellular glucose 6-phosphate nor xanthan production. The intracellular availability of glucose 6-phosphate is proposed as a rate-limiting factor in xanthan production, and it may be possible to increases production of xanthan by modulating the activities of enzymes in central carbon metabolism.

Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of glutamyl-tRNA synthetase (Xoo1504) from Xanthomonas oryzae pv. oryzae.

The gltX gene from Xanthomonas oryzae pv. oryzae (Xoo1504) encodes glutamyl-tRNA synthetase (GluRS), one of the most important enzymes involved in bacterial blight (BB), which causes huge production losses of rice worldwide. GluRS is a class I-type aminoacyl-tRNA synthetase (aaRS) that is primarily responsible for the glutamylation of tRNA(Glu). It plays an essential role in protein synthesis, as well as the regulation of cells, in all organisms. As it represents an important target for the development of new antibacterial drugs against BB, determination of the three-dimensional structure of GluRS is essential in order to understand its catalytic mechanism. In order to analyze its structure and function, the gltX gene was cloned and the GluRS enzyme was expressed, purified and then crystallized. A GluRS crystal belonging to the monoclinic space group C2 diffracted to 2.8 A resolution and had unit-cell parameters a = 186.8, b = 108.4, c = 166.1 A, beta = 96.3 degrees . The unit-cell volume of the crystal allowed the presence of six to eight monomers in the asymmetric unit, with a corresponding Matthews coefficient (V(M)) range of 2.70-2.02 A(3) Da(-1) and a solvent-content range of 54.5-39.3%.

Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of leucine aminopeptidase (LAP) from the pepA gene of Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes the serious disease bacterial blight in rice. The pepA (Xoo0834) gene from Xoo is one of around 100 genes that have been selected for the design of antibacterial drugs. The pepA gene encodes leucine aminopeptidase (LAP), an exopeptidase that catalyzes the hydrolysis of leucine residues from the N-terminus of a protein or peptide. This enzyme was expressed in Escherichia coli, purified and crystallized, and preliminary X-ray structural studies have been carried out. The LAP crystal diffracted to 2.6 A resolution and belonged to the cubic space group P2(1)3. The unit-cell volume of the crystal was compatible with the presence of two monomers in the asymmetric unit.

Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of beta-ketoacyl-ACP synthase III (FabH) from Xanthomonas oryzae pv. oryzae.

The bacterial beta-ketoacyl-ACP synthase III (KASIII) encoded by the gene fabH (Xoo4209) from Xanthomonas oryzae pv. oryzae, a plant pathogen, is an important enzyme in the elongation steps of fatty-acid biosynthesis. It is expected to be one of the enzymes responsible for bacterial blight (BB), a serious disease that results in huge production losses of rice. As it represents an important target for the development of new antibacterial drugs against BB, determination of the crystal structure of the KAS III enzyme is essential in order to understand its reaction mechanism. In order to analyze the structure and function of KAS III, the fabH (Xoo4209) gene was cloned and the enzyme was expressed and purified. The KASIII crystal diffracted to 2.05 A resolution and belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 69.8, b = 79.5, c = 62.3 A. The unit-cell volume of the crystal is compatible with the presence of a single monomer in the asymmetric unit, with a corresponding Matthews coefficient V(M) of 2.27 A(3) Da(-1) and a solvent content of 45.8%.

Mutational analysis of the gum gene cluster required for xanthan biosynthesis in Xanthomonas oryzae pv oryzae.

Genome sequence analysis of Xanthomonas oryzae pv. oryzae has revealed a cluster of 12 ORFs that are closely related to the gum gene cluster of Xanthomonas campestris pv. campestris. The gum gene cluster of X. oryzae encodes proteins involved in xanthan production; however, there is little experimental evidence supporting this. In this study, biochemical analyses of xanthan produced by a defined set of X. oryzae gum mutant strains allowed us to preliminarily assign functions to most of the gum gene products: biosynthesis of the pentasaccharide repeating unit for GumD, GumM, GumH, GumK, and GumI, xanthan polymerization and transport for GumB, GumC, GumE, and GumJ, and modification of the pentasaccharide repeating unit for GumF, GumG, and GumL. In addition, we found that the exopolysaccharides are essential but not specific for the virulence of X. oryzae.

Predicting the interactome of Xanthomonas oryzae pathovar oryzae for target selection and DB service.

BACKGROUND: Protein-protein interactions (PPIs) play key roles in various cellular functions. In addition, some critical inter-species interactions such as host-pathogen interactions and pathogenicity occur through PPIs. Phytopathogenic bacteria infect hosts through attachment to host tissue, enzyme secretion, exopolysaccharides production, toxins release, iron acquisition, and effector proteins secretion. Many such mechanisms involve some kind of protein-protein interaction in hosts. Our first aim was to predict the whole protein interaction pairs (interactome) of Xanthomonas oryzae pathovar oryzae (Xoo) that is an important pathogenic bacterium that causes bacterial blight (BB) in rice. We developed a detection protocol to find possibly interacting proteins in its host using whole genome PPI prediction algorithms. The second aim was to build a DB server and a bioinformatic procedure for finding target proteins in Xoo for developing pesticides that block host-pathogen protein interactions within critical biochemical pathways. DESCRIPTION: A PPI network in Xoo proteome was predicted by bioinformatics algorithms: PSIMAP, PEIMAP, and iPfam. We present the resultant species specific interaction network and host-pathogen interaction, XooNET. It is a comprehensive predicted initial PPI data for Xoo. XooNET can be used by experimentalists to pick up protein targets for blocking pathological interactions. XooNET uses most of the major types of PPI algorithms. They are: 1) Protein Structural Interactome MAP (PSIMAP), a method using structural domain of SCOP, 2) Protein Experimental Interactome MAP (PEIMAP), a common method using public resources of experimental protein interaction information such as HPRD, BIND, DIP, MINT, IntAct, and BioGrid, and 3) Domain-domain interactions, a method using Pfam domains such as iPfam. Additionally, XooNET provides information on network properties of the Xoo interactome. CONCLUSION: XooNET is an open and free public database server for protein interaction information for Xoo. It contains 4,538 proteins and 26,932 possible interactions consisting of 18,503 (PSIMAP), 3,118 (PEIMAP), and 8,938 (iPfam) pairs. In addition, XooNET provides 3,407 possible interaction pairs between two sets of proteins; 141 Xoo proteins that are predicted as membrane proteins and rice proteomes. The resultant interacting partners of a query protein can be easily retrieved by users as well as the interaction networks in graphical web interfaces. XooNET is freely available from http://bioportal.kobic.kr/XooNET/.

Expression, crystallization and preliminary X-ray crystallographic analysis of XometC, a cystathionine gamma-lyase-like protein from Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight of rice (Oryza sativa L.), one of the most devastating diseases of rice in most rice-growing countries. XometC, a cystathionine gamma-lyase (CGL) like protein that is an antibacterial drug-target protein against Xoo, was cloned, expressed, purified and crystallized. CGL catalyzes the second step in the reverse-transsulfuration pathway, which is essential for the metabolic interconversion of the sulfur-containing amino acids cysteine and methionine. Crystals of two different shapes, plate-shaped and pyramid-shaped, diffracted to 2.9 and 3.2 A resolution and belonged to the primitive orthogonal space group P2(1)2(1)2(1) and the tetragonal space group P4(1) (or P4(3)), with unit-cell parameters a = 73.0, b = 144.9, c = 152.3 A and a = b = 78.2, c = 300.7 A, respectively. For the P2(1)2(1)2(1) crystals, three or four monomers exist in the asymmetric unit with a corresponding V(M) of 3.02 or 2.26 A(3) Da(-1) and a solvent content of 59.3 or 45.7%. For the P4(1) (or P4(3)) crystals, four or five monomers exist in the asymmetric unit with a corresponding V(M) of 2.59 or 2.09 A(3) Da(-1) and a solvent content of 52.5 or 40.6%.

Expression, crystallization and preliminary X-ray crystallographic analysis of Xoo2316, a predicted 6-phosphogluconolactonase, from Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, which is one of the most devastating diseases of rice (Oryza sativa L.) in many rice-growing countries. The coding sequence of Xoo2316 (a predicted 6-phosphogluconolactonase; 6PGL) from Xoo was cloned and expressed in Escherichia coli. 6PGL is an enzyme that is involved in the second step of the pentose phosphate pathway, which is essential for the synthesis of nucleotide sugars and NADPH, the main source of reducing power. The protein was purified and crystallized in order to elucidate the molecular basis for its enzymatic reaction. Native crystals diffracted to 2.4 A resolution and belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 40.0, b = 65.1, c = 78.8 A. A monomer exists in the asymmetric unit with a corresponding V(M) of 1.93 A(3) Da(-1) and a solvent content of 36.5%.

Expression, crystallization and preliminary X-ray crystallographic analysis of peptide deformylase from Xanthomonas oryzae pv. oryzae.

Peptide deformylase (PDF) catalyzes the removal of the N-formyl group from the N-terminus of newly synthesized polypeptides; this process is crucial for cell survival. As it is an antibacterial drug target against Xanthomonas oryzae pv. oryzae (Xoo), PDF from Xoo was cloned, expressed, purified and crystallized. Native PDF crystals diffracted to 2.7 A resolution and belonged to the hexagonal space group P6(1)22, with unit-cell parameters a = b = 59.0, c = 266.3 A. One monomer is present in the asymmetric unit, with a corresponding crystal volume per protein weight of 3.50 A(3) Da(-1) and a solvent content of 64.9%.

Expression, crystallization and preliminary X-ray crystallographic analysis of Xoo0352, D-alanine-D-alanine ligase A, from Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight (BB), which is one of the most devastating diseases of rice in most rice-growing countries. D-Alanine-D-alanine ligase A (DdlA), coded by the Xoo0352 gene, was expressed, purified and crystallized. DdlA is an enzyme that is involved in D-alanine metabolism and the biosynthesis of an essential bacterial peptidoglycan precursor, in which it catalyzes the formation of D-alanyl-D-alanine from two D-alanines, and is thus an attractive antibacterial drug target against Xoo. The DdlA crystals diffracted to 2.3 A resolution and belonged to the primitive tetragonal space group P4(3)2(1)2, with unit-cell parameters a = b = 83.0, c = 97.6 A. There is one molecule in the asymmetric unit, with a corresponding V(M) of 1.88 A(3) Da(-1) and a solvent content of 34.6%. The initial structure was determined by molecular replacement using D-alanine-D-alanine ligase from Staphylococcus aureus (PDB code 2i87) as a template model.

Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of 3-dehydroquinate synthase, Xoo1243, from Xanthomonas oryzae pv. oryzae.

The disease bacterial blight results in serious production losses of rice in Asian countries. The aroB gene encoding dehydroquinate synthase (DHQS), which is a potential antibiotic target, was identified from the plant-pathogenic bacterium Xanthomonas oryzae pv. oryzae (Xoo). DHQS plays an essential role in the synthesis of aromatic compounds in the shikimate pathway. The aroB gene (Xoo1243) was cloned from Xoo and the corresponding DHQS protein was subsequently overexpressed in Escherichia coli. The purified protein was crystallized using the hanging-drop vapour-diffusion method and yielded crystals that diffracted to 2.5 A resolution. The crystals belonged to the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = b = 118.2, c = 98.2 A. According to a Matthews coefficient calculation, the crystal contained two molecules in the asymmetric unit, with a corresponding V(M) of 2.06 A(3) Da(-1) and a solvent content of 40.4%.

Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of malonyl-CoA-acyl carrier protein transacylase (FabD) from Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight in rice, which is one of the most devastating diseases in rice-cultivating countries. The Xoo0880 (fabD) gene coding for a malonyl-CoA-acyl carrier protein transacylase (MCAT) from Xoo was cloned and expressed in Escherichia coli. MCAT is an essential enzyme that catalyzes a key reaction of fatty-acid synthesis in bacteria and plants: the conversion of malonyl-CoA to malonyl-acyl carrier protein. The FabD enzyme was purified and crystallized in order to elucidate its three-dimensional structure and to determine its enzymatic reaction mechanism and biological importance. The crystal obtained diffracted to 1.9 A resolution and belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 41.4, b = 74.6, c = 98.5 A. According to Matthews coefficient calculations, the crystallographic structure contains only one monomeric unit in the asymmetric unit with a V(M) of 2.21 A(3) Da(-1) and a solvent content of 44.3%.

Functional analysis of pilQ gene in Xanthomonas oryzae pv. oryzae, bacterial blight pathogen of rice.

Bacterial blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is the most devastating bacterial disease in rice. A virulence-attenuated mutant strain HNU89K9 of X. oryzae pv. oryzae (KACC10331), with a transposon insertion in the pilQ gene was used for this study. The pilQ was involved in the gene cluster pilMNOPQ of the Xoo genome. Growth rate of the pilQ mutant was similar to that of wild-type. At level of amino acids, PilQ of Xoo showed that a high sequence identities more than 94% and 70% to Xanthomonas species and to Xyllela fastidiosa, respectively but a low sequence homology less than 30% to other bacterial species. The twitching motility forming a marginal fringe on PSA media was observed on colony of the wild-type strain KACC10331, but not in mutant HNU89K9. Wild-type Xoo cells formed a biofilm on the surface of the PVC plastic test tube, while the mutant strain HNU89K9 did not form a biofilm. The results suggest that the pilQ gene of X. oryzae pv. oryzae plays a critical role in pathogenicity, twitching motility, and biofilm formation.

Analysis of Genetic and Pathogenic Diversity of Ralstonia solanacearum Causing Potato Bacterial Wilt in Korea.

The Ralstonia solanacearum species complex (RSSC) can be divided into four phylotypes, and includes phenotypically diverse bacterial strains that cause bacterial wilt on various host plants. This study used 93 RSSC isolates responsible for potato bacterial wilt in Korea, and investigated their phylogenetic relatedness based on the analysis of phylotype, biovar, and host range. Of the 93 isolates, twenty-two were identified as biovar 2, eight as biovar 3, and sixty-three as biovar 4. Applied to the phylotype scheme, biovar 3 and 4 isolates belonged to phylotype I, and biovar 2 isolates belonged to phylotype IV. This classification was consistent with phylogenetic trees based on 16S rRNA and egl gene sequences, in which biovar 3 and 4 isolates clustered to phylotype I, and biovar 2 isolates clustered to phylotype IV. Korean biovar 2 isolates were distinct from biovar 3 and 4 isolates pathologically as well as genetically - all biovar 2 isolates were nonpathogenic to peppers. Additionally, in host-determining assays, we found uncommon strains among biovar 2 of phylotype IV, which were the tomato-nonpathogenic strains. Since tomatoes are known to be highly susceptible to RSSC, to the best of our knowledge this is the first report of tomato-nonpathogenic potato strains. These results imply the potential prevalence of greater RSSC diversity in terms of host range than would be predicted based on phylogenetic analysis.

Analysis of virulence and growth of a purine auxotrophic mutant of Xanthomonas oryzae pathovar oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight of rice. A random insertional mutant library of Xoo KACC10331 was constructed using a Tn5-derived transposon, and the virulence of the mutants against the susceptible rice cultivar IR24 was assayed. After the virulence assay, the M793 (purD::Tn5) mutant that had reduced virulence against the rice plants was isolated. Thermal asymmetric interlaced-PCR and sequence analysis revealed that the transposon was inserted into the purD gene (encodes a phosphoribosylamine-glycine ligase) of the M793 mutant. The reverse transcriptase-PCR assay revealed that the mutation of the purD gene did not affect the expression of other purine biosynthesis genes. However, the M793 mutant required exogenous purines and thiamine for growth in minimal media. These results indicate that the purD gene plays a crucial role in the growth and virulence of Xoo.

The genome sequence of Xanthomonas oryzae pathovar oryzae KACC10331, the bacterial blight pathogen of rice.

The nucleotide sequence was determined for the genome of Xanthomonas oryzae pathovar oryzae (Xoo) KACC10331, a bacterium that causes bacterial blight in rice (Oryza sativa L.). The genome is comprised of a single, 4 941 439 bp, circular chromosome that is G + C rich (63.7%). The genome includes 4637 open reading frames (ORFs) of which 3340 (72.0%) could be assigned putative function. Orthologs for 80% of the predicted Xoo genes were found in the previously reported X.axonopodis pv. citri (Xac) and X.campestris pv. campestris (Xcc) genomes, but 245 genes apparently specific to Xoo were identified. Xoo genes likely to be associated with pathogenesis include eight with similarity to Xanthomonas avirulence (avr) genes, a set of hypersensitive reaction and pathogenicity (hrp) genes, genes for exopolysaccharide production, and genes encoding extracellular plant cell wall-degrading enzymes. The presence of these genes provides insights into the interactions of this pathogen with its gramineous host.