In-depth identification of pathways related to cisplatin-induced hepatotoxicity through an integrative method based on an informatics-assisted label-free protein quantitation and microarray gene expression approach.

Cisplatin is used widely for treatment of a variety of cancer diseases. Recently, however, the use of cisplatin is restricted because of its adverse effects such as hepatotoxicity. There is no study with current proteomics technology to evaluate cisplatin-induced hepatotoxicity, even if some studies have reported on the hepatotoxicity. In this study, proteomic as well as genomic analyses have been used for identification of proteins and genes that respond to cisplatin treatment in rat primary hepatocytes. To investigate the hepatotoxic effects of cisplatin, rat primary hepatocytes were treated with an IC(20) concentration for 24 h. From proteomic analysis based on label-free quantitation strategy, cisplatin induced 76 up-regulated and 19 down-regulated proteins among 325 distinct proteins. In the mRNA level, genomic analysis revealed 72 up-regulated and 385 down-regulated genes in the cisplatin-treated group. Based on these two analyses, 19 pathways were commonly altered, whereas seven pathways were identified only by proteomic analysis, and 19 pathways were identified only by genomic analysis. Overall, this study explained the mechanism of cisplatin-induced hepatotoxicity with two points of view: well known pathways including drug metabolism, fatty acid metabolism, and glycolysis/TCA cycle and little known pathways including urea cycle and inflammation metabolism, for hepatotoxicity of other toxic agents. Up-regulated proteins detected by proteomic analysis in the cisplatin-treated group: FBP1 (fructose 1,6-bisphosphatase 1), FASN (fatty acid synthase), CAT (catalase), PRDX1 (peroxiredoxin-1), HSPD1 (60-kDa heat shock protein), MDH2 (malate dehydrogenase 2), and ARG1 (arginase 1), and also down-regulated proteins in the cisplatin-treated group: TPM1 (tropomyosin 1), TPM3 (tropomyosin 3), and CTSB (cathepsin B), were confirmed by Western blot analysis. In addition, up-regulated mRNAs detected by microarray analysis in the cisplatin-treated group: GSTA2, GSTT2, YC2, TXNRD1, CYP2E1, CYP2C13, CYP2D1, ALDH17, ARG1, ARG2, and IL-6, and also down-regulated mRNAs: CYP2C12, CYP26B1, TPM1, and TPM3, were confirmed by RT-PCR analysis. In case of PRDX1, FASN, and ARG1, they were further confirmed by immunofluorescence analysis. Through the integrated proteomic and genomic approaches, the present study provides the first pathway map related to cisplatin-induced hepatotoxicity, which may provide new insight into the mechanism of hepatotoxicity.

Antioxidant mechanism of isoflavone metabolites in hydrogen peroxide-stimulated rat primary astrocytes: critical role of hemeoxygenase-1 and NQO1 expression.

The brain is highly vulnerable to oxidative stress, thus controlling oxidative stress is considered to be an important therapeutic target for neurodegenerative diseases. In this study, we found that two isoflavone metabolites (tectorigenin and glycitein) inhibited hydrogen peroxide-induced reactive oxygen species (ROS) generation and subsequent cell death in rat primary astrocytes. The isoflavone metabolites increased the expression of phase II antioxidant enzymes, such as hemeoxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1), and pre-treatment of cells with their specific inhibitors or small interfering RNA (siRNA) reversed the antioxidant and cytoprotective effects of isoflavones. The results suggest that the antioxidant/cytoprotective effects of isoflavone metabolites are at least because of increased HO-1 and NQO1 expression. Further mechanistic studies revealed that isoflavones increase the binding of transcription factors [nuclear factor-E2-related factor 2 (Nrf2) and c-Jun] to the antioxidant response element (ARE) on HO-1 and NQO1 promoters. Down-regulation of Nrf2 and/or c-Jun using dominant-negative mutants (DNMs) or siRNA diminished the expression of HO-1 and NQO1, suggesting that Nrf2 and c-Jun are key transcription factors modulating HO-1/NQO1 expression. Moreover, PI3 kinase and mitogen-activated protein kinase (MAPK) signaling pathways were shown to be involved in HO-1 and/or NQO1 expression by isoflavones. Our data collectively suggest that HO-1 and NQO1 play a critical role in antioxidant effects of isoflavone metabolites in rat brain astrocytes.

Metallopharmaceuticals based on silver(I) and silver(II) polydiguanide complexes: activity against burn wound pathogens.

OBJECTIVES: The in vitro pharmacodynamics of silver(I) and silver(II) complexes of a polydiguanide ligand, chlorhexidine, were assayed to examine the value of the bactericidal endpoint as an alternative means of evaluating their antibacterial activities against burn wound pathogens. METHODS: Synthesis of silver(I) chlorhexidine [Ag(I)CHX] was accomplished by in situ precipitation of the complex from a feebly acidic or neutral aqueous solution of AgNO(3) and chlorhexidine, whereas silver(II) chlorhexidine [Ag(II)CHX] was synthesized by oxidation of Ag(I), followed by complexation of the oxidized metal with chlorhexidine. Their antibacterial potencies were assessed in vitro by determining the MICs and MBCs for four Gram-positive and four Gram-negative bacteria. Time-kill assays using three different concentrations of these agents were also performed. RESULTS: The MICs of Ag(I)CHX and Ag(II)CHX were much lower than those of chlorhexidine, AgNO(3) and silver sulfadiazine. The time-kill study provided quantitative information on actual times required to reach the bactericidal endpoint using a particular concentration of the active agent. The lethality rates of Ag(I)CHX and Ag(II)CHX against the tested bacteria were 2× to 8× faster than those of chlorhexidine or AgNO(3) at a concentration equal to or 4× MIC. CONCLUSIONS: Ag(I)CHX and Ag(II)CHX showed superior antibacterial activity and faster killing kinetics compared with chlorhexidine and AgNO(3). These complexes may serve as new-generation antibacterial agents in wound care.

Aminoacyl-tRNA synthetase-interacting multifunctional proteins (AIMPs): a triad for cellular homeostasis.

Aminoacyl-tRNA synthetases (ARSs) are highly conserved for efficient and precise translation of genetic codes. In higher eukaryotic systems, several different ARSs including glutamyl-prolyl-, isoelucyl-, leucyl-, methionyl-, glutaminyl-, lysyl-, arginyl-, and aspartyl-tRNA synthetase form a macromolecular protein complex with three nonenzymatic cofactors (AIMP1/p43, AIMP2/p38, and AIMP3/p18). Although the structure and functional implications for this complex formation are not completely understood, rapidly accumulating evidences suggest that this complex would work as a molecular hub linked to the multiple signaling pathways that involve the components of enzymes and cofactors. In this article, the roles of three nonenzymatic components of the multi-tRNA synthetase complex in the assembly of the components and in cell regulation are addressed.

Extended spectrum of quinolone resistance, even to a potential latter third-generation agent, as a result of a minimum of two GrlA and two GyrA alterations in quinolone-resistant Staphylococcus aureus.

BACKGROUND: This study was performed to determine the extended spectrum of quinolone resistance caused by increased mutations within the target enzymes of quinolones. METHODS: The minimum inhibitory concentrations (MICs) for ciprofloxacin, sparfloxacin, trovafloxacin and DW286 were determined against 98 ciprofloxacin-resistant Staphylococcus aureus strains. Also, PCR-amplified grlA, grlB, gyrA and gyrB DNA fragments were sequenced and amino acid changes were analyzed. RESULTS: The MIC(50) values of quinolones decreased with later-generation compounds, i.e. >or=64 microg/ml for ciprofloxacin, 16 microg/ml for sparfloxacin, 2 microg/ml for trovafloxacin and 0.25 microg/ml for DW286. Combinations of amino acid changes within GrlA (Ser-80, Tyr-83 or Glu-84), GrlB (Pro-451, Pro-585 or Asp-432) and GyrA (Ser-84, Ser-85 or Glu-88) were constructed. The combination of Ser-80-->Phe within GrlA and Ser-84-->Leu within GyrA was the fundamental combination in alterations involved in ciprofloxacin resistance, and additional alterations extended quinolone resistance. CONCLUSION: A larger number of alterations within GrlA and GyrA further extended the spectrum of quinolone resistance.

Synthesis of highly antibacterial nanocrystalline trivalent silver polydiguanide.

Highly monodispersed nanoparticles of a trivalent silver polydiguanide complex are synthesized by oxidation of the monovalent silver, followed by stabilization of the oxidized higher-valent metal through complexation with a polydiguanide ligand in a reverse microemulsion at room temperature. The synthesized nanoparticles have excellent photostability and displayed superior antibacterial activity toward Gram-positive and Gram-negative prokaryotes of clinical interest in vitro compared to silver sulfadiazine. These nanoparticles may serve as a new generation antibacterial metallopharmaceutical in wound care.

Development of TaqMan probe-based real-time PCR method for erm(A),erm(B), and erm(C), rapid detection of macrolide-lincosamide-streptogramin B resistance genes, from clinical isolates.

To achieve more accurate and rapid detection of macrolidelincosamide- streptogramin B resistance genes, erm(A), erm(B), and erm(C), we developed a TaqMan probe-based real-time PCR (Q-PCR) method and compared it with conventional PCR (C-PCR), which is the most widely using erm gene identification method. The detection limit of Q-PCR was 5 fg of genomic DNA or 5-8 CFU of bacterial cells of Staphylococcus aureus. The utilization of Q-PCR might shorten the time to erm detection from 3-4 h to about 50 min. These data indicated that Q-PCR assay appears to be not only highly sensitive and specific, but also the most rapid diagnostic method. Therefore, the appropriate application of the Q-PCR assay will permit rapid and accurate identification of erm genes from clinical and other samples.

Translational attenuation and mRNA stabilization as mechanisms of erm(B) induction by erythromycin.

Translational attenuation has been proposed to be the mechanism by which the erm(B) gene is induced. Here, we report genetic and biochemical evidence, obtained by using erythromycin as the inducing antibiotic, that supports this hypothesis. We also show that erythromycin increases the level of the erm(B) transcript by stalling the ribosome on the leader mRNA and thereby facilitating the stabilization and processing of the mRNA. Erythromycin-induced mRNA stabilization and processing were observed with an ochre stop at codons 11 to 13 of the leader but not with an ochre stop at codon 10. This suggests that erythromycin does not stall the ribosome before codon 11 of the leader reaches the aminoacyl site. Secondary structure analyses of the erm(B) transcripts by in vitro and in vivo chemical probing techniques identified conformational changes in the transcripts that result from induction by erythromycin. These findings demonstrate that stalling of erythromycin-bound ribosomes at leader codon 11 causes the refolding of mRNA into a conformation in which the translational initiation site for the structural gene is unmasked and renders erm(B) translationally active.

Foggy D-shaped zone of inhibition in Staphylococcus aureus owing to a dual character of both inducible and constitutive resistance to macrolide-lincosamide-streptogramin B.

OBJECTIVES: We identified Staphylococcus aureus strains having blunt inhibitory zones around the clindamycin or josamycin discs proximal to the erythromycin disc filled with slight bacterial growth. This ambiguous phenotype was termed as 'macrolide-lincosamide-streptogramin B antimicrobial (MLS(B)) resistance having a foggy D-shaped inhibitory zone' (fMLS(B)), and we tried to analyse its molecular mechanisms. METHODS: Forty-one clinically isolated strains of fMLS(B) S. aureus were studied. The regulatory region of the erm(A) gene, which was found to be the only molecular mechanism of fMLS(B), was sequenced. Then, beta-galactosidase assays were performed to observe their expression patterns through the nucleotide sequential alteration. RESULTS: According to the sequencing electropherogram, a grouping was made of a homogeneous nucleotide sequence group (73.2%) and a heterogeneous nucleotide sequence group (26.8%). The former group was composed of a 25 bp tandem duplication type and a 25 bp tandem triplication type. Their beta-galactosidase activity was similar to that of constitutive MLS(B) resistance due to its high basal level. Nevertheless, the predicted mRNA secondary structure of the regulatory region maintains the stem-loops of inducible wild-type erm(A), and thereby its inducible character might be expected in vivo. Strains in the latter group were proven to have two different erm(A) genes, and then dual effect of expression was observed. CONCLUSIONS: The ambiguous phenotype of fMLS(B) is due to its possessing the dual character of inducible and constitutive expression of erm(A). The dual character is due to having one erm(A) gene of dual character or coexistence of two characterized erm(A) genes simultaneously.

Molecular analysis of constitutive mutations in ermB and ermA selected in vitro from inducibly MLSB-resistant enterococci.

Frequencies of spontaneous mutation from inducible resistance to constitutive resistance were determined for the four clinical isolates of erythromycin-resistant enterococci, including one isolate with ermB gene and three clinical isolates with ermA gene. The rate of ermB mutation was higher than that of ermA mutation by more than 10 fold. Sequence analysis of the regulatory regions of erm genes revealed that mutation type of ermB was just point mutation, by contraries the mutation type of ermA was either deletion or tandem duplication. These results showed distinct characteristics in mutation patterns of ermB and ermA.

Molecular mechanism of cofilin dephosphorylation by ouabain.

We previously reported that phosphorylated cofilin-triosephosphate isomerase (TPI) complex interacts with Na,K-ATPase and enhances the pump activity through the phosphorylation of cofilin via Rho-mediated signaling pathway. In this study, we tested the hypothesis that the dephosphorylation of cofilin may be induced through Na,K-ATPase inhibition by ouabain. The phosphorylation level of cofilin by ouabain which decreases in a time- and dose-dependent manner in various human cell lines, remains unchanged by pretreatment with Src inhibitor, PP2; epidermal growth factor receptor (EGFR) inhibitor, AG1478; Raf-1 kinase (Raf) inhibitor, GW5074; and ERK kinase (MEK) inhibitor, PD98059, and by transfection of Ras dominant negative mutant (RasN17). This suggests that ouabain dephosphorylates cofilin through the Src/EGFR/Ras/Raf/MEK pathway. Ouabain activates Ras/Raf/MEK pathway, but down-regulates Rho kinase (ROCK)/LIM kinase (LIMK)/cofilin pathway, implying that there may be a cross-talk by ouabain between the Ras/Raf/MEK and the ROCK/LIMK/cofilin pathways. Immunofluorescence and flow cytometry suggest that ouabain-induced active form of cofilin may be involved in cytoskeletal reorganization and cell volume regulation. Thus, these findings demonstrate a new molecular mechanism for the dephosphorylation of cofilin through the inhibition of Na,K-ATPase by ouabain.

Comparison of antibiotic effect and corneal epithelial toxicity of levofloxacin and moxifloxacin in vitro.

PURPOSE: To compare the bacterial susceptibility and corneal epithelial toxicity of levofloxacin and moxifloxacin in the human corneal epithelial cells (HCECs). METHODS: We used 2 types of strains, ie, American Type Culture Collection strains and resistant strains. The former included Staphylococcus aureus, coagulase-negative staphylococci, Pseudomonas aeruginosa, and Serratia marcescens, The latter were methicillin-resistant Staphylococcus aureus; methicillin-resistant, coagulase-negative Staphylococcus sp.; and ciprofloxacin-resistant P. aeruginosa. The HCECs were incubated with each bacterial population for 1 hour and exposed to both antibiotics for 1 hour. The colony-forming units of viable bacteria per well were expressed as base 10 logarithms. To determine corneal epithelial toxicity, we exposed the HCECs to each antibiotic agent, and the viable epithelial cells were quantified by the MTT assay. We also observed the wound healing rate of injured HCECs cultured in each antibiotic agent for 24 hours. RESULTS: In bacterial susceptibility testing of antibiotics, levofloxacin was less effective for Serratia marcescens than moxifloxacin (P < 0.05). However, both moxifloxacin and levofloxacin showed the same efficacy against Gram-positive bacteria, P. aeruginosa, and resistant strains (P > 0.05). Moxifloxacin showed a higher toxicity than levofloxacin when the HCECs were exposed to the respective antibiotics for 2 and 24 hours (P < 0.05). The moxifloxacin inhibited the effect of wound healing in HCEC injury, but levofloxacin did not (P < 0.05). CONCLUSIONS: There was no significant difference in antibiotic effects between moxifloxacin and levofloxacin on most bacterial strains, except for Serratia marcescens. On the other hand, levofloxacin seemed to be safer than moxifloxacin in HCECs.

Activities of clindamycin, synercid, telithromycin, linezolid, and mupirocin against Gram-positive coccal strains resistant to erythromycin in Korea.

The antibacterial activities of clindamycin, synercid, telithromycin, linezolid and mupirocin were evaluated against erythromycin-resistant Gram-positive coccal clinical isolates collected in Korean hospitals. In Staphylococcus aureus, synercid, linezolid and mupirocin were the most active agents. Against coagulase-negative staphylococci (CNS), synercid, linezolid and mupirocin were also active. Telithromycin and synercid resistance was common against enterococci, only linezolid and mupirocin were active. The reason of low activity of telithromycin against staphylococci and enterococci is because most of the isolates were constitutively resistant to erythromycin. Synercid, telithromycin, linezolid and mupirocin were active against streptococci.

Long-acting interferon-alpha 2a modified with a trimer-structured polyethylene glycol: preparation, in vitro bioactivity, in vivo stability and pharmacokinetics.

The proper selection of size and shape for polyethylene glycol (PEG) is one of the most important points in PEGylation technology. Therefore, PEGs of various sizes and shapes have been widely developed to endow specific properties. In this study, a unique, trimer-structured, 43 kDa PEG was conjugated to interferon-alpha 2a (IFN) by forming an amide bond to improve the pharmacokinetic properties and minimize the loss of IFN bioactivity. Mono-PEGylated IFN (PEG(3)-IFN) prepared by utilizing this unique PEG was purified and characterized by cation-exchange chromatography and MALDI-TOF mass spectrometry. The in vitro bioactivity, in vivo stability, and pharmacokinetics of PEG(3)-IFN were examined and compared to those of native IFN. PEG(3)-IFN exhibited comparable in vitro bioactivities to native IFN and an excellent stability of the conjugation linkage in rat serum and various organs following subcutaneous injection. Furthermore, it showed slow absorption and markedly reduced clearance in rats, thereby increasing the biological half-life by about 40-fold compared to that of native IFN. This is the first report on the application of unique, trimer-structured PEG to bioactive proteins. The results suggest that unique, trimer-structured 43 kDa PEG can provide some advantages to improve the pharmacokinetic properties and to maintain the bioactivity of therapeutic proteins in clinical use.

ErmK leader peptide : amino acid sequence critical for induction by erythromycin.

The ermK gene from Bacillus lichenformis encodes an inducible rRNA methylase that confers resistance to the macrolide-lincosamide-streptogramin B antibiotics. The ermK mRNA leader sequence has a total length of 357 nucleotides and encodes a 14-amino acid leader peptide together with its ribosome binding site. The secondary structure of ermK leader mRNA and a leader peptide sequence have been reported as the elements that control expression. In this study, the contribution of specific leader peptide amino acid residues to induction of ermK was studied using the PCR-based megaprimer mutation method. ermK methylases with altered leader peptide codons were translationally fused to E. coil beta-galactosidase reporter gene. The deletion of the codons for Thr-2 through Ser-4 reduced inducibility by erythromycin, whereas that for Thr-2 and His-3 was not. The replacement of the individual codons for Ser-4, Met-5 and Arg-6 with termination codon led to loss of inducibility, but stop mutation of codon Phe-9 restored inducibility by erythromycin. Collectively, these findings suggest that the codons for residue 4, 5 and 6 comprise the critical region for induction. The stop mutation at Leu-7 expressed constitutively ermK gene. Thus, ribosome stalling at codon 7 appears to be important for ermK induction.

Increased antibacterial activity of DW286, a novel fluoronaphthyridone antibiotic, against Staphylococcus aureus strains with defined mutations in DNA gyrase and topoisomerase IV.

To investigate the activity of DW286, a new fluoronaphthyridone, the quinolone resistance determining regions (QRDRs) of gyrA, gyrB, grlA and grlB genes in 64 Staphylococcus aureus clinical isolates were analyzed and the MICs of DW286 and comparator quinolones determined. Double and triple mutants in gyrA and grlA were resistant to ciprofloxacin, sparfloxacin, trovafloxacin and gemifloxacin but susceptible to DW286 (MIC 0.25-0.5 mg/l). The fourth alteration, Ser85Pro of GyrA was required to make a strain resistant to DW286 (MIC 4-32 mg/l). For a strain with the mutations at GyrA Ser84Leu and GrlA Ser80Phe, the MBC of DW286 was two-fold higher than its corresponding MIC, in contrast to ciprofloxacin which was not bactericidal.

Role of disulfide bond of arylsulfate sulfotransferase in the catalytic activity.

Bacterial arylsulfate sulfotransferase (ASST) catalyzes the transfer of sulfate group from a phenyl sulfate ester to a phenolic acceptor. The promoter region and the transcription start sites of Enterobacter amnigenus astA have been determined by primer extension analysis. Northern blot analysis resolved two mRNA species with lengths of 3.3 and 2.0 kb, which correspond to the distances between the transcriptional initiation sites and the two inverted repeat sequences (IRSs). By length, the 3.3 kb RNA could comprise the three-gene (astA with dsbA and dsbB) operon. ASST has three highly conserved cysteine residues. Reducing and nonreducing SDS-PAGE and activity staining showed that disulfide bond is needed for the activity of the enzyme. To identify the cysteine residues responsible for the disulfide bond formation, a series of Cys to Ser mutants has been constructed and the enzymatic activity was measured. Based on the results, we assumed that the first cysteine (Cys349) might be involved in disulfide bond mainly with the second cysteine (Cys445) and result in active conformation.

Further modification of the Hodge test to screen AmpC beta-lactamase (CMY-1)-producing strains of Escherichia coli and Klebsiella pneumoniae.

Cefoxitin-resistant Escherichia coli and Klebsiella pneumoniae are widespread in Korea. Significant proportions of them are considered to be CMY-1 producers. For effective screening of CMY-1 producers, the Hodge test was modified by using a cefoxitin disk and the performance was evaluated. The sensitivity and specifity of the test were 100% and 94.9%, respectively. The test was easier to perform than the three-dimensional extract test. This modified test should be suitable for screening CMY-1-producing strains of E. coli and K. pneumoniae.

Hypocholesterolemic activity of Bifidobacteria isolated from a healthy Korean.

This study was undertaken to investigate the hypocholesterolemic activity of Bifidobacteria (B. breve K-110, B. breve K-111, and B. infantis K-525) isolated from a healthy Korean. The administration of B. breve K-110 and K-111 with a high cholesterol diet significantly protected the increase of serum total cholesterol and LDL cholesterol relative to that of a high cholesterol diet alone. Such a diet supplemented with 0.5% B. breve K-111 decreased serum total cholesterol and LDL cholesterol to 57 and 55%, respectively. The administration of Bifidobacteria also significantly inhibited the lipid-deposited surface in the aorta. The normalizing activity of serum cholesterol level in cholesterolemic rats was accelerated by Bifidobacteria. The normalizing activity of B. breve K-111 on serum cholesterol level was superior to that of B. breve K-110. These results suggest that Bifidobacteria in the human intestine play a role in the prophylactics of arteriosclerosis.

Alterations in regulatory regions of erm(B) genes from clinical isolates of enterococci resistant to telithromycin.

We determined rates of resistance to the ketolide telithromycin in 56 Enterococcus faecalis isolates and 44 Enterococcus faecium isolates collected from hospitals in Korea between 2005 and 2006. Twenty nine (51.8%) isolates of E. faecalis and 35 (79.5%) isolates of E. faecium were resistant to telithromycin (minimum inhibitory concentrations, ≥ 4 µg/mL). All of the telithromycin-resistant E. faecalis carried the erm(B) gene only. Of the telithromycin-resistant E. faecium, 29 resistant strains carried erm(B) only, the other six carried erm(A) and erm(B) together. The nucleotide sequence of the erm(B) regulatory regions from 29 E. faecalis and 29 E. faecium isolates with erm(B) only was analyzed. Five types of alterations were detected. The first and second types had point mutations that destabilize the secondary structure of erm(B) mRNA sequestering the translation initiation region of the structural gene. The third type was identical to erm(Bv1), a previously reported variant of erm(B) with different induction specificity. The fourth and fifth types had point mutations within the critical sequence for induction and a point mutation destabilizing the stem-loop of erm(B) mRNA sequestering the translation initiation region of the structural gene.