Molecular cloning,expression, and functional characterization of a 2Cys-peroxiredoxin in Chinese cabbage

A cDNA (C2C-Prx) corresponding to a 2Cys-peroxiredoxin (2Cys-Prx) was isolated from a leaf cDNA library of Chinese cabbage. The predicted amino acid sequence of C2C-Prx has 2 conserved cysteines and several peptide domains present in most of the 2Cys-Prx subfamily members. It shows the highest sequence homology to the 2Cys-Prx enzymes of spinach (88%) and Arabidopsis (86%). Southern analysis using the cDNA insert of C2C-Prx revealed that it consists of a small multigene family in Chinese cabbage genome. RNA blot analysis showed that the gene was predominantly expressed in the leaf tissue of Chinese cabbage seedlings, but the mRNA was generally expressed in most tissues of mature plant, except roots. The expression of C2C-Prx was slightly induced by treatment with H₂O₂ (100M) or Fe³⁺/O₂/DTT oxidation system, but not by ABA (50 M) or GA₃ (10 M). The C2C-Prx is encoded as a preprotein of 273 amino acids containing a putative chloroplast-targeting signal of 65 amino acids at its N-terminus. The N-terminally truncated recombinant protein (C2C-Prx) migrates as a dimer in a non-reducing SDS-polyacrylamide gel and as a monomer in a reducing condition. The C2C-Prx shows no immuno cross-reactivity to antiserum of the yeast thiol-specific antioxidant protein, and vice versa. The C2C-Prx prevents the inactivation of glutamine synthetase and the DNA cleavage in the metal-catalyzed oxidation system. In the yeast thioredoxin system containing thioredoxin reductase, thioredoxin, and NADPH, the C2C-Prx exhibits peroxidase activity on H₂O₂.     

Activated liver X receptors stimulate adipocyte differentiation through induction of peroxisome proliferator-activated receptor gamma expression

Liver X receptors (LXRs) are nuclear hormone receptors that regulate cholesterol and fatty acid metabolism in liver tissue and in macrophages. Although LXR activation enhances lipogenesis, it is not well understood whether LXRs are involved in adipocyte differentiation. Here, we show that LXR activation stimulated the execution of adipogenesis, as determined by lipid droplet accumulation and adipocyte-specific gene expression in vivo and in vitro. In adipocytes, LXR activation with T0901317 primarily enhanced the expression of lipogenic genes such as the ADD1/SREBP1c and FAS genes and substantially increased the expression of the adipocyte-specific genes encoding PPARγ (peroxisome proliferator-activated receptor γ) and aP2. Administration of the LXR agonist T0901317 to lean mice promoted the expression of most lipogenic and adipogenic genes in fat and liver tissues. It is of interest that the PPARγ gene is a novel target gene of LXR, since the PPARγ promoter contains the conserved binding site of LXR and was transactivated by the expression of LXRα. Moreover, activated LXRα exhibited an increase of DNA binding to its target gene promoters, such as ADD1/SREBP1c and PPARγ, which appeared to be closely associated with hyperacetylation of histone H3 in the promoter regions of those genes. Furthermore, the suppression of LXRα by small interfering RNA attenuated adipocyte differentiation. Taken together, these results suggest that LXR plays a role in the execution of adipocyte differentiation by regulation of lipogenesis and adipocyte-specific gene expression.     

Investigation of extended-spectrum beta-lactamases produced by clinical isolates of Klebsiella pneumoniae and Escherichia coli in Korea

AIMS: Isolates obtained from various regions in Korea in 2002 were identified and their susceptibility to extended-spectrum cephalosporins, monobactams and/or cephamycins was studied along with any production of extended-spectrum beta-lactamases (ESBLs). METHODS AND RESULTS: Bacteria identified by the conventional techniques and Vitek GNI card were Klebsiella pneumoniae and Escherichia coli. Using disk diffusion and double-disk synergy tests, we found that 39.2% of strains produced ESBLs. About 52% of isolates transferred resistance to ceftazidime by conjugation. Banding patterns of PCR amplification with the designed primers showed that 837- and 259-bp fragments specific to bla(TEM) genes were amplified in 63.3% of strains. 929- and 231-bp fragments (bla(SHV)), 847- and 520-bp fragments (bla(CMY)), 597- and 858-bp fragments (bla(CTX-M)) were amplified in 61.5, 17.3 and 7.7% of strains respectively. About 51.9% of strains contained more than two types of beta-lactamase genes. Especially, one strain contained bla(TEM), bla(CMY) and bla(CTX-M) genes. SIGNIFICANCE: Resistance mechanisms to beta-lactams, comprising mostly ESBL production, lead to the resistance against even recently developed beta-lactams in enterobacteria, which is now a serious threat to antibiotic therapy. The high prevalence of bla(CMY) genes and multidrug-resistant genes may also make therapeutic failure and lack of eradiation of these strains by extended-spectrum cephalosporins or cephamycins.     

Oxidative modification of neurofilament-L by the Cu,Zn-superoxide dismutase and hydrogen peroxide system

Neurofilament-L (NF-L) is a major element of neuronal cytoskeletons and known to be important for their survival in vivo. Since oxidative stress might play a critical role in the pathogenesis of neurodegenerative diseases, we investigated the role of Cu,Zn-superoxide dismutase (SOD) in the modification of NF-L. When disassembled NF-L was incubated with Cu,Zn-SOD and H2O2, the aggregation of protein was proportional to the concentration of hydrogen peroxide. Cu,Zn-SOD/H2O2-mediated modification of NF-L was significantly inhibited by radical scavenger, spin trap agents and copper chelators. Dityrosine crosslink formation was obtained in Cu,Zn-SOD/H2O2-mediated NF-L aggregates. Antioxidant molecules, carnosine and anserine significantly inhibited the aggregation of NF-L and the formation of dityrosine. This study suggests that copper-mediated NF-L modification may be closely related to oxidative reactions which play a critical role in neurodegenerative diseases.     

Dendritic cells loaded with exogenous antigen by electroporation can enhance MHC class I–mediated antitumor immunity

To develop an efficient antitumor immunotherapy, we have examined if dendritic cells (DCs) loaded with soluble antigens by electroporation present more antigens via the MHC (major histocompatibility complex) class I pathway, which mediate a cytotoxic T-cell response. DCs loaded with ovalbumin (OVA) by electroporation presented more MHC class I–restricted determinants compared with DCs pulsed with OVA. When electroporated DCs were pulsed with OVA for additional times, both MHC class I– and II–restricted presentation of OVA were increased compared with each single procedure, including electroporation or simple pulse. Immunization with DCs loaded with OVA by electroporation induced higher cytotoxicity of splenocytes to E.G7 cells, a clone of EL4 cells transfected with an OVA cDNA, than immunization with DCs pulsed with OVA. In the animal study, immunization with DCs loaded with OVA or tumor cell lysates by electroporation induced an effective antitumor immunity against tumor of E.G7 cells or Lewis lung carcinoma cells, respectively. In addition, immunization with DCs loaded with antigen by combination of electroporation and pulse, completely protected mice from tumor formation, and prolonged survival, in both tumor models. These results demonstrated that electroporation would be a useful way to enhance MHC class I–mediated antitumor immunity without functional deterioration, and that the combination of electroporation and pulse could be a simple and efficient antigen-loading method and consequently lead to induction of strong antitumor immunity.Abbreviations DCs dendritic cells - MHC major histocompatibility complex - OVA ovalbumin - TAA tumor-associated antigen - CTL cytotoxic T lymphocyte - LDH lactate dehydrogenase     

Interaction between a negative regulator (Msa2/Nrd1) and a positive regulator (Cpc2) of sexual differentiation in Schizosaccharomyces pombe

The sexual differentiation of Schizosaccharomyces pombe is controlled by many cellular components which have not been fully characterized. We isolated a gene called msa2 as a multi-copy suppressor of a sporulation abnormal mutant (sam1). Msa2p is identical with Nrd1p which has been characterized as a factor that blocks the onset of sexual differentiation. The yeast two-hybrid system was used to identify Cpc2p, a fission yeast homolog of the RACK1 protein, that interacted with Msa2p/Nrd1p. We confirmed that Msa2p/Nrd1p interacted with Cpc2p in S. pombe cells. An epistatic analysis of msa2/nrd1 and cpc2 suggests that Msa2p/Nrd1p was an upstream regulator for Cpc2p. A localization analysis of Cpc2p and Msa2p/Nrd1p indicates that both proteins were predominantly localized in the cytoplasm. The interaction of negative regulator Msa2p/Nrd1p with positive regulator Cpc2p suggests a new regulatory circuit in the sexual differentiation of S. pombe.     

Structural and biochemical implications of single amino acid substitutions in the nucleotide-dependent switch regions of the nitrogenase Fe protein from<Emphasis Type="Italic"> Azotobacter vinelandii</Emphasis>

The structures of nitrogenase Fe proteins with defined amino acid substitutions in the previously implicated nucleotide-dependent signal transduction pathways termed switch I and switch II have been determined by X-ray diffraction methods. In the Fe protein of nitrogenase the nucleotide-dependent switch regions are responsible for communication between the sites responsible for nucleotide binding and hydrolysis and the [4Fe-4S] cluster of the Fe protein and the docking interface that interacts with the MoFe protein upon macromolecular complex formation. In this study the structural characterization of the Azotobacter vinelandii nitrogenase Fe protein with Asp at position 39 substituted by Asn in MgADP-bound and nucleotide-free states provides an explanation for the experimental observation that the altered Fe proteins form a trapped complex subsequent to a single electron transfer event. The structures reveal that the substitution allows the formation of a hydrogen bond between the switch I Asn39 and the switch II Asp125. In the structure of the native enzyme the analogous interaction between the side chains of Asp39 and Asp125 is precluded due to electrostatic repulsion. These results suggest that the electrostatic repulsion between Asp39 and Asp125 is important for dissociation of the Fe protein:MoFe protein complex during catalysis. In a separate study, the structural characterization of the Fe protein with Asp129 substituted by Glu provides the structural basis for the observation that the Glu129-substituted variant in the absence of bound nucleotides has biochemical properties in common with the native Fe protein with bound MgADP. Interactions of the longer Glu side chain with the phosphate binding loop (P-loop) results in a similar conformation of the switch II region as the conformation that results from the binding of the phosphate of ADP to the P-loop.