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Human pyridoxal 5'-phosphate phosphatase (PLPP), also known as a chronophin, is a phosphatase belonging to subfamily II of the HAD phosphatases, characterized by a large cap domain. As a member of the subfamily, its cap-open conformation is expected for substrate binding. We determined apo and PLP-bound PLPP/chronophin structures showing a cap-closed conformation. The active site, in which a PLP molecule was found, is too small to accommodate a phospho-cofilin peptide, the substrate of chronophin. A conformational change to a cap-open conformation may be required for substrate binding. The core and cap domains are joined through linker peptide hinges that change conformation to open the active site. The crystal structures reveal that a disulphide bond between the cap and core domains restricts the hinge motion. The enzyme displays PLP dephosphorylation activity in the cap-closed conformation with the disulphide bond and even in the crystal state, in which repositioning of the cap and core domains is restricted. Structural analysis suggests that a small substrate such as PLP can bind to the active site through a small movement of a local motif. However, a change to the cap-open conformation is required for binding of larger substrates such as phosphopeptides to the active site.

Levels of O-GlcNAc transferase (OGT) and hyper-O-GlcNAcylation expression levels are associated with cancer pathogenesis. This study aimed to find conditions that maximize the therapeutic effect of cancer and minimize tissue damage by combining an OGT inhibitor (OSMI-1) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We found that OSMI-1 treatment in HCT116 human colon cancer cells has a potent synergistic effect on TRAIL-induced apoptosis signaling. Interestingly, OSMI-1 significantly increased TRAIL-mediated apoptosis by increasing the expression of the cell surface receptor DR5. ROS-induced endoplasmic reticulum (ER) stress by OSMI-1 not only upregulated CHOP-DR5 signaling but also activated Jun-N-terminal kinase (JNK), resulting in a decrease in Bcl2 and the release of cytochrome c from mitochondria. TRAIL induced the activation of NF-κB and played a role in resistance as an antiapoptotic factor. During this process, O-GlcNAcylation of IκB kinase (IKK) and IκBα degradation occurred, followed by translocation of p65 into the nucleus. However, combination treatment with OSMI-1 counteracted the effect of TRAIL-mediated NF-κB signaling, resulting in a more synergistic effect on apoptosis. Therefore, the combined treatment of OSMI-1 and TRAIL synergistically increased TRAIL-induced apoptosis through caspase-8 activation. Conclusively, OSMI-1 potentially sensitizes TRAIL-induced cell death in HCT116 cells through the blockade of NF-κB signaling and activation of apoptosis through ER stress response.

The precise mechanism of hepatic cirrhosis remains largely unclear. In particular, a potential regulatory mechanism by which protein kinase C-delta (PKC&delta, ) affects profibrogenic gene expression involved in hepatic cirrhosis has never been explored. In the present study, we investigated whether PKC&delta, activation is involved in liver inflammatory fibrosis in both lipopolysaccharide (LPS)-treated RAW 264.7 and CCl4-treated mice. PKC&delta, was strongly activated by LPS or CCl4 treatment and consequently stimulated nuclear factor (NF)-&kappa, B inflammatory response. Interestingly, the activation of PKC&delta, negatively regulated sirtuin-1 (SIRT1) expression, whereas PKC&delta, suppression by PKC&delta, peptide inhibitor V1-1 or siRNA dramatically increased SIRT1 expression. Furthermore, we showed that the negative regulation of PKC&delta, leads to a decrease in SIRT1 expression. To our knowledge, these results are the first demonstration of the involvement of PKC&delta, in modulating NF-&kappa, B through SIRT1 signaling in fibrosis in mice, suggesting a novel role of PKC&delta, in inflammatory fibrosis. The level of NF-&kappa, B p65 in the nucleus was also negatively regulated by SIRT1 activity. We showed that the inhibition of PKC&delta, promoted SIRT1 expression and decreased p65 levels in the nucleus through deacetylation. Moreover, the inactivation of PKC&delta, with V1-1 dramatically suppressed the inflammatory fibrosis, indicating that PKC&delta, represents a promising target for treating fibrotic diseases like hepatic cirrhosis.

Chronic alcohol consumption causes hepatic steatosis, which is characterized by a considerable increase in free fatty acid (FFA) and triglyceride levels. To identify the possible proteins involved in the progression to alcoholic hepatosteatosis, we performed proteomic analysis on livers of mice exposed to alcohol. 2D-based proteomic analysis revealed that EtOH exposure in mice changed the expression of 43 proteins compared with that in mice fed a normal diet (ND). The most notable protein changes were proteins involved in Met metabolism and oxidative stress, most of which were significantly downregulated in alcohol-exposed animals. Although non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD) seem to share the same molecular processes, the difference between these conditions is still unclear. To address this question, we explored the features of alcoholic hepatosteatosis that were different compared with those of methionine and choline deficient (MCD) diet-induced mice with nonalcoholic liver damage. Although most of the differentially expressed proteins associated with ALD did not significantly differ from those of NAFLD, nine proteins showed considerably different patterns. Of these, ornithine aminotransferase, vitamin D binding protein, and phosphatidylethanolamine-binding protein were considerably upregulated in ALD mice, compared to that in NAFLD and ND mice. However, other proteins including inorganic pyrophosphatase were differentially regulated in MCD mice; however, they did not differ significantly between the alcoholic model and ND control mice. These results suggested that the identified proteins might be useful candidate markers to differentiate ALD from NAFLD. J. Cell. Biochem. 118: 1189-1200, 2017. © 2016 Wiley Periodicals, Inc.

Alternative splicing is a major mechanism regulating pattern of gene expression through the production of multiple mRNAs from a single gene transcript. Any misregulation can cause various human diseases and also have severe effects on embryogenesis. SRSF1 is one of the critical factors regulating alternative splicing at many stages of vertebrate development and any disturbance in SRSF1 leads to serious consequences. In current study, we investigated the effects of loss of the SRSF1 gene using antisense morpholino oligonucleotides (MO) in Xenopus embryogenesis. It is evident from the results of RT-PCR and whole-mount in situ hybridization that SRSF1 is a maternal gene having strong expression in head, eyes and central nervous system. Moreover, SRSF1 morphants exhibited malformed phenotypes, including miscoiled guts, heart and cartilage formation, edema in the head and heart, and small eyes. Especially, in SRSF1 morphants, bone cartilage formation was reduced in the brain and Nkx-2.5 expression was dramatically reduced in the heart of SRSF1 morphants. In addition, a dramatic reduction in functional chordin RNA in SRSF1 morphants was observed suggesting that chordin is one of the targets of SRSF1. Thus, we concluded that SRSF1 is an essential factor for pattern formation including heart, cartilage and germ layers through the regulation of specific genes.

Peroxiredoxin5 (Prdx5), a thioredoxin peroxidase, is an antioxidant enzyme that is widely studied for its antioxidant properties and protective roles in neurological and cardiovascular disorders. This study is aimed at investigating the functional significance of Prdx5 in mitochondria and at analyzing its roles in ciliogenesis during the process of vertebrate development.We found that several Prdx genes were strongly expressed in multiciliated cells in developing Xenopus embryos, and their peroxidatic functions were crucial for normal cilia development. Depletion of Prdx5 increased levels of cellular reactive oxygen species (ROS), consequently leading to mitochondrial dysfunction and abnormal cilia formation. Proteomic and transcriptomic approaches revealed that excessive ROS accumulation on Prdx5 depletion subsequently reduced the expression level of pyruvate kinase (PK), a key metabolic enzyme in energy production. We further confirmed that the promotor activity of PK was significantly reduced on Prdx5 depletion and that the reduction in PK expression and its promoter activity led to ciliary defects observed in Prdx5-depleted cells.Our data revealed the novel relationship between ROS and Prdx5 and the consequent effects of this interaction on vertebrate ciliogenesis. The normal process of ciliogenesis is interrupted by the Prdx5 depletion, resulting in excessive ROS levels and suggesting cilia as vulnerable targets of ROS.Prdx5 plays protective roles in mitochondria and is critical for normal cilia development by regulating the levels of ROS. The loss of Prdx5 is associated with excessive production of ROS, resulting in mitochondrial dysfunction and aberrant ciliogenesis.

The mechanisms underlying the progression to non-alcoholic steatohepatitis (NASH) remain to be elucidated. In the present study, we aimed to identify the proteins involved in the pathogenesis of liver tissue inflammation and to investigate the effects of silibinin, a natural polyphenolic flavonoid, on steatohepatitis. We performed comparative proteomic analysis using methionine and choline-deficient (MCD) diet-induced NASH model mice. Eighteen proteins were identified from the two-dimensional proteomic analysis, which are not only differentially expressed, but also significantly improved, by silibinin treatment. Interestingly, seven of these proteins, including keratin cytoskeletal 8 and 18, peroxiredoxin-4, and protein disulfide isomerase, are known to undergo GlcNAcylation modification, most of which are related to structural and stress-related proteins in NASH model animals. Thus, we primarily focused on how the GlcNAc modification of these proteins is involved in the progression to NASH. Remarkably, silibinin treatment alleviates the severity of hepatic inflammation along with O-GlcNAcylation in steatohepatitis. In particular, the reduction of inflammation by silibinin is due to the inhibition of the O-GlcNAcylation-dependent NF-&kappa, B-signaling pathway. Therefore, silibinin is a promising therapeutic agent for hyper-O-GlcNAcylation as well as NASH.

Background Lysine-specific histone demethylase 5C (KDM5C) belongs to the jumonji family of demethylases and is specific for the di- and tri-demethylation of lysine 4 residues on histone 3 (H3K4 me2/3). KDM5C is expressed in the brain and skeletal muscles of humans and is associated with various biologically significant processes. KDM5C is known to be associated with X-linked mental retardation and is also involved in the development of cancer. However, the developmental significance of KDM5C has not been explored yet. In the present study, we investigated the physiological roles of KDM5C during Xenopus laevis embryonic development. Results Loss-of-function analysis using kdm5c antisense morpholino oligonucleotides indicated that kdm5c knockdown led to small-sized heads, reduced cartilage size, and malformed eyes (i.e., small-sized and deformed eyes). Molecular analyses of KDM5C functional roles using whole-mount in situ hybridization, β-galactosidase staining, and reverse transcription-polymerase chain reaction revealed that loss of kdm5c resulted in reduced expression levels of neural crest specifiers and genes involved in eye development. Furthermore, transcriptome analysis indicated the significance of KDM5C in morphogenesis and organogenesis. Conclusion Our findings indicated that KDM5C is associated with embryonic development and provided additional information regarding the complex and dynamic gene network that regulates neural crest formation and eye development. This study emphasizes the functional significance of KDM5C in Xenopus embryogenesis; however, further analysis is needed to explore the interactions of KDM5C with specific developmental genes. Electronic supplementary material The online version of this article (10.1186/s13072-018-0241-x) contains supplementary material, which is available to authorized users.

Methylglyoxal (MG), a metabolite of glucose, is the major precursor of protein glycation and induces apoptosis. MG is associated with neurodegeneration, including oxidative stress and impaired glucose metabolism, and is efficiently metabolized to S-D-lactoylglutathione by glyoxalase (GLO). Although GLO has been implicated as being crucial in various diseases including ischemia, its detailed functions remain unclear. Therefore, we investigated the protective effect of GLO (GLO1 and GLO2) in neuronal cells and an animal ischemia model using Tat-GLO proteins. Purified Tat-GLO protein efficiently transduced into HT-22 neuronal cells and protected cells against MG- and H2O2-induced cell death, DNA fragmentation, and activation of caspase-3 and mitogen-activated protein kinase. In addition, transduced Tat-GLO protein increased D-lactate in MG- and H2O2-treated cells whereas glycation end products (AGE) and MG levels were significantly reduced in the same cells. Gerbils treated with Tat-GLO proteins displayed delayed neuronal cell death in the CA1 region of the hippocampus compared with a control. Furthermore, the combined neuroprotective effects of Tat-GLO1 and Tat-GLO2 proteins against ischemic damage were significantly higher than those of each individual protein. Those results demonstrate that transduced Tat-GLO protein protects neuronal cells by inhibiting MG- and H2O2-mediated cytotoxicity in vitro and in vivo. Therefore, we suggest that Tat-GLO proteins could be useful as a therapeutic agent for various human diseases related to oxidative stress including brain diseases.

CTGF (connective tissue growth factor) is widely regarded as an important amplifier of the profibrogenic action of TGF-β (transforming growth factor β) in a variety of tissues, although the precise mechanism of how the TGF-β signalling pathways modulate CTGF expression remains unclear. In the present study, the role of PKCδ (protein kinase Cδ) in TGF-β1-mediated CTGF expression was investigated using HepG2 cells. TGF-β1 treatment specifically elevated PKCδ activation and CTGF expression. In contrast, blockade of PKCδ by the selective inhibitor Rottlerin or by siRNA knockdown significantly reduced TGF-β1-induced CTGF production. The regulatory mechanism was further demonstrated in HepG2 cells whereby TGF-β1-induced PKCδ activation negatively regulated the nuclear levels of PPM1A (protein phosphatase, Mg2+/Mn2+ dependent, 1A) through the RhoA/ROCK (Rho-associated kinase) pathway. Moreover, we showed that both Smad signalling and the PKCδ pathway appeared to be stimulated by TGF-β1 in parallel. Time course assessments indicated that PKCδ signalling may have a function in maintaining nuclear phospho-Smads at a maximal level. The collective results of the present study demonstrated that PKCδ-stimulated RhoA/ROCK activation resulted in a reduction in PPM1A, thereby up-regulating Smad-dependent gene induction for extended periods. These findings indicated that PKCδ plays a critical role in TGF-β1-induced CTGF production in HepG2 cells.

Although doxorubicin (Dox)-induced oxidative stress is known to be associated with cytotoxicity, the precise mechanism remains unclear. Genotoxic stress not only generates free radicals, but also affects actin cytoskeleton stability. We showed that Dox-induced RhoA signaling stimulated actin cytoskeleton alterations, resulting in central stress fiber disruption at early time points and cell periphery cortical actin formation at a later stage, in HeLa cells. Interestingly, activation of a cofilin phosphatase, chronophin (CIN), was initially evoked by Dox-induced RhoA signaling, resulting in a rapid phosphorylated cofilin turnover leading to actin cytoskeleton remodeling. In addition, a novel interaction between CIN and 14-3-3ζ was detected in the absence of Dox treatment. We demonstrated that CIN activity is quite contrary to 14-3-3ζ binding, and the interaction leads to enhanced phosphorylated cofilin levels. Therefore, initial CIN activation regulation could be critical in Dox-induced actin cytoskeleton remodeling through RhoA/cofilin signaling. [BMB Reports 2017; 50(6): 335-340].

FK506 binding protein 12 (FK506BP) belongs to a family of immunophilins, and is involved in multiple biological processes. However, the function of FK506BP in corneal disease remains unclear. In this study, we examined the protective effects on dry eye disease in a Botulinum toxin A (BTX-A) induced mouse model, using a cell-permeable PEP-1-FK506BP protein. PEP-1-FK506BP efficiently transduced into human corneal epithelial cells in a time- and dose-dependent manner, and remained stable in the cells for 48 h. In addition, we demonstrated that topical application of PEP-1-FK506BP was transduced into mouse cornea and conjunctiva by immunohistochemistry. Furthermore, topical application of PEP-1-FK506BP to BTX-A-induced mouse model markedly inhibited expression levels of pro-inflammatory cytokines such as interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha) and macrophage inhibitory factor (MIF) in corneal and conjunctival epithelium. These results suggest PEP-1-FK506BP as a potential therapeutic agent for dry eye diseases. [BMB Reports 2013; 46(2): 124-129]

Heat shock proteins (HSPs) are a highly conserved family of proteins that are induced in response to various environmental stressors including reactive oxygen species. HSP27 is a chaperone protein with the ability to increase cell survival in response to oxidative stress. Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons. Although the mechanism of PD remains unclear, oxidative stress is known to be important in its pathogenesis. This study investigated the protective effects of PEP-1-HSP27 on neuronal damage induced by 1-methyl-4-phenyl pyridinium (MPP(+) ) in SH-SY5Y cells and in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. PEP-1-HSP27 rapidly entered the cells and protected them against MPP(+) -induced toxicity by inhibiting the reactive oxygen species levels and DNA fragmentation. Furthermore, transduced PEP-1-HSP27 prevented dopaminergic neuronal cell death in the substantia nigra of MPTP-induced PD mouse models. These results demonstrate that PEP-1-HSP27 provides a potential strategy for therapeutic delivery against various diseases and is a potential tool for the treatment of PD.

This study investigated the preventive effect of ribosomal protein S3 (rpS3) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema in mice. A cell permeable expression vector PEP-1-rpS3 was constructed. Topical application of the vector markedly inhibited TPA-induced expression levels of cyclooxygenase-2 (COX-2) and pro-inflammatory cytokines. Application of PEP-1-rpS3 also resulted in a significant reduction in the activation of nuclear factor-kappa B (NF-kB) and mitogen-activated protein kinase (MAPK) in TPA-treated ears. These results indicate that PEP-1-rpS3 inhibits inflammatory response cytokines and enzymes by blocking NF-kB and MAPK, prompting the suggestion that PEP-1-rpS3 can be used as a therapeutic agent against skin inflammation.

Antioxidant enzymes are considered to have beneficial effects against various diseases mediated by reactive oxygen species (ROS). Ischemia is characterized by both oxidative stress and changes in the antioxidant defense system. Catalase (CAT) and superoxide dismutase (SOD) are major antioxidant enzymes by which cells counteract the deleterious effects of ROS. To investigate the protective effects of CAT, we constructed PEP-1-CAT cell-permeative expression vectors. When PEP-1-CAT fusion proteins were added to the culture medium of neuronal cells, they rapidly entered the cells and protected them against oxidative stress-induced neuronal cell death. Immunohistochemical analysis revealed that PEP-1-CAT prevented neuronal cell death in the hippocampus induced by transient forebrain ischemia. Moreover, we showed that the protective effect of PEP-1-CAT was observed in neuronal cells treated with PEP-1-SOD. Therefore, we suggest that transduced PEP-1-CAT and PEP-1-SOD fusion proteins could be useful as therapeutic agents for various human diseases related to oxidative stress, including stroke.

Febrile seizure (FS) is the most common type of seizure that occurs during early childhood. It has been proposed that atypical FS (prolonged, multiple, or lateralized) results in the development of recurrent complex partial seizures accompanied by Ammon's horn sclerosis or mesial temporal sclerosis, which is the most common of the intractable epilepsy. To elucidate the characteristics of epileptogenesis or acquired epilepsy following FS, we performed prospective long-term studies using hyperthermia-induced seizure model. Rat pups (postnatal 11 day old) were induced to hyperthermia (41-43 degrees C in core temperature) by exposure to a 175 W mercury vapor lamp. Six-nine weeks after hyperthermic seizure, the dentate gyrus showed impairments of paired-pulse inhibitions and excitability ratio. In addition, newly generated granule cells and synaptogenesis were observed in this region. Ten-twelve weeks after hyperthermic seizure, animals (approximately 68%) showed electroencephalographic seizure activity with increased VGLUT-1 immunoreactivity in the dentate gyrus. Parvalbumin immunoreactivity was markedly reduced in the hilus. These findings indicate that in this model the epileptogenic changes in the dentate gyrus may be based on the persistent alterations in excitability via neurogenesis, synaptogenesis, and impaired GABA(B) receptor-mediated inhibition.

Reactive oxygen species contribute to the development of various human diseases. Ischemia is characterized by both significant oxidative stress and characteristic changes in the antioxidant defense mechanism. Heat shock protein 27 (HSP27) has a potent ability to increase cell survival in response to oxidative stress. In the present study, we have investigated the protective effects of PEP-1–HSP27 against cell death and ischemic insults. When PEP-1–HSP27 fusion protein was added to the culture medium of astrocyte and primary neuronal cells, it rapidly entered the cells and protected them against cell death induced by oxidative stress. Immunohistochemical analysis revealed that, when PEP-1–HSP27 fusion protein was intraperitoneally injected into gerbils, it prevented neuronal cell death in the CA1 region of the hippocampus in response to transient forebrain ischemia. Our results demonstrate that transduced PEP-1–HSP27 protects against cell death in vitro and in vivo, and suggest that transduction of PEP-1–HSP27 fusion protein provides a potential strategy for therapeutic delivery in various human diseases in which reactive oxygen species are implicated, including stroke.

Reactive oxygen species generated under oxidative stress are involved in neuronal diseases, including ischemia. Glutathione S-transferase pi (GSTpi) is a member of the GST family and is known to play important roles in cell survival. We investigated the effect of GSTpi against oxidative stress-induced hippocampal HT-22 cell death, and its effects in an animal model of ischemic injury, using a cell-permeable PEP-1-GSTpi protein. PEP-1-GSTpi was transduced into HT-22 cells and significantly protected against H2O2-treated cell death by reducing the intracellular toxicity and regulating the signal pathways, including MAPK, Akt, Bax, and Bcl-2. PEP-1-GSTpi transduced into the hippocampus in animal brains, and markedly protected against neuronal cell death in an ischemic injury animal model. These results indicate that PEP-1-GSTpi acts as a regulator or an antioxidant to protect against oxidative stress-induced cell death. Our study suggests that PEP-1-GSTpi may have potential as a therapeutic agent for the treatment of ischemia and a variety of oxidative stress-related neuronal diseases. [BMB Reports 2016; 49(7): 382-387]

The epileptic hippocampus shows differential paired-pulse responses between the dentate gyrus and the CA1 region. However, little data are available to explain this phenomenon. In the present study, we identified the relationship between regional differences of paired-pulse response and voltage gated Cl − channel 2 (CLC-2)/vesicular GABA transport (VGAT) expression in a pilocarpine-induced rat model. During epileptogenic periods, paired-pulse inhibitions in the dentate gyrus and the CA1 region were markedly reduced. After recurrent seizure onset, paired-pulse inhibition in the dentate gyrus was markedly enhanced, while that in the CA1 region more reduced. Unlike VGAT, CLC-2 immunoreactivity was markedly reduced in the hippocampus during epileptogenic periods and was re-enhanced only in the dentate gyrus after recurrent seizure onset. Linear regression analysis showed an inverse proportional relationship between alterations in CLC-2 immunoreactivity and changes in normalized population spike amplitude ratio within the CA1 region and the dentate gyrus. Therefore, our findings suggest that the regionally specific alterations in CLC-2 immunoreactivity after SE may determine the properties of paired-pulse responses in the hippocampus of the pilocarpine-induced rat epilepsy model.

Parkinson's disease (PD) is a common neurodegenerative disorder and is characterized by the progressive loss of dopaminergic neurons in the substantia nigra. Although many studies showed that the aggregation of alpha-synuclein might be involved in the pathogenesis of PD, its protective properties against oxidative stress remain to be elucidated. In this study, human wild type and mutant alpha-synuclein genes were fused with a gene fragment encoding the nine amino acid transactivator of transcription (Tat) protein transduction domain of HIV-1 in a bacterial expression vector to produce a genetic in-frame WT Tat-alpha-synuclein (wild type) and mutant Tat-alpha-synucleins (mutants; A30P and A53T), respectively, and we investigated the protective effects of wild type and mutant Tat-alpha-synucleins in vitro and in vivo. WT Tat-alpha-synuclein rapidly transduced into an astrocyte cells and protected the cells against paraquat induced cell death. However, mutant Tat-alpha-synucleins did not protect at all. In the mice models exposed to the herbicide paraquat, the WT Tat-alpha-synuclein completely protected against dopaminergic neuronal cell death, whereas mutants failed in protecting against oxidative stress. We found that these protective effects were characterized by increasing the expression level of heat shock protein 70 (HSP70) in the neuronal cells and this expression level was dependent on the concentration of transduced WT Tat-alpha-synuclein. These results suggest that transduced Tat-alpha-synuclein might protect cell death from oxidative stress by increasing the expression level of HSP70 in vitro and in vivo and this may be of potential therapeutic benefit in the pathogenesis of PD.

Native low-density lipoprotein (LDL) and oxidized LDL (oxLDL) possess a wide variety of biological properties, and play a central role in atherogenesis. In this study, we used a proteomic analysis of human monocyte THP-1 cells induced with oxLDL or with LDL, to identify proteins potentially involved in atherosclerotic processes. Of the 2500 proteins detected, 93 were differentially expressed as a result of priming with LDL or oxLDL. The proteins were unambiguously identified by comparing the masses of their tryptic peptides with those of all known proteins using MALDI-TOF MS and the NCBI database. The largest differences in expression were observed for vimentin (94-fold increase), meningioma-expressed antigen 6 (48-fold increase), serine/threonine protein phosphatase 2A (40-fold increase), and beta-1,3-galactosyltransferase (15-fold increase). In contrast, the abundance of an unnamed protein product and phosphogluconate dehydrogenase decreased 30-fold and 25-fold, respectively. The expression of some selected proteins was confirmed by Western blot and RT-PCR analyses. The proteins identified in this study are attractive candidates for further biomarker research. This description of the altered protein profiles induced by oxLDL in human monocytes will support functional studies of the macrophage-derived foam cells involved in the pathogenesis of atherosclerosis.

Pyridoxine-5-P oxidase catalyses the terminal step in the biosynthesis of pyridoxal-5-P, the biologically active form of vitamin B6 which acts as an essential cofactor. Here, a human brain pyridoxine-5-P oxidase gene was fused with a gene fragment encoding the HIV-1 Tat protein transduction domain (RKKRRQRRR) in a bacterial expression vector to produce a genetic in-frame Tat-pyridoxine-5-P oxidase fusion protein. Expressed and purified Tat-pyridoxine-5-P oxidase fusion protein transduced efficiently into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced Tat-pyridoxine-5-P oxidase protein showed catalytic activity and was stable for 48 h. Moreover, the formation of pyridoxal-5-P was increased by adding exogenous Tat-pyridoxine-5-P oxidase to media pre-treated with the vitamin B6 precursor pyridoxine. In addition, the intracellular concentration of pyridoxal-5-P was markedly increased when Tat-pyridoxal kinase was transduced together with Tat-pyridoxine-5-P oxidase into cells.These results suggest that the transduction of Tat-pyridoxine-5-P oxidase fusion protein presents a means of regulating the level of pyridoxal-5-P and of replenishing this enzyme in various neurological disorders related to vitamin B6.

In the present study, age-related changes of pyridoxal 5′-phosphate (PLP) synthesizing enzymes, pyridoxal kinase (PLK) and pyridoxine 5′-phosphate oxidase (PNPO), their protein contents and activities were examined in the gerbil hippocampus proper. Significant age-dependent changes in PLK and PNPO immunoreactivities were found in the CA1 region, but not in the CA2/3 region. In the postnatal month 1 (PM 1) group, PLK and PNPO immunoreactivities were detected mainly in the stratum pyramidale of the CA1 region. PLK and PNPO immunoreactivities and their protein contents were highest in the PM 6 group, showing that many CA1 pyramidal cells had strong PLK and PNPO immunoreactivities. Thereafter, PLK and PNPO immunoreactivities started to decrease and were very low at PM 24. Alterations in the change patterns in protein contents and total activities of PLK and PNPO corresponded to the immunohistochemical data, but their specific activities were not altered in any experimental group. Based on double immunofluorescence study, PLK and PNPO immunoreactive cells in the strata oriens and radiatum were identified as GABAergic cells. Therefore, decreases of PLK and PNPO in the hippocampal CA1 region of aged brains may be involved in aging processes related with γ-aminobutyric acid (GABA) function.

The succinic semialdehyde dehydrogenase gene (SSADH; EC 1.2.1.24) from human brain was cloned and overexpressed in Escherichia coli . Based on SDS–PAGE, the apparent molecular mass of subunit was 54 kDa, in good agreement with the theoretical size. The purified SSADH appears to be a tetramer of identical subunits. The specific activity of the recombinant protein was 1.82 μmol NADH formed min −1 mg −1 and the optimal pH was found to be 8.5. The Michaelis constants K m for succinic semialdehyde and NAD + were 6.3 and 125 μM, respectively. Initial velocity studies show NADH to be a competitive inhibitor with respect to NAD + , but to be non-competitive inhibitor with respect to succinic semialdehyde. The overexpression of SSADH in E. coli and one-step purification of the highly active SSADH will facilitate further biochemical studies on this enzyme. In addition, an mRNA master dot-blot for multiple human tissues provided a complete map of the tissue distribution for SSADH. The major sites of SSADH expression are liver, skeletal muscle, kidney, and brain. The data indicate that mRNA expression of SSADH is ubiquitous, but highly regulated at the level of transcription in a tissue-specific manner.

Myo-inositol monophosphate phosphatase (IMPP) is a key enzyme in the phosphoinositide cell-signaling system. This study found that incubating the IMPP from a porcine brain with pyridoxal-5'-phosphate (PLP) resulted in a time-dependent enzymatic inactivation. Spectral evidence showed that the inactivation proceeds via the formation of a Schiff's base with the amino groups of the enzyme. After the sodium borohydride reduction of the inactivated enzyme, it was observed that 1.8 mol phosphopyridoxyl residues per mole of the enzyme dimer were incorporated. The substrate, myo-inositol-1-phosphate, protected the enzyme against inactivation by PLP. After tryptic digestion of the enzyme modified with PLP, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. Amino acid sequencing of the peptide identified a portion of the PLP-binding site as being the region containing the sequence L-Q-V-S-Q-Q-E-D-I-T-X, where X indicates that phenylthiohydantoin amino acid could not be assigned. However, the result of amino acid composition of the peptide indicated that the missing residue could be designated as a phosphopyridoxyl lysine. This suggests that the catalytic function of IMPP is modulated by the binding of PLP to a specific lysyl residue at or near its substrate-binding site of the protein.

Vitamin E and probucol are well-known antioxidants that prevent cells from the oxidative stress, which is a risk factor of atherosclerosis. Male rabbits were fed either 0.03% vitamin E or 0.05% probucol in a 0.5% high-cholesterol (HC) diet for 8 weeks. Vitamin E and probucol significantly suppressed an increase in plasma total-cholesterol (total-C) and low-density lipoprotein cholesterol compared to HC-control group. However, plasma high-density lipoprotein-cholesterol (HDL-C) and HDL-C/total-C ratio levels and plasma paraoxonase activity were only significantly higher in vitamin E group after 8 weeks. Hepatic ACAT activity was significantly lower in both vitamin E and probucol groups than in HC-control group, while HMG-CoA reductase activity was the highest only in the probucol group. Total fecal sterol content was significantly higher in probucol and vitamin E groups than in the two control groups. Some atherogenic signs were discovered in the aortic fatty streak of HC-control group, yet not in other groups. Hepatic mRNA expressions of apo B-100 and apo C-III were significantly lower in probucol group than in other groups. Vitamin E supplementation was found to alter the plasma HDL-C-related factors; meanwhile, probucol supplementation was very effective in enhancing cholesterol metabolism, except for a negative effect that reduced plasma HDL-C concentration.

Oligoribonucleotides containing 8-oxo-7,8-dihydroguanosine (8-oxoG) and 8-oxo-7,8-dihydro-2'-O-methylguanosine (8-oxoG-Me) were synthesized. The base pairing properties of 8-oxoG and 8-oxoG-Me in oligoribonucleotide in cDNA synthesis by reverse transcriptases were studied. dCMP was preferentially incorporated into the site opposite 8-oxoG or 8-oxoG-Me than into other dNMPs. TMP and dCMP were inserted preferentially into sites opposite 8-oxoG or 8-oxoG by reverse transcriptases. HIV-RT did not incorporate TMP, but RAV2-RT incorporated 50% more TMP than dCMP into the site opposite 8-oxoG. In the site opposite 8-oxoG-Me TMP was substantially incorporated by HIV-RT or RAV2-RT. Thermodynamic analysis of the DNA.RNA heteroduplex containing 8-oxoG revealed that 8-oxoG and 8-oxoG-Me formed base pairs with cytidine and thymidine with similar stability. The thermodynamic parameter (DeltaG degrees ) demonstrated that the formation of duplexes between 8-oxoG or 8-oxoG-Me and cytidine or thymidine is more thermodynamically favorable than with adenosine and guanosine. However, differences in the melting temperature and DeltaG degrees 's of 8-oxoG/dC and 8-oxoG/T were much smaller than between G/dC and G/T. CD spectra showed that DNA . RNA containing 8-oxoG or 8-oxoG-Me duplexes showed similarities between the A-type RNA and B-type DNA conformations.

We used a combined computer and biochemical approach to characterize human pyridoxine 5'-phosphate oxidase (PNPO). The human PNPO gene is composed of seven exons and six introns, and spans approximately 8 kb. All exon/intron junctions contain the gt/ag consensus splicing site. The absence of TATA-like sequences, the presence of Sp1-binding sites and more importantly, the presence of CpG islands in the regulatory region of the PNPO gene are characteristic features of housekeeping genes. Northern blot analyses showed two species of poly(A)(+) RNA of approximately 2.4 and approximately 3.4 kb at identical intensity, whereas Western blot analysis showed that no protein isoform exists in any of the tissues examined. PCR-based analysis led to the idea that two messages are transcribed from a single copy gene, and that the size difference is due to differential usage of the polyadenylation signal. The major sites of PNPO expression are liver, skeletal muscle and kidneys while a very weak signal was detected in lung. The mRNA master dot-blot for multiple human tissues provided a complete map of the tissue distribution not only for PNPO but also for pyridoxal kinase and pyridoxal phosphatase. The data indicate that mRNA expression of all three enzymes essential for vitamin B(6) metabolism is ubiquitous but is highly regulated at the level of transcription in a tissue-specific manner. In addition, human brain PNPO cDNA was expressed in Escherichia coli, and the roles of both the N- and C-terminal regions were studied by creating sequential truncation mutants. Our results showed that deletion of the N-terminal 56 residues affects neither the binding of coenzyme nor catalytic activity.

In the present study, we investigated site-specific expressions of GABA(A) and GABA(B) receptor subunits in the seizure-sensitive (SS) and seizure-resistant (SR) gerbil hippocampus to elucidate the function of the gamma-aminobutyric acid (GABA) receptor in seizure activity in this animal. There were no differences of the immunoreactivities of GABA(B) receptor and some GABA(A) receptor subunits (alpha3, alpha4, pan beta and delta) in the hippocampus between SR and SS gerbils. The alpha1 subunit expression was mainly detected in interneurons of stratum radiatum and hilar region of dentate gyrus in the SR gerbil. However, in SS gerbil, interneurons were nearly devoid of alpha1 subunit immunoreactivity and mainly detected in the molecular layer of dentate gyrus. In SR gerbil, alpha2 subunit immunoreactivity was detected in Ammon's horn, particularly in the CA2 region. In SS gerbil, granule cell layer of the dentate gyrus in SS gerbil showed strong alpha2 subunit immunoreactivity. The distribution of alpha5 and gamma2 subunit immunoreactivity in the hippocampus was similarly detected in SR and SS gerbil. However, alpha5 immunodensity of SR gerbil was slightly lower than that of SS gerbil in CA1 region and was slightly strong than that of SS gerbil in subiculum. These differences in distribution of GABA(A) receptor, not GABA(B) receptor, in the SR and SS gerbil hippocampus may indicate that abnormal hyperactive neuronal discharges are occurred in SS gerbil, which presumably result in spontaneous and repetitive seizure activity in this animal.

Pyridoxal phosphatase catalyzes the dephosphorylation of pyridoxal 5′-phosphate (PLP) and pyridoxine 5′-phosphate. A human brain cDNA clone was identified to the PLP phosphatase on the basis of peptide sequences obtained previously. The cDNA predicts a 296-amino acid protein with a calculated Mr of 31698. The open reading frame is encoded by two exons located on human chromosome 22q12.3, and the exon-intron junction contains the GT/AG consensus splice site. In addition, a full-length mouse PLP phosphatase cDNA of 1978 bp was also isolated. Mouse enzyme encodes a protein of 292 amino acids with Mr of 31512, and it is localized on chromosome 15.E1. Human and mouse PLP phosphatase share 93% identity in protein sequence. A BLAST search revealed the existence of putative proteins in organism ranging from bacteria to mammals. Catalytically active human PLP phosphatase was expressed in Escherichia coli, and characteristics of the recombinant enzyme were similar to those of erythrocyte enzyme. The recombinant enzyme displayed Km and kcat values for pyridoxal of 2.5 μm and 1.52 s–1, respectively. Human PLP phosphatase mRNA is differentially expressed in a tissue-specific manner. A single mRNA transcript of 2.1 kb was detected in all human tissues examined and was highly abundant in the brain. Obtaining the molecular properties for the human PLP phosphatase may provide new direction for investigating metabolic pathway involving vitamin B6.

We investigated the distribution of somatostatin receptors (SSTs) in the hippocampi of SR (seizure-resistant) and SS (seizure-sensitive) gerbils in order to characterize the alterations in SST expressions induced by seizure activity. SST2A immunodensity in the hippocampus of SS gerbils was lower than that of SR gerbils, though its localization in the hippocampus was similar in both SR and SS gerbils. SST3 immunodensity in the hippocampus of SS gerbils was lower than in SR gerbils. In SR gerbils, strong SST4 immunoreactivity was detected in the dentate gyrus and in the CA3 region, in contrast little immunoreactivity was detected in these regions in SS gerbils. In SR and SS gerbils, the strong SST5 immunoreactivity in the hippocampus was also detected in the stratum oriens of the CA2-3 regions and the septal area of CA1 region. However, SST5 immunodensity in the stratum radiatum in SS gerbils was lower than in SR gerbils. These results are the first comprehensive description of the distribution of SSTs in the normal and epileptic hippocampus of gerbils, and suggest that these alterations in the hippocampus of the SS gerbil may be related with a regulatory mechanism for seizure activity in these seizure prone animals.

In the present study, the distribution of glutamic acid decarboxylase (GAD) isoforms in the hippocampus of the Mongolian gerbil and its association with different sequelae of spontaneous seizure were investigated to identify the roles of balance of GAD isoforms in the epileptogenesis and the recovery mechanisms in these animals. The GAD67/GAD65 ratio in the hippocampus of pre-seizure seizure sensitive (SS) gerbil was approximately 3.5-fold higher as compared to seizure resistant (SR) gerbil. Following seizure, this ratio shifted to the level of SR gerbils up to 12 h postical. Therefore, the mismatched GAD67/GAD65 ratio (imbalance of GAD isoform expressions) in the hippocampus of SS gerbil implies that GABAergic neurons may be highly activated in order to regulate the increased neuronal excitability. In addition, the alteration in this ratio after seizure may be the compensatory response for reduction of epileptic activity in this animal.

To elucidate the role of endogenous transforming growth factor (TGF)-β2 on human osteoblast cell, antisense phosphorothioate oligonucleotides (S-ODNs) complementary to regions in mRNA of TGF-β2 were synthesized and examined their effects on TGF-β2 production and cell proliferation in a human osteoblast cell line ROS 17/2. Antisense S-ODNs were designated for three different target regions in the mRNA of TGF-β2. Among several antisense S-ODN analyzed, an oligonucleotide (AS-11) complementary to the translation initiation site of mRNA of TGF-β2 demonstrated a selective and strong inhibitory effect on TGF-β2 production in osteoblast cells. Other antisense S-ODNs which were designated for other regions in mRNA of TGF-β2 and one- or three-base mismatched analogs of AS-11 showed little or much less antisense activities than AS-11. Therefore, the most effective target site in mRNA of TGF-β2 is at the initiation codon region. The antisense effects of AS-11 were observed without reduction of levels of mRNA of TGF-β2. Furthermore, the inhibition of TGF-β2 expression by antisense S-ODN appeared to enhance cell proliferation, demonstrating the growth inhibitory effect of autocrine TGF-β2 in osteoblast cells.

The pathogenesis of non-alcoholic steatohepatitis (NASH) is not fully understood. In the present study, both in vitro and in vivo vimentin expression and secretion in NASH were investigated. The exposure of palmitate and lipopolysaccharide (LPS) to HepG2 cells enhanced caspase-3 activity and vimentin expression, respectively. The combined effects of both treatments on vimentin expression and caspase-3 activation appeared to be synergic. In contrast, blockade of caspase-3 activity by zVADfmk resulted in a significant reduction of cleaved vimentin and secreted vimentin into the culture supernatant. Similarly, lipid accumulation and inflammation occurred in mice fed a methionine-choline-deficient diet; thus, vimentin expression and serum cleaved vimentin levels were increased. However, vimentin was not significantly upregulated, and no cleavage occurred in mice fed a high-fat diet. It was conclusively determined that lipid accumulation in hepatocytes induces apoptosis through a caspase-3 dependent pathway; whereas, LPS stimulates vimentin expression, leading to its cleavage and secretion. Increased vimentin fragment levels indicated the existence of substantial hepatocellular death via an apoptotic mechanism. [BMB Reports 2014; 47(8): 457-462]

Parkinson’s disease (PD) is a well known neurodegenerative disorder characterized by selective loss of dopaminergic neurons in the substantia nigra pars compact (SN). Although the exact mechanism remains unclear, oxidative stress plays a critical role in the pathogenesis of PD. DJ-1 is a multifunctional protein, a potent antioxidant and chaperone, the loss of function of which is linked to the autosomal recessive early onset of PD. Therefore, we investigated the protective effects of DJ-1 protein against SH-SY5Y cells and in a PD mouse model using a cell permeable Tat-DJ-1 protein. Tat-DJ-1 protein rapidly transduced into the cells and showed a protective effect on 6-hydroxydopamine (6-OHDA)-induced neuronal cell death by reducing the reactive oxygen species (ROS). In addition, we found that Tat-DJ-1 protein protects against dopaminergic neuronal cell death in 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP)-induced PD mouse models. These results suggest that Tat-DJ-1 protein provides a potential therapeutic strategy for against ROS related human diseases including PD.

Fluorescence techniques have been used to investigate the interaction of bovine 70-kDa heat shock cognate protein (Hsc 70) with small molecular weight peptides and myo-inositol monophosphatase. The emission properties of Hsc 70 remain invariant upon addition of ATP. The results of steady-state fluorescence indicate that the tryptophan residues of Hsc 70 are exposed to the rapidly relaxing aqueous solvent. Binding of residues 1-20 of ribonuclease A (RNase S-peptide) to Hsc 70 causes protein fluorescence quenching which was used to determine a dissociation constant Kd = 2.7 microM for the binary Hsc 70.RNase S-peptide complex. The octapeptide corresponding to the NH2-terminal portion of sickle cell hemoglobin recognizes Hsc 70 and binds with a Kd = 9.3 microM. Binding of RNase S-peptide to Hsc 70 produces a small enhancement of ATPase activity. Unfolded myo-inositol monophosphatase, tagged with the fluorescent probe 5-[2-(2-iodoacetamido)ethylamino]-1-naphthalenesulfonic acid, recognizes Hsc 70; the formation of a stable complex was detected by steady-state emission anisotropy measurements. The rate and extent of recovery of catalytic activity of unfolded myo-inositol monophosphatase is not influenced by Hsc 70. It is suggested that interaction of Hsc 70 with unfolded proteins in the cell may be able to delay the formation of misfolded structures.

Annexin-1 (ANX1) is an anti-inflammatory protein as well as an important modulator in inflammation. However, the precise action of ANX1 remains unclear. To elucidate the protective effects of ANX1 on lipopolysaccharide (LPS)-induced murine macrophage Raw 264.7 cells, we constructed a cell-permeable Tat-ANX1 protein. The transduced Tat-ANX1 protein markedly inhibited the expression of cyclooxygenase-2, production of prostaglandin E(2), and generation of pro-inflammatory cytokines in the cells. Furthermore, transduced Tat-ANX1 protein caused a significant reduction in the activation of nuclear factor- kappa B (NF-kB) and mitogen-activated protein kinase (MAPK). The results indicate that Tat-ANX1 inhibits the production of inflammatory response cytokines and enzymes by blocking NF-kB and MAPK. Therefore, Tat-ANX1 protein may be useful as a therapeutic agent against various inflammatory diseases.

During germination and early growth of the seedling, storage proteins are degraded by proteases. Currently, limited information is available on the degradation of storage proteins in the soybean during germination. In this study, a combined two-dimensional gel electrophoresis and mass spectrometry approach was utilized to determine the proteome profile of soybean seeds (Glycine max L.; Eunhakong). Comparative analysis showed that the temporal profiles of protein expression are dramatically changed during the seed germination and seedling growth. More than 80% of the proteins identified were subunits of glycinin and β-conglycinin, two major storage proteins. Most subunits of these proteins were degraded almost completely at a different rate by 120h, and the degradation products were accumulated or degraded further. Interestingly, the acidic subunits of glycinin were rapidly degraded, but no obvious change in the basic chains. Of the five acidic subunits, the degradation of G2 subunit was not apparently affected by at least 96h but the levels decreased rapidly after that, while no newly appearing intermediate was detected upon the degradation of G4 subunit. On the other hand, the degradation of β-conglycinin during storage protein mobilization appeared to be similar to that of glycinin but at a faster rate. Both α and α' subunits of β-conglycinin largely disappeared by 96h, while the β subunits degraded at the slowest rate. These results suggest that mobilization of subunits of the storage proteins is differentially regulated for seed germination and seedling growth. The present proteomic analysis will facilitate future studies addressing the complex biochemical events taking place during soybean seed germination.

myo-Inositol monophosphatase isolated from pig brain is a very stable dimeric protein characterized by a rotational correlation time of 30 ns. The unfolding and dissociation of the dimeric enzyme (58 kDa) by guanidine hydrochloride have been investigated at equilibrium. The overall process was reversible as judged from the complete recovery of catalytic activity after dilution of guanidine hydrochloride-treated samples. Unfolding of myo-inositol monophosphatase was monitored by circular dichroism, fluorescence, and steady state emission anisotropy. A folded, monomeric form of the monophosphatase was not detected by the method of denaturant gel filtration. Uncoupled dissociation and unfolding of the oligomeric enzyme could not be demonstrated. The circular dichroism and emission anisotropy results are consistent with a model in which the dimeric protein unfolds in a single cooperative transition from folded dimer to two unfolded monomers.

Obesity causes changes in fatty acid metabolism that consequently leads to fatty liver. To identify the possible proteins involved in the processes of obesity, we performed a proteomic analysis of obesity-induced mouse liver. Male C57BL/6J mice that were fed a high-fat diet (HFD) for 24wk, developed hepatic steatosis characterized by considerable increase in free fatty acid (FFA) and triglyceride levels. Body weights were measured weekly and other measurements at weeks 2, 6, 12, 16, and 24. 2-D-based proteomic analysis revealed that, compared with the normal diet (ND) (n 550), high-fat diet (n 550) changed the expression of 12 protein (8 up and 4 downregulated, by a 1.5 fold change and more, po0.05). The most pronounced difference was observed in intermediate microfilament (IF) cytoskeleton proteins. In particular, vimentin (vim) as well as cytokeratins (CK-8 and CK-18) were significantly upregulated in obese animals. Moreover, the level of caspase-generated IF fragment was also positively correlated with the degree of steatosis. The results suggest a significant alteration in IF organization during the development of hepatic steatosis leading to inflammation. The expression profile of selected proteins including vim was validated by Western blot, microarray analysis, and hepatocyte morphology by immunohistochemistry. Our results suggest that vim, like CK-18, may be a useful marker for predicting obesity and liver disease.

To confirm the roles of D-serinergic gliotransmission in epilepsy, we investigated the relationship between spatiotemporally specific glial responses and the D-serine/serine racemase system in mesial temporal structures following status epilepticus (SE). In control animals, D-serine and serine racemase immunoreactivities were detected mainly in astrocytes. After SE, D-serine and serine racemase immunoreactivities were increased in astrocytes. Double-immunofluorescence study revealed that up-regulation of serine racemase immunoreactivity was relevant not to D-serine immunoreactivity but to nestin or vimentin immunoreactivity. Neither D-serine nor serine racemase was found in naive or reactive microglia. In addition, phosphorylated N-methyl-D-aspartate (NMDA) receptor subunit 1 (pNR1-Ser896) immunoreactivity in the hippocampus was increased compared with controls. Increased D-serine immunoreactivity showed direct correlation with the phosphorylation of Ser896 of NR1. Given the findings of our previous study, these findings suggest that D-serine and serine racemase in astrocytes may play roles in neuronal hyperexcitability via a cooperative activation of NMDA receptors. Furthermore, serine racemase may be involved in migration and differentiation of immature astrocytes, which is relevant to reactive astrogliosis.

Hyperglycemia-induced oxidative stress is widely recognized as a key mediator in the pathogenesis of diabetic nephropathy, a complication of diabetes. We found that both expression and enzymatic activity of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) were upregulated in the renal cortexes of diabetic rats and mice. Similarly, IDPc was induced in murine renal proximal tubular OK cells by high hyperglycemia, while it was abrogated by co-treatment with the antioxidant N-Acetyl-Cysteine (NAC). In OK cells, increased expression of IDPc by stable transfection prevented hyperglycemia-mediated reactive oxygen species (ROS) production, subsequent cellular oxidative stress and extracellular matrix accumulation, whereas these processes were all stimulated by decreased IDPc expression. In addition, production of NADPH and GSH in the cytosol was positively correlated with the expression level of IDPc in OK cells. These results together indicate that upregulation of IDPc in response to hyperglycemia might play an essential role in preventing the progression of diabetic nephropathy, which is accompanied by ROS-induced cellular damage and fibrosis, by providing NADPH, the reducing equivalent needed for recycling reduced glutathione and low molecular weight antioxidant thiol proteins.

5-Hydroxyuridine (5-OHU) is a major lesion of uridine and cytosine produced in RNA by various chemical oxidants. To elucidate its biochemical and biophysical effects on RNA replication, the site-specifically modified oligoribonucleotides containing 5-OHU were synthesized with C5-hydroxy-5'-O-DMTr-2'-TBDMS-uridine phosphoramidite using automated solid phase synthesis. The base-pairing properties of nucleotides opposite 5-OHU in 24 mer oligoribonulcleotides with dNTP were studied using three reverse transcriptases (Super-Script(TM)II-, AMV-, MMLV-RT) in cDNA synthesis. Adenine as well as guanine was incorporated preferentially by all reverse transcriptases. In the UV-melting temperature experiment, the results from the relative stabilities of the base pairs were A : 5-OHU > G : 5-OHU > T : 5-OHU approximately C : 5-OHU. Circular Dichroism (CD) studies showed that DNA-RNA containing 5-OHU heteroduplexes exhibit a similar conformation between the A-type RNA and B-type DNA. These results suggest that 5-OHU from oxidative damage was mainly influenced by adenine mismatch.

Familial Amyotrophic lateral sclerosis (FALS) is a progressive neurodegenetative disorder induced by mutations of the SOD1 gene. Heat shock protein 27 (HSP27) is well-defined as a stress-inducible protein, however the its role in ALS protection has not yet been established. To investigate the role HSP27 may have in SOD1 mutant-mediated apoptosis, human SOD1 or HSP27 genes were fused with a PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame fusion protein, which was then transduced into cells. We found the purified PEP-1-HSP27 fusion proteins can be transduced efficiently into neuronal cells and protect against cell death by enhancing mutant SOD1 activity. Moreover, transduced PEP-1-HSP27 efficiently prevents protein aggregation produced by oxidative stress. These results suggest that transduced HSP27 fusion protein may be explored as a potential therapeutic agent for FALS patients.

Brain pyridoxine-5-P oxidase is activated by the tryptophan metabolites 3-hydroxyanthranilate and 3-hydroxykynurenine. 3-Hydroxyanthranilate at concentrations of 0.03 mM relieves the inhibition elicited by accumulation of the substrate pyridoxine-5-P (Ki = 60 microM). The results of fluorometric measurements indicate that four molecules of 3-hydroxyanthranilate bind to the dimeric enzyme (56 kDa) with an association constant of 5.5 x 10(4) M-1. Differential spectral measurements failed to detect any direct interaction between the cofactor FMN and the effector 3-hydroxyanthranilate. These results are consistent with the hypothesis that the effector molecules bind to sites of the dimeric protein distinct from the cofactor site. Limited chymotrypsin digestion of pyridoxine-5-P oxidase yields catalytically active species that are no longer susceptible to activation by 3-hydroxykynurenine. A polypeptide of 16 kDa containing FMN and endowed with full catalytic activity was isolated by ion-exchange chromatography. It is postulated that the structural domain associated with catalytic activity composes approximately one-half of the molecular mass of pyridoxine-5-P oxidase (28 kDa), whereas the remaining portion of the macromolecule contains regulatory binding sites.

Epilepsy is characterized by the presence of spontaneous episodes of abnormal neuronal discharges and its pathogenic mechanisms remain poorly understood. Recently, we found that the expression of creatine kinase (CK) was markedly decreased in an epilepsy animal model using proteomic analysis. A human CK gene was fused with a HIV-1 Tat peptide to generate an in-frame Tat-CK fusion protein. The purified Tat-CK fusion protein was efficiently transduced into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced Tat-CK fusion protein was stable for 48 h. Moreover, the Tat-CK fusion protein markedly increased endogenous CK activity levels within the cells. These results suggest that Tat-CK provides a strategy for the therapeutic delivery of proteins in various human diseases including the delivery of CK for potential epilepsy treatment.

The invasion of monocytes through the endothelial wall of arteries and their transformation from macrophage into form cells has been implicated as a critical initiating event in atherogenesis. Human THP-1 monocytic cells can be induced to differentiate into macrophages by phorbol myristate acetate (PMA) treatment, and can be converted into foam cells by exposure to oxidized low-density lipoprotein (oxLDL). To identify proteins potentially involved in atherosclerotic processes, we performed a proteomic analysis of THP-1 macrophages exposed to oxLDL generated by treatment with native LDL with hypochlorous acid/hypochlorite (HOCl/OCl(-)). We detected more than a thousand proteins, of which 104 differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and the NCBI database. The largest differences in expression were observed for bifunctional purine biosynthesis protein, vacuolar protein sorting 33A, breast carcinoma amplified sequence, adenine phosphoribosyltransferase, and tropomyosin alpha 3 chain. Interestingly, many apoptotic proteins such as lamin B1, poly (ADP-ribose) polymerase, Bcl-2 related protein A1 and vimentin were identified by MALDI-TOF analysis. Identities were confirmed by matching the sequence of several tryptic peptides using MALDI-TOF/TOF MS, Western blot analyses and immunofluorescent microscopy. The data described here will contribute to establishing a functional profile of the human macrophage proteome. Furthermore, the proteins identified in this study are attractive candidates for further biomarkers involved in the pathogenesis of atherosclerosis.

Pyridoxal-5'-phosphate phosphatase (PLPP) catalyzes the dephosphorylation of pyridoxal-5'-phosphate (PLP). A human brain PLPP gene was fused with a PEP-1 peptide and produced a genetic in-frame PEP-1-PLPP fusion protein. The purified PEP-1-PLPP fusion protein was efficiently transduced into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced PEP-1-PLPP fusion protein was stable for 36 h. The concentration of PLP was markedly decreased by the addition of exogenous PEP-1-PLPP to media pretreated with the vitamin B(6) precursors; pyridoxine, pyridoxal kinase and pyridoxine-5'-phosphate oxidase into cells. The results suggest that the transduction of the PEP-1-PLPP fusion protein can be one mode of PLP level regulation, and to replenish this enzyme in the various neurological disorders related to vitamin B(6).

In protein therapy, it is important for exogenous protein to be delivered into the target subcellular localization. To transduce a therapeutic protein into its specific subcellular localization, we synthesized nuclear localization signal (NLS) and membrane translocation sequence signal (MTS) peptides and produced a genetic in-frame SOD fusion protein. The purified SOD fusion proteins were efficiently transduced into mammalian cells with enzymatic activities. Immunofluorescence and Western blot analysis revealed that the SOD fusion proteins successfully transduced into the nucleus and the cytosol in the cells. The viability of cells treated with paraquat was markedly increased by the transduced fusion proteins. Thus, our results suggest that these peptides should be useful for targeting the specific localization of therapeutic proteins in various human diseases.