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ABSTRACT: Class IV polyhydroxyalkanoate (PHA) synthase from Bacillus cereus YB-4 (PhaRC(YB4)) or B. megaterium NBRC15308(T) (PhaRC(Bm)) was expressed in Ralstonia eutropha PHB(-)4 to compare the ability to produce PHA and the substrate specificity of PhaRCs. PhaRC(YB4) produced significant amounts of PHA and had broader substrate specificity than PhaRC(Bm).
Bioscience Biotechnology and Biochemistry 08/2011; 75(8):1615-7. · 1.28 Impact Factor
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ABSTRACT: Polyhydroxyalkanoate (PHA)-producing Bacillus strains possess class IV PHA synthases composed of two subunit types, namely, PhaR and PhaC. In the present study, PHA synthases from Bacillus megaterium NBRC15308(T) (PhaRC(Bm)), B. cereus YB-4 (PhaRC(YB4)), and hybrids (PhaR(Bm)C(YB4) and PhaR(YB4)C(Bm)) were expressed in Escherichia coli JM109 to characterize the molecular weight of the synthesized poly(3-hydroxybutyrate) [P(3HB)]. PhaRC(Bm) synthesized P(3HB) with a relatively high molecular weight (M(n) = 890 × 10(3)) during 72 h of cultivation, whereas PhaRC(YB4) synthesized low-molecular-weight P(3HB) (M(n) = 20 × 10(3)). The molecular weight of P(3HB) synthesized by PhaRC(YB4) decreased with increasing culture time and temperature. This time-dependent behavior was observed for hybrid synthase PhaR(Bm)C(YB4), but not for PhaR(YB4)C(Bm). These results suggest that the molecular weight change is caused by the PhaC(YB4) subunit. The homology between PhaCs from B. megaterium and B. cereus YB-4 is 71% (amino acid identity); however, PhaC(YB4) was found to have a previously unknown effect on the molecular weight of the P(3HB) synthesized in E. coli.
Biomacromolecules 06/2011; 12(7):2660-6. · 5.48 Impact Factor
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ABSTRACT: Recently, many people have developed rheumatoid arthritis (RA), and prednisolone (PD) is often used for treatment; however, long use and a large dose of PD can cause toxic side effects. In this study, in order to enhance the therapeutic effects and to suppress the toxic side effects, the conjugate (GC-SP) was prepared by coupling between glycol-chitosan (GC) and succinyl-prednisolone (SP). The drug-release properties of GC-SP were examined and analyzed kinetically. The plasma concentration-time profiles of GC-SP and released PD were investigated after i.v. injection to normal rats, and their pharmacokinetic profiles were analyzed. PD was stable and released gradually (ca. 1%/h) from GC-SP at physiological pH, while PD was unstable at basic pH and the release from GC-SP was accelerated at basic pH. GC-SP showed good systemic retention (more than 16-fold area under the plasma concentration-time curve (AUC) as compared to PD alone), and released PD gradually in vivo. The in vivo release rate was calculated to be much faster than the in vitro rate. From these results, it is expected that GC-SP will be accumulated at inflammatory sites based on enhanced permeability and retention (EPR) effects, and release PD there effectively.
International journal of pharmaceutics 03/2011; 410(1-2):17-22. · 2.96 Impact Factor
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Michiya Takada,
Yoshiyuki Ban,
Gou Yamamoto,
Toshihiko Ueda, Yuta Saito,
Eiichi Nishimura,
Kunimi Fujisawa,
Ryohei Koide,
Masakazu Mizutani,
Tadahiko Kozawa,
Yuji Shiraishi,
Yasuhiko Bando,
Tetsuhiko Tachikawa,
Tsutomu Hirano
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ABSTRACT: Diabetes can lead to serious microvascular complications including proliferative diabetic retinopathy (PDR), the leading cause of blindness in adults. Recent studies using gene array technology have attempted to apply a hypothesis-generating approach to elucidate the pathogenesis of PDR, but these studies rely on mRNA differences, which may or may not be related to significant biological processes. To better understand the basic mechanisms of PDR and to identify potential new biomarkers, we performed shotgun liquid chromatography (LC)/tandem mass spectrometry (MS/MS) analysis on pooled protein extracts from neovascular membranes obtained from PDR specimens and compared the results with those from non-vascular epiretinal membrane (ERM) specimens. We detected 226 distinct proteins in neovascular membranes and 154 in ERM. Among these proteins, 102 were specific to neovascular membranes and 30 were specific to ERM. We identified a candidate marker, periostin, as well as several known PDR markers such as pigment epithelium-derived factor (PEDF). We then performed RT-PCR using these markers. The expression of periostin was significantly up-regulated in proliferative membrane specimens. Periostin induces cell attachment and spreading and plays a role in cell adhesion. Proteomic analysis by LC/MS/MS, which permits accurate quantitative comparison, was useful in identifying new candidates such as periostin potentially involved in the pathogenesis of PDR.
Biochemical and Biophysical Research Communications 08/2010; 399(2):221-6. · 2.48 Impact Factor
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ABSTRACT: Polyhydroxyalkanoate (PHA) synthases catalyze chain transfer (CT) reaction after polymerization reaction of PHA by transferring PHA chain from PHA synthase to a CT agent, resulting in covalent bonding of CT agent to PHA chain at the carboxyl end. Previous studies have shown that poly(ethylene glycol) (PEG) is an effective exogenous CT agent. This study aimed to compare the effects of PEG on CT reaction during poly[(R)-3-hydroxybutyrate] [P(3HB)] synthesis by using six PHA synthases in Escherichia coli JM109. The synthesized P(3HB) polymers were characterized in terms of molecular weight and end-group structure. Supplementation of PEG to the culture medium reduced P(3HB) molecular weights by up to 96% due to PEG-induced CT reaction. The P(3HB) polymers were subjected to (1)H NMR analysis to confirm the formation of a covalent bond between PEG and P(3HB) chain at the carboxyl end. This study revealed the reactivity of PHA synthases to PEG with respect to CT reaction in E. coli.
Applied Microbiology and Biotechnology 07/2010; 87(4):1427-35. · 3.42 Impact Factor
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ABSTRACT: We previously reported that the adhesion of gastric carcinoma cells to the peritoneum mediated by the alpha3beta1 integrin-laminin interaction is a key step in the initial process of peritoneal metastatic dissemination. Carcinoma cells subsequently invade through the intercellular gaps of mesothelial linings. In this study, we examined the role of the interaction of carcinoma cells with laminin-5, which is a major component of submesothelial basement membranes and serves as a high-affinity ligand for alpha3beta1 integrin, in carcinoma cell invasion. Human gastric carcinoma cell lines (MKN1, GT3TKB, and NUGC-4) adhered in an alpha3beta1 integrin-dependent manner to the extracellular matrix deposited by peritoneal mesothelial cells. An in vitro invasion assay using the Boyden chamber system revealed that MKN1 cell migration through the membranes increased when the membranes were coated with matrices produced by mesothelial cells or with laminin-5-containing Matrigel as compared to Matrigel alone. The cell migration promoted by laminin-5-containing Matrigel was inhibited by the presence of anti-alpha3 integrin antibody. When MKN1 cells were cultured in a laminin-5-coated plate, these cells were promoted to produce matrix metalloproteinase (MMP)-9, as assessed by gelatin zymography, enzyme-linked immunosorbent assay, and reverse transcription-polymerase chain reaction. These results suggest that the production of MMP-9 by MKN1 cells was potentiated by the alpha3beta1 integrin-laminin-5 interaction, which facilitated their invasion via degradation of the matrix.
Clinical and Experimental Metastasis 03/2010; 27(4):197-205. · 3.52 Impact Factor
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ABSTRACT: To study NAD(P)H oxidase-dependent outcomes after oxygen stresses that are similar to those experienced by preterm infants today using a rat model of retinopathy of prematurity.
Within 4 hours of birth, pups and their mothers were cycled between 50% and 10% oxygen daily for 14 days and were returned to room air (21% O2, 50/10 oxygen-induced retinopathy [OIR]) or supplemental oxygen (28% O2, 50/10 OIR+SO) for 4 days. Pups received intraperitoneal injections of the specific NAD(P)H oxidase inhibitor apocynin (10 mg/kg/d) or of PBS from postnatal day (P)12 to P17, and some received intraperitoneal injections of hypoxyprobe before kill. Intravitreous neovascularization (IVNV), avascular/total retinal areas, vascular endothelial growth factor (VEGF), NAD(P)H oxidase activity, or hypoxic retina (conjugated hypoxyprobe) were determined in neurosensory retinas. Human retinal microvascular endothelial cells (RMVECs) treated with apocynin or control were exposed to 1% or 21% O2 and assayed for phosphorylated (p-)Janus kinase (JNK) and NAD(P)H oxidase activity.
Retinas from 50/10 OIR+SO had increased NAD(P)H oxidase activity and lower VEGF than did retinas from 50/10 OIR. Apocynin treatment reduced the IVNV area and hypoxic retina in 50/10 OIR+SO. RMVECs treated with 1% O2 had increased p-JNK compared with RMVECs exposed to room air.
Different oxygen stresses activate NAD(P)H oxidase to varying degrees to trigger disparate pathways (angiogenesis or apoptosis). The oxygen stresses and outcomes used in this study are relevant to human ROP and may explain some of the complexity in the pathophysiology of ROP resulting from oxygen exposure.
Investigative Ophthalmology & Visual Science 05/2008; 49(4):1591-8. · 3.60 Impact Factor
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ABSTRACT: The invasive and metastatic potentials of hepatocellular carcinoma are positively correlated with the expression level of alpha3beta1 integrin, a high-affinity adhesion receptor for laminin isoforms including laminin-5. In this study, we investigated changes in the adhesive and invasive behaviors of human HCC HepG2 cells after transfection with cDNA for alpha3 integrin in order to elucidate the direct involvement of this integrin in these cellular processes. We introduced cDNA for splice variants of alpha3 integrin (alpha3A and alpha3B) into the cells, and selected two transfectant clones (HepG2-3A and HepG2-3B), which express the alpha3A and alpha3B integrins, respectively. Both transfectant cells adhered almost equally to laminin-5-coated plates in an alpha3 integrin-dependent manner, indicating that transfected alpha3Abeta1 and alpha3Bbeta1 integrins were functionally active in these cells. The migratory and invasive potentials of the transfectant cells were assessed by scratch wound assay and in vitro chemoinvasion assay. The results demonstrated that the migration of HepG2-3A and HepG2-3B cells but not of mock transfectant (HepG2-M) cells was stimulated on the plates coated with laminin-5. Furthermore, HepG2-3A and HepG2-3B cells were found to be more invasive into laminin-5-containing matrices than were HepG2-M cells. These results strongly suggest that enhanced expression of alpha3beta1 integrin on HCC cells is directly involved in their malignant phenotypes such as invasion and metastasis.
Biochimica et Biophysica Acta 04/2008; 1780(3):564-70. · 4.66 Impact Factor
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Yuta Saito,
Yasuko Hasebe-Takenaka,
Toshihiko Ueda,
Takako Nakanishi-Ueda,
Shotaro Kosuge,
Masaki Aburada,
Tsutomu Shimada,
Yukinobu Ikeya,
Hidetoshi Onda,
Hirotsugu Ogura,
Yoko Taguchi,
Hajime Yasuhara,
Ryohei Koide
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ABSTRACT: This study aimed to investigate the preventive effects of green tea fractions (GTFs) on rat model of oxygen-induced retinopathy (OIR). Neonatal Sprague-Dawley rats were exposed to daily cycles of 80% oxygen (20.5 h), ambient air (0.5 h), and progressive return to 80% oxygen (3 h) until postnatal day 12 (P12), then the rats were placed in ambient air until P18. The green tea was fractionated by DM-A50, DM-W, M-B, and M-W. The rats were treated once daily from P6 to P17 by gastric gavage of GTFs (0.05 or 0.01 g/ml) or distilled water (DW) at 50 microl/10 g body weight. On P18, the rats were sacrificed and the retinal samples were collected. The retinal neovascularization (NV) was scored and avascular areas (AVAs) were measured as a % of total retinal area (%AVAs) in ADPase stained retinas. The NV scores in 0.01 g/ml M-W were significantly lower than those in DW. The %AVAs in 0.05 g/ml DM-A50 and in 0.05 g/ml and 0.01 g/ml M-W were significantly lower than those in DW. There were less catechins, and less caffeine in M-W fraction compared with other GTFs, suggesting components of green tea except for catechins and caffeine might suppress the neovascularization in rat model of OIR.
Journal of Clinical Biochemistry and Nutrition 08/2007; 41(1):43-9. · 1.98 Impact Factor
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Hirotsugu Ogura,
Takako Nakanishi-Ueda,
Toshihiko Ueda,
Shinichi Iwai,
Seiichi Uchida, Yuta Saito,
Yoko Taguchi,
Hajime Yasuhara,
Donald Armstrong,
Katsuji Oguchi,
Ryohei Koide
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ABSTRACT: The aim of this study was to investigate the effect of A-3922, a dihydrobenzofuran derivative, on linoleic acid hydroperoxide (LHP)-induced corneal neovascularization (NV) in a rabbit model. Male New Zealand rabbits received intraperitoneal (i.p.) injections of 10 or 30 mg/kg per day A-3922 or its vehicle as control for 3 days. One day after i.p. injections, LHP was injected with a 30-gauge needle into the corneal stroma of the superior quadrant 4.5-mm below the limbus. Photographs of the vessels were taken for digital analysis with a surgical microscope. Vascular endothelial growth factor (VEGF) was measured using an immunoassay kit, and matrix metalloproteinase (MMP)-9 was measured by gelatin zymography in corneal samples. At 7 days post-LHP injection, the total vessel length was 26.7 +/- 3.8 mm in the control animals (n = 8), 16.1 +/- 0.8 mm in the A-3922 (10 mg/kg)-treated group (n = 5), and 11.4 +/- 2.1 mm in the 30 mg/kg group (n = 8, P<0.01 vs control), respectively. After LHP injection, the content of VEGF and MMP-9 activity were increased in the superior cornea, but these were not influenced by A-3922 treatments. These results indicate that LHP-induced corneal NV is inhibited by treatment with A-3922 and therefore may represent a potential pharmacological intervention for ocular neovascularization disorders.
Journal of Pharmacological Sciences 03/2007; 103(2):234-40. · 2.08 Impact Factor
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ABSTRACT: To study the mechanisms of action of the antioxidants, n-acetylcysteine (NAC), and the nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase oxidase inhibitor, apocynin, on intravitreous neovascularization (IVNV), and retinal avascularity in a rat model of retinopathy of prematurity (ROP).
Newborn rats exposed to oxygen-induced retinopathy underwent intraperitoneal (IP) injections of NAC (150 mg/kg) at post-natal day (p)2, p6, p10 (early NAC-treated), or p12 through p17 (late NAC-treated), apocynin (10 mg/kg) from p12 through p17, or phosphate buffered saline (PBS; controls). Lipid hydroperoxide (LHP) was measured in early NAC-treated oxygen-induced retinopathy (OIR) at p7, p14 and p18. Pups were placed in room air at p14. At p18, retinal flat mounts were scored for IVNV and avascular/total retinal area, or retinas were assayed for cleaved caspase-3 and vascular endothelial growth factor (VEGF) protein. In non-injected OIR pups, retinas were assayed for gp91(phox). Cryosections were stained with isolectin B4, cleaved caspase-3, CD68, CD31, gp91(phox), neuron-glial antigen 2 (NG-2), or anti-glial fibrillary acidic protein (GFAP) and visualized with confocal microscopy.
LHP increased over time in retinas from OIR exposed pups in association with IVNV. Early NAC-treated retinas had significantly reduced LHP compared to PBS-control at p18 (p<0.012). However, neither early nor late treatment with NAC had an effect on IVNV or retinal avascularity. Although apocynin had no effect on IVNV, it reduced both avascular retina (p=0.017) and retinal cleaved caspase-3 determined by western blot (p=0.021). In cryosections from OIR eyes, cleaved caspase-3 positive cells co-labeled with some lectin-stained vessels, NG2 labeled cells, and with GFAP positive cells in the inner nuclear layer. We found that the intravascular expression of gp91(phox) co-localized mostly with CD31 and some CD68 positive cells.
Our results do not support the antioxidant properties of NAC as effective in reducing IVNV or avascular retina in the 50/10 OIR rat model. Apocynin reduced avascularity and apoptosis in the OIR model perhaps through pathways triggered by ROS generation but upstream from LHP production. Further study and consideration may be given to apocynin or NAD(P)H oxidase inhibitors as adjunctive therapy for ROP to reduce the avascular retina.
Molecular vision 02/2007; 13:840-53. · 2.20 Impact Factor
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ABSTRACT: To study the effects of intravitreous triamcinolone acetonide (TA) on neovascularization (NV), capillary density, and retinal endothelial cell (REC) viability in a model of oxygen-induced retinopathy (OIR).
Newborn rats exposed to OIR underwent intravitreous injections (right eye) at day 14 to achieve intravitreous concentrations of: dexamethasone (DEX) (0.3 mg/mL), triamcinolone (TA; 0.4-4 mg/mL), or PBS. Animals were removed to room air and at day 18, retinal flatmounts were assayed for clock hours of NV, percent peripheral avascular retina, capillary density, apoptosis, and VEGF protein. At day 15, retinas were assayed for insulin-like growth factor (IGF)-1 receptor phosphorylation (IGF-1Rphos). Human RECs exposed to TA were assayed for trypan blue exclusion or activated caspase-3.
TA but not DEX or PBS reduced NV (ANOVA, P < 0.001), capillary density (ANOVA, P < 0.001), and systemic weight gain (ANOVA, P = 0.002). VEGF protein was not different between TA- and PBS-injected or noninjected groups. Apoptosis was not increased in vivo or in vitro between groups, but there was a dose-dependent toxic effect of TA on cultured RECs (P < 0.001). At day 15, retinas from the 4 mg/mL TA-injected OIR group had a trend toward reduced IGF-1Rphos compared with room air-raised PBS- or non-injected OIR groups.
TA caused dose-dependent reductions in NV, retinal vascularization, and systemic weight gain associated with a reduction in IGF-1Rphos. Long-term studies are needed to assess TA toxicity in vivo. TA doses should be carefully considered before administering the drug in diseases with ongoing retinal vascular development, such as retinopathy of prematurity.
Investigative Ophthalmology & Visual Science 11/2006; 47(11):4975-82. · 3.60 Impact Factor
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ABSTRACT: The invasive and metastatic potentials of hepatocellular carcinoma (HCC) are positively correlated with the expression level of alpha3beta1 integrin, a high-affinity adhesion receptor for laminin isoforms. Transforming growth factor (TGF)-beta1 stimulates non-invasive HCC cells to acquire invasive phenotypes in association with the enhanced expression of alpha3 integrin. In this study, we investigated the molecular mechanism underlying the upregulation of alpha3beta1 integrin by TGF-beta1 in non-invasive HepG2 HCC cells. The treatment of HepG2 cells with TGF-beta1 induced the expression of alpha3 integrin and potentiated these cells to adhere to laminin-5 and to migrate through laminin-5-coated membranes. The promoter activity was measured by luciferase assay with a series of deletion constructs of the 5'-flanking region of the mouse alpha3 integrin gene, and the results showed that the -260/-119 region (relative to the major transcription start site) contained elements responsive to TGF-beta1 stimulation. The introduction of mutations into the putative consensus binding sequence for the Ets-family of transcription factors located at -133 greatly decreased the promoter activity responding to TGF-beta1 stimulation. The nuclear proteins extracted from TGF-beta1-stimulated HepG2 cells yielded a larger amount of DNA-nuclear protein complexes than did those extracted from unstimulated cells, as determined by an electrophoretic mobility shift assay using an oligonucleotide containing the Ets-site as a probe. These results suggest that TGF-beta1 stimulates HepG2 cells to express a higher level of alpha3 integrin by transcriptional upregulation via Ets transcription factors and to exhibit a more invasive phenotype.
Clinical and Experimental Metastasis 02/2005; 22(7):539-48. · 3.52 Impact Factor
