Purification and Characterization,of Rice Bran Lipase II

A species of rice bran lipase (lipase II) was purified by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-cellulose, Sephadex G–75 and CH-Sephadex C–50. Both polyacrylamide disc electrophoresis and ultracentrifugation demonstrated that the enzyme protein is homogeneous. The isoelectric point of the enzyme was 9.10 by ampholine electrophoresis. The sedimentation coefficient of the enzyme was evaluated to be 2.60 S, and the molecular weight to be 33,300 according to Archbald’s method. The enzyme showed the optimum pH between 7.5 and 8.0, and the optimum temperature at about 27°C. It was stable over the pH range from 5 to 9.5 and below 30°C. In substrate specificity, the enzyme exhibited a high specificity toward triglycerides having short-carbon chain fatty acids, although it was capable of hydrolyzing the ester bonds in the rice and olive oil.     

Quantification of Chitinase mRNA Levels in Human and Mouse Tissues by Real-Time PCR: Species-Specific Expression of Acidic Mammalian Chitinase in Stomach Tissues

Chitinase hydrolyzes chitin, which is an N-acetyl-D-glucosamine polymer that is present in a wide range of organisms, including insects, parasites and fungi. Although mammals do not contain any endogenous chitin, humans and mice express two active chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). Because the level of expression of these chitinases is increased in many inflammatory conditions, including Gaucher disease and mouse models of asthma, both chitinases may play important roles in the pathophysiologies of these and other diseases. We recently established a quantitative PCR system using a single standard DNA and showed that AMCase mRNA is synthesized at extraordinarily high levels in mouse stomach tissues. In this study, we applied this methodology to the quantification of chitinase mRNAs in human tissues and found that both chitinase mRNAs were widely expressed in normal human tissues. Chit1 mRNA was highly expressed in the human lung, whereas AMCase mRNA was not overexpressed in normal human stomach tissues. The levels of these mRNAs in human tissues were significantly lower than the levels of housekeeping genes. Because the AMCase expression levels were quite different between the human and mouse stomach tissues, we developed a quantitative PCR system to compare the mRNA levels between human and mouse tissues using a human-mouse hybrid standard DNA. Our analysis showed that Chit1 mRNA is expressed at similar levels in normal human and mouse lung. In contrast, the AMCase expression level in human stomach was significantly lower than that expression level observed in mouse stomach. These mRNA differences between human and mouse stomach tissues were reflecting differences in the chitinolytic activities and levels of protein expression. Thus, the expression level of the AMCase in the stomach is species-specific.     

Depression of Complement Regulatory Factors in Rat and Human Renal Grafts Is Associated with the Progress of Acute T-Cell Mediated Rejection

Background

The association of complement with the progression of acute T cell mediated rejection (ATCMR) is not well understood. We investigated the production of complement components and the expression of complement regulatory proteins (Cregs) in acute T-cell mediated rejection using rat and human renal allografts.

Methods

We prepared rat allograft and syngeneic graft models of renal transplantation. The expression of Complement components and Cregs was assessed in the rat grafts using quantitative real-time PCR (qRT-PCR) and immunofluorescent staining. We also administered anti-Crry and anti-CD59 antibodies to the rat allograft model. Further, we assessed the relationship between the expression of membrane cofactor protein (MCP) by immunohistochemical staining in human renal grafts and their clinical course.

Results

qRT-PCR results showed that the expression of Cregs, CD59 and rodent-specific complement regulator complement receptor 1-related gene/protein-y (Crry), was diminished in the rat allograft model especially on day 5 after transplantation in comparison with the syngeneic model. In contrast, the expression of complement components and receptors: C3, C3a receptor, C5a receptor, Factor B, C9, C1q, was increased, but not the expression of C4 and C5, indicating a possible activation of the alternative pathway. When anti-Crry and anti-CD59 mAbs were administered to the allograft, the survival period for each group was shortened. In the human ATCMR cases, the group with higher MCP expression in the grafts showed improved serum creatinine levels after the ATCMR treatment as well as a better 5-year graft survival rate.

Conclusions

We conclude that the expression of Cregs in allografts is connected with ATCMR. Our results suggest that controlling complement activation in renal grafts can be a new strategy for the treatment of ATCMR.     

Fatty acids increase the circulating levels of oxidative stress factors in mice with diet-induced obesity via redox changes of albumin

Plasma concentrations of free fatty acids are increased in metabolic syndrome, and the increased fatty acids may cause cellular damage via the induction of oxidative stress. The present study was designed to determine whether the increase in fatty acids can modify the free sulfhydryl group in position 34 of albumin (Cys34) and enhance the redox-cycling activity of the copper-albumin complex in high-fat diet-induced obese mice. The mice were fed with commercial normal diet or high-fat diet and water ad libitum for 3 months. The high-fat diet-fed mice developed obesity, hyperlipemia, and hyperglycemia. The plasma fatty acid/albumin ratio also significantly increased in high-fat diet-fed mice. The increased fatty acid/albumin ratio was associated with conformational changes in albumin and the oxidation of sulfhydryl groups. Moreover, an ascorbic acid radical, an index of redox-cycling activity of the copper-albumin complex, was detected only in the plasma from obese mice, whereas the plasma concentrations of ascorbic acid were not altered. Plasma thiobarbituric acid reactive substances were significantly increased in the high-fat diet group. These results indicate that the increased plasma fatty acids in the high-fat diet group resulted in the activated redox cycling of the copper-albumin complex and excessive lipid peroxidation.     

Backbone resonance assignment for the outer membrane lipoprotein receptor LolB from Escherichia coli

LolB, which is anchored to the outer membrane of Gram-negative bacteria, receives outer-membrane-directed lipoproteins from LolA, and incorporates them into the outer membrane. We established backbone resonance assignments of ²H/¹³C/¹⁵N labeled LolB from Escherichia coli.     

Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-suppression technology

We developed a rapid single nucleotide polymorphism (SNP) detection system named smart amplification process version 2 (SMAP 2). Because DNA amplification only occurred with a perfect primer match, amplification alone was sufficient to identify the target allele. To achieve the requisite fidelity to support this claim, we used two new and complementary approaches to suppress exponential background DNA amplification that resulted from mispriming events. SMAP 2 is isothermal and achieved SNP detection from whole human blood in 30 min when performed with a new DNA polymerase that was cloned and isolated from Alicyclobacillus acidocaldarius (Aac pol). Furthermore, to assist the scientific community in configuring SMAP 2 assays, we developed software specific for SMAP 2 primer design. With these new tools, a high-precision and rapid DNA amplification technology becomes available to aid in pharmacogenomic research and molecular-diagnostics applications.     

Plasminogen activator inhibitor-1 deficiency suppresses osteoblastic differentiation of mesenchymal stem cells in mice

Plasminogen activator inhibitor-1 (PAI-1) is known as an inhibitor of fibrinolytic system. Previous studies suggest that PAI-1 is involved in the pathogenesis of osteoporosis induced by ovariectomy, diabetes, and glucocorticoid excess in mice. However, the roles of PAI-1 in early-stage osteogenic differentiation have remained unknown. In the current study, we investigated the roles of PAI-1 in osteoblastic differentiation of mesenchymal stem cells (MSCs) using wild-type (WT) and PAI-1-deficient (PAI-1 KO) mice. PAI-1 mRNA levels were increased with time during osteoblastic differentiation of MSCs or mesenchymal ST-2 cells. However, the increased PAI-1 levels declined at the mineralization phase in the experiment using MC3T3-E1 cells. PAI-1 deficiency significantly blunted the expression of osteogenic gene, such as osterix and alkaline phosphatase enhanced by bone morphogenetic protein (BMP)-2 in bone marrow-derived MSCs (BM-MSCs), adipose-tissue-derived MSCs (AD-MSCs), and bone marrow stromal cells of mice. Moreover, a reduction in endogenous PAI-1 levels by small interfering RNA significantly suppressed the expression of osteogenic gene in ST-2 cells. Plasmin did not affect osteoblastic differentiation of AD-MSCs induced by BMP-2 with or without PAI-1 deficiency. PAI-1 deficiency and a reduction in endogenous PAI-1 levels did not affect the phosphorylations of receptor-specific Smads by BMP-2 and transforming growth factor-β in AD-MSCs and ST-2 cells, respectively. In conclusion, we first showed that PAI-1 is crucial for the differentiation of MSCs into osteoblasts in mice.     

Effect of anti-regucalcin antibody on neutral phosphatase activity in rat liver cytosol: Involvement of endogenous regucalcin

The effect of anti-regucalcin monoclonal antibody on neutral phoshatase activity in rat liver cytosol was investigated. Phosphotyrosine, phosphoserine, and phosphothreonine were used as the substrate toward phosphatase asssy. Liver cytosolic phosphatase activity with three phosphoaminoacids was significantly increased in the presence of anti-regucalcin antibody (100 and 200 ng/ml) in the enzyme reaction mixture with calcium chloride (0.1 mM) or EGTA (1.0 mM). The effect of anti-regucalcin antibody was completely abolished in the presence of exogenous regucalcin (1.0 M), indicating the involvement of endogenous regucalcin. The anti-regucalcin anti body- increased phosphatase activity was not significantly altered in the presence of trifluoperazine (20 M), an antagonist of calmodulin, or akadaic acid (10 M), an inhibitor of protein phosphatase, although these inihibitors caused a slight decrease in liver cytosolic phosphatase activity. The effect of endogenous regucalcin might be not related to calmodulin, and it was insensitive to okadaic acid. The present findings suggest that endogenous regucalcin is involved in the regulation of protein phasphatase in rat liver cytoplasm.     

Overexpression of regucalcin suppresses cell death and apoptosis in cloned rat hepatoma H4-II-E cells induced by lipopolysaccharide, PD 98059, dibucaine, or Bay K 8644

The effect of regucalcin, a regulatory protein in intracellular signaling pathway, on cell death was investigated by using the cloned rat hepatoma H4-II-E cells overexpressing regucalcin. The hepatoma cells (wild-type) and stable regucalcin (RC)/pCXN2 transfectants were cultured for 72 h in medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured for 12-72 h in medium without FBS containing either vehicle or lipopolysaccharide (LPS; 0.1 or 1.0 microg/ml). The number of wild-type cells was significantly decreased by culture for 24 or 48 h in the presence of LPS (0.1 or 1.0 microg/ml). The effect of LPS (0.1 or 1.0 microg/ml) in decreasing the number of hepatoma cells was significantly prevented in transfectants overexpressing regucalcin. However, the culture with LPS (0.1 or 1.0 microg/ml) for 72 h caused a significant decrease in cell number of transfectants. Ca(2+)/calmodulin-dependent nitric oxide (NO) synthase activity was significantly decreased by culture with LPS (1.0 microg/ml) for 24-72 h of wild-type cells. This decrease was significantly prevented in transfectants. LPS (0.1 or 1.0 microg/ml)-induced decrease in the number of wild-type cells was significantly prevented by culture with caspase-3 inhibitor (10(-8) M). Moreover, the number of wild-type cells was significantly decreased by culture with PD 98059 (10(-6) M), dibucaine (10(-6) M), or staurosporine (10(-6) M), which is an inhibitor of various protein kinases. The effect of PD 98059 or dibucaine on the number of wild-type cells was not observed in transfectants, although the effect of staurosporine was seen in transfectants. Culture with Bay K 8644 (2.5 x 10(-6) M), an agonist of Ca(2+) entry in cells, caused a significant decrease in the number of wild-type cells. Such an effect was not seen in transfectants. The presence of LPS did not significantly decrease the number of wild-type cells in the presence of Bay K 8644. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with Bay K 8644, and this DNA fragmentation was significantly prevented in transfectants. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell death induced by LPS or various intracellular signaling-related factors.     

Enzymatic characterization of scytalone dehydratase Val75Met variant found in melanin biosynthesis dehydratase inhibitor (MBI-D) resistant strains of the rice blast fungus

Carpropamid ((1RS,3SR)-2,2-dichloro-N-[(R)-1-(4-chlorophenyl)ethyl]-1-ethyl-3-methylcyclopropanecarboxamide) is a potent chemical against the rice blast fungus, Pyricularia oryzae. In 2001, isolates of the fungus with reduced sensitivity to this fungicide appeared in Saga Prefecture of Japan and were regarded as a potential threat to rice protection by carpropamid. The cause of the resistance has been identified genetically as a point mutation resulting in the Val75Met change in scytalone dehydratase, the primary target of the fungicide. We constructed an overexpression system of the variant enzyme and characterized the kinetics in the catalysis and the inhibition by carpropamid isomers. The variant enzyme retained a significant level of enzymatic activity. Inhibition of the variant enzyme by carpropamid was more than 200-fold reduced in comparison with that of the wild-type. Based on the results, here we propose possible mechanisms of the carpropamid-resistance of the variant enzyme in retaining the normal enzymatic activity.