Comprehensive study of domain rearrangements of single-chain bispecific antibodies to determine the best combination of configurations and microbial host cells.

Small bispecific antibodies (bsAbs) are important therapeutic molecules and represent the first bsAb format approved by the United States Food and Drug Administration. Diabody (Db), a small bsAb format, has four possible domain orders; we previously reported the differences in the expression levels and cancer growth inhibition effects upon rearranging the domain order of this format. However, there have been no comprehensive reports on domain rearrangements of bispecific single-chain Db (scDb) and tandem single-chain Fv (taFv), which are widely used bsAb formats. In this study, we designed all possible domain orders for scDb and taFv (each with eight variants) with identical Fv pairs and individually expressed all 16 variants using Escherichia coli, Pichia pastoris, and Brevibacillus choshinensis. Comprehensive investigations showed that the intrinsic functions of the variants were similar to each other, regardless of the expression host system, but expression levels varied depending on the format as well as on the host cell. Among the 16 variants, we found a promising candidate that exhibited high activity and productivity. Furthermore, we determined that B. choshinensis is an attractive expression host because of its secretory production of recombinant proteins.

The production of ß-glucosidases by Fusarium proliferatum NBRC109045 isolated from Vietnamese forest.

Fusarium proliferatum NBRC109045 is a filamentous fungus isolated from Vietnamese forest due to high production of ß-glucosidases. Production of the enzyme was studied on varied carbon source based mediums. The highest activity was obtained in medium containing 1% corn stover + 1% wheat bran (3.31 ± 0.14 U/ml). It is interesting to note that glucose (0.69 ± 0.02 U/ml) gave higher activity and just followed by cellobiose among the di- and mono-saccharides, which is generally regarded as a universal repressor of hydrolases. We improved the zymogram method to prove that in response to various carbon sources, F. proliferatum could express various ß-glucosidases. One of the ß-glucosidases produced by F. proliferatum growing in corn stover + wheat bran based medium was partially purified and proved to have high catalytic ability.

5-Hydroxy-eicosapentaenoic acid is an endogenous GPR119 agonist and enhances glucose-dependent insulin secretion.

GPR119 is one of the G-protein-coupled receptors expressed in pancreatic ß-cells and intestinal endocrine cells. Since agonists to GPR119 stimulate glucose-dependent insulin secretion, GPR119 agonists are anticipated to promote anti-diabetic effects and control of glucose homeostasis. Here, we reported that an omega-3 unsaturated fatty acid metabolite, 5-hydroxy-eicosapentaenoic acid (5-HEPE), was a potent agonist for GPR119 and enhanced glucose-dependent insulin secretion. 5-HEPE stimulated cAMP accumulation in mouse MIN6 insulinoma cells and human HuTu80 intestinal adenocarcinoma cells. These effects were blunted by GPR119-specific siRNA. Recombinant GPR119 also responded to 5-HEPE as well as authentic agonists. Several previous reports have indicated the beneficial biological effects of omega-3 unsaturated fatty acids, and epidemiological studies have suggested that these fatty acids plays a protective role against diabetes. However, the molecular pharmacology and receptor identifications of omega-3 unsaturated fatty acids and their metabolites have not yet been well investigated. It is hoped that our findings will encourage novel investigations into the molecular relationships between omega-3 fatty acids and diabetes.

Establishment of a prediction method for premature rupture of membranes in term pregnancy using active ceruloplasmin in cervicovaginal secretion as a clinical marker.

AIM: Term pregnancy complicated by premature rupture of membranes (PROM) is thought to be associated in part with subclinical infection, and places mothers and neonates at an increased risk for several complications. Therefore, perinatal care would be greatly helped if a reliable clinical measure were available for predicting the incidence of PROM. METHODS: One hundred and ninety-six pregnant women who consented to enter this study were screened using a method developed to assess active ceruloplasmin in cervicovaginal secretion as a clinical marker for predicting the incidence of PROM. Cervicovaginal secretions were obtained from the cervical canal at about 36 weeks of pregnancy. The active ceruloplasmin level in the cervicovaginal secretion was measured using an original enzyme-linked immunoabsorbent assay. RESULTS: Of 196 women, 27 women (13.8%) developed PROM and 169 women (86.2%) did not develop PROM. Active ceruloplasmin in the cervicovaginal secretion was significantly higher in the PROM group than in the non-PROM group (P < 0.001). Analysis using receiver-operating characteristic curves showed that the active ceruloplasmin level (1420.0 ng/mL) proved to be the proper cut-off value to best predict the incidence of PROM. CONCLUSION: Active ceruloplasmin in the cervicovaginal secretion might be a reliable clinical marker for term PROM.

The effect of Au injection on the ceruloplasmin, metallothionein and 8-hydroxydeoxyguanosine of rat serum, kidney and liver.

The present study was carried out to investigate the effect of gold (Au) injection on copper (Cu) and two types of ceruloplasmin (Cp), total Cp (ID1) and active Cp (ID2), metallothionein (MT) in the serum, kidney and liver, and 8-hydroxydeoxyguanosine (8-OHdG) in the rat kidney. The Cu contents in sera and kidneys of Au-injected rats were 1.7 and 5.5 times higher than those in sera and kidneys of control rats, respectively. The most of Cu in the sera of the control rats or Au-injected rats were observed in the Cp fractions from a Sephacryl S-200 column. The Cu concentration in the Cp fractions was increased by Au injection. Significant increases of ID1 and ID2 were found in the sera of the control rats and Au-injected rats, while there was no significant difference in those concentrations of livers or kidneys between the control rats and Au-injected rats. Our results indicated that the most of Cp existed as active ID1. The immunoreactivity of 8-OHdG was located in the cortex of the Au-injected rat. These results indicated that the oxidative DNA damage occurred in the renal cortex of the Au-injected rat and the localization of DNA damage did not coincide with that of Cu-MT. These findings suggest that the oxidative DNA damage in the kidneys of rats injected with Au is associated with Cu except Cu-MT.