Development of high-throughput screening system by single-cell reaction using microchamber array chip

To demonstrate the practical use of a novel high-throughput screening system by a single cell reaction for the rapid screening of combinatorially mutated beta-glucosidase 1 from Aspergillus oryzae, a yeast cell chip microchamber array in combination with yeast cell surface engineering was successfully developed.     

Enhanced antibody-dependent cellular phagocytosis by chimeric monoclonal antibodies with tandemly repeated Fc domains

We previously reported that chimeric monoclonal antibodies (mAbs) with tandemly repeated Fc domains, which were developed by introducing tandem repeats of Fc domains downstream of 2 Fab domains, augmented binding avidities for all Fcγ receptors, resulting in enhanced antibody (Ab)-dependent cellular cytotoxicity. Here we investigated regarding Ab-dependent cellular phagocytosis (ADCP) mediated by these chimeric mAbs, which is considered one of the most important mechanisms that kills tumor cells, using two-color flow cytometric methods. ADCP mediated by T3-Ab, a chimeric mAb with 3 tandemly repeated Fc domains, was 5 times more potent than that by native anti-CD20 M-Ab (M-Ab hereafter). Furthermore, T3-Ab-mediated ADCP was resistant to competitive inhibition by intravenous Ig (IVIG), although M-Ab-mediated ADCP decreased in the presence of IVIG. An Fcγ receptor-blocking study demonstrated that T3-Ab mediated ADCP via both FcγRIA and FcγRIIA, whereas M-Ab mediated ADCP exclusively via FcγRIA. These results suggest that chimeric mAbs with tandemly repeated Fc domains enhance ADCP as well as ADCC, and that Fc multimerization may significantly enhance the efficacy of therapeutic Abs.     

Study on detection of allergenic substances (egg and milk) in processed meat products and frozen foods

Allergenic substances (egg and milk) were measured in processed meat products and frozen foods of which the milk and egg ingredient ratios and manufacturing processes were clearly identified. An ovomucoid ELISA kit gave good results in the detection of egg ingredients. With an ovalbumin ELISA kit and egg ELISA kit, results for 6 of 16 foods were negative, but better results were obtained when a protein denaturant was added to the extraction buffer. Occurrence of contamination in the egg ingredient manufacturing line was confirmed in frozen foods. For milk ingredients, good results were obtained by ELISA using two kinds of kits (a casein ELISA kit and milk ELISA kit) described as being suitable for detection of allergenic substances. Allergenic substances were identified by Western blot analysis in all of the foods containing egg and milk ingredients.     

Relationship between Pectic Substances and Strand Separation of Cooked Spaghetti Squash--Part 2. Changes in Firmness,Histological Structure and Pectic Substances during Soaking in Pectin Extractants

The flesh of spaghetti squash separates into strands when cooked. The purpose of this paper is to investigate the cause of strand separation （during cooking） by soaking for 24 h at 35 ℃ in solutions with three kinds of pectin extractant. The changes in strand separation, firmness, histological structure and the pectin of flesh during soaking in 0.01 N HCI solution （pH 2.0）, 0.035 M ammonium oxalate solution （pH 4.0） or 2% sodium hexametaphosphate solution （pH 4.0） were investigated. When flesh was soaked in the HCI solution, the separation into strands and removal of calcium and magnesium were greater than that soaked in other pectin extractants. High methoxyl pectin was extracted by soaking in HC1 solution （pH 2.0） due to removal of polyvalent cations. This result shows that high methoxyl pectin glues strands together in the flesh of spaghetti squash. The shape of the cells which constituted strands was round; on the other hand, that of cells surrounded strands was elongated. When cooked in boiling water or soaked at pH 2.0, the shape of the former cells was maintained, but the latter cells, which contributed to adhesion between strands, broke down. Thus, the flesh separated into strands. When flesh was boiled for 15-30 min, pectin degraded and dissolved in the cooking solution; consequently, the flesh separated into strands and also the middle lamella of cell walls of strands separated. However, pectin remaining in strands maintained their crispness.     

Secular variation of BHC in the paper of books

In order to consider the distribution, migration and accumulation of the organochlorine compounds in cities, hexachlorocyclohexane (BHC) levels were measured in the paper of books. BCH was always present in books published between 1927 and 1972; the highest concentration was observed in the books published in 1946 but it was not detected (below 1 ng/g) in the paper of books published in 1973. The secular variation of BHC content in the paper of books is considered as an indicator for the use of BHC in Japan.     

Formation of magnetite by bacteria and its application

Magnetic particles offer high technological potential since they can be conveniently collected with an external magnetic field. Magnetotactic bacteria synthesize bacterial magnetic particles (BacMPs) with well-controlled size and morphology. BacMPs are individually covered with thin organic membrane, which confers high and even dispersion in aqueous solutions compared with artificial magnetites, making them ideal biotechnological materials. Recent molecular studies including genome sequence, mutagenesis, gene expression and proteome analyses indicated a number of genes and proteins which play important roles for BacMP biomineralization. Some of the genes and proteins identified from these studies have allowed us to express functional proteins efficiently onto BacMPs, through genetic engineering, permitting the preservation of the protein activity, leading to a simple preparation of functional protein-magnetic particle complexes. They were applicable to high-sensitivity immunoassay, drug screening and cell separation. Furthermore, fully automated single nucleotide polymorphism discrimination and DNA recovery systems have been developed to use these functionalized BacMPs. The nano-sized fine magnetic particles offer vast potential in new nano-techniques.     

Specific determination of bromate in bread by ion chromatography with ICP-MS

A sensitive method for detecting bromate in bread by ion chromatography with inductively-coupled plasma mass spectrometry (IC/ICP-MS) was developed. Bromate was extracted from bread with water. The clean-up procedure included a 0.2 micron filter, a C18 cartridge for defatting, a silver cartridge to remove halogen anions, a centrifugal ultrafiltration unit to remove proteins, and a cation-exchange cartridge to remove silver ions. A 500 microL sample solution was applied to IC/ICP-MS. The detection limit and the quantitation limit of bromate in the solution were 0.3 ng/mL and 1.0 ng/mL, expressed as HBrO3, respectively, which corresponded to 2 ng/g and 5 ng/g, respectively, in bread. Recovery of bromate was about 90%, and the CV was about 2%. Based on the detection limit in solution and recovery from bread, the detection limit of bromate in bread was estimated to be 2 ng/g.     

Content composition and antioxidant activity of isoflavones in commercial and homemade soymilk and tofu

BACKGROUND: Isoflavones, found in soymilk and tofu, are one of the phytochemicals in soy‐based products that may promote good health. Homemade tofu and various homemade soymilk samples were made using different soaking, grinding, and cooking methods. The homemade samples were compared to commercial tofu and soymilk for total isoflavone content and composition as well as their antioxidant capacity. All samples were freeze‐dried and extracted with a 58% acetonitrile solution which was subsequently used to determine the isoflavone content by reverse‐phase high‐performance liquid chromatography. The antioxidant activity of extracts was determined using a modified 2,2‐azino‐bis‐(3‐ethylbenzthiazoline‐6‐sulfonic acid) method and total antioxidant capacity was reported as ascorbic acid equivalents. RESULTS: The total isoflavone, aglycone, and antioxidant levels were significantly higher in homemade soymilk and tofu (1571 µg) than in commercial samples. Homemade soymilk made by the extended boiling method yielded the highest total isoflavone (2567 µg) and glucoside (1525 µg) content. A strong positive correlation was observed between the total isoflavone, aglycone conjugates, and genistein series concentration and antioxidant capacity of soymilk. CONCLUSION: Increased moist heating time yielded the highest concentration of total isoflavones as well as aglycone conjugates and the genistein series. Increasing the duration of boiling can increase the isoflavone content of both homemade and commercial soymilk and tofu. Copyright © 2007 Society of Chemical Industry     

Effect of an additionally introduced degQ gene on di-d-fructofuranosyl 2,6′:2′,6 anhydride (DFA IV) production by recombinant Bacillus subtilis in a single culture production system

Di-d-fructofuranosyl 2,6′:2′,6 anhydride (DFA IV) was produced directly from sucrose using a single culture of recombinant Bacillus subtilis 168 carrying the levan fructotransferase (lft) gene. In this study, three plasmids carrying the degQ36 gene, which is a degQ allele of B. subtilis (degQ36) with a degQ36 mutation on its promoter, were constructed to overproduce intact DegQ in B. subtilis 168. The transformant B. subtilis/pHT-D36 (with the degQ36 gene) consumed sucrose and produced levan at a higher rate than B. subtilis/pHT43 (without the degQ36 gene). The transformant B. subtilis/pLFT-GD36, carrying the lft and degQ36 genes, also consumed sucrose at a higher rate and produced more DFA IV than B. subtilis/pLFT-G, carrying the lft but without the degQ36 gene. B. subtilis/pLFT-GD36 produced 43.5 g/l of DFA IV and consumed 240 g/l of sucrose (96% of added sucrose) by 72 h of cultivation, whereas B. subtilis/pLFT-G produced 23.4 g/l of DFA IV with 76.9 g/l of sucrose still remaining in the system. Sucrose-inducible expression vectors were also constructed, which made it possible to produce DFA IV without IPTG induction. Using these vectors, sucrose consumption rates were enhanced and DFA IV production was increased upon introduction of the degQ36 gene. From these results, it can be concluded that the additionally introduced regulatory gene, degQ, was able to stimulate sucrose conversion to levan, and therefore increased DFA IV production in this system.     

Conjugation of quantum dots and JT95 IgM monoclonal antibody for thyroid carcinoma without abolishing the specificity and activity of the antibody

Among the immunoglobulins, IgM class-antibodies are now considered to be potent immunological reagents for anticancer remedies. However, only a few reports are available about the effective labeling of IgM with enzymes, fluorescence, or other bioreactive reagents. Here, we report an effective application of luminescent semiconductive nanoparticles, quantum dots (QDs), as a labeling material of the IgM antibody. The CdSe carboxyl QDs were reacted with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfo- succinimide in 2-(morpholino) ethanesulfonic acid. The reacted QDs were then coupled to JT95 IgM antibody, which recognizes thyroid carcinoma associated antigen. The specificity and activity of the conjugates were tested by immunoblot, immunoquantitive assay and immunohistological imaging. The QDs were firmly conjugated with JT95 IgM monoclonal antibody. In immunoblot assay, QD-JT95 conjugates directly detected the target molecules without obstructing the binding site. In immunoquantitive assay, the conjugates could quantify the antigen in the range of 1.56-100 μg/mL. Also, QDs-labeled antibody detected the antigen on plasma membrane. Our results demonstrate that labeling of JT95 and other IgM class antibodies with QDs is feasible. This approach may be an important method for the medical application of IgM in the diagnosis and treatment of cancers.