Degranulation and expression of cytokines were modulated by diazinon in activated mast cells.

Diazinon is an organophosphorus (OP) insecticides used in agriculture, home gardening and indoor pest control in Japan. It can activate macrophages and induce pro-inflammatory responses and has been reported to cause airway hyper-reactivity, suggesting the possibility of asthma exacerbation from exposure to OP insecticides. Despite the correlation between insecticide use and the pathogenesis of allergic diseases, there have been no reports on the effects of diazinon on mast cell function.

Decrease in cholesterol in the cell membrane is essential for Nrf2 activation by quercetin.

Quercetin is a flavonoid with various cytoprotective effects. We previously reported that quercetin exerts anti-allergic, anti-oxidative, and anti-fibrotic activities via the induction of heme oxygenase (HO)-1. However, the mechanisms by which quercetin induces HO-1 to exhibit cytoprotective effects are poorly understood.

Inhibition of HIV replication by a CD4-reactive Fab of an IgM clone isolated from a healthy HIV-seronegative individual.

HIV replication is restricted by some anti-CD4 mouse mAb in vitro and in vivo. However, a human monoclonal anti-CD4 Ab has not been isolated. We screened EBV-transformed peripheral B cells from 12 adult donors for CD4-reactive Ab production followed by functional reconstitution of Fab genes.

Production of Fab fragment corresponding to surface protein antigen of Streptococcus mutans serotype c-derived peptide by Escherichia coli and cultured tobacco cells.

The cDNA of a mouse Fab fragment was cloned from a hybridoma cell line that produces a mouse monoclonal antibody, KH5, that reacts with the peptide fragment of the surface protein antigen of Streptococcus mutans serotype c (PAc). After transfection with cDNA, recombinant Fab fragments were produced by Escherichia coli (T15 Fab) and cultured tobacco cells (X253 and X262 Fabs). The antipeptide activities of T15 and X253 were similar to that of KH5.

Immune deficiency enhances expression of recombinant human antibody in mice after nonviral in vivo gene transfer.

A cDNA encoding human antibody against hepatitis B virus was expressed in normal and severe combined immune deficiency (SCID) mice to clarify whether or not host immune status affects circulating levels of the recombinant human antibody (RhAb) after nonviral in vivo gene transfer. For transferring genes, either electroporation (EP) or hydrodynamics-based transfection (HD) was employed. The former was applied to the leg muscle to express the gene, while the latter primarily targeted foreign gene expression in the liver.

Production and characterization of recombinant human anti-HBs Fab antibodies.

Recombinant human Fab antibodies were generated with different reactivities against the hepatitis B virus surface (HBs) antigen. To isolate the antibodies, a method was used that combined transformation of human B cells by Epstein-Barr virus (EBV) infection with a primer-vector system developed for isolating DNA fragments of human Ig Fab portions. With this method, monoclonal and oligoclonal cell lines producing anti-HBs antibodies were established and three anti-HBs Fab antibodies were isolated from two of these cell lines.

Transgenic plant-derived pharmaceuticals - the practical approach?

Production of biopharmaceuticals in transgenic plants would involve the creation of a new industry. Those transgenic plants, including staple food crops, could provide many benefits to people all over the world. However, the new industry might require a strict regulation system.

Transgenic tobacco cells producing the human monoclonal antibody to hepatitis B virus surface antigen.

The recombinant human monoclonal antibody (MAb) against hepatitis B virus (HBV) surface antigen (HBsAg) was expressed in tobacco suspension cultures. The parental CL4MAb was produced by the Epstein-Barr virus (EBV) transformed human cell line TAPC301-CL4. The CL4MAb cDNA was introduced into tobacco suspension cells by Agrobacterium mediated transformation.

Bacterial expression of a human monoclonal antibody-alkaline phosphatase conjugate specific for Entamoeba histolytica.

We previously produced human monoclonal antibody Fab fragments specific to Entamoeba histolytica in Escherichia coli. In order to use these Fab fragments for diagnostic purposes, an expression vector to produce a fusion protein of Fab and alkaline phosphatase (PhoA) in E. coli was designed and constructed.

Carbohydrate-dependent signaling from the phosphatidylglucoside-based microdomain induces granulocytic differentiation of HL60 cells.

Glycosphingolipids form glycosphingolipid signaling microdomains. Here, we report an unrecognized type of phosphatidylglucoside (PhGlc)-based lipid microdomain in HL60 cells. Treatment of cells with rGL-7, which preferentially reacts with PhGlc, induced differentiation of HL60 cells.

Recombinant antibody Fab against the hypervariable region 1 of hepatitis C virus blocks the virus adsorption to susceptible cells in vitro.

Antibodies against hypervariable region 1 (HVR1) of hepatitis C virus (HCV) are putatively considered to be neutralizing. We previously found that monoclonal antibodies (mAbs) (30F1 and 30F3) against the HVR1 of HCV neutralize HCV in vitro. To develop potentially therapeutic molecules against HCV, we cloned cDNAs of antibody Fab fragments from the mouse hybridoma cells secreting these two mAbs.

Cloning and expression of human anti-tumor necrosis factor-alpha monoclonal antibodies from Epstein-Barr virus transformed oligoclonal libraries.

Peripheral blood was obtained from a healthy human volunteer and transformed with Epstein-Barr virus (EBV). This produced an oligoclonal cell library in culture medium that was screened by ELISA for anti-human tumor necrosis factor-alpha (TNFalpha) activity. RNA from two positive clones was applied to RT-PCR using antibody-specific primers, and the light (kappa and lambda) and heavy chain genes (gamma and mu) were cloned into the plasmid vector pFab1-His2.

Bacterial expression of a human recombinant monoclonal antibody fab fragment against hepatitis B surface antigen.

The Fab fragment was cloned from the monoclonal cell line TAPC301-CL4, which was produced using the Epstein-Barr virus (EBV) transformation method. This cell line produces a human monoclonal antibody (CL4MAb) against the hepatitis B surface antigen (HBsAg). This MAb was shown to have hepatitis B virus (HBV) neutralizing activity in chimpanzees.

Preparation of recombinant human monoclonal antibody Fab fragments specific for Entamoeba histolytica.

Genes coding for human antibody Fab fragments specific for Entamoeba histolytica were cloned and expressed in Escherichia coli. Lymphocytes were separated from the peripheral blood of a patient with an amebic liver abscess. Poly(A)+ RNA was isolated from the lymphocytes, and then genes coding for the light chain and Fd region of the heavy chain were amplified by a reverse transcriptase PCR.

Bacterial expression of a neutralizing mouse monoclonal antibody Fab fragment to a 150-kilodalton surface antigen of Entamoeba histolytica.

A mouse monoclonal antibody (MAb) (EH3015, IgG1 with a K light chain) prepared by hybridoma technology recognizes a 150-kD surface antigen of Entamoeba histolytica and inhibits adherence and cytotoxicity of the ameba to mammalian cells. The genes encoding the light chain and the Fd region of the heavy chain of the MAb were cloned and expressed in Escherichia coli. The plasmid used was designed for the expression of Fab with a hexa-histidine tag in the periplasmic space.

Use of a glycoprotein gB promoter for expression of genes inserted into the human cytomegalovirus genome.

We attempted to utilize the human cytomegalovirus (HCMV) as an expression vector by replacing the dispensable genes of the viral genome with foreign genes. The selection of a promoter to be fused to the foreign gene is important to achieve a high expression rate in the recombinant virus. We selected the glycoprotein B (gB) promoter of HCMV as a target of analysis because gB is one of the most abundantly synthesized components in cell culture.

A micromethod for protein assay using a light microscope.

A micromethod which utilizes a protein-dye binding reaction on an acetate membrane and a light microscope with an automatic exposure instrument has been developed for measuring small amounts of protein from limited biological materials. After discoidal cellulose acetate membranes (2.0 mm diameter), which absorbed 0.

Human monoclonal anti-HCMV neutralizing antibody from phage display libraries.

Human cytomegalovirus (HCMV) infection in immunocompromised patients causes considerable morbidity and mortality. Although ganciclovir prophylaxis reduces the incidence of HCMV disease, severe side effects raise serious problems. Thus, the development of new strategies for prophylaxis are clearly needed, and human monoclonal antibodies offer a potential alternative.

[Neutralizing human antibody specific for human cytomegalovirus in E. coli expression system].

We isolated neutralizing human Fab fragment specific for human cytomegalovirus (HCMV) by phage display system. Fab libraries were constructed from peripheral lymphocyte of healthy individual. In several clones reacted for HCMV-infected HEL cells, one clone, designated 13-3, stained HCMV infected cells at 96 hrs post infection.

Purification and cDNA cloning of evolutionally conserved larval cuticle proteins of the silkworm, Bombyx mori.

A specific set of structural proteins termed larval cuticle proteins (LCPs) accumulates in integuments during larval development of the silkworm, Bombyx mori. Two major larval cuticle proteins, LCP17 and LCP22, were purified from the guanidine hydrochloride extract of the larval cuticle, and specific antibodies were raised against these proteins. Immunoblot analysis revealed that both LCPs are actively synthesized during larval intermolt stages, whereas the LCP17 epitope is also slightly but significantly detectable in pupal integuments.

Identification of mutation sites of a temperature-sensitive mutant of HCMV DNA polymerase activity.

The human cytomegalovirus (strain Towne) temperature-sensitive mutant ts 256 exhibits a virus specific DNA polymerase-negative phenotype. The position of the mutation of ts 256 was determined by three step marker-rescue assays to be within a 5.1 kb XbaI-BamHI fragment between map unit 0.