Masataka Takekoshi

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. This was manifested by the appearance of nitroblue tetrazolium-positive cells together with CD38 expression and c-Myc down-regulation. We determined the molecular mechanisms underlying early stages of signal transduction. rGL-7 treatment induced rapid tyrosine phosphorylation of Src family protein kinases Lyn and Hck. Reduction of endogenous cholesterol after application of methyl-β-cyclodextrin suppressed rGL-7-stimulated tyrosine phosphorylation. Phosphorylated proteins and PhGlc colocalized in the Triton X-100 insoluble, light buoyant density fraction after sucrose gradient ultracentrifugation of HL60 cell lysates. This suggests PhGlc-based microdomain is involved in GL-7 signaling. Ligation of known components of microdomains, such as sphingomyelin and ganglioside GM1, with corresponding antibodies failed to induce differentiation and tyrosine phosphorylation. These results show that PhGlc constitutes a previously undescribed lipid signaling domain, and the glucose residue of PhGlc is critical for organization of the carbohydrate-dependent signaling domain involved in cellular differentiation of HL60 cells.

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. We describe the cloning, using the phage display system, of a recombinant human Fab fragment against HCMV. A phage display library with 4 x 10(6) clones was panned three times against lysates of HCMV-infected cells, and screened by ELISA. Of six antigen-binding clones, one monoclonal antibody reacted strongly to HCMV. In immunostaining analysis, this Fab was able to stain HCMV-infected cells from 24 h post-infection (pi) through to 96 h pi, but not at 6 h pi. In the presence of cytosine arabinoside, HCMV-infected cells were not stained, even at 24 h pi. These results indicate that an HCMV protein that was recognized by the Fab was synthesized in the late phase of infection. In addition, this Fab exhibited neutralizing activity: at 1 microg/ml it reduced HCMV plaque formation by 50%. The Fab was able to neutralize three HCMV strains, but it did not neutralize HSV-1 or -2 infection.

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. The Fab fragment was produced by subjecting the heavy and light chain antibody genes of the TAPC301‐CL4 cell line to reverse transcription‐polymerase chain reaction, cloning the products in the plasmid vector pFab1‐His2 and introducing the plasmid into bacteria. Sequence analyses of the CL4Fab fragment revealed that the light and heavy chains belong to the Vk3a and VH3 groups of the immunoglobulin (Ig) family, respectively. An enzyme‐linked immunosorbent assay confirmed that specificity of the recombinant CL4Fab antibody against HBsAg was the same as that of the parental MAb. Flow cytometric analysis using PLC/PRF/5 (Alexander) cells, which express HBsAg, showed the reactivities of the CL4MAb and CL4Fab antibody were the same. These results suggest that the recombinant CL4Fab antibody produced by Escherichia coli using the new vector‐primer system developed for human IgG Fab fragments has a very high affinity for the HBsAg and may be useful clinically. A source for generation of human MAb for human therapy with very stable and specific expression was thus produced by isolating antibodies from EBV‐transformed cell lines. J. Med. Virol. 58:338–345, 1999.

A new human cytomegalovirus isolate has an invertible subsegment within its L component producing eight genome isomers

A HindIII cleavage map of the genome DNA of a new isolate of human cytomegalovirus (HCMV), strain Tanaka, was constructed by cosmid cloning and Southern blot hybridization of virion DNA. The genome was found to be unique in that its long (L) component was composed of two subsegments, L1 and L2, and subsegment L2 underwent inversion relative to L1 at high frequency. In addition to the normal inversions of the L and short (S) components, this produced eight genome isomers. The novel invertible subsegment was flanked by an inverted sequence distinct from the inversion-specific a sequence present in the terminal and junction regions of the genome.

Inducible expression of a foreign gene inserted into the human cytomegalovirus genome

We previously described the insertion of a foreign gene into a non-essential region of human cytomegalovirus (HCMV) by homologous recombination. Here we report insertion of the Escherichia coli lacZ gene downstream of the mouse metallothionein promoter into the HindIII-O region of HCMV by replacement-type recombination. Expression of the lacZ gene in the recombinant was independent of viral growth, but dependent on induction by heavy metals. Of several metals tested for beta-galactosidase induction and also for their toxicity to HEL cells, Zn was found to be the most suitable for use as an inducer. In HEL cells infected with the recombinant in the presence of 50 microsM-Zn, beta-galactosidase activity was maximal 3 days after infection, and reached levels 27 times higher than the value obtained in the absence of Zn.

Bacterial Expression of a Human Monoclonal Antibody-Alkaline Phosphatase Conjugate Specific for Entamoeba histolytica

ABSTRACT 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. The E. coli PhoA gene was fused to the 3′ terminus of the gene encoding the heavy-chain Fd region. The kappa and Fd genes from a previously prepared antibody clone, CP33, which is specific for the 260-kDa lectin of E. histolytica, were used as human antibody genes. When the fusion protein of CP33 and PhoA was incubated with paraformaldehyde-fixed trophozoites of E. histolytica and developed with a substrate, the trophozoites appeared to be stained. These results demonstrate the feasibility of bacterial expression of a human monoclonal antibody-PhoA conjugate specific for E. histolytica and that the antibody can be used to detect E. histolytica antigen without the use of chemically conjugated secondary antibodies.

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. Fab fragments produced in Escherichia coli were purified by a single step of nickel-chelate affinity chromatography via a hexa-histidine tag. The specificity of the Fabs was confirmed by competition ELISA, BIAcore analysis, and N-terminal amino acid sequencing. The binding constant for the interaction with HVR1 was 1.39 nM for Fab 30F1 and 3.96 nM for Fab 30F3. The HCV capture assay and inhibition of HCV adsorption test demonstrated that both Fabs had neutralizing activity. The data may be useful for designing immunological therapy of HCV.

Cleavage maps of human cytomegalovirus genome (strain towne) determined by the use of cosmid cloning system

SummaryVirion DNA of human cytomegalovirus strain Towne was partially digested with endonuclease Hind III and fragments larger than 29 kbp were ligated to cosmid pHC79. The whole viral DNA sequence has been cloned in large overlapping segments carried by 32 recombinant cosmid clones (a pIT series). A whole set of Hind III fragments has also been cloned (a pHI series). By using these, we have constructed corrected cleavage maps of strain Towne DNA for Hind III, Bam H I, EcoR I and Xba I.

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. It is probable that such a strict system would not be acceptable to Japan or to most developing countries. Many countries should use non-food crops for production of biopharmaceuticals and take on more simple systems. The new industry must develop strategies for promoting the benefits of transgenic plant-derived biopharmaceuticals on both the domestic and worldwide scales.

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

SummaryThe 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 kbXbaI-BamHI fragment between map unit 0.33 and 0.35 in aHindIII-D fragment located in a long unique region. Nucleotide sequencing showed that the 5.1 kb fragment contained three open reading frames corresponding to those of the genes for UL52, UL53 and UL54 (DNA polymerase gene), respectively, of strain AD169. The functions of UL52 and UL53 are unknown. Comparison of the DNA sequences of the 5.1 kb fragments of the wild-type and ts 256 mutant revealed two base changes within UL53 and UL54, respectively, which result in amino acid substitutions. The mutation in the UL54 gene was located within a distinct conserved region VI common to α-like DNA polymerases, suggesting that this base change would be responsible for DNA negative phenotype.