Hisataka Ohta

Molecular and biological characterization of fusion regulatory proteins (FRPs): anti-FRP mAbs induced HIV-mediated cell fusion via an integrin system.

Anti‐FRP mAbs induced polykaryocyte formation of U2ME‐7 cells (CD4+U937 cells transfected with the HIV gp160 gene). Anti‐FRP‐1 mAb immunoprecipitated gp80‐85, gp120 and homodimers of these peptides, and anti‐FRP‐2 mAb reacted with gp135 identically to the alpha 3 subunit of integrin. Both anti‐FRP‐1 and anti‐FRP‐2 mAb‐induced cell fusion was blocked by anti‐beta 1 integrin antibody, fibronectin or inhibiting anti‐FRP‐1 antibody. Therefore, anti‐FRP mAbs were thought to induce the fusion via an integrin system(s). FRP‐mediated fusion was temperature, cytoskeleton, energy and Ca2+ dependent. These experiments showed a possible regulatory function of cell fusion by an integrin system(s).

Molecular cloning and expression of the cDNA coding for a new member of the S100 protein family from porcine cardiac muscle

We isolated a new calcium‐binding protein from porcine cardiac muscle by calcium‐dependent hydrophobic and dye‐affinity chromatography. It showed an apparent molecular weight of 11 000 on SDS‐PAGE. Amino acid sequence determination revealed that the protein contained two calcium‐binding domains of the EF‐hand motif. The cDNA gene coding for this protein was cloned from the porcine lung cDNA library. Sequence analysis of the cloned cDNA showed that the protein was composed of 99 amino acid residues and its molecular weight was estimated to be 11 179. Immunological and functional characterization showed that the recombinant S100C protein expressed in Escherichia coli was identical to the natural protein. Homologies to calpactin light chain, S100α and β protein were 41.1%, 40.9% and 37.5%, respectively. The protein was expressed at high levels in lung and kidney, and low levels in liver and brain. The tissue distribution was apparently different from those of the other S100 protein family. These results indicate that this protein represents a new member of the S100 protein family, and thus we refer to it as S100C protein.

HN proteins of human parainfluenza type 4A virus expressed in cell lines transfected with a cloned cDNA have an ability to induce interferon in mouse spleen cells

Primary monkey kidney cells infected with human parainfluenza type 4A virus (HPIV-4A) were treated with various concentrations of formaldehyde. Formaldehyde (0.275%) treatment completely blocked virus production. However, when mouse spleen cells were cocultured with the fixed virus-infected cells, interferon was produced in the culture fluid. On the other hand, when mouse spleen cells were incubated with the fixed virus-infected cells in the presence of anti-HPIV-4A antiserum or a mixture of anti-HN protein monoclonal antibodies, interferon activity could scarcely be detected in the culture fluid. These findings indicated that the fixed virus-infected cells had an ability to induce interferon in mouse spleen cells and that the HN protein was related to interferon induction. Subsequently, a recombinant plasmid was constructed by inserting the cDNA of the HN gene of HPIV-4A into a pcDL-SR alpha expression vector. Mouse spleen cells produced interferon when cocultured with COS7 cells transfected with the recombinant plasmid, but did not when cocultured with COS7 cells transfected with the vector alone. Furthermore, we established HeLa cells constitutively expressing HPIV-4A HN (HeLa-4aHN cells) or F protein (HeLa-4aF cells). Type I (alpha/beta) interferon was detected in culture fluids of mouse spleen cells with HeLa-4aHN cells, but was not detected in those with HeLa-4aF cells. Therefore, it was concluded that the HN glycoproteins on the cell surface were sufficient for interferon induction to occur.

Sequence determination of the P gene of simian virus 41: presence of irregular deletions near the RNA-editing sites of paramyxoviruses

The complete nucleotide sequence of the P gene of simian virus 41 (SV41) was determined. The gene was found to be 1406 nucleotides long and to contain a relatively small open reading frame encoding a cysteine-rich V protein with a calculated M(r) of 24076. We have demonstrated that RNA-editing events occur in SV41 P gene transcripts and that the ratio of edited mRNAs to faithfully copied mRNA (P-mRNA:V-mRNA) is about 1:5 at either 24 or 40 h post-infection. The mRNA with two G insertions was capable of encoding a P protein of 395 amino acids with a predicted M(r) of 41,992. A kinetic study of P and V proteins by Western blot analysis showed that in virus-infected cells the amounts of both proteins were almost equal although the V-mRNA was considerably more abundant than the P-mRNA. Alignment of the SV41 P and V proteins with those of nine other paramyxoviruses demonstrated that irregular gaps were present around the RNA-editing sites.

Sequence analyses of human parainfluenza virus type 4A and type 4B fusion proteins

cDNAs encoding human parainfluenza virus type 4A and type 4B (hPIV-4A and -4B) fusion (F) proteins were cloned and sequenced. The predicted amino acid sequences of the F proteins had similar characteristic traits to those reported for the F proteins of other paramyxoviruses. They were more closely related to the F proteins of simian virus 5 (SV5), mumps virus (MuV), hPIV-2 and Newcastle disease virus (NDV) than to the F proteins of hPIV-1, hPIV-3, Sendai virus (SV) and measles virus (MV). In addition, hPIV-4A, hPIV-4B, SV5 and MuV shared a common feature of genomic organization: there was a small ORF between the F and haemagglutinin-neuraminidase (HN)-coding sequences, implying a common ancestry.

Selective low level of protein kinase C isozyme in a tumor promoter-dependent mouse leukemia cell line.

It was found that the activity of protein kinase C in the tumor promoter-dependent cell line (A65T) was significantly lower than that in the independent cell line (A65IND) which was mutated from the dependent cell line. On the other hand, there was no significant difference between these cell lines with regard to cAMP-dependent protein kinase activity. It was found that the maximal binding capacity of [20-3H]phorbol-12,13-dibutyrate of the tumor promoter-dependent cells is lower than that of the independent cells with similar affinities of the two cell lines. Moreover, we found that the level of immunoreactive antigen with monoclonal antibody for type III of protein kinase C in A65T cells was significantly lower than that in the A65IND cells. Thus, this selective lower level of type III of protein kinase C in A65T cells, as compared with A65IND cells means that this difference may be linked to its tumor promoter-dependent cell proliferation.