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Biomedical subjects

Y Kameoka

Publications and source records attributed to Y Kameoka.

At least 19 recordsLinked to original sources
PubMed2004

Production of virus-specific antiserum corresponding to sequences in the lactate dehydrogenase-elevating virus (LDV) ORF6 protein.

The elucidation of the antigenic structure of the envelope proteins of Arteriviridae which includes lactate dehydrogenase-elevating virus (LDV) will provide further understanding of a mechanism of strict host cell specificity. To analyze the linkage between LDV envelope proteins, M/VP-2 and VP-3, which may play an important role in viral infectivity, we generated specific antibody against M/VP-2 that has not been reported in previous studies. A synthetic polypeptide corresponding to the C-terminal region of LDV strain C (LDV-C) ORF6, which encodes M/VP-2, was chemically synthesized and coupled to keyhole limpet hemocyanin (KLH). The peptide was immunogenic in rabbits and induced antibody specific for viral protein. Western blotting and immunofluorescence analysis of virion M/VP-2 in infected macrophages showed that the antibody was able to react specifically with authentic virion protein. The immunoreactive antibody against LDV M/VP-2 described in this study will be useful for further studies of the specific roles of the envelope proteins in arterivirus assembly and infectivity.

Animals↗
PubMed2004

Analysis of protein expression by mammalian cell lines stably expressing lactate dehydrogenase-elevating virus ORF 5 and ORF 6 proteins.

Lactate dehydrogenase-elevating virus (LDV) has a strict species-specificity. Because only a subset of mouse primary macrophages have been identified that can support LDV replication in vitro, the precise molecular mechanism of viral entry and replication remains unclear. To analyze the LDV envelope proteins, which probably mediate viral attachment to the host cell, we developed a mammalian system for stable co-expression of LDV open reading frame (ORF) 5- and ORF 6-encoded proteins (ORF 5 and ORF 6 proteins), which correspond to envelope VP-3 and M/VP-2, respectively, and compared these expressed proteins to the native ones. Western blotting analysis combined with N-glycanase digestion revealed that ORF 5 and ORF 6 proteins were similar in size to native VP-3 and M/VP-2, and that ORF 5 protein was N-glycosylated, like the native VP-3. Immunofluorescence microscopy revealed that both ORF 5 and ORF 6 proteins were distributed throughout the cytoplasm and were colocalized in most cells. Moreover, ORF 5 protein was localized both in the perinuclear region and the Golgi complex and transported to the cell surface. This mammalian expression system in which the exogenously expressed proteins closely resemble the native proteins will provide the experimental basis for further studies of the interactions between LDV envelope proteins and host cells.

Animals↗
PubMed2003

Activated leucocytes express and secrete macrophage inflammatory protein-1alpha upon interaction with synovial fibroblasts of rheumatoid arthritis via a beta2-integrin/ICAM-1 mechanism.

OBJECTIVE: To examine the expression and regulation of chemotactic factor, macrophage inflammatory protein-1alpha (MIP-1alpha) by fibroblast-like synoviocytes (FLS), monocytes and polymorphonuclear neutrophils (PMN) isolated from the synovial fluid (SF) of rheumatoid arthritis (RA) patients. METHODS: Monocytes or PMN obtained from RA SF were co-cultured with unstimulated semiconfluent RA FLS. Culture supernatants were assayed for MIP-1alpha by enzyme-linked immunosorbent assay. The expression of MIP-1alpha mRNA and protein was also determined by Northern blot analyss and immunohistochemistry respectively. RESULTS: Interaction of activated leucocytes with FLS synergistically increased MIP-1alpha expression and secretion via a mechanism mediated by beta2-integrin/ intercellular adhesion molecule 1. CONCLUSION: MIP-1alpha expression within inflamed joints appears to be regulated not only by inflammatory cytokines but also by the physical interaction of activated leucocytes and FLS, and plays a crucial role in the progression and maintenance of RA synovitis.

Arthritis, Rheumatoid↗
PubMed2002

Polymerase chain reaction (PCR)-based molecular discrimination between Paragonimus westermani and P. miyazakii at the metacercarial stage.

The metacercariae of the lung flukes, Paragonimus westermani and P. miyazakii, are of known medical importance as the pathogens causing human paragonimiasis. They are both found in the same freshwater crab species in Japan and are morphologically quite similar. The aim of the present study was to establish molecular methods for accurate discrimination between individual metacercariae of the two species. In the first step, we amplified and sequenced the second internal transcribed spacer (ITS2) region of ribosomal DNA. Searches of nucleotide databases revealed that the ITS2 sequences generated from the metacercarial DNA were identical to those previously reported for the adults of the respective species. Utilizing a nucleotide difference between the two species, we have established two PCR-based techniques; PCR-restriction fragment length polymorphism and direct PCR-amplification using species-specific primers. Both techniques showed that the individual metacercariae of the two species could be unequivocally discriminated from one another. The present results suggest that the ITS2 region is useful for species discrimination irrespective of the life cycle stages of the lung flukes. Established techniques can thus be used for epidemiological investigations of the prevalence of human lung fluke metacercariae.

Animals↗
PubMed2001

Molecular characterization of Cryptosporidium isolates obtained from human and bovine infections in Japan.

Cryptosporidium oocysts, morphologically identified as Cryptosporidium parvum, were isolated from 22 human and 14 bovine cases in Japan, and were genotyped by means of a PCR/RFLP analysis of the polythreonine gene. DNA profiles of human isolates gave three distinct genotypes, namely an anthroponotic genotype 1, zoonotic genotype 2 and a new genotype. Isolates from bovine samples gave zoonotic genotype 2. The unusual genotype of Cryptosporidium was isolated from the feces of three immunologically healthy adults, and was further characterized by the sequence analysis of the 18S rRNA gene. The third genotype was identified as Crypto sporidium meleagridis, demonstrating that C. meleagridis, which occurs worldwide, has the potential to infect humans regardless of their immunological condition.

Animals↗
PubMed2001

Characterization of Sri Lanka rabies virus isolates using nucleotide sequence analysis of nucleoprotein gene.

Thirty-four suspected rabid brain samples from 2 humans, 24 dogs, 4 cats, 2 mongooses, I jackal and I water buffalo were collected in 1995-1996 in Sri Lanka. Total RNA was extracted directly from brain suspensions and examined using a one-step reverse transcription-polymerase chain reaction (RT-PCR) for the rabies virus nucleoprotein (N) gene. Twenty-eight samples were found positive for the virus N gene by RT-PCR and also for the virus antigens by fluorescent antibody (FA) test. Rabies virus isolates obtained from different animal species in different regions of Sri Lanka were genetically homogenous. Sequences of 203 nucleotides (nt)-long RT-PCR products obtained from 16 of 27 samples were found identical. Sequences of 1350 nt of N genes of 14 RT-PCR products were determined. The Sri Lanka isolates under study formed a specific cluster that included also an earlier isolate from India but did not include the known isolates from China, Thailand, Malaysia, Israel, Iran, Oman, Saudi Arabia, Russia, Nepal, Philippines, Japan and from several other countries. These results suggest that one type of rabies virus is circulating among human, dog, cat, mongoose, jackal and water buffalo living near Colombo City and in other five remote regions in Sri Lanka.

Animals↗
PubMed2000

Variation in sic gene encoding complement-inhibiting protein of Streptococcus pyogenes serotype M1 isolates in Japan.

DNA sequencing of the gene encoding a complement-inhibiting protein of Streptococcus pyogenes (streptococcal inhibitor of complement, Sic) was carried out on 49 strains of S. pyogenes serotype M1. Those strains were obtained from patients and asymptomatic carriers in Japan from 1969 to 1997 and had various pulsed-field gel electrophoresis (PFGE) patterns. Four identical polymorphic sites were found in the strains with the same PFGE pattern (Ia), but not in those giving the pattern IIa. The other identical sites were found in the strains with the PFGE pattern IIa, but not in those with the pattern Ia. These observations suggest that each of PFGE patterns was restricted to a set of variation in the sic gene.

Alleles↗
PubMed1999

In vitro processing of amyloid precursor protein by cathepsin D.

The formation of beta A4 amyloid in the brains of individuals with Alzheimer's disease requires the proteolytic cleavage of amyloid precursor protein. Several lines of evidence suggest that cathepsin D, the major lysosomal/endosomal aspartic protease, may be involved in this process. In this work, we used a sensitive in vitro method of detection to investigate the role of cathepsin D in the proteolytic processing of a 100-amino acid C-terminal fragment (C100) inclusive of beta A4 and cytoplasmic domain of APP. Digestion of C100 with cathepsin D resulted in cleavage at the amyloidogenic gamma-cleavage sites. This occurred preferentially at Thr43-Val44 and at Ala42-Thr43, generating full length beta A4 43 and beta A4 42 amyloid peptides, respectively. Cathepsin D was also found to cleave the substrate at the following nonamyloidogenic sites; Leu34-Met35, Thr48-Leu49 and Leu49-Val50. A high concentration of cathepsin D resulted in cleavage also occurring at Phe19-Phe20, Phe20-Ala21 and Phe93-Phe94 of the C100, suggesting that these sites are somewhat less sensitive to the action of cathepsin D. Digestion of C100 using different solublizing agents indicated that the cleavage of C100 by cathepsin D is greatly influenced by the structural integrity of the substrate. However, our results suggest that cathepsin D could generate the pathogenic beta A4 amyloid peptides from its precursor in vitro, which may indicate a role in the amyloidogenesis of Alzheimer's disease.

Alzheimer Disease↗
PubMed1998

RP58 associates with condensed chromatin and mediates a sequence-specific transcriptional repression.

An approximately 120-amino acid domain present generally at the NH2 termini, termed the POZ domain, is highly conserved in various proteins with zinc finger DNA binding motifs. We have isolated a novel protein sharing homology with the POZ domain of a number of zinc finger proteins, including the human BCL-6 protein. By using a binding site selection technique (CAST), a high affinity binding site of the protein was determined to be (A/C)ACATCTG(G/T)(A/C), containing the E box core sequence motif. The protein was shown to repress transcription from a promoter linked to its target sequences and was hence named RP58 (Repressor Protein with a predicted molecular mass of 58 kDa). Immunogold electron microscopic study revealed that almost all RP58 is localized in condensed chromatin regions. These observations demonstrate for the first time that a protein mediating a sequence-specific transcriptional repression associates with highly condensed chromatin. We suggest that RP58 may be involved in a molecular link between sequence-specific transcriptional repression and the organization of chromosomes in the nucleus.

Amino Acid Sequence↗
PubMed1998

Molecular cloning, structural characterization, and chromosomal mapping of the human LECT2 gene.

We originally isolated LECT2 (leukocyte cell-derived chemotaxin 2) as a 16-kDa secreted protein having a human neutrophil chemotactic activity, then cloned human and bovine LECT2 cDNAs and demonstrated the liver-specific expression of the protein. LECT2 is thought to be a multifunctional protein, because it was recently found to be identical to chondromodulin-II a growth stimulator of chondrocyte cells. We report here the cloning and the structural analysis of the human LECT2 gene. The gene spans approximately 8 kb and consists of four exons and three introns. Primer extension analysis revealed that several transcription initiation sites occur within 70-230 nucleotides upstream of the translation initiation codon. Several transcriptional control sequences relevant to the liver-specific expression have been identified at the 5' untranslated region of the human LECT2 gene. The human LECT2 gene was mapped to chromosome 5q31.1-q32 by fluorescence in situ hybridization. This region contains a cluster of cytokine genes including IL-4, IL-5, and IL-9.

Base Sequence↗
PubMed1998

Changes in serum levels of sialoglycoproteins in mice exposed to UV-B radiation.

In the acute phase reaction, hepatocytes in the liver are activated and increase the plasma levels of acute phase reactants. Our previous study has shown that plasma sialic acid, an acute phase reactant, was increased following exposure of mice to UV-B radiation. Plasma sialic acid is derived from many plasma components. To clarify the type of plasma sialic acid that is increased by exposure to UV-B radiation, we performed two-dimensional electrophoresis and staining for sialic acid. Consequently, the increases in haptoglobin and hemopexin were marked and 90% or more of the increased sialic acid was derived from these two glycoproteins after exposure to UV radiation. The increase in alpha1-acid glycoprotein levels was slight and did not contribute to the total increase in plasma sialic acid after exposure to UV radiation. Plasma levels of several proteins including antichymotrypsin (ACT), were reduced following exposure to UV radiation. The discrepancy between our results and published ones regarding ACT levels are discussed in terms of the type of cytokine.

Acute-Phase Proteins↗
PubMed1997

cDNA cloning and chromosomal mapping of genes encoding novel protein kinases termed PKU-alpha and PKU-beta, which have nuclear localization signal.

We have cloned cDNAs for novel serine/threonine protein kinases (PK), termed PKU-alpha and PKU-beta, by screening a bacteriophage expression library for kinase activity. Sequence analysis of PKU-alpha and PKU-beta genes revealed that their open reading frames (ORF) were 2151 and 2361 nucleotides (nt) encoding polypeptides of 717 and 787 amino acid (aa) residues, respectively. The deduced aa sequences of PKU-alpha and PKU-beta contained typical serine/threonine PK domains at the C-terminal region and were 86% identical to each other, indicating that they belong to the same PK family. Northern analysis reveals that they are expressed in nearly all human tissues and in cultured cells. The genes for PKU-alpha and PKU-beta were mapped to chromosome 17q23 and 8p12-p22, respectively, by fluorescence in situ hybridization. The proteins encoded by both cDNAs contain a putative nuclear localization signal (NLS) in their N-terminal region. These signals are likely to function in nuclear localization. Glutathione S-transferase (GST)-fusions to regions of PKU-alpha and beta containing the NLS were efficiently localized to the nucleus. In addition, PKU-beta transiently expressed in COS-1 cells was predominantly nuclear. PKU-alpha and PKU-beta differ: a consensus sequence for a nt binding motif is present near the NLS of PKU-beta. These results suggest that PKU-alpha and beta may phosphorylate serine and/or threonine residues on similar proteins, but their activities are regulated through distinct interactions with a nuclear component.

Amino Acid Sequence↗
PubMed1997

Nucleoprotein gene analysis of fixed and street rabies virus variants using RT-PCR.

A simple and rapid single-step reverse transcriptase-polymerase chain reaction (RT-PCR) was used to investigate the nucleoprotein (N) gene of 11 rabies viruses. A conserved set of RT-PCR primers was designed to amplify the most variable region in the N gene. N gene regions were amplified from 6 fixed laboratory viruses, 4 street viruses from dogs in Thailand, and a horse in Zambia. Sequences of the amplified products, together with the database of 91 additional sequences, were analyzed by using PILEUP program of the GCG package. The rabies viruses grouped into at least 9 distinct clusters by < 90% nucleotide similarity of the N gene region: I (4 isolates, USA), II (2 isolates, South America), III (3 isolates, Africa), IV (52 strains, Europe, Middle East, Africa and South America), V (16 isolates, North America and Arctic), VI (17 isolates, Africa), VII (1 isolate, Africa), VIII (6 isolates, Thailand and Malaysia) and IX (1 isolate, Sri Lanka). A unique group of rabies viruses from Thailand and clusters of isolates corresponding to their geographic origin also were determined. The simple and rapid single-step RT-PCR proved to be useful for identifying rabies viruses, and for grouping the viruses into clades by sequence analysis.

Animals↗
PubMed1997

A novel cDNA clone of Schistosoma japonicum encoding the 34,000 Dalton eggshell precursor protein.

A cDNA clone encoding the 34 kDa eggshell protein of Schistosoma japonicum was isolated from an adult female cDNA library with a rabbit antiserum raised against the 34 kDa female worm fraction. A 230 bp-insert of this clone (Sj23A) was introduced in frame into the expression plasmid vector, pMAL-c2, and the recombinant fusion protein of the Sj23A transiation product was induced in Escherichia coli. The antiserum raised against the recombinant protein reacted only with the native 34 kDa protein of mature female worms, which localized in the vitelline cells of the vitelline glands. By reverse transcription-polymerase chain reaction (RT-PCR) analysis, it was found that the gene corresponding to the Sj23A was expressed exclusively in mature female worms. The clone Sj23A showed a high degree of homology to the genes for the eggshell precursor proteins of Fasciola hepatica. At the deduced polypeptide level, the Sj23A also had similarities with the F. hepatica-protein sequence, the amino acid composition [high glycine (16%), lysine (12%) and tyrosine (11%)] and the presence of tyrosine residues flanked by glycine. The clone Sj23A also shared an extensive sequence homology with 3 S. mansoni expression sequence tags (ESTs). The present results suggest that the protein encoded by the female-specific Sj23A gene of S. japonicum is widely conserved in trematodes and plays a significant role as a precursor involved in eggshell formation.

Amino Acid Sequence↗
PubMed1996

Human rasGTPase-activating protein (human counterpart of GAP1m): sequence of the cDNA, primary structure of the protein, production and chromosomal localization.

When cDNA encoding rat rasGTPase-activating protein (rat GAP1m) was used as a probe, two partial cDNA clones of a human counterpart of rat GAP1m were isolated from a cDNA library derived from growth-arrested normal human ectocervical epithelial cells. One clone was found to be a cDNA of premature mRNA with two introns. A complete cDNA of human GAP1m was constructed by a series of reverse transcription-polymerase chain reaction (RT-PCR) using total RNA from human epidermoid carcinoma A431 cells. Human GAP1m shows 87.7% nucleotide identity to rat GAP1m in open reading frame and encodes an 850-amino acid protein that shows 89.2% identity to rat GAP1m. A 100-kDa protein was detected in A431 cells by Western blotting with anti-rat GAP1m antibody. The human GAP1m gene was mapped to chromosome 3q24-q26.

Animals↗
PubMed1996

Application of long PCR method of identification of variations in nucleotide sequences among varicella-zoster virus isolates.

Restriction fragment length polymorphism (RFLP) analysis of whole viral DNA of varicella-zoster virus (VZV) requires the time-consuming and laborious preparation of a large amount of purified viral DNA. RFLP analysis of small DNA fragments amplified by PCR was developed as an alternative method. However, its use has been limited because of the small number of variations in VZV. To overcome these drawbacks and to identify variations in VZV, we developed an RFLP analysis method combined with the long PCR method which has recently been developed for the amplification of DNA fragments between 5 and 35 kb in length. We amplified three DNA regions ranging from 6.8 to 11.4 kb and demonstrated that RFLP analyses of these regions allowed for the classification of 40 VZV isolates in Japan into 17 groups. One-fourth of the isolates contained a nucleotide difference of C versus T, which abolished the StyI site at position 76530; this alteration was linked to the reported PstI site polymorphism at position 69349 (nucleotide positions are based on those of strain Dumas). Nucleotide sequence variation in the examined regions among VZV isolates in Japan was estimated at roughly less than 0.05%, confirming the previously proposed idea that VZV is genetically stable and not highly diversified. Our method will be useful for studies of the molecular epidemiology of VZV.

Adolescent↗