Biomedical subjects

Masanori Kasahara

Publications and source records attributed to Masanori Kasahara.

At least 19 recordsLinked to original sources

PubMed2007

Enhanced production of p24 Gag protein in HIV-1-infected rat cells fused with uninfected human cells.

Although many human molecules have been suggested to affect replication of human immunodeficiency virus type 1 (HIV-1), the distribution of such cofactors in human cell types is not well understood. Rat W31/D4R4 fibroblasts expressing human CD4 and CXCR4 receptors were infected with HIV-1. The provirus was integrated in the host genome, but only a limited amount of p24 Gag protein was produced in the cells and culture supernatants. Here we found that p24 production was significantly increased by fusing HIV-1-infected W31/D4R4 cells with uninfected human cell lines of T-cell, B-cell, or macrophage lineages. These findings suggest that human cellular factors supporting HIV-1 replication are distributed widely in cells of lymphocyte and macrophage lineages. We also examined whether the amount of p24 produced by rat-human hybrid cells was correlated with expression levels of specific human genes. The results suggested that HP68 and MHC class II transactivator (CIITA) might up- and down-regulate p24 production, respectively. It was also suggested that HIV-1 replication is affected by molecules other than those examined in this study, namely, cyclin T1, cyclin-dependent kinase 9, CRM1, HP68, and CIITA.

Animals↗

PubMed2007

Evidence for the involvement of testicular protein CRISP2 in mouse sperm-egg fusion.

CRISP2, originally known as Tpx-1, is a cysteine-rich secretory protein specifically expressed in male haploid germ cells. Although likely to be involved in gamete interaction, evidence for a functional role of CRISP2 in fertilization still remains poor. In the present study, we used a mouse model to examine the subcellular localization of CRISP2 in sperm and its involvement in the different stages of fertilization. Results from indirect immunofluorescence and protein extraction experiments indicated that mouse CRISP2 is an intraacrosomal component that remains associated with sperm after capacitation and the acrosome reaction (AR). In vitro fertilization assays using zona pellucida-intact mouse eggs showed that an antibody against the protein significantly decreased the percentage of penetrated eggs, with a coincident accumulation of perivitelline sperm. The failure to inhibit zona pellucida penetration excludes a detrimental effect of the antibody on sperm motility or the AR, supporting a specific participation of CRISP2 at the sperm-egg fusion step. In agreement with this evidence, recombinant mouse CRISP2 (recCRISP2) specifically bound to the fusogenic area of mouse eggs, as previously reported for rat CRISP1, an epididymal protein involved in gamete fusion. In vitro competition investigations showed that incubation of mouse zona-free eggs with a fixed concentration of recCRISP2 and increasing amounts of rat CRISP1 reduced the binding of recCRISP2 to the egg, suggesting that the proteins interact with common complementary sites on the egg surface. Our findings indicate that testicular CRISP2, as observed for epididymal CRISP1, is involved in sperm-egg fusion through its binding to complementary sites on the egg surface, supporting the idea of functional cooperation between homologous molecules to ensure the success of fertilization.

Animals↗

PubMed2006

Cooperation of multiple chaperones required for the assembly of mammalian 20S proteasomes.

The 20S proteasome is a catalytic core of the 26S proteasome, a central enzyme in the degradation of ubiquitin-conjugated proteins. It is composed of 14 distinct gene products that form four stacked rings of seven subunits each, alpha(1-7)beta(1-7)beta(1-7)alpha(1-7). It is reported that the biogenesis of mammalian 20S proteasomes is assisted by proteasome-specific chaperones, named PAC1, PAC2, and hUmp1, but the details are still unknown. Here, we report the identification of a chaperone, designated PAC3, as a component of alpha rings. Although it can intrinsically bind directly to both alpha and beta subunits, PAC3 dissociates before the formation of half-proteasomes, a process coupled with the recruitment of beta subunits and hUmp1. Knockdown of PAC3 impaired alpha ring formation. Further, PAC1/2/3 triple knockdown resulted in the accumulation of disorganized half-proteasomes that are incompetent for dimerization. Our results describe a cooperative system of multiple chaperones involved in the correct assembly of mammalian 20S proteasomes.

Cell Line↗

PubMed2006

Structural diversity of the hagfish variable lymphocyte receptors.

Variable lymphocyte receptors (VLRs) are recently discovered leucine-rich repeat (LRR) family proteins that mediate adaptive immune responses in jawless fish. Phylogenetically it is the oldest adaptive immune receptor and the first one with a non-immunoglobulin fold. We present the crystal structures of one VLR-A and two VLR-B clones from the inshore hagfish. The hagfish VLRs have the characteristic horseshoe-shaped structure of LRR family proteins. The backbone structures of their LRR modules are highly homologous, and the sequence variation is concentrated on the concave surface of the protein. The conservation of key residues suggests that our structures are likely to represent the LRR structures of the entire repertoire of jawless fish VLRs. The analysis of sequence variability, prediction of protein interaction surfaces, amino acid composition analysis, and structural comparison with other LRR proteins suggest that the hypervariable concave surface is the most probable antigen binding site of the VLR.

Amino Acid Sequence↗

PubMed2006

MHC class I-like MILL molecules are beta2-microglobulin-associated, GPI-anchored glycoproteins that do not require TAP for cell surface expression.

MILL (MHC class I-like located near the leukocyte receptor complex) is a family of MHC class I-like molecules encoded outside the MHC, which displays the highest sequence similarity to human MICA/B molecules among known class I molecules. In the present study, we show that the two members of the mouse MILL family, MILL1 and MILL2, are GPI-anchored glycoproteins associated with beta2-microglobulin (beta2m) and that cell surface expression of MILL1 or MILL2 does not require functional TAP molecules. MILL1 and MILL2 molecules expressed in bacteria could be refolded in the presence of beta2m, without adding any peptides. Hence, neither MILL1 nor MILL2 is likely to be involved in the presentation of peptides. Immunohistochemical analysis revealed that MILL1 is expressed in a subpopulation of thymic medullary epithelial cells and a restricted region of inner root sheaths in hair follicles. The present study provides additional evidence that MILL is a class I family distinct from MICA/B.

Animals↗

PubMed2006

Variable domains in hagfish: NICIR is a polymorphic multigene family expressed preferentially in leukocytes and is related to lamprey TCR-like.

The jawless vertebrates, represented by hagfish and lampreys, are the most advanced animals that apparently lack T cell and B cell receptors. As such, they offer unique opportunities for understanding the evolution of antigen receptors and variable (V)-type immunoglobulin (Ig)-like domains. In the present study, we describe four hagfish Ig superfamily (IgSF) members carrying V-type domains. None of them appeared to have direct counterparts in jawed vertebrates, indicating that many IgSF molecules have either evolved independently in jawed and jawless vertebrates or diverged to the extent that clear homology is no longer recognizable. One of the members encoded a molecule closely related to the previously described membrane protein designated novel ITAM (immunoreceptor tyrosine-based activation motif)-containing IgSF receptors (NICIR). We show here that NICIR is a polymorphic multigene family with at least three members and is expressed predominantly in peripheral blood leukocytes. Phylogenetic analysis indicates that among known proteins, NICIR is most closely related to the lamprey molecule recently proposed to be a potential ancestor of T cell receptors.

Amino Acid Sequence↗

PubMed2006

Role of neuronal interferon-gamma in the development of myelopathy in rats infected with human T-cell leukemia virus type 1.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of not only adult T-cell leukemia but also HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Among the rat strains infected with HTLV-1, chronic progressive myelopathy, named HAM rat disease, occurs exclusively in WKAH rats. In the present study, we found that HTLV-1 infection induces interferon (IFN)-gamma production in the spinal cords of HAM-resistant strains but not in those of WKAH rats. Neurons were the major cells that produced IFN-gamma in HTLV-1-infected, HAM-resistant strains. Administration of IFN-gamma suppressed expression of pX, the gene critically involved in the onset of HAM rat disease, in an HTLV-1-immortalized rat T-cell line, indicating that IFN-gamma protects against the development of HAM rat disease. The inability of WKAH spinal cord neurons to produce IFN-gamma after infection appeared to stem from defects in signaling through the interleukin (IL)-12 receptor. Specifically, WKAH-derived spinal cord cells were unable to up-regulate the IL-12 receptor beta2 gene in response to IL-12 stimulation. We suggest that the failure of spinal cord neurons to produce IFN-gamma through the IL-12 pathway is involved in the development of HAM rat disease.

Animals↗

PubMed2005

CD4+/CD8+ macrophages infiltrating at inflammatory sites: a population of monocytes/macrophages with a cytotoxic phenotype.

We found a population of nonlymphoid cells expressing both CD4 and CD8 in peripheral blood mononuclear cells (PBMCs) of human T-cell leukemia virus type-I pX transgenic rats with autoimmune diseases. These cells, which showed a monocytic phenotype, were also found in wild-type rats, and their number increased by adjuvant-assisted immunization. GM-CSF increased the number of these double-positive (DP) monocytes in PBMCs. Consistent with the idea that DP monocytes differentiate into DP macrophages at sites of inflammation, we found infiltration of DP macrophages at the site of myosin-induced myocarditis in wild-type rats; these cells exhibited a T-helper 1 (Th1)-type cytokine/chemokine profile and expressed high levels of Fas ligand, perforin, granzyme B, and NKR-P2 (rat orthologue of human NKG2D). Adoptive transfer of GFP-positive spleen cells confirmed hematogenous origin of DP macrophages. DP monocytes had a cytotoxic phenotype similar to DP macrophages, indicating that this phenotypic specialization occurred before entry into a tissue. In line with this, DP monocytes killed tumor cells in vitro. Combined evidence indicates that certain inflammatory stimuli that induce GM-CSF trigger the expansion of a population of DP monocytes with a cytotoxic phenotype and that these cells differentiate into macrophages at inflammatory sites. Interestingly, human PBMCs also contain DP monocytes.

Adjuvants, Immunologic↗

PubMed2005

Chicken CD1 genes are located in the MHC: CD1 and endothelial protein C receptor genes constitute a distinct subfamily of class-I-like genes that predates the emergence of mammals.

Mammals have several major histocompatibility complex (MHC) class-I-like genes. Although some of them are assumed to have originated before the emergence of mammals, the origin of class-I-like genes is poorly understood. We analyzed here the recently released chicken draft genome sequence and identified two families of class-I-like genes: CD1 and PROCR (the gene for the endothelial protein C receptor). Chickens have two CD1 genes, designated CD1.1 and CD1.2, located in tandem approximately 840 bp apart from each other. Chicken CD1.1 and CD1.2 are neither group 1- nor group 2-like, indicating that the two groups of CD1 emerged in a mammalian lineage. Although the database provides no information as to their chromosomal localization, we found that chicken CD1 genes are located adjacent to the previously characterized MHC B system contig on chromosome 16. We confirmed the linkage of CD1 to the B system by dual-color fluorescence in situ hybridization. Chickens have a single copy of PROCR. Among known class-I-like genes, PROCR is most closely related to CD1, indicating that CD1 and PROCR constitute a distinct subfamily of class-I-like genes that predates the emergence of mammals.

Amino Acid Sequence↗

PubMed2005

Variable lymphocyte receptors in hagfish.

A previously uncharacterized type of variable lymphocyte receptors (VLR) was identified recently in the Sea lamprey. This jawless vertebrate generates an extensive VLR repertoire through differential insertion of neighboring diverse leucine-rich repeat (LRR) cassettes into an incomplete germ-line VLR gene. We report here VLR homologs from two additional lamprey species and the presence of two types of VLR genes in hagfish, the only other order of contemporary jawless vertebrates. As in the Sea lamprey, the incomplete hagfish germ-line VLR-A and -B genes are modified in lymphocyte-like cells to generate highly diverse repertoires of VLR-A and -B proteins via a presently undetermined mechanism. This jawless-fish mode of VLR diversification starkly contrasts with the rearrangement of Ig V(D)J gene segments used by all jawed vertebrates to produce diverse repertoires of T and B lymphocyte antigen receptors. The development of two very different strategies for receptor diversification at the dawn of vertebrate evolution approximately 500 million years ago attests to the fitness value of a lymphocyte-based system of anticipatory immunity.

Amino Acid Motifs↗

PubMed2005

Hagfish leukocytes express a paired receptor family with a variable domain resembling those of antigen receptors.

Jawed vertebrates are equipped with TCR and BCR with the capacity to rearrange their V domains. By contrast, jawless vertebrates, represented by hagfish and lampreys, apparently lack such receptors. We describe in this study a family of hagfish genes carrying a single V-type domain resembling those of TCR/BCR. This multigene family, which we call agnathan paired receptors resembling Ag receptors (APAR), is expressed in leukocytes and predicted to encode a group of membrane glycoproteins with organizations characteristic of paired Ig-like receptors, consisting of activating and inhibitory forms. APAR has a J region in its V-type domain, and its V and J regions are encoded in a single exon. Thus, APAR is a member of the emerging families of diversified, innate immune-type receptors with TCR/BCR-like V-type domains and has many of the features expected for a primordial TCR/BCR-like receptor. The extracellular domain of APAR may be descended from a V-type domain postulated to have acquired recombination signal sequences in a jawed vertebrate lineage.

Amino Acid Sequence↗

PubMed2004

Natural killer cell receptors in the horse: evidence for the existence of multiple transcribed LY49 genes.

In rodents, the Ly49 family encodes natural killer (NK) receptors interacting with classical MHC class I molecules, whereas the corresponding receptors in primates are members of the killer cell immunoglobulin-like receptor (KIR) family. Recent evidence indicates that the cattle, domestic cat, dog, and pig have a single LY49 and multiple KIR genes, suggesting that predominant NK receptors in most non-rodent mammals might be KIR. Here, we show that the horse has at least six LY49 genes, five with an immunoreceptor tyrosine-based inhibition motif (ITIM) and one with arginine in the transmembrane region. Interestingly, none of the horse KIR-like cDNA clones isolated by library screening encoded molecules likely to function asNK receptors; four types of clones were KIR-Ig-like transcript (KIR-ILT) hybrids and contained premature stop codons and/or frameshift mutations, and two putative allelic sequences predicting KIR3DL molecules had mutated ITIM. To our knowledge, this is the first report suggesting that non-rodent mammals may use LY49 as NK receptors for classical MHC class I. We also show that horse spleen expresses ILT-like genes with unique domain organizations. Radiation hybrid mapping and fluorescence in situ hybridization localized horse LY49 and KIR/ILT genes to chromosomes 6q13 and 10p12, respectively.

Amino Acid Sequence↗

PubMed2004

Comparative genomics of the Mill family: a rapidly evolving MHC class I gene family.

Mill (MHC class I-like located near the leukocyte receptor complex) is a novel family of class I genes identified in mice that is most closely related to the human MICA/B family. In the present study, we isolated Mill cDNA from rats and carried out a comparative genomic analysis. Rats have two Mill genes orthologous to mouse Mill1 and Mill2 near the leukocyte receptor complex, with expression patterns similar to those of their mouse counterparts. Interspecies sequence comparison indicates that Mill is one of the most rapidly evolving class I gene families and that non-synonymous substitutions occur more frequently than synonymous substitutions in its alpha 1 domain, implicating the involvement of Mill in immune defenses. Interestingly, the alpha 2 domain of rat Mill2 contains a premature stop codon in many inbred strains, indicating that Mill2 is not essential for survival. A computer search of the database identified a horse Mill-like expressed sequence tag, indicating that Mill emerged before the radiation of mammals. Hence, the failure to find Mill in human indicates strongly that it was lost from the human lineage. Our present work provides convincing evidence that Mill is akin to the MICA/B family, yet constitutes a distinct gene family.

Amino Acid Sequence↗

PubMed2004

Transcriptome analysis of hagfish leukocytes: a framework for understanding the immune system of jawless fishes.

Jawless fishes occupy a critical phylogenetic position in understanding the origin of the adaptive immune system. Here, we performed large-scale expressed sequence tag analysis of leukocytes isolated from the inshore hagfish Eptatretus burgeri. Although we found many immunity-related genes such as those involved in lymphocyte or hematopoietic cell signaling and development as well as cytokine and cytokine receptor genes, MHC molecules or antigen receptors were not identified. We characterized two hagfish cDNAs that closely resembled mammalian proteins with essential roles in adaptive immunity, one encoding a GATA3-like molecule and another encoding a Bruton's tyrosine kinase (Btk)-like molecule. The GATA3-like gene of hagfish was equidistant from GATA3 and GATA2 in jawed vertebrates. Similarly, the hagfish Btk-like molecule was not Btk itself, but qualified as a pre-duplicated form of Btk and Bmx in jawed vertebrates. In total, our work provides circumstantial evidence that adaptive immunity is unique to jawed vertebrates.

Amino Acid Sequence↗

PubMed2004

On the origins of the adaptive immune system: novel insights from invertebrates and cold-blooded vertebrates.

When and how adaptive immunity emerged is one of the fundamental questions in immunology. Accumulated evidence suggests that the key components of adaptive immunity, rearranging receptor genes and the MHC, are unique to jawed vertebrates. Recent studies in protochordates, in particular, the draft genome sequence of the ascidian Ciona intestinalis, are providing important clues for understanding the origin of antigen receptors and the MHC. We discuss a group of newly identified protochordate genes along with some cold-blooded vertebrate genes, the ancestors of which might have provided key elements of antigen receptors. The organization of the proto-MHCs in protochordates provides convincing evidence that the MHC regions of jawed vertebrates emerged as a result of two rounds of chromosomal duplication.

Animals↗

PubMed2004

Construction of a bacterial artificial chromosome library from the inshore hagfish, Eptatretus burgeri: A resource for the analysis of the agnathan genome.

The jawless fish occupy an important phylogenetic position for understanding the evolution of body plans, the origin of adaptive immunity and genome evolution in chordates. We describe here the construction of a large-insert bacterial artificial chromosome (BAC) library from the inshore hagfish, Eptatretus burgeri. The BAC library contains 93,978 clones with an average insert size of 100 kb and is estimated to represent threefold genome-equivalent coverage. The library was organized in three-dimensional pools to facilitate screening by PCR. We have screened this library by PCR and isolated several BAC clones; the average number of positive clones was compatible with the estimated genome coverage of the library. This BAC library, constructed for the first time from the jawless fish, should serve as a useful resource for the scientific community.

Animals↗

PubMed2003

Identification of the rat IgA Fc receptor encoded in the leukocyte receptor complex.

Human FcalphaRI (CD89) is a myeloid-specific IgA Fc receptor encoded in the leukocyte receptor complex. Thus far, no gene coding for FcalphaRI has been identified in mice. Here, we show that, unlike mice, rats have the gene ( Fcar) coding for FcalphaRI. The rat Fcar gene has an exon-intron structure essentially identical to that of the human counterpart and is encoded in the leukocyte receptor complex on Chromosome 1. Southern blot analysis using the rat Fcar as a probe revealed hybridizing bands in Chinese and Syrian hamsters and gerbils, but not in mice, indicating that Fcar was lost in the lineage leading to mice after the divergence of rats and mice. Identification of FcalphaRI in rats should facilitate the elucidation of the in vivo role of this receptor.

Amino Acid Sequence↗

PubMed2003

Genomic analysis of immunity in a Urochordate and the emergence of the vertebrate immune system: "waiting for Godot".

Genome-wide sequence analysis in the invertebrate chordate, Ciona intestinalis, has provided a comprehensive picture of immune-related genes in an organism that occupies a key phylogenetic position in vertebrate evolution. The pivotal genes for adaptive immunity, such as the major histocompatibility complex (MHC) class I and II genes, T-cell receptors, or dimeric immunoglobulin molecules, have not been identified in the Ciona genome. Many genes involved in innate immunity have been identified, including complement components, Toll-like receptors, and the genes involved in intracellular signal transduction of immune responses, and show both expansion and unexpected diversity in comparison with the vertebrates. In addition, a number of genes were identified which predicted integral membrane proteins with extracellular C-type lectin or immunoglobulin domains and intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based activation motifs (ITAMs) (plus their associated signal transduction molecules), suggesting that activating and inhibitory receptors have an MHC-independent function and an early evolutionary origin. A crucial component of vertebrate adaptive immunity is somatic diversification, and the recombination activating genes (RAG) and activation-induced cytidine deaminase (AID) genes responsible for the Generation of diversity are not present in Ciona. However, there are key V regions, the essential feature of an immunoglobulin superfamily VC1-like core, and possible proto-MHC regions scattered throughout the genome waiting for Godot.

Animals↗