[MHC class I deficiency].
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ATP Binding Cassette Transporter, Subfamily B, Mem↗
Biomedical subjects
T Morio
Publications and source records attributed to T Morio.
[MHC class II deficiency (CIITA/RFX deficiency)].
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DNA-Binding Proteins↗
LbrA, a protein predicted to have a role in vesicle trafficking, is necessary for normal morphogenesis in Polysphondylium pallidum.
The fruiting body of Polysphondylium pallidum is composed of whorls of branches along the axis of a central stalk. In the course of fruiting body formation, the interval between neighboring whorls, and the number and the spacing of branches in a whorl are highly regulated. In this study, using the REMI (restriction-enzyme-mediated integration) insertional mutagenesis method, we obtained a mutant (strain M2323) with longer branches than those of the wild-type strain PN500. The sequence analyses revealed the presence of an ORF of 206 aa residues (23 kDa) near the vector insertion site. Disruption of the gene, lbrA (long branch A), by homologous recombination causes the same phenotype as that of M2323. A lbrA transcript is expressed maximally at the early aggregation stage in the parental strain, but is not detectable in the REMI mutant. A homology search showed that LbrA is a member of the p24 family proteins, which have been proposed to function as receptors for cargo proteins that are transported by COP I- (coat protein I) and/or COP II-coated vesicles between the endoplasmic reticulum and the Golgi complex. As far as we know, this is the first paper to show that a p24 family member is implicated in morphogenesis.
Amino Acid Sequence↗
Proposed standard for human blood vitamin B1 value using HPLC. The Committee for Vitamin Laboratory Standards, Japan.
Standard reference ranges for all laboratory test values are mandatory. This study was designed to establish a reference range for blood vitamin B1 levels, since the normal range has not been determined in the Japanese population. We founded the Japan Committee for Vitamin Laboratory Standards, which was incorporated with the Vitamin Society of Japan and the Japanese Society of Nutrition and Food Science. We standardized whole blood vitamin B1 levels using three HPLC techniques (post-column reverse-phase HPLC, pre-column reverse-phase HPLC, and precolumn GP-HPLC). The reference range was obtained in 54 volunteers administered a 1,800 kcal diet with 2 mg of vitamin B1 (1.74 mg measured) daily to avoid marginal vitamin B1 deficiency in the population. The range for each assay was 26-47, 28-51, and 28-56 ng/ml, respectively. Our data suggest that 26-28 ng/ml is the lower limit of normal for whole blood vitamin B1, but further studies in a larger population are needed in order to obtain more definitive results.
Chromatography, High Pressure Liquid↗
The pleckstrin homology domain of the Wiskott-Aldrich syndrome protein is involved in the organization of actin cytoskeleton.
In this study, we investigated the role of the pleckstrin homology (PH) domain of the Wiskott-Aldrich syndrome protein (WASP) in the regulation of actin cytoskeleton, which is defective in patients with Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT). Overexpression of the WASP in COS-7 cells cultured in the presence of fetal calf serum (FCS) resulted in large cluster formation of polymerized actin and WASP in the cytoplasm. In contrast, when the WASP transfected cells were cultured in the absence of FCS, activation with PMA or EGF was required to induce cluster formation. Overexpression of WASP with a missense mutation in the N-terminus of the PH domain failed to induce the large cluster formation in COS-7 cells even in the presence of FCS. We also found that phosphatidylinositol 4,5-bisphosphate (PIP(2)), which is known to regulate the actin cytoskeleton, binds to the PH domain of WASP, and the binding was abolished by the introduction of a missense mutation into the N-terminus but not the C-terminus of the PH domain. Together with the observations that most of the missense mutations observed in patients with WAS and XLT are located within the PH domain, these results indicate that the PH domain of WASP plays important roles in the regulation of actin cytoskeleton and suggested that the binding of PIP(2) to the PH domain is necessary for WASP to function properly.
Actins↗
Ku in the cytoplasm associates with CD40 in human B cells and translocates into the nucleus following incubation with IL-4 and anti-CD40 mAb.
CD40 plays a critical role in survival, growth, differentiation, and class switching of B lymphocytes. Although Ku is required for immunoglobulin class switching, how CD40 signal transduction is coupled to Ku is still unknown. Here, we show that CD40 directly interacts with Ku through the membrane-proximal region of cytoplasmic CD40. Ku was confined to the cytoplasm in human primary B cells, and the engagement of CD40 on the B cells cultured in the presence of IL-4 resulted in the dissociation of Ku from CD40, translocation of Ku into the nucleus, and increase in the activity of DNA-dependent protein kinase. These findings indicate that Ku is involved in the CD40 signal transduction pathway and may play an important role in the CD40-mediated events.
Antibodies, Monoclonal↗
WASP is involved in proliferation and differentiation of human haemopoietic progenitors in vitro.
The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, immunodeficiency and eczema. X-linked thrombocytopenia (XLT) is a mild form of WAS with isolated thrombocytopenia. Both phenotypes are caused by mutation of the Wiskott-Aldrich syndrome protein (WASP) gene. In this study we investigated the role of WASP in the differentiation of CD34-positive (CD34+) cells isolated from the bone marrow of patients with WAS (n = 5) or with XLT (n = 4). Megakaryocyte colony formation was significantly decreased in patients with WAS when compared with normal controls. The formation of granulocyte-macrophage colonies and erythroid bursts were also decreased in WAS patinets. In contrast, in XLT patients, formation of all these colonies was normal. However, in vitro proplatelet formation of megakaryocytes induced by thrombopoietin was markedly decreased in both XLT and WAS. Electron microscopic examination revealed that megakaryocytes obtained from WAS or XLT patients grown in vitro had abnormal morphologic features, which seemed to be caused by defective actin cytoskeletal organization, including labyrinth-like structures of the demarcation membrane system and deviated distribution of the alpha-granules and demarcation membrane system. These observations indicate that WASP is involved in the proliferation and differentiation of CD34+ haemopoietic progenitor cells probably by its participation in signal transduction and in the regulation of the cytoskeleton.
Blood Platelets↗
[The molecular basis of primary immunodeficiency syndrome and the current therapeutic approach for the disease].
The development of genetic tools in recent years has enabled characterization of the molecular basis of several genetic disorders of the human system. These findings have led to better understanding of the physiology of the human system as well as to the establishment of improved diagnosis tools offering new perspectives in genetic counseling and therapeutics. Recently, genetic disorders of the regulation of the immune system have been described both in humans and mice. These diseases are characterized by defective control of activation/proliferation of lymphocytes and macrophages. These findings opened a new field in immunological research. Detailed analysis of the molecular basis and functional characterization of the affected gene product may help to understand the phenotype of the patients. Considerable progress has been made in the therapeutics for these patients including unrelated bone marrow transplantation, cord blood stem cell transplantation, and gene therapy. Ex vivo expanded activated T cell transfusion, a new therapeutic approach to control opportunistic infection, may be effective for some patients with impaired T cell function.
Animals↗
The Dictyostelium developmental cDNA project: generation and analysis of expressed sequence tags from the first-finger stage of development.
In an effort to identify and characterize genes expressed during multicellular development ill Dictyostelium, we have undertaken a cDNA sequencing project. Using size-fractionated subsets of cDNA from the first finger stage, two sets of gridded libraries were constructed for cDNA sequencing. One, library S, consisting of 9984 clones, carries relatively short inserts, and the other, library L, which consists of 8448 clones, has longer inserts. We sequenced all the selected clones in library S from their 3'-ends, and this generated 3093 non-redundant, expressed sequence tags (ESTs). Among them, 246 ESTs hit known Dictyostelium genes and 910 showed significant similarity to genes of Dictyostelium and other organisms. For library L, 1132 clones were randomly sequenced and 471 non-redundant ESTs were obtained. In combination, the ESTs from the two libraries represent approximately 40% of genes expressed in late development, assuming that the non-redundant ESTs correspond to independent genes. They will provide a useful resource for investigating the genetic networks that regulate multicellular development of this organism.
Animals↗
Functional analysis of peripheral blood B cells in patients with X-linked agammaglobulinemia.
X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disease caused by mutations of Bruton tyrosine kinase (Btk); Btk plays an essential role in the development of mature B cells. However, small numbers of B cells ("leaky B cells") are present in the peripheral blood of most XLA patients. In this study, we analyzed the function of these leaky B cells obtained from XLA patients. Enough numbers of B cells were available for analysis from five of nine XLA patients originally screened. Sequence analysis revealed missense mutations of Btk in four of the five XLA patients. No mutation was found in the coding region of Btk in one patient. Western blotting and/or flow cytometric analysis failed to detect Btk protein in all five patients. B cells isolated from peripheral blood of these XLA patients were CD5-, CD20+, CD19+, and CD21-. If stimulated with anti-CD40 and IL-4, XLA B cells proliferated normally and produced significant amounts of IgE. Anti-CD40 stimulation of XLA B cells resulted in normal expression of CD23. In addition, three of the five XLA patients studied were immunized with bacteriophage phiX174 and produced low but detectable levels of antiphage-specific Ab. Similarly, X-linked immunodeficiency mice, which carry a missense mutation in Btk, produced substantial amounts of antiphage Ab. These results indicate that CD40 signaling is intact in B cells lacking demonstrable Btk, and that leaky B cells in XLA patients can proliferate, undergo isotype switching, and differentiate into specific Ab-producing cells.
Adolescent↗
A ribosomal protein gene cluster is encoded in the mitochondrial DNA of Dictyostelium discoideum: UGA termination codons and similarity of gene order to Acanthamoeba castellanii.
We sequenced a region of about 14.5 kb downstream from the ribosomal protein L11 gene (rpl11) in the mitochondrial DNA (54+/-2 kb) of the cellular slime mold Dictyostelium discoideum. Sequence analysis revealed that eleven ribosomal protein genes and six open reading frames (ORFs) formed a cluster arranged in the order: rpl11-orf189-rps12-rps7-rpl2-rps19-+ ++orf425-orf1740-rpl16-rpl14-orf188- rps14-rps8-rpl6-rps13-orf127-orf796. This order was very similar to that of homologous genes in Acanthamoeba castellanii mitochondrial DNA. The N-terminal region of ORF425 and the C-terminal region of ORF1740 had partial similarities to the S3 ribosomal protein of other organisms. The termination codons of rpl16 and orf188 were UGA, which has not hitherto been found in genes encoded in D. discoideum mitochondrial DNA.
Acanthamoeba↗
Characterization of a novel small RNA encoded by Dictyostelium discoideum mitochondrial DNA.
In this study, we analyzed a mitochondrial small (ms) RNA in Dictyostelium discoideum, which is 129 nucleotides long and has a GC content of only 22.5%. In the mitochondrial DNA, a single-copy gene (msr) for the ms RNA was located downstream of the gene for large-subunit rRNA. The location of msr was similar to that of the 5S rRNA gene in prokaryotes and chloroplasts, but clearly different from that in mitochondria of plants, liverwort and the chlorophycean alga Prototheca wikerhamii, in which small-subunit rRNA and 5S rRNA genes are closely linked. THe primary sequence of ms rRNA showed low homology with mitochondrial 5S rRNA from plants, liverwort and the chlorophycean alga, but the proposed secondary structure of ms RNA was similar to that of cytoplasmic 5S rRNA. In addition, ms RNA showed a highly conserved GAAC sequence in the same loop as in common 5S rRNA. However, ms RNA was detected mainly in the mitochondrial 25,000 x g supernatant fraction which was devoid of ribosomes. It is possible that ms RNA is an evolutionary derivative of mitochondrial 5S rRNA.
Animals↗
[A case of fasciitis associated with Basedow's disease and polymyositis].
A 39-year-old female suffered from diffuse goiter, palpitation, finger tremor and body weight loss for about one year. Then she developed acute onset of myalgia and swelling of calves, and muscle weakness of proximal limbs. She could not walk because of myalgia and muscle weakness, and was admitted to our hospital 4 days after the onset of muscle symptoms. On admission, her pulse was 110 per minute and she had finger tremor of 11-12 Hz. The thyroid gland was markedly and diffusely enlarged with an elastic soft surface. She presented muscle weakness of proximal limbs and neck, and had intermittent swelling and myalgia on calves. Deep tendon reflexes were increased in all extremities. The erythrocyte sedimentation rate was 22 mm per hour. Eosinophilia was not recognized. Serum CK level was elevated to 671 IU/l. Serum free T3 was higher than 21.7 pg/ml and free T4 was also elevated to 10.19 ng /dl. Serum TSH was lower than 0.05 microU/ml and thyroid stimulating antibody was 1,302.0%. Muscle biopsy of her left gastrocnemius muscle revealed markedly hypertrophic fascia with inflammatory cellular infiltration on HE staining. Inflammatory change was also recognized in muscle tissue and in perivascular region of perimysium. Variation of fiber size, necrotic fibers, and central nuclei were also seen. From these clinical and laboratory findings she was diagnosed as having Basedow's disease associated with fasciitis and polymyositis. Her thyroid function was improved by anti-thyroid drug, and swelling and myalgia of sural regions and weakness of proximal limbs were also improved by steroid therapy. Only one case of Basedow's disease associated with fasciitis and seven cases of that associated polymyositis have so far been reported. This is the first case report of fasciitis associated with Basedow's disease and polymyositis.
Adult↗
A site-specific DNA endonuclease specified by one of two ORFs encoded by a group I intron in Dictyostelium discoideum mitochondrial DNA.
The second intron (DdOX1/2.2) of Dictyostelium discoideum cytochrome oxidase subunit 1/2 fused gene has two free-standing ORF genes (Dd ai2a and Dd ai2b) in a loop, which have similar amino acid sequences and are homologous to aI4 DNA endonuclease (I-SceII) of Saccharomyces cerevisiae. To elucidate the functions of these ORFs, we cloned the ORFs into an expression vector and introduced the composite vectors into E. coli. The expression of Dd ai2a in E. coli caused growth inhibition and degradation of the E. coli genomic DNA. To determine whether Dd ai2a protein is a homing type DNA endonuclease, the ability to cleave the homing site of its intron in vivo was examined. Dd ai2a cleaved only one strand of intronless DNA sequence at the site which coincides with the I-SceII cleavage recognition site. We suppose that Dd ai2a functions actually as a homing type DNA endonuclease in D. discoideum mitochondria by virtue of other factors. To obtain further information about the relationship between the existence of introns and the mating system, we carried out in vitro self-splicing assay and polymerase chain reaction analysis using 13 strains of the cellular slime mold.
Animals↗
A mutation in the cAMP signaling pathway affects sexual development of Dictyostelium discoideum.
Amoebae of cellular slime molds have two developmental modes, asexual fruiting body formation and sexual macrocyst formation. How developmental choice is made is an interesting subject of wide importance. Light exposure and dry conditions are favorable for asexual development, while conditions of darkness and high humidity are so for sexual development. In Dictyostelium discoideum, the latter conditions enhance zygote formation, which determines the fate of surrounding cells for sexual development. Here, a mutant (TMC1) defective in the post-fusion aggregation of cells during sexual development is described. This mutant is also aggregationless in asexual development, and the level of cyclic adenosine monophosphate (cAMP) receptor is reduced. Correspondingly, a series of existing mutants with defects in cAMP signaling pathways showed the same sexual phenotype as TMC1. These results suggest that molecular mechanisms of development are shared by the two alternative developmental modes.
Animals↗
HIV glycoprotein gp120 inhibits TCR-CD3-mediated activation of fyn and lck.
HIV major glycoprotein gp120 interacts with CD4 molecules and perturbs signaling through the TCR-CD3 complex. We examined the effects of gp120 on TCR-CD3-induced phosphorylation and activation of the src-type protein tyrosine kinases (PTK), fyn and lck. gp120 caused minimal changes in lck phosphorylation or lck enzymatic activity, but preincubation of Jurkat cells with gp120 for 20 min strongly inhibited TCR-CD3-mediated phosphorylation and activation of lck and fyn, as well as phosphorylation of CD3 zeta. Inhibition of TCR-CD3 signaling in T cells preincubated with gp120 was paralleled by inhibition of T cell proliferation to the antigen tetanus toxoid. Neither surface CD4 expression nor CD4-lck association was affected by gp120. Furthermore, gp120 inhibited lck phosphorylation induced by cross-linking of TCR-CD3 and CD4 suggesting that the inhibition of lck phosphorylation could not be simply accounted for by sequestration of CD4 molecules. gp120 selectively enhanced the phosphorylation of the lck peptide containing the autoinhibitory tyrosine residue Tyr505 relative to the lck peptide containing the positive regulatory residue Tyr394, suggesting that a qualitative alteration in lck may underlie the inhibition of TCR-CD3 signaling by gp120.
CD4 Antigens↗
Decreased alpha/beta heterodimer among CD8 molecules of peripheral blood T cells in Wiskott-Aldrich syndrome.
Cell surface CD8 molecules exist as either alpha/alpha homodimers or alpha/beta heterodimers, and most CD8+ T cells that undergo differentiation in the thymus express alpha/beta heterodimers. CD8 molecules on peripheral blood lymphocytes of patients with Wiskott-Aldrich syndrome (WAS), an X-linked immunodeficiency disease, have now been shown to be composed mostly of alpha/alpha homodimers, while the total number of lymphocytes was maintained. This observation may indicate aberrant differentiation of CD8+ T cells in the thymus of WAS. Decreased CD8alpha/beta heterodimer expression, and the relative increase of CD8+ T cells expressing CD8alpha/alpha homodimer, may explain a part of the defective lymphocyte functions in WAS patients.
Aging↗
Characterization of a 23-kDa protein associated with CD40.
CD40 is a 45-kDa glycoprotein member of the tumor necrosis factor receptor (TNFR) family expressed on B cells, thymic epithelial cells, dendritic cells, and some carcinoma cells. The unique capacity of CD40 to trigger immunoglobulin isotype switching is dependent on the activation of protein-tyrosine kinases, yet CD40 possesses no kinase domain and no known consensus sequences for binding to protein-tyrosine kinases. Recently, an intracellular protein (CD40bp/LAP-1/CRAF-1) which belongs to the family of TNFR-associated proteins was reported to associate with CD40. We describe a 23-kDa cell surface protein (p23) which is specifically associated with CD40 on B cells and on urinary bladder transitional carcinoma cells. Protein microsequencing revealed that p23 shows no homology to any known protein. A rabbit antibody raised against a peptide derived from p23 recognized a 23-kDa protein in CD40 immunoprecipitates. In contrast to CD40bp/LAP-1/CRAF-1, p23 was not associated with TNFR p80 (CD120b). These findings suggest that p23 is a novel member of the CD40 receptor complex.
B-Lymphocytes↗