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Biomedical subjects

H Kagawa

Publications and source records attributed to H Kagawa.

At least 19 recordsLinked to original sources

Genome structure, mapping and expression of the tropomyosin gene tmy-1 of Caenorhabditis elegans.

The complete tropomyosin gene, designated tmy-1, of Caenorhabditis elegans was recovered by genome walking from a fragment that was obtained by exon-expression cloning using specific cloning using specific anti-tropomyosin antiserum as a probe. The genome structure of the tmy-1 gene has been determined by combining the DNA sequences of cDNA clones with those of the genomic fragments. The single-copy gene spans approximately 13 kb and include 14 exons. Comparison of cDNA and genomic sequences demonstrates that three isoforms are encoded by the gene tmy-1. Homology of the 27 C-terminal amino acid residues to those of Drosophila and vertebrates suggest that these may be the body wall, pharyngeal and non-muscle types. Tissue-specific expression of the tmy-1 gene was determined by microinjection of a promoter/lacZ fusion gene and with immunohistochemistry by using affinity-purified tissue-specific anti-tropomyosins. The 5' end promoter common to CeTMI and CeTMII is expressed in the body wall muscles, vulva, anus muscles and male tail muscles. Control sequences of the 5' end promoter are located 660 to 800 bp upstream of the initial methionine codon. The third isoform, CeTMIII, encoding 256 amino acids residues was expressed in the pharyngeal muscles by the promoter in the third intron. The mRNA of CeTMIII was trans-spliced with SL1 and SL2. These results allow is to solve the question of what is common from this worm to vertebrates, and also what are the cross-species complexities and the tissue-specific differences of tropomyosins. The tmy-1 gene is located on the C. elegans genomic YAC grid near the right end of chromosome I, in the region on the lev-11 gene.

Alternative Splicing

Relationship of microparticles with beta 2-glycoprotein I and P-selectin positivity to anticardiolipin antibodies in immune thrombocytopenic purpura.

We investigated the association of beta 2-glycoprotein I and P-selectin with platelet-derived microparticles in 48 patients with immune thrombocytopenic purpura and 20 normal controls using two-color flow cytometric analysis. In addition, anticardiolipin antibodies were detected by an enzyme-linked immunosorbent assay. Platelet microparticles from the patients showed a higher positivity for beta 2-glycoprotein I than those from the normal controls (23.1 +/- 15.4% vs. 5.3 +/- 3.1%, p < 0.01), but this positivity was not related to the presence of platelet-associated IgG or to the severity of thrombocytopenia. In the 18 patients with more than 20% P-selectin-positive microparticles, beta 2-glycoprotein I positivity was significantly higher than in the 30 patients with less than 20% P-selectin-positive microparticles (37.1 +/- 20.5% vs. 21.5 +/- 17.3%, p < 0.01). In addition, anticardiolipin antibodies were detected in eight patients, and they had a significantly higher level of beta 2-glycoprotein I-positive microparticles than the patients without such antibodies (42.0 +/- 22.9% vs. 22.6 +/- 18.9%, p < 0.05). Our results suggest that anticardiolipin antibodies activate platelets in immune thrombocytopenic purpura and cause the generation of microparticles rich in beta 2-glycoprotein I and P-selectin. These microparticles may then act to regulate coagulation abnormalities in patients with anticardiolipin antibodies.

Adolescent

Platelet-derived microparticles may influence the development of atherosclerosis in diabetes mellitus.

We investigated the association between low-density lipoprotein (LDL), triglycerides, and platelet activation in 18 patients with hypertension age 41-64 years and 18 with diabetes mellitus aged 43-70 years. Platelet P-selectin positivity and the microparticle level (indicators of activation) were both significantly higher in the diabetics than in healthy controls (P-selectin: 28.0% +/- 7.5% vs. 7.3% +/- 4.2%, P < 0.001; microparticles: 1900 +/- 966 vs. 526 +/- 158/10(4) platelets, P < 0.01). In contrast, there was no significant increase of either parameter in the patients with hypertension. Plasma microparticle levels were also significantly greater in the diabetics with high LDL levels than in those with low LDL levels (2375 +/- 949 vs. 1519 +/- 796/10(4) platelets, P < 0.05), and in those with high rather than low triglyceride levels (2188 +/- 845 vs. 1492 +/- 783/10(4) platelets, P < 0.05). However, platelet positivity for P-selectin was not significantly different between these two subgroups. Microparticle and P-selectin levels both showed no significant difference between the hypertensive patients with high and low LDL or triglyceride levels. These results suggest that platelet-derived microparticles may participate in the development or progression of atherosclerosis in patients with diabetes mellitus.

Adult

Significance of cytokines and CD68-positive microparticles in immune thrombocytopenic purpura.

We investigated the significance of cytokines (soluble interleukin-2 receptor, granulocyte-macrophage colony-stimulating factor, interleukin-6, and interferon-gamma) and CD68-positive microparticles in immune thrombocytopenic purpura. Cytokines were measured by enzyme-linked immunosorbent assay and microparticles were detected by flow cytometry. CD68 expression by histiocytic U937 cells incubated with lipopolysaccharide or cytokines was also assessed in a control study. The level of CD68-positive microparticles was significantly higher in the patients with thrombocytopenia than in normal controls (p < 0.01). The soluble interleukin-2 receptor level was also significantly higher in patients than in controls (p < 0.01), but the other cytokines did not show a significant difference. However, patients with severe thrombocytopenia (platelet count > 20,000/microliters) had significantly higher levels of granulocyte-macrophage colony-stimulating factor and interleukin-6 than the controls (p < 0.05). When opsonized platelets were incubated with activated U937 cells, lipopolysaccharide and granulocyte-macrophage colony-stimulating factor caused an increase of CD68-positive microparticles in the supernatant. These results suggest that granulocyte-macrophage colony-stimulating factor is released by activated T cells in immune thrombocytopenic purpura and activates monocyte/macrophage phagocytosis, resulting in an increase of circulating CD68-positive microparticles and enhanced platelet destruction.

Adolescent

[Detection of soluble interleukin-2 receptor in idiopathic thrombocytopenic purpura].

We measured soluble interleukin-2 receptor (sIL-2 R) in serum samples from 57 patients with idiopathic thrombocytopenic purpura (ITP). The sIL-2 R level was significantly increased in the ITP patients (481.3 +/- 378.5 U/ml) compared with controls (176.2 +/- 66.9 U/ml) (p < 0.001), and was significantly higher in 8 patients positive for hepatitis C virus (HCV) antibody positive (1,140.7 +/- 194.3 U/ml) than in 49 HCV-antibody negative patients (378.9 +/- 278.6 U/ml) (p < 0.0001). There was also a significant difference between the HCV-antibody negative ITP patients and the controls (p < 0.01). Elevated sIL-2 R levels correlated with the CD 4/8 ratio (p < 0.05), but not with the platelet count or the level of platelet-associated IgG. The increase of sIL-2 R in ITP may be related to the immunological abnormalities underlying this disease.

Adult

Development of maturational competence of oocytes of red seabream, Pagrus major, after human chorionic gonadotropin treatment in vitro requires RNA and protein synthesis.

The effects of human chorionic gonadotropin (HCG) and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (DHP) on in vitro germinal vesicle breakdown (GVBD) in oocytes obtained at different times of day from a daily spawning marine teleost, the red seabream Pagrus major, were investigated. Oocytes isolated at 0800 hr underwent GVBD in response to HCG (10 IU/ml) but not to DHP (10 ng/ml). GVBD could be induced in oocytes isolated at 1600 hr by either HCG or DHP. Oocytes underwent GVBD in response to DHP after DHP-insensitive oocytes were incubated with HCG for 30, 60, or 120 min. The effects of actinomycin D (a transcriptional inhibitor) and cycloheximide (a translational inhibitor) on HCG- and DHP-induced GVBD were also investigated. Actinomycin D (1 microgram/ml) totally inhibited HCG-induction of GVBD in oocytes isolated at 0600 and 0800 hr. Actinomycin D also significantly inhibited GVBD induced by HCG in combination with DHP in oocytes taken at 0600 and 0800 hr. Cycloheximide (1 microgram/ml) completely inhibited the inducation of GVBD by HCG or DHP, alone or in combination, in oocytes obtained at 0600, 1000, and 1400 hr. These results indicate that gonadotropin induces maturational competence (responsiveness to maturation-inducing steroid) in oocytes of red seabream. These results also suggest that gonadotropin-induced synthesis of new protein through a mechanism dependent on RNA is essential for the development of maturational competence in oocytes of red seabream.

Animals

Amyloid beta-protein precursor-rich platelet microparticles in thrombotic disease.

We investigated the association of amyloid beta-protein precursor (APP) and platelet derived microparticles in 20 normal controls and 91 patients with various diseases causing a thrombotic tendency. Compared with the controls, the mean percentage of APP-positive microparticles was significantly greater in the patients with cerebral infarction (39.1 +/- 17.7%, p < 0.001), diabetes (31.1 +/- 12.6%, p < 0.001), and uremia (30.1 +/- 14.7%, p < 0.01), but not in those with hypertension (8.2 +/- 6.3%, p = NS). Sixteen patients with cerebral infarction, 20 with diabetes, and 11 with uremia had microparticles with very high APP levels. In normal controls, 7.2 +/- 3.7% of the microparticles were positive for P-selectin, while the percentage in cerebral infarction, diabetes, uremia, and hypertension was respectively 43.5 +/- 15.1%, 40.0 +/- 12.8%, 31.8 +/- 12.2%, and 11.6 +/- 7.3%. There was a significant correlation between P-selectin and APP positivity of microparticles. Our results suggest that microparticle APP may have a regulatory influence on coagulation abnormalities.

Amyloid beta-Protein Precursor

Characterization of proteins released from legume seeds in hot water.

When immersed in water at 50-60 degrees, mature soybean seeds release a large amount of protein. The major protein released was basic 7S globulin (Bg), which is present in the cotyledons of soybean seeds. The released Bg consisted of the 27,000 and 16,000 subunits which were linked by disulphide bonding and glycosylated. The released Bg exhibited an identical structure with the mature Bg which was synthesized in the normal developing seeds. Proteins like Bg were also found to be released into hot water from the seeds of legume species such as azuki-bean, cowpea, mung-bean and winged-bean. Besides Bg and Bg-like proteins, a few proteins including the 9,000 hydrophobic protein in soybean, ubiquitin in cowpea and mung-bean, and Kunitz trypsin inhibitor in winged-bean, were released from the seeds in hot water.

Amino Acid Sequence

[Assessment of ovarian tumors by transvaginal scanning and clinical significance of ultrasonic examination in postoperative follow up].

A hundred and sixty cases of ovarian tumor were preoperatively examined by transvaginal (TV) and Transabdominal (TA) scanning and accuracy of the assessment for histologic type was compared. We also examined 56 cases of postoperative ovarian cancer by ultrasonography to detect recurrent lesions. Positive predictive values for serous cysts and endometrial cysts were higher with TV than TA. Sensitivity for cancer was also high, but false negative rate was lower with TV than TA. The results showed that scatter from small particles and small mural echo in the cystic mass are visualized more clearly with TV, so that TV is more useful for assessing histological type of ovarian tumors. Sensitivity and specificity of ultrasonographic examination for detecting intraperitoneal recurrent lesions were 88.2% and 97.4%, respectively. Those for extraperitoneal lesions were 86.7% and 97.6%. Ultrasonography is suggested to be useful for detecting recurrence ovarian cancer especially for experitoneal lesions, as laparoscopy is not effective for such lesions.

Female

Single charge change on the helical surface of the paramyosin rod dramatically disrupts thick filament assembly in Caenorhabditis elegans.

Charge interactions between alpha-helical coiled-coil proteins have been postulated to determine the alignment of many filamentous proteins, such as myosin heavy-chain rod, paramyosin and alpha-keratin. Here we determined the sequence changes in nine mutations in the unc-15 paramyosin gene of Caenorhabditis elegans, including one nonsense, four missense, one deletion and three suppressor mutations. These mutation sites were located on a molecular model, constructed by optimizing charge interactions between paramyosin rods. Remarkably, single charge reversals (e.g., glutamic acid to lysine) were found that either disrupted or restored filament assembly in vivo. The positions of the mutations within the paramyosin molecule support the models of paramyosin assembly and further suggest that the C-terminal region containing a cluster of five mutations, and a site interacting with it, play a key role in assembly. One amino acid substitution in this C-terminal region, in which there is a "weak spot", led to a loss of reactivity with one monoclonal anti-paramyosin antibody. The results demonstrate how a single amino acid substitution can alter the assembly properties of alpha-helical molecules.

Actin Cytoskeleton

Peptide sequences for sucrose splitting and glucan binding within Streptococcus sobrinus glucosyltransferase (water-insoluble glucan synthetase).

The gene encoding glucosyltransferase responsible for water-insoluble glucan synthesis (GTF-I) of Streptococcus sobrinus (formerly Streptococcus mutans 6715) was cloned, expressed, and sequenced. A gene bank from S. sobrinus 6715 DNA was constructed in vector pUC18 and screened with anti-GTF-I antibody to detect clones producing GTF-I peptide. Five immunopositive clones were isolated, all of which produced peptides that bound alpha-1,6 glucan. GTF-I activity was found in only two large peptides: one stretching over the full length of the GTF-I peptide and composed of about 1,600 amino acid residues (AB1 clone) and the other lacking about 80 N-terminal residues and about 260 C-terminal residues (AB2 clone). A deletion study of the AB2 clone indicated that specific glucan binding, which is essential for water-insoluble glucan synthesis, was lost prior to sucrase activity with an increase in deletion from the 3' end of the GTF-I gene. These results suggest that the GTF-I peptide consists of three segments: that for sucrose splitting (approximately 1,100 residues), that for glucan binding (approximately 240 residues), and that of unknown function (approximately 260 residues), in order from the N terminus. The primary structure of the GTF-I peptide, deduced by DNA sequencing of the AB1 clone, was found to be very similar to that of the homologous protein from another strain of S. sobrinus.

Amino Acid Sequence

Fusion protein based epitope mapping of the MPB57 protein from Mycobacterium bovis BCG and its epitope insertion into the native protein.

The gene coding for the 12-kDa protein (MPB57) of Mycobacterium bovis BCG has recently been cloned and sequenced (R. Yamaguchi, K. Matsuo, A. Yamazaki, S. Nagai, K. Terasaka, and T. Yamada. 1988. FEBS Lett. 240: 115-117). To map linear B-cell epitopes by beta-galactosidase fusion proteins, we have constructed convenient vectors (pUR278S, pUR288S, and pUR289S) with the SmaI site. Based on recognition by polyclonal antibodies, two epitope regions on the MPB57 protein were identified, both of which corresponded to the amino acid sequences Glu20 to Val45 (26 residues, epitope I region) and Ile78 to Leu86 (9 residues, epitope II). Complementary oligonucleotides encoding epitope II were synthesized, polymerized by a ligase reaction, inserted into the native MPB57 protein gene, and expressed in Escherichia coli, giving rise to epitope-inserted proteins. Their stability and potential uses are described.

Amino Acid Sequence

Plasma coagulation and fibrinolysis parameters in patients with collagen diseases, and analysis of the multimeric structure of von Willebrand factor (vWF).

We studied the relationship between vascular complications and coagulation and fibrinolysis parameters in 75 subjects with collagen diseases. Thirty normal healthy persons served as controls. We found that patients with collagen diseases were in a state of a hypercoagulation and hyperfibrinolysis. In SLE (systemic lupus erythematosus) in particular, coagulation and fibrinolysis parameters appeared to be indices of vascular complications. Increases in the levels of thrombin-antithrombin III complex (TAT) and alpha 2-plasmin inhibitor-plasmin (PIP) were particularly associated with proteinuria, while increases in fibrinopeptide A (FPA) levels were associated with Raynaud's phenomenon. Administration of glucocorticoid seemed to improve the hypercoagulation and hyperfibrinolytic states of patients with collagen diseases. Analysis of the multimeric structure of von Willebrand factor (vWF) revealed a tendency for large and intermediate multimers (LIM) of plasma vWF to increase in SLE patients with accompanying vascular complications, whereas such increases were not observed in SLE patients without any vascular complications. Therefore, analysis of the multimeric structure of vWF appeared to be a useful indicator of vascular complications in collagen diseases.

Adult

Paramyosin gene (unc-15) of Caenorhabditis elegans. Molecular cloning, nucleotide sequence and models for thick filament structure.

Paramyosin is a major structural component of thick filaments isolated from many invertebrate muscles. The Caenorhabditis elegans paramyosin gene (unc-15) was identified by screening with specific antibodies an "exon-expression" library containing lacZ/nematode gene fusions. Short probes recovered from the library were used to identify bacteriophage lambda and cosmid clones that encompass the entire paramyosin (unc-15) gene. From these clones, numerous subclones containing epitopes reacting with anti-paramyosin sera were obtained, providing strong evidence that the initial cloned fragment was, in fact, derived from the structural gene for paramyosin. The complete nucleotide sequence of a 12 x 10(3) base-pair region spanning the gene was obtained. The gene is composed of ten short exons encoding a protein of 866 [corrected] amino acid residues. Paramyosin is highly similar to residues 267 to 1089 of myosin heavy chain rods. For most of its length, paramyosin appears to form an alpha-helical coiled-coil and shows the expected heptad repeat of hydrophobic amino acid residues and the 28-residue repeat of charged amino acids characteristic of myosin heavy chain rods. However, paramyosin differs from myosin in having non-helical extensions at both the N and C termini and an additional "skip" residue that interrupts the 28-residue repeat. The distribution of charges along the length of the paramyosin rod is also significantly different from that of myosin heavy chain rods. Potential charge-mediated interactions between paramyosin rods and between paramyosin and myosin rods were calculated using a model successfully applied previously to the analysis of the myosin rod sequences. Myosin rods aligned in parallel show optimal charge-charge interactions at multiples of 98 residue staggers (i.e. at axial displacements of multiples of 143 A). Paramyosin rods, in contrast, appear to interact optimally at parallel staggers of 493 residues (i.e. at axial displacements of 720 A) but show only weak interaction peaks at 98 or 296 residues. Similar calculations suggest optimal interactions between paramyosin molecules and myosin rods and in their anti-parallel alignments. The implications of these results for the structure of the bare zone and the assembly of nematode thick filaments are discussed.

Animals