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Biomedical subjects

S Yoshitake

Publications and source records attributed to S Yoshitake.

At least 73 records · Page 4Linked to original sources
PubMed1987

Hemophilia B (factor IXSeattle 2) due to a single nucleotide deletion in the gene for factor IX.

To understand the molecular basis for hemophilia B in patients with little or no circulating Factor IX antigen, a patient who had less than 0.2% circulating Factor IX antigen (Factor IXSeattle 2) was selected for analysis of his Factor IX gene. Genomic DNA fragments from the abnormal gene were cloned into bacteriophage lambda vectors and recombinant phage were identified using radiolabeled genomic probes obtained from the normal Factor IX gene. The exons and flanking regions of the abnormal gene were sequenced by the dideoxy chain-termination method and this sequence was compared with that of the normal gene. Only one significant difference was observed, the deletion of a single adenine nucleotide in exon V. This resulted in a frameshift that converted an aspartic acid at position 85 in the protein to a valine and the formation of a stop signal at position 86. These data indicate that the gene for Factor IXSeattle 2 codes for an 85 residue polypeptide that terminates after the first epidermal growth factor domain. Thus, the putative Factor IXSeattle 2 polypeptide lacks the second epidermal growth factor domain, the activation peptide, and the catalytic domain present in the normal protein. This provides an explanation for the coagulation disorder in this patient and represents the first report of a single nucleotide deletion and frameshift resulting in hemophilia B.

Adenine↗
PubMed1986

Human liver fibronectin complementary DNAs: identification of two different messenger RNAs possibly encoding the alpha and beta subunits of plasma fibronectin.

Human fibronectin polymorphism arises from variation in the C-terminal region [e.g., Sekiguchi, K., Siri, A., Zardi, L., & Hakomori, S. (1985) J. Biol. Chem. 260, 5105-5114]. In order to verify the chemical basis of the fibronectin polymorphism, cDNAs encoding the C-terminal region of human liver fibronectin have been isolated, sequenced, and compared with cDNAs encoding so-called "cellular fibronectin" (i.e., fibronectin produced by cultured cells in vitro). Among the five independent cDNAs thus isolated, two cDNAs, named pLF2 and pLF4, differed in the nucleotide sequence at the "type III connecting segment" (IIIcs) region. pLF4 contained 192 bases in this region whereas pLF2 completely lacked these bases. S1 mapping analysis indicated that both cDNAs with and without the 192 bases are faithful copies of two fibronectin mRNA species abundantly present in human liver. Comparison of the liver cDNAs with those coding for cellular fibronectin indicates that the latter cDNAs contain the 75-base and/or 93-base extra segments at the 5' and 3' boundaries of the 192-base IIIcs region. These extra segments have the consensus sequences for the 3' splice sites at their 3' ends, suggesting that fibronectin mRNAs with partial or complete deletion of the IIIcs sequence result from alternative splicing of a primary RNA transcript. Liver fibronectin cDNAs also lacked the 270-based "extra domain" (ED) segment present in some, but not all, cDNAs encoding cellular fibronectin. Thus, cellular fibronectin appears to have three extra peptide segments, encoded by the 75-base and 93-base segments in the IIIcs region and by the 270-base ED region, that are mostly absent in the liver fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Amino acid sequence of human histidine-rich glycoprotein derived from the nucleotide sequence of its cDNA.

A lambda gt 11 library containing cDNA inserts prepared from human liver mRNA has been screened with an affinity-purified antibody to human histidine-rich glycoprotein (HRG) and then with a restriction fragment isolated from the 5' end of the largest cDNA insert obtained by antibody screening. A number of positive clones were identified and shown to code for HRG by DNA sequence analysis. A total of 2067 nucleotides were determined by sequencing 3 overlapping cDNA clones, which included 121 nucleotides of 5'-noncoding sequence, 54 nucleotides coding for a leader sequence of 18 amino acids, 1521 nucleotides coding for the mature protein of 507 amino acids, a stop codon of TAA, and 352 nucleotides of 3'-noncoding sequence followed by a poly(A) tail of 16 nucleotides. The length of the noncoding sequence of the 3' end differed in several clones, but each contained a polyadenylylation or processing sequence of AATAAA followed by a poly(A) tail. More than half of the amino acid sequence of HRG consisted of five different types of internal repeats. Within the last 3 internal repeats (type V), there were 12 tandem repetitions of a 5 amino acid segment with a consensus sequence of Gly-His-His-Pro-His. This repeated portion, referred to as a "histidine-rich region", contained 53% histidine and showed a high degree of similarity to a histidine-rich region of high molecular weight kininogen.

Amino Acid Sequence↗
PubMed1986

A more sensitive and less time-consuming sandwich enzyme immunoassay for insulin in human serum with less serum interference.

This paper describes an improved sandwich enzyme immunoassay for insulin in human serum. The detection limit was significantly improved from 0.1 mU/L to 0.02 mU/L, by incubation with guinea-pig anti-insulin Fab'-peroxidase conjugate in the presence of normal guinea-pig F(ab')2 to reduce the non-specific binding of the conjugate. The degree of serum interference was considerably reduced by coating polystyrene balls with acid-treated anti-insulin IgG and by incubating the polystyrene balls with serum samples at a lower temperature for a shorter time. The time for incubation with anti-insulin Fab'-peroxidase conjugate was also shortened. As a result, the volume of serum that could be used increased from 10 microliters to 50 microliters, and the time for immuno-reactions was reduced from 8 h to 4 h. Applicability of the present improvement to enzyme immunoassay of other antigens in human serum is discussed.

Humans↗
PubMed1985

Nucleotide sequence of the gene for human factor IX (antihemophilic factor B).

Two different human genomic DNA libraries were screened for the gene for blood coagulation factor IX by employing a cDNA for the human protein as a hybridization probe. Five overlapping lambda phages were identified that contained the gene for factor IX. The complete DNA sequence of about 38 kilobases for the gene and the adjacent 5' and 3' flanking regions was established by the dideoxy chain termination and chemical degradation methods. The gene contained about 33.5 kilobases of DNA, including seven introns and eight exons within the coding and 3' noncoding regions of the gene. The eight exons code for a prepro leader sequence and 415 amino acids that make up the mature protein circulating in plasma. The intervening sequences range in size from 188 to 9473 nucleotides and contain four Alu repetitive sequences, including one in intron A and three in intron F. A fifth Alu repetitive sequence was found immediately flanking the 3' end of the gene. A 50 base pair insert in intron A was found in a clone from one of the genomic libraries but was absent in clones from the other library. Intron A as well as the 3' noncoding region of the gene also contained alternating purine-pyrimidine sequences that provide potential left-handed helical DNA or Z-DNA structures for the gene. KpnI repetitive sequences were identified in intron D and the region flanking the 5' end of the gene. The 5' flanking region also contained a 1.9-kb HindIII subfamily repeat. The seven introns in the gene for factor IX were located in essentially the same position as the seven introns in the gene for human protein C, while the first three were found in positions identical with those in the gene for human prothrombin.

Amino Acid Sequence↗
PubMed1985

The nucleotide sequence of the gene for human protein C.

A human genomic DNA library was screened for the gene for protein C by using a cDNA probe coding for the human protein. Three different overlapping lambda Charon 4A phage were isolated that contain inserts for the gene for protein C. The complete sequence of the gene was determined by the dideoxy method and shown to span about 11 kilobases of DNA. The coding and 3' noncoding portion of the gene consists of eight exons and seven introns. The eight exons code for a preproleader sequence of 42 amino acids, a light chain of 155 amino acids, a connecting dipeptide of Lys-Arg, and a heavy chain of 262 amino acids. The preproleader sequence and the connecting dipeptide are removed during processing, resulting in the mature protein composed of a heavy and a light chain held together by a disulfide bond. The heavy chain also contains the catalytic region for the serine protease. Two Alu sequences and two homologous repeats of about 160 nucleotides were found in intron E. The seven introns in the gene for protein C are located in essentially the same positions in the amino acid sequence as the seven introns in the gene for human factor IX, while the first three introns in protein C are located in the same positions as the first three in the gene for human prothrombin.

Amino Acid Sequence↗
PubMed1985

An intragenic deletion of the factor IX gene in a family with hemophilia B.

A family of seven patients severely afflicted with hemophilia B has been studied for their factor IX genes through the use of factor IX cDNA and genomic DNA probes. The patients had detectable (less than 10% of normal) factor IX antigen in urine and no detectable inhibitors in sera to factor IX protein. Based on the DNA hybridization analysis, these patients showed a partial intragenic deletion in their factor IX gene. The deletion included two exons (exons V and VI) coding for the amino acid sequence from number 85 to 195 of the factor IX protein. The deleted portion of the gene contained the entire factor IX activation peptide. The length of the deletion was estimated to be 10 +/- 0.3 kilobase pairs. This specific gene has been named FIXSeattle. In this family both the deletion and a Taq 1 restriction fragment length polymorphism can be used as a useful marker for accurate detection of female carriers of the deficient factor IX gene.

Chromosome Deletion↗
PubMed1983

Use of inorganic salts to minimize serum interference in a sandwich enzyme immunoassay for human growth hormone using Fab'-horseradish peroxidase conjugate.

In a sandwich enzyme immunoassay (EIA) for human growth hormone (hGH) with anti-hGH Fab'-peroxidase conjugate, the effects of inorganic salts on serum interference were examined, and serum interference was eliminated by incubation of serum samples with anti-hGH IgG-coated polystyrene balls in the presence of 0.4 mol/l NaCl, avoiding the need for hGH-free serum. The sensitivity for hGH was 60 fg/tube or 3 ng/l of serum. No cross-reaction was observed with prolactin, chorionic gonadotropin or luteinizing, thyroid-stimulating and follicle-stimulating hormones. The coefficients of within-assay and between-assay variations were 2.8-6.5% and 4.8-8.7%, respectively. The regression equation and correlation coefficient to radioimmunoassay (RIA) were y(EIA) = 0.89x(RIA) + 0.11 and 0.98 (n = 100), respectively. hGH levels in normal male and female adult serum taken between 9:00 and 10:00 a.m. after overnight fasting and 1 h rest were 312 ng/l (range 53-940 ng/l; n = 10) and 662 ng/l (112-2195 ng/l; n = 13), respectively.

Adult↗
PubMed1983

Preparation of monomeric Fab'-horseradish peroxidase conjugate using thiol groups in the hinge and its evaluation in enzyme immunoassay and immunohistochemical staining.

Horseradish peroxidase and Fab' were conjugated by using thiol groups in the hinge of Fab'. Maleimide or pyridyl disulfide groups were introduced into peroxidase by treatment with N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate or N-succinimidyl 3-(2-pyridyldithio)propionate and were allowed to react with thiol groups in the hinge of Fab'. The conjugates were obtained in high yields with a minimal polymerization and without impairing the activities of peroxidase and antibodies, and were superior to those prepared using amino groups of Fab' by the glutaraldehyde and periodate methods in performing sandwich enzyme immunoassay and immunohistochemical staining. The conjugate yield was higher in the maleimide method than in the pyridyl disulfide method.

Enzyme-Linked Immunosorbent Assay↗
PubMed1983

An enzyme-linked immunoassay for the measurement of circulating immune complexes.

A solid-phase, enzyme-linked immunoassay for the quantitation of circulating immune complexes was developed using C1q. In this assay, the immune complexes in human serum are adhered to C1q-coated polystyrene tubes and then detected by a beta-D-galactosidase-conjugated monovalent fragment (Fab') of rabbit (anti-human IgG, gamma chain-specific) IgG. The enzyme activity bound to the tubes was determined fluorometrically using 4-methylumbelliferyl-beta-D-galactopyranoside as substrate. The sensitivity of this assay was as little as 100 ng/tube, which corresponded to 6 micrograms/ml when 16.7 microliter of serum was assayed. The specificity of the assay was demonstrated by the following observations: (1) absence of cross-reaction of monomeric IgG; (2) non-detectability of immune complex in most of the sera from normal subjects; (3) parallelism of the standard curve with dilution of reference serum. The precision of the assay was proved by the demonstration of sufficient within-assay coefficients of variation (10.7-14.9%).

Animals↗