Biomedical subjects

H Yamagata

Publications and source records attributed to H Yamagata.

At least 55 records · Page 3Linked to original sources

PubMed1998

Molecular cloning and characterization of a cDNA encoding asparagine synthetase from soybean (Glycine max L.) cell cultures.

A cDNA encoding glutamine-dependent asparagine synthetase was isolated from dark-adapted Glycine max cell culture. The deduced amino acid sequence showed 76-89% identity with other plant sequences. The gene for asparagine synthetase is expressed predominantly in shoots as compared to roots of etiolated plants and the level of expression decreases following light treatment, suggesting that the gene expression is down-regulated by light.

Amino Acid Sequence↗

PubMed1998

Rice bifunctional alpha-amylase/subtilisin inhibitor: characterization, localization, and changes in developing and germinating seeds.

A bifunctional alpha-amylase/subtilisin inhibitor (RASI) was purified to electrophoretic homogeneity from rice (Oryza sativa L.) bran. Its molecular mass was 21 kDa by SDS-PAGE and its isoelectric point was 9.05. Purified RASI inhibited subtilisin Carlsberg strongly and inhibited alpha-amylase from germinating rice seeds weakly. It inhibited rice alpha-amylase more than barley alpha-amylase, and the inhibition of rice alpha-amylase was greater at higher pHs. RASI did not inhibit trypsin, chymotrypsin, cucumisin, or mammalian alpha-amylase. The RASI was in the outermost part of the rice grain and its subcellular site seemed to be aleurone particles in aleurone cells. SDS-PAGE and western blotting showed that RASI was synthesized in the late milky stage in developing seeds, and it remained fairly constant during the first 7 days of germination.

Blotting, Western↗

PubMed1998

[A case of mild-type myotonic dystrophy with dementia and severe arteriosclerosis obliterans].

A 70-year-old man, with mild-type myotonic dystrophy (MyD) diagnosed by molecular genetic analysis when he was 68 years old, complained of worsening intermittent claudication during the past 2 years. Doppler examination revealed severe stenosis and obstruction in his leg arteries, which we diagnosed as arteriosclerosis obliterans (ASO). We then found him to be suffering from dementia, which was confirmed by dementia scale tests (Mini Mental State, 20/30; Hasegawas' Dementia Scale-Revision, 15/30). Even in mild-type MyD, as MyD is one of the progeria syndromes, the abnormal genes of MyD may accelerate the aging processes.

Aged↗

PubMed1997

Phytochrome-regulated repression of gene expression requires calcium and cGMP.

The plant photoreceptor phytochrome A utilizes three signal transduction pathways, dependent upon calcium and/or cGMP, to activate genes in the light. In this report, we have studied the phytochrome A regulation of a gene that is down-regulated by light, asparagine synthetase (AS1). We show that AS1 is expressed in the dark and repressed in the light. Repression of AS1 in the light is likely controlled by the same calcium/cGMP-dependent pathway that is used to activate other light responses. The use of the same signal transduction pathway for both activating and repressing different responses provides an interesting mechanism for phytochrome action. Using complementary loss- and gain-of-function experiments we have identified a 17 bp cis-element within the AS1 promoter that is both necessary and sufficient for this regulation. This sequence is likely to be the target for a highly conserved phytochrome-generated repressor whose activity is regulated by both calcium and cGMP.

Aspartate-Ammonia Ligase↗

PubMed1997

Cloning and analysis of a cDNA encoding a two-domain hemoglobin chain from the water flea Daphnia magna.

A cDNA encoding a two-domain hemoglobin (Hb) chain of Daphnia magna was cloned and its nucleotide (nt) sequence of 1261 bp was determined. The nt sequence contained 74 bp of the leader sequence, 1047 bp of an open reading frame (ORF), and 119 bp of the 3'-untranslated region (UTR), excluding the polyadenylation tail. A sequence, AATACA, located 24 bp upstream from the polyA sequence was considered to be a polyadenylation signal. cDNA-derived amino acid (aa) sequence revealed that D. magna Hb chain is synthesized as a secretory precursor with a signal peptide of 18 aa. Mature D. magna Hb chain consists of 330-aa residues with a calculated molecular weight of 36227, which is composed of two large repeated domains, domain 1 and 2. Several key aa that are invariant in all or most of other Hb and required for functional heme-binding are conserved in each of the two domains. The N-terminal extension (pre-A segment) of domain 1 was unusually long and contained an unusual threonine-rich sequence. The homology between the aa sequences of the two domains (24% identity) was much lower than that observed in other two-domain Hb chains from clams or nematode. Hb mRNA level in D. magna reared under low oxygen concentration was more than 12 times higher than that in D. magna reared with sufficient aeration, indicating that the expression of Hb gene is regulated by mRNA level.

Amino Acid Sequence↗

PubMed1997

Cloning of dnaK and dnaJ homologous genes from a purple non-sulfur bacterium Rhodopseudomonas species.

The dnaK and dnaJ genes were isolated as a cluster from a purple non-sulfur phototrophic bacterium, Rhodopseudomonas species No. 7 by a polymerase chain reaction (PCR) based method. The deduced products of dnaK (631 amino acids) and dnaJ (379 amino acids) were 67% and 56% identical to the respective Escherichia coli gene products. The functions of DnaK and DnaJ could be confirmed by complementation of the respective E. coli mutants.

Amino Acid Sequence↗

PubMed1997

Efficient production of a functional mouse/human chimeric Fab' against human urokinase-type plasminogen activator by Bacillus brevis.

Expression/secretion vectors for the production of Fab' and single-chain (sc) Fab' by Bacillus brevis have been constructed. For the production of Fab', the cDNAs encoding the L chain and Fd' fragment (Fd with the hinge region) of a mouse-human chimeric Fab' against human urokinase-type plasminogen activator were fused directly with the translation-start and signal-peptide-encoding regions of the mwp gene, the gene for one of the major cell-wall proteins of Bacillus brevis. The two fused genes were placed tandemly downstream from the promoter of the cell-wall protein gene operon (cwp) of B. brevis. For the production of scFab', the two cDNAs were linked with a synthetic oligonucleotide encoding a flexible peptide linker of 17 or 24 amino acids, and fused with the translation start and signal-peptide-encoding regions of the mwp gene. Fab' was efficiently produced by B. brevis, being accumulated at a level of 100 mg/l in the culture medium in a simple shake-flask culture, which is the highest level obtained so far for a gram-positive bacterium. On the other hand, the scFab' remained at a level of a few milligrams per liter in the culture medium. The Fab' produced by B. brevis showed comparable antigen-binding activity to that of the parental antibody. The L chain and Fd' fragment, constituting the Fab', had the correct N-terminal amino acid sequences. These results indicate that B. brevis is a very promising host for the production of native Ig fragments.

Amino Acid Sequence↗

PubMed1997

Haplotype analysis of congenital myotonic dystrophy patients from asymptomatic DM father.

We report a family with congenital myotonic dystrophy (CDM) transmitted from an asymptomatic DM father; we analyzed the haplotype of this family by using polymorphism within and by flanking the DM protein kinase locus. One patient with congenital DM was homozygous for all markers studied, except for the expanded CTG repeats. Two other patients with congenital DM were heterozygous. One patient with congenital DM who was homozygous had greater clinical severity and more expanded CTG repeats than other CDM patients who were heterozygous. The asymptomatic father had a DM protomutation.

Child↗

PubMed1997

Structural and functional analysis of the phosphoenolpyruvate carboxylase gene from the purple nonsulfur bacterium Rhodopseudomonas palustris No. 7.

The ppc gene, encoding phosphoenolpyruvate carboxylase (PEPC), from Rhodopseudomonas palustris No. 7 was cloned and sequenced. Primer extension analysis identified a transcriptional start site 42 bp upstream of the ppc initiation codon. An R. palustris No. 7 PEPC-deficient strain showed a slower doubling time compared with the wild-type strain either anaerobically in the light or aerobically in the dark, when pyruvate was used as a carbon source.

Amino Acid Sequence↗

PubMed1997

Secretion of human interleukin-2 in biologically active form by Bacillus brevis directly into culture medium.

We constructed an efficient system for synthesis and secretion of human interleukin-2 (IL-2) by Bacillus brevis. The secretion vector we constructed had strong promoters and contained the region coding for the signal peptide of the gene for B. brevis 47 cell-wall protein, followed directly by the gene encoding mature IL-2. Modification of the signal peptide and use of a protease-deficient mutant of B. brevis HPD31 increased productivity. When the signal peptide was more basic near its amino terminal and more hydrophobic in the middle region, IL-2 production increased 20 fold. Production by the mutant harboring the secretion vector was four fold that of the parent harboring the same plasmid. The yield of IL-2 increased further to 0.12 g/liter, when cultural conditions were made optimal, such by the addition of Tween 40 to the medium. The IL-2 produced by B. brevis had the same biological activity as authentic IL-2. Biologically active human IL-2 was produced efficiently and secreted directly into the medium by B. brevis.

Amino Acid Sequence↗

PubMed1997

Molecular cloning and characterization of a cDNA encoding early light-inducible protein from soybean (Glycine max L.).

A cDNA for early light-inducible protein (ELIP) was isolated from Glycine max L. and the nucleotides were sequenced. The cDNA encodes a 192-residue polypeptide of 20.3 kDa. The deduced amino acid sequence included a transit peptide in the amino-terminus and three hydrophobic regions long enough to span the thylakoid membranes, and had a high similarity to those of ELIPs and related polypeptides from other plants.

Amino Acid Sequence↗

PubMed1997

[Molecular genetics of myotonic dystrophy--population genetics of the CTG repeat expansion of the MTPK gene].

The CTG repeat number in the 3'-untranslated region of the myotonin protein kinase (MTPK) gene varies between 5 and 37 in normal individuals, whereas myotonic dystrophy (DM) patients have expansions from 50 to 3000 copies. However little is known about the molecular mechanisms or the genetic control of the expansion of triplet repeats. To explain the dynamic mutation mechanism and high prevalence in the population, slippage theory, multistep model and meiotic drive hypothesis have been proposed. Recent studies have shown that repeat expansion may affect neighboring genes (59 gene and DMAHP gene), or exert its effect at the RNA level by modulating the binding of (CUG)n-RNA binding proteins which are required for the maturation, stability and translation of specific mRNAs.

Genetics, Population↗

PubMed1996

The efficient production of human epidermal growth factor by Bacillus brevis.

A system has been developed for the efficient production of heterologous proteins using Bacillus brevis as a host that secretes large amounts of cell wall protein into the medium. The promoter region and signal peptide-encoding region of the cell wall protein gene were used to construct an expression-secretion vector. Bacterial proteins such as amylases can be produced in large amounts by this system (1 g/l or more), but mammalian proteins such as human alpha-amylase are produced at a low level (one or two orders of magnitude less than for bacterial proteins). The highly efficient secretion of human epidermal growth factor (h-EGF, more than 1 g/l) was obtained with B. brevis HPD31 as the host and plasmid pHY481, derived from B. brevis 481, as the vector. Recombinant hEGF was purified easily from the culture supernatant by two steps. Purified hEGF had the identical NH2-terminal amino acid sequence and COOH-terminal amino acid sequence with those of the authentic hEGF, and it was fully active in biological assays. This recombinant hEGF has been shown to be successful for biological wool harvesting (CSIRO, Australia). These results, in combination with previous results, indicate that foreign proteins of diverse origins can be produced efficiently as functional proteins in B. brevis.

Amino Acid Sequence↗

PubMed1996

Heterologous expression and site-directed mutagenesis studies on the activation mechanism and the roles of the basic residues in the prosegment of aspergillopepsinogen I.

To study the structure/function relationship of the prosegment of aspartic proteinase, a putative proform of aspergillopepsin I (or proteinase B) from Aspergillus niger var. macrosporus was expressed by Escherichia coli, refolded in vitro, and purified. The conversion of the purified proenzyme (aspergillopepsinogen I, proproteinase B) into the active mature form occurred at pH < or = 4.5 and was completely inhibited by pepstatin A, a specific inhibitor for aspartic proteinase, suggesting autoprocessing. The N-terminus of this mature form was Glu67 (numbering in preproform), which was different from the N-terminal Ser70 of native proteinase B although there was no significant difference in enzymatic activity. During the conversion, two intermediates were observed on SDS/PAGE, indicating a stepwise mechanism. The Lys56-Phe57 sequence seems to be a counterpart of the Lys-Tyr pair highly conserved in the prosequences of aspartic proteinases. When the mutant proenzyme (K56N), in which Lys56 was replaced with Asn by site-directed mutagenesis, was allowed to refold under various conditions, no significant potential activity could be obtained. Proproteinase B was also expressed by Bacillus brevis HPD31. This system required no in vitro refolding to obtain potentially active proenzyme, which was secreted into the culture medium (30-120 mg/l) and had the same properties with that obtained by the E. coli system. The K56N mutant prepared by this system also had no potential activity, and was rapidly digested by incubation with native proteinase B, suggesting that the mutant did not fold correctly. On the other hand, the K56R mutant (Lys56-Arg) was potentially active. These results indicated that Lys56 is essential for the folding through electrostatic interaction with the catalytic Asp residues in the active site although it may be replaced with Arg. In the presence of a low concentration of pepstatin A, an incompletely processed form with N-terminal Ser53 was obtained. Further, the R52Q (Arg52-->Glin) mutant showed no processing but was converted to the active mature form by incubation with the native enzyme. Therefore, the cleavage between Arg52 and Ser53 is considered to be the initial and essential step of the autoactivation. The R26Q, K27Q, R36Q, K40Q, R42Q, and K66Q mutants were also potentially active. The K66Q mutant was processed to a form with N-terminal Ala55.

Amino Acid Sequence↗

PubMed1996

Association of CTG repeats and the 1-kb Alu insertion/deletion polymorphism at the myotonin protein kinase gene in the Japanese population suggests a common Eurasian origin of the myotonic dystrophy mutation.

We have studied linkage disequilibrium between CTG repeats and an Alu insertion/deletion polymorphism at the myotonin protein kinase gene (DMPK) in 102 Japanese families, of which 93 were affected with myotonic dystrophy (DM). All of the affected chromosomes are in complete linkage disequilibrium with the Alu insertion allele. Among the normal chromosomes, alleles of CTG repeats 5 and > or = 17 are exclusively associated with the insertion allele. On the other hand, intermediate alleles of 11-16 repeats show a significantly greater association with the deletion allele. A strikingly similar pattern of linkage disequilibrium observed in European populations suggests a common origin of the DM mutation in the Japanese and European populations.

Asian People↗

PubMed1996

Late development of anti-La/SS-B antibodies in a patient with Sjogren's syndrome and high titer anti-Ro/SS-A antibodies.

The frequent coexistence of anti-Ro and anti-La autoantibodies is well described, however, there is little evidence of sequential development of these two autoantibodies. We report a case of typical Sjogren's syndrome with high titer anti-Ro antibodies, who subsequently developed anti-La antibodies later in the course. This case suggests that the anti-La antibodies may actually follow the anti-Ro antibodies in some cases as hypothesized in the concept of linked set of autoantibodies, analogous to development of anti-Sm in certain anti-nRNP antibody positive SLE patients and animal models.

Antibodies, Antinuclear↗

PubMed1996

Restricted heterogeneity and changing spectrotypes in autoantibodies to La/SS-B.

Isoelectric focusing (IEF) spectrotype of specific immunoglobulins has been studied as a marker for B-cell clonality. In the present study, the spectrotype of anti-La antibodies in human autoimmune sera were analyzed by newly developed IEF sandwich assay in which focused total immunoglobulin on filter papers are incubated with crude antigen followed by horse-radish peroxidase-labeled anti-La antibodies. The anti-La spectrotypes contained oligoclonal bands, the positions and patterns of which are different in each patient, suggesting the preferential expansion of limited numbers of anti-La producing B-cell clones unique to individual patients. Furthermore, the bands on anti-La spectrotype in sequentially obtained sera changed continuously, suggesting alteration in the expanding anti-La producing clones. These may reflect affinity maturation and/or diversification of the B-cell epitopes involving somatic mutation.

Antibodies, Antinuclear↗

PubMed1996

An intergenerational contraction of the CTG repeat in Japanese myotonic dystrophy.

We present the first report of a Japanese family with myotonic dystrophy (DM) that showed an intergenerational contraction of the CTG repeat. The size of the expanded CTG repeats was 3.2 kb for the father and 2.2 kb for the daughter, indicating that the expansion decreased during transmission from the father to the daughter. Despite the CTG repeat contraction, the daughter showed earlier age of onset than the father. However, she appeared to be less severely affected than the father. We discuss the correlation between the CTG repeat contraction and the clinical phenotype. The presence of the CTG repeat contraction in Japanese DM is important for genetic counseling of Japanese DM families.

Adult↗