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Biomedical subjects

Y Fujiki

Publications and source records attributed to Y Fujiki.

At least 127 records · Page 7Linked to original sources
PubMed1990

Topogenesis of peroxisomal proteins.

Molecular and biochemical analysis of the biogenesis of peroxisomes has made rapid progress in recent years. Research on the mechanism of targeting of peroxisomal proteins has revealed that many, but not all, peroxisomal proteins have a conserved tripeptide motif in their carboxy-terminal portions which is required for entry into peroxisomes; the topogenic signal mechanism thus differs in these instances from those employed in mitochondria and endoplasmic reticulum. Other factors involved in peroxisome biogenesis are also coming to light.

Amino Acid Sequence↗
PubMed1990

Changes of acetylcholine and choline concentrations in cerebrospinal fluids of normal subjects and patients with dementia of Alzheimer-type.

Acetylcholine (ACh) and choline (Ch) in cerebrospinal fluid from 29 normal volunteers and 7 patients with Alzheimer-type dementia (DAT) were examined using high-performance liquid chromatography with electrochemical detector coupled with liquid cation-exchange method. In normal volunteers, ACh concentration was decreased significantly from 40-50 years and Ch concentration was increased significantly from 50-60 years. CSF from patients with DAT revealed high Ch concentration and the increase was statistically significant while ACh concentration in CSF of DAT did not show a significant difference with that of normal volunteers. This Ch augmentation may suggest a disturbance in utilization of Ch for ACh synthesis and may become an useful indicator for organic changes in central cholinergic system.

Acetylcholine↗
PubMed1990

Isolation and characterization of Chinese hamster ovary cell mutants defective in assembly of peroxisomes.

We made use of autoradiographic screening to isolate two Chinese hamster ovary (CHO) cell mutants deficient in peroxisomal dihydroxyacetonephosphate acyltransferase, a key enzyme for the biosynthesis of ether glycerolipids such as plasmalogens. Morphological analysis revealed no evidence of peroxisome in these mutants. Catalase was as active as in the normal cells but was not sedimentable. Pulse-chase radiolabeling experiments and cell-free translation of RNA demonstrated that acyl-CoA oxidase, the first enzyme of the peroxisomal beta-oxidation system, was synthesized as the 75-kD form but was not converted to 53- and 22-kD mature components that were present in the wild-type CHO cells; rather, degradation was apparent. Peroxisomal thiolase was synthesized as in normal cells but remained as a larger, 44-kD precursor, whereas maturation to the 41-kD enzyme was detected in the wild-type cells. The peroxisomal 70-kD integral membrane protein was also equally synthesized, as in the wild-type cells, and was not degraded. These results suggest that assembly of the peroxisomes is defective in the mutants, whereas the synthesis of peroxisomal proteins appears to be normal. Cell-fusion studies revealed that the two mutants are recessive to the wild-type CHO cells and belong to different complementation groups. Thus, these mutants presumably contain different lesions in gene(s) encoding factor(s) required for peroxisome assembly.

Acetyl-CoA C-Acetyltransferase↗
PubMed1990

Molecular cloning and sequence of the complementary DNA encoding human mitochondrial acetoacetyl-coenzyme A thiolase and study of the variant enzymes in cultured fibroblasts from patients with 3-ketothiolase deficiency.

Complementary DNAs encoding the precursor of human hepatic mitochondrial acetoacetyl-CoA thiolase (T2) (EC 2.3.1.9) were cloned and sequenced. The cDNA inserts in these clones were 1,518 bases in length when overlapped, and encoded the 427-amino acid precursor of this enzyme (45,199 mol wt). This amino acid sequence included a 33-residue leader peptide moiety and a 394-amino acid subunit of the mature enzyme (41,385 mol wt). The T2 gene expression in fibroblasts from four patients with 3-ketothiolase deficiency was analyzed by Northern blotting. The T2 mRNA in all four cell lines had the same 1.7 kb as that of the control. However, the amounts of T2 mRNA differed: the content was reduced in two cell lines (cases 1 and 3), whereas it was within a normal range in others (cases 2 and 4). Pulse labeling followed by subcellular fractionation revealed that the T2 proteins in the fibroblasts from these patients are present in the mitochondria. These results suggest that different mechanisms are involved in the enzyme defects in the four patients.

Acetyl-CoA C-Acetyltransferase↗
PubMed1990

[A three-headed SPECT system with high resolution and high sensitivity: application to myocardial imaging].

A three-headed single-photon emission computed tomography (SPECT) system was developed, and the fundamental SPECT performance and clinical applications were investigated. The full width at half maximum (FWHM) of the SPECT system is 10.8 mm at the center of rotation with a radius of 20 cm. In clinical applications, 201Tl myocardial images with the three-headed system demonstrated a distincter and thinner myocardium compared to those with the dual-headed system. The right ventricular wall was observed even in patients without right ventricular overload. Owing to both the increased sensitivity and resolution, the three-headed system has high performance capability in clinical use such as ECG-gating and dynamic studies.

Coronary Disease↗
PubMed1989

Molecular cloning and nucleotide sequence of cDNA encoding the entire precursor of rat mitochondrial acetoacetyl-CoA thiolase.

cDNA clones for rat mitochondrial acetoacetyl-CoA thiolase were isolated and sequenced. The most 5'-extended clone (RT2-6) consisted of 1,460 bases and contained a 1,272-base open reading frame encoding a polypeptide of 424 amino acid residues. A coupled in vitro transcription/translation analysis of RT2-6 revealed that RT2-6 encodes the entire precursor of this enzyme. The amino-terminal sequence and amino acid composition of the purified enzyme agreed with the primary structure deduced from the cDNA. The calculated molecular masses of the precursor and the subunit of the mature enzyme are 44,694 and 41,364 Da, respectively. The primary structure of this enzyme was compared with those of four other thiolases (rat mitochondrial and peroxisomal 3-ketoacyl-CoA thiolases, acetoacetyl-CoA thiolase of Zoogloea ramigera, and cytosolic acetoacetyl-CoA thiolase of Saccharomyces uvarum). Marked homology between any two of them (34-51% identity) indicates that the genes of thiolases have evolved from a common ancestral gene. It has been reported that this enzyme has two isoenzymes A and B. However, the purified isoenzymes were indistinguishable from each other in some analyses. Though 17 independent cDNA clones were isolated, no definite evidence indicating the presence of different cDNAs was found.

Acetyl-CoA C-Acetyltransferase↗
PubMed1989

Biosynthesis of nonspecific lipid transfer protein (sterol carrier protein 2) on free polyribosomes as a larger precursor in rat liver.

The biosynthesis of nonspecific lipid transfer protein (nsLTP) was investigated. Total RNA of rat liver was translated in a rabbit reticulocyte lysate cell-free protein-synthesizing system with [35S]methionine as label. The immunoprecipitation of translation products with affinity-purified anti-nsLTP antibody yielded 14.5- and 60-kDa [35S]polypeptides. The molecular mass of the former polypeptide was approximately 1.5 kDa larger than that of the purified mature nsLTP (13 kDa). The site of synthesis of nsLTP was studied by in vitro translation of free and membrane-bound polyribosomal RNAs followed by immunoprecipitation. mRNA for both the 14.5- and 60-kDa polypeptides were found predominantly in the free polyribosomal fraction in both normal and clofibrate-treated rats. Clofibrate, a hypolipidemic drug that proliferates peroxisomes, did not increase the relative amount of nsLTP mRNA in rat liver. Pulse-chase experiments in rat hepatoma H-35 cells suggested that nsLTP was synthesized as a larger precursor of 14.5 kDa and converted to a mature form of 13 kDa. We have recently shown that nsLTP is highly concentrated in peroxisomes in rat hepatocytes [Tsuneoka et al. (1988) J. Biochem. 104, 560-564]. Taken together, these results suggest that nsLTP is synthesized as a larger precursor of 14.5 kDa on cytoplasmic free polyribosomes, then post-translationally transported to peroxisomes, where the precursor is presumably proteolytically processed to its mature form of 13 kDa. The relationship between the 13-kDa nsLTP and the 60-kDa polypeptide is also discussed.

Animals↗
PubMed1989

The X gene of hepatitis B virus induced growth stimulation and tumorigenic transformation of mouse NIH3T3 cells.

To examine the transforming potential of the X gene product of hepatitis B virus (HBV), the X-gene-containing region (referred to as the HBx region) was introduced into mouse NIH3T3 cells. Each transformed cell line expressed X-coding mRNA at a different level. A positive correlation was found between the level of X-coding mRNA and the saturation density of the cells. The HBx-transformed cell lines exhibited X protein production and tumor formation in nude mice. The function of HBV in oncogenesis may involve the continuous expression of the X-gene-coded product in the HBV DNA-integrated cells.

Animals↗
PubMed1989

Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus.

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.

Acyl Coenzyme A↗
PubMed1988

Nonspecific lipid transfer protein (sterol carrier protein-2) is located in rat liver peroxisomes.

Intracellular localization of nonspecific lipid transfer protein (nsLTP) in rat hepatocytes was investigated by immunoblot analysis of the subcellular fractions and immunoelectron microscopy, using affinity-purified antibody against nsLTP. Immunoblot analysis showed that the protein exists in the peroxisomal and cytosolic fractions. Further study indicated that nsLTP exists in the soluble subfraction of the peroxisomes. Immunoelectron microscopic observation revealed that nsLTP is highly concentrated in the matrices of the peroxisomes. From these results, we concluded that nsLTP mainly exists in the matrix of the peroxisomes. The role of nsLTP is discussed.

Animals↗
PubMed1987

Isolation of cDNA clones coding for peroxisomal proteins of Candida tropicalis: identification and sequence of a clone for catalase.

A cDNA library, complementary to mRNAs of alkane-grown Candida tropicalis, was screened by differential DNA dot-blot hybridization with [32P]cDNA reverse-transcribed from mRNA of alkane-grown cells or from cells in which peroxisome formation was repressed by growth on glucose. 9% of the library encodes alkane-induced sequences. The cell-free translation products of eight hybrid-selected mRNAs were characterized by SDS-polyacrylamide gel electrophoresis and fluorography: most of them are probably peroxisomal proteins. Among these, a catalase clone was identified by immunoprecipitation of the translation product with anti-catalase. The clone was sequenced: the inferred amino acid sequence is homologous to the carboxytermini of mammalian and Saccharomyces cerevisiae catalases. C. tropicalis catalase mRNA is 1.7-1.8 kb long by Northern analysis, of which 1.5-1.6 kb is required to code for the 57 kDa polypeptide. Catalase mRNA (assayed by dot-blot hybridization) is strikingly induced in C. tropicalis by growth on alkanes, suggesting that peroxisome induction is transcriptionally regulated. This sublibrary of alkane-induced, mostly peroxisomal clones, together with a recently developed cell-free peroxisome protein import assay, will permit investigation of the targeting of proteins to peroxisomes.

Amino Acid Sequence↗
PubMed1987

Effects of changes in Ca2+ concentration of the culture medium on the in vitro development of 11-day mouse embryos.

The present study was designed to examine the effects of changes in Ca2+ concentration in a culture medium on the development of 11-day mouse embryos (plug day = day 0). The embryos were individually cultured for 24 hours by rotating bottles using a culture medium of 25% rat serum and 75% Waymouth's medium. The Ca2+ concentration was changed by adding EGTA or CaCl2 to the Waymouth's medium. The Ca2+ concentrations examined were 0.5, 1.0, 1.5, 2.0, and 3.0 mM in 95% oxygen and 1.0, 2.0, and 3.0 mM in 60% oxygen. Development of embryos was evaluated on the basis of heartbeat, survival, growth, and differentiation. In 95% oxygen, the best survival was obtained at 1.5 mM Ca2+, while growth and differentiation were little affected by changes in Ca2+ concentrations except at 0.5 mM. In 60% oxygen, rhythmical heartbeats were difficult to maintain in many of the embryos at all Ca2+ concentrations examined, although reasonable development was obtained at 3.0 mM Ca2+. The optimal conditions were a culture medium containing 1.5 mM Ca2+ and an oxygen concentration of 95%. The addition of Waymouth's medium to the rat serum is useful in controlling the Ca2+ concentration of the culture medium.

Animals↗