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Biomedical subjects

Y Fujiki

Publications and source records attributed to Y Fujiki.

At least 145 records · Page 8Linked to original sources
PubMed1986

Induction, identification, and cell-free translation of mRNAs coding for peroxisomal proteins in Candida tropicalis.

Peroxisomes have been purified from Candida tropicalis grown on oleic acid and shown to be nearly pure by marker enzyme analysis, electron microscopy, and comparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They contain approximately 20 polypeptides, among which acyl-CoA oxidase, trifunctional hydratase-dehydrogenase-epimerase, and catalase have been identified. Rabbit antisera have been elicited that react with these three proteins. When C. tropicalis is grown on alkanes, a dozen mRNAs are strikingly induced. Nine of the 12 induced mRNAs code for polypeptides that comigrate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with peroxisomal proteins, among which three have been identified immunochemically as the acyl-CoA oxidase, the trifunctional protein, and catalase. These results indicate that some genes coding for peroxisomal proteins are strongly expressed during growth of C. tropicalis on alkanes. The data are consistent with evidence in other species that peroxisomes form by the post-translational incorporation of newly made proteins into pre-existing peroxisomes, generally without proteolytic processing, followed by peroxisome division.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1986

Presence of the peroxisomal 22-kDa integral membrane protein in the liver of a person lacking recognizable peroxisomes (Zellweger syndrome).

Peroxisomes have not been detected in liver and kidney of patients with Zellweger syndrome. Some peroxisome proteins are missing; others are present in normal amounts but are located in the cytosol. We have prepared an antiserum against the 22-kDa integral membrane protein characteristic of rat liver peroxisomes. The antiserum crossreacts with the human liver counterpart, which likewise has a mass of 22 kDa. By immunoblot analysis, we demonstrate that the 22-kDa protein is present in normal amount in Zellweger liver and is integral to a membrane. The result suggests that peroxisome membranes are assembled in Zellweger syndrome but may be defective for the import of matrix proteins. As a result, newly synthesized proteins are left in the cytosol, where some persist and others are degraded. Lacking their usual content, such aberrant peroxisomal membranes would be unrecognizable morphologically. Immunoblot analyses also showed that the peroxisomal hydratase-dehydrogenase is deficient in Zellweger kidney as well as liver, but catalase is present in both organs.

Catalase↗
PubMed1986

[CRF-like immunoreactivities in mammalian brains examined with a radioimmunoassay for CRF].

A sensitive and useful radioimmunoassay for CRF was established using synthetic ovine CRF. CRF-like immunoreactivities of hypothalami in the bovine, human, rat, guinea pig, and mouse could be detected with this radioimmunoassay. A variety of CRF-like immunoreactivities of hypothalami in the mammalian brains led us to discuss a putative species difference of amino-acid structure in CRF. In the bovine, CRF-like immunoreactivities of the frontal cortex, caudate nucleus, putamen, and hippocampus could be detected in addition to CRF-like immunoreactivity of hypothalamus. These observations suggested a wide distribution of CRF in the central nervous system, and gave room for discussion about a physiological role of extrahypothalamic CRF. Gel chromatographic studies revealed a high-molecular-weight form of CRF-like immunoreactivity in the bovine frontal cortical profile that was not found in the hypothalamic profile. The results show a multiplicity of CRF-like immunoreactivity in the bovine brain.

Animals↗
PubMed1985

Post-translational import of fatty acyl-CoA oxidase and catalase into peroxisomes of rat liver in vitro.

Total polysomal RNA of rat liver was translated in vitro in a rabbit reticulocyte lysate system. The translation products were mixed with a postnuclear supernatant fraction of rat liver and incubated post-translationally at 26 degrees C for 15-60 min. The import assay mixture was separated into a particulate fraction and supernatant by centrifugation, both of which were analyzed by immunoprecipitation with a goat antibody against rat liver peroxisomal proteins, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography. One peroxisomal translation product (Mr 72,000) appeared in the particulate fraction, was partly proteinase K-resistant, and addition of detergents prior to proteolysis abolished this resistance. In isopycnic centrifugation of the uptake assay mixture, the protease-resistant 35S-polypeptide of Mr 72,000 cosedimented with the peroxisomes. This translation product was identified immunochemically as fatty acyl-CoA oxidase; both before and after import it was indistinguishable in size from subunit A of the purified enzyme by prolonged sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the cell-free translation products were incubated with highly purified peroxisomes, 35S-catalase entered peroxisomes (by the criterion of protease resistance), and its entry was stimulated by the addition of a high speed supernatant (cytosolic) fraction of rat liver. These results demonstrate the post-translational import into peroxisomes in vitro of at least two cell-free translation products.

Acyl-CoA Oxidase↗
PubMed1985

Synthesis of 3-ketoacyl-CoA thiolase of rat liver peroxisomes on free polyribosomes as a larger precursor. Induction of thiolase mRNA activity by clofibrate.

The site of synthesis and induction by clofibrate of peroxisomal 3-ketoacyl-CoA thiolase (acetyl-CoA acyltransferase; EC 2.3.1.16) was investigated. Free and membrane-bound polyribosomal RNA species from the livers of normal rats and rats treated with clofibrate, a hypolipidaemic drug that causes marked proliferation of peroxisomes, were translated in a nuclease-treated rabbit reticulocyte-lysate cell-free protein-synthesizing system with [35S]methionine as label. The cell-free translation products were immunoprecipitated with monospecific X rabbit anti-thiolase serum and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and fluorography. Thiolase mRNA was found predominantly in free polyribosomes, in both normal and clofibrate-treated rats. Clofibrate treatment increased mRNA activity for thiolase approx. 20-fold. The translation product of clofibrate-induced thiolase mRNA migrated slightly faster in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis than did the translation product of normal thiolase mRNA. Both the normal and the clofibrate-induced translation products were approx. 6000 Da larger than the 41000-Da subunit of the purified enzyme. Immunoblot analysis of liver homogenates, isolated peroxisomes and the purified enzyme indicated that the thiolase subunit was approx. 41000 Da in all samples, ruling out proteolysis during the purification of thiolase. Thiolase biogenesis thus differs from that of rat liver peroxisomal proteins studied previously in that it is synthesized as a larger precursor, implying post-translational import of thiolase into peroxisomes with proteolytic processing. Clofibrate apparently alters the size as well as the amount of the translation product.

Acetyl-CoA C-Acyltransferase↗
PubMed1985

Cloning of cDNA coding for peroxisomal acyl-CoA oxidase from the yeast Candida tropicalis pK233.

Candida tropicalis pK233 cells were grown with n-alkanes as carbon source to induce the synthesis of peroxisomal proteins and the proliferation of peroxisomes. Poly-(A)+ RNA was isolated and used to construct a cDNA library by insertion of double-stranded reverse transcripts into the Pst I site of pBR322 followed by cloning in Escherichia coli. Clones complementary to mRNAs induced by growth on alkanes were selected by differential DNA dot-blot analysis using [32P]cDNA reverse-transcribed from poly(A)+ RNA of glucose-grown cells (which contain few peroxisomes) or of alkane-grown cells. Among these clones, one containing a 1.7-kilobase insert coding for acyl-CoA oxidase (the first enzyme in the peroxisomal Beta-oxidation pathway) was identified by hybridization-selection translation and immunoprecipitation. By RNA blot analysis, the acyl-CoA oxidase mRNA was estimated to be approximately equal to 2.2 kilobases long, of which 2.1 kilobases are required to code for the approximately equal to 76-kDa protein. Since the mRNA is polyadenylylated, there appears to be little additional nontranslated region. Cell-free mRNA translation and RNA dot-blot hybridization analyses demonstrated that, whereas glucose-grown C. tropicalis contained little or no acyl-CoA oxidase mRNA, alkane-grown cells contained so much of this mRNA as to make acyl-CoA oxidase one of the major in vitro translation products.

Acyl-CoA Oxidase↗
PubMed1985

Partial disassembly of peroxisomes.

Rat liver peroxisomes were subjected to a variety of procedures intended to partially disassemble or damage them; the effects were analyzed by recentrifugation into sucrose gradients, enzyme analyses, electron microscopy, and SDS PAGE. Freezing and thawing or mild sonication released some matrix proteins and produced apparently intact peroxisomal "ghosts" with crystalloid cores and some fuzzy fibrillar content. Vigorous sonication broke open the peroxisomes but the membranes remained associated with cores and fibrillar and amorphous matrix material. The density of both ghosts and more severely damaged peroxisomes was approximately 1.23. Pyrophosphate (pH 9) treatment solubilized the fibrillar content, yielding ghosts that were empty except for cores. Some matrix proteins such as catalase and thiolase readily leak from peroxisomes. Other proteins were identified that remain in mechanically damaged peroxisomes but are neither core nor membrane proteins because they can be released by pyrophosphate treatment. These constitute a class of poorly soluble matrix proteins that appear to correspond to the fibrillar material observed morphologically. All of the peroxisomal beta-oxidation enzymes are located in the matrix, but they vary greatly in how easily they leak out. Palmitoyl coenzyme A synthetase is in the membrane, based on its co-distribution with the 22-kilodalton integral membrane polypeptide.

Animals↗
PubMed1985

Zellweger syndrome: biochemical and morphological studies on two patients treated with clofibrate.

Two infants with Zellweger syndrome (cerebro-hepato-renal syndrome) have been studied biochemically and morphologically. Peroxisomal enzymes involved in respiration, fatty acid beta-oxidation, and plasmalogen biosynthesis were assessed. In liver, catalase was present in normal amounts but was located in the cell cytosol. Dihydroxyacetone phosphate acyltransferase activity was less than one-tenth of normal. The amount of the bifunctional protein catalyzing two beta-oxidation reactions was found by immunoblotting to be greatly reduced. Catalase activity was normal in intestine. D-Amino acid oxidase was subnormal in kidney. The observed enzyme deficiencies may plausibly explain many of the metabolite imbalances observed clinically. Morphologically, peroxisomes were absent from liver. In intestine, normal peroxisomes were also missing, but some rare, smaller (0.04-0.13 micrometer) bodies were seen with a slight positive cytochemical reaction for catalase. These results, together with current concepts of peroxisome biogenesis, suggest but do not prove, that the primary defect in Zellweger syndrome may be in peroxisome assembly. The infants were treated with clofibrate, but it was ineffectual as assessed biochemically, morphologically, and clinically.

Abnormalities, Multiple↗
PubMed1984

Heating RNA before cell-free translation is essential for the efficient and reproducible synthesis of several peroxisomal proteins.

Total RNA, extracted with guanidinium thiocyanate from liver of clofibrate-treated rats, was translated in vitro. Heating the RNA at 60 degrees C for 5 min before translation increased the synthesis of three peroxisomal polypeptides 10-100-fold. Preproalbumin synthesis increased 10-fold. Total incorporation of [35S]methionine into proteins merely doubled. Heating is essential for reproducible and adequate translation of mRNAs coding for peroxisomal and some other proteins.

Animals↗
PubMed1984

The effect of 5-bromodeoxyuridine on mouse embryos during neurulation in vitro.

Mouse embryos explanted at various stages during neurulation were cultured for 20-28 h in the presence of 25-900 micrograms/ml of 5-bromodeoxyuridine (BUdR). BUdR strongly inhibited closure of the cranial neural tube, which was found to be stage-dependent. When mouse embryos were exposed to BUdR after development of the concave curvature in the neuroepithelium of the midbrain to the upper hindbrain regions, they became insensitive to the drug-induced open cranial neural tube. Histological observations showed that BUdR interfered with interkinetic migration and cytokinesis of the neuroepithelial cells. These cellular abnormalities were not dependent on the morphological development of the cranial neural folds. The 3H-BUdR experiment confirmed that the label was mostly incorporated into the DNA fraction.

Abnormalities, Drug-Induced↗
PubMed1984

Synthesis of a major integral membrane polypeptide of rat liver peroxisomes on free polysomes.

The manner of synthesis and assembly of the peroxisomal membrane proteins is unknown. Understanding these processes is essential to an understanding of the formation of the organelle. We have investigated the biogenesis of the previously identified major 21.7-kDa integral peroxisomal membrane polypeptide [Fujiki, Y., Fowler, S., Shio, H., Hubbard, A. L. & Lazarow, P. B. (1982) J. Cell Biol. 93, 103-110]. This protein was purified to apparent homogeneity and used to elicit a rabbit antiserum. In immunoblotting analysis, antibody bound only to the 22-kDa membrane polypeptide present exclusively in peroxisomal membranes. Total rat liver RNA was translated in a nuclease-treated rabbit reticulocyte cell-free protein-synthesizing system. The in vitro translation product, isolated by means of the antibody and Staphylococcus aureus cells, comigrated with the mature 22-kDa polypeptide in NaDodSO4/PAGE. Analysis of the translation products of RNAs from free and membrane-bound polysomes indicated that the mRNA for the 22-kDa membrane polypeptide is found predominantly in free polysomes. The results imply post-translational insertion of the membrane polypeptide into the peroxisomal membrane without proteolytic processing and suggest that peroxisomes, like mitochondria and chloroplasts, form by fission from preexisting organelles.

Animals↗
PubMed1984

Acyl-Coa oxidase and hydratase-dehydrogenase, two enzymes of the peroxisomal beta-oxidation system, are synthesized on free polysomes of clofibrate-treated rat liver.

We investigated the site of synthesis of two abundant proteins in clofibrate-induced rat hepatic peroxisomes. RNA was extracted from free and membrane-bound polysomes, heated to improve translational efficiency, and translated in the mRNA-dependent, reticulocyte-lysate-cell-free, protein-synthesizing system. The peroxisomal acyl-CoA oxidase and enoyl-CoA hydratase-beta-hydroxyacyl-CoA dehydrogenase 35S-translation products were isolated immunochemically, analyzed by SDS PAGE and fluorography, and quantitated by densitometric scanning. The RNAs coding for these two peroxisomal proteins were found predominantly on free polysomes, and the translation products co-migrated with the mature proteins. As in normal rat liver, preproalbumin and catalase were synthesized mainly by membrane-bound and by free polysomes, respectively. mRNAs for a number of minor 35S-translation products also retained by the anti-peroxisomal immunoadsorbent were similarly found on free polysomes. These results, together with previous data, allow the generalization that the content proteins of rat liver peroxisomes are synthesized on free polysomes, and the data imply a posttranslational packaging mechanism for these major content proteins.

Acyl-CoA Oxidase↗
PubMed1984

[Effect of local sustained stress on general oxygen consumption in belt conveyer workers].

The modern mechanization and rationalization of production procedures have recently reduced the intensity of work in most factories. However, it has been pointed out that monotonous inactive work and/or co-operative work can in some sense impose stronger mental and physical stresses which may induce ill health in workers. In this study, assuming the assembly and adjustment of videotape-recorders on a belt conveyor to be representative of monotonous inactive and co-operative work, changes in oxygen consumption, heart rate, blood pressure, body temperature, critical flicker fusion and subjective symptoms of fatigue throughout a workday have been followed for six female workers engaged in this type of work. The main results obtained were as follows: The average oxygen consumption of six workers was 230 ml/min, almost unchanged during the work. The belt conveyor work indicated the metabolic rate of 0.3 in terms of relative metabolic rate. The net daily energy expenditure due to the work was only about 100 kcal. The average heart rate during assembly and adjustment were respectively, about 1.4 and 1.2 times the average heart rate at rest. The blood pressure and the rate of oxygen removal increased slightly as the work progressed in assembly work, but did not increase at all in adjustment work. The critical flicker fusion and body temperature showed little change during the work.

Adult↗