Search PubMed⌕ Search

Biomedical subjects

Y Fujiki

Publications and source records attributed to Y Fujiki.

At least 19 recordsLinked to original sources
PubMed1992

Carboxyl-terminal consensus Ser-Lys-Leu-related tripeptide of peroxisomal proteins functions in vitro as a minimal peroxisome-targeting signal.

The minimal sequence requirement for a peroxisome-targeting signal was investigated using an in vitro import system. Carboxyl-terminal sequences Ser-Lys-Leu (SKL) and Leu-Gln-Ser-Lys-Leu (LQSKL) of acyl-CoA oxidase (AOX) directed to peroxisomes the fused proteins with import-incompetent forms of AOX and catalase that had been truncated, implying that the SKL tripeptide functions as a targeting signal. Elimination of the entire SKL sequence or deletion of any 1 or 2 amino acids in the sequence abolished the import activity of AOX. Substitution of alanine for serine did not affect the import activity. Topogenic activity was retained when lysine was mutated to either arginine or histidine, whereas mutation to glutamic acid completely abolished the activity. A synthetic peptide comprising the carboxyl-terminal 10 amino acid residues of AOX inhibited the import of the authentic AOX polypeptide, whereas other peptides in which SKL was mutated, deleted, or internally located were not effective. The uptake of AOX was little affected by the peptide with an amidated alpha-carboxyl group. These results strongly suggest that the carboxyl-terminal SKL motif sequence (Ser/Ala)-(Lys/Arg/His)-Leu functions as a topogenic signal in translocation of proteins into peroxisomes, requiring the whole tripeptide sequence with a free alpha-COOH group at the carboxyl terminus.

Acyl-CoA Oxidase↗
PubMed1992

A human gene responsible for Zellweger syndrome that affects peroxisome assembly.

The primary defect arising from Zellweger syndrome appears to be linked to impaired assembly of peroxisomes. A human complementary DNA has been cloned that complements the disease's symptoms (including defective peroxisome assembly) in fibroblasts from a patient with Zellweger syndrome. The cause of the syndrome in this patient was a point mutation that resulted in the premature termination of peroxisome assembly factor-1. The homozygous patient apparently inherited the mutation from her parents, each of whom was heterozygous for that mutation.

Amino Acid Sequence↗
PubMed1992

Complementation study of peroxisome-deficient disorders by immunofluorescence staining and characterization of fused cells.

Genetic heterogeneity in peroxisome-deficient disorders, including Zellweger's cerebrohepatorenal syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease, was investigated. Fibroblasts from 17 patients were fused using polyethylene glycol, cultivated on cover slips, and the formation of peroxisomes in the fused cells was visualized by immunofluorescence staining, using anti-human catalase IgG. Two distinct staining patterns were observed: (1) peroxisomes appeared in the majority of multinucleated cells, and (2) practically no peroxisomes were identified. Single step 12-(1'-pyrene) dodecanoic acid/ultraviolet (P12/UV)-selection confirmed that the former groups were resistant to this selection, most of the surviving cells contained abundant peroxisomes, and the latter cells died. In the complementary matching, [1-14C]lignoceric acid oxidation and the biosynthesis of peroxisomal proteins were also normalized. Five complementation groups were identified. Group A: Zellweger syndrome and infantile Refsum disease; Groups B, C and D: Zellweger syndrome; Group E: Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease. We compared these groupings with those of Roscher and identified eight complementation groups. There was no obvious relation between complementation groups and clinical phenotypes. These results indicate that the transport, intracellular processing and function of peroxisomal proteins were normalized in the complementary matching and that at least eight different genes are involved in the formation of normal peroxisomes and in the transport of peroxisomal enzymes.

Adrenoleukodystrophy↗
PubMed1992

Animal cell mutants represent two complementation groups of peroxisome-defective Zellweger syndrome.

Generalized peroxisome-deficient disorders including cerebro-hepato-renal Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease are autosomal recessive diseases, where catalase-containing particles (peroxisomes) are morphologically absent. We previously isolated two Chinese hamster ovary (CHO) cell mutants (Z24 and Z65) that resemble the fibroblasts from patients with such diseases, in their defective peroxisome assembly (Tsukamoto, T., S. Yokota, and Y. Fujiki. 1990. J. Cell Biol. 110:651-660). Here we report isolation by the P9OH/UV method of a peroxisome-deficient CHO mutant, ZP92, of the third complementation group distinct from those of Z24 and Z65. Peroxisomal membrane ghosts were noted by immunochemical staining in all of the CHO mutants. Complementation analysis by cell fusion of the CHO mutants with cultured fibroblasts from patients with generalized peroxisomal disorders revealed that two CHO mutants (Z24 and ZP92) represent the human complementation groups, E (the same as group 1 in the U.S.) and C (the same as group 4), respectively. These CHO cell mutants are an apparently relevant animal cell model for studies on the molecular bases and primary defects of human peroxisome-deficient diseases.

Animals↗
PubMed1992

Localization of nonspecific lipid transfer protein (nsLTP = sterol carrier protein 2) and acyl-CoA oxidase in peroxisomes of pigment epithelial cells of rat retina.

We investigated the localization of nonspecific lipid transfer protein (nsLTP) in rat retina, especially in the pigment epithelial (RPE) cells, by the avidin-biotin-peroxidase complex method on cryosections for light microscopy and by the cryoimmunogold method for electron microscopy. Light microscopic observation revealed that the RPE, inner segment layer, nerve fiber layer, and Müller cells contain nsLTP. In the RPE cells gold particles were exclusively concentrated in the small peroxisomes (microperoxisomes; 0.1-0.3 micron in diameter), which were identified by double staining using anti-nsLTP and anti-catalase antibodies. In the peroxisomes gold particles were distributed homogeneously in the matrices and no preferential binding to the limiting membrane was observed. Acyl-CoA oxidase was also localized in the matrices of the peroxisomes. We suggest that the peroxisomes in RPE cells play important roles in the metabolism of lipids of the outer segment disk membranes, especially in the beta-oxidation of polyunsaturated long-chain and very long-chain fatty acids, such as docosahexaenoic acid which is composed of approximately one third of fatty acids in the disk membranes.

Acyl-CoA Oxidase↗
PubMed1992

[Determination of acetone, methanol, and methyl ethyl ketone in urine using head-space gas chromatography (HS.GC)].

Using HS.GC, We have succeeded in simultaneous determination of Ac, MeOH and MEK in urine without any complicated pretreatment or correction by internal standard. Moreover, in order to lower the detection limits of these materials, study was made on the salting out effect using 14 kinds of salts. As pretreatment, 2.0 ml of urine, 3.0 g of sodium sulfate and small sized magnetic stirrer are put into vial, which is sealed by septum. This is then heated for 10 min in warm bath of 50 degrees C. In order to dissolve the added salts as much as possible, the specimen is stirred by the stirrer. After cooling the liquid to room temperature, the specimen is analysed by HS.GC. The results showed that sodium sulfate was excellent synthetically. 1) Using the urine of workers not exposed to organic solvents three kinds of urine having specific gravity of 1.010, 1.024 and 1.034 were prepared and mixed standard organic solvents (Ac, MeOH and MEK) were added. Recovery percentages and coefficients of variation were calculated. The results showed that recovery percentages ranged from 92.0 to 101.7% and coefficients of variation from 0.2 to 4.6%. 2) The regression equations of standard curves were satisfactory with y = 9053x - 200(r = 0.999, n = 12) for Ac, y = 801x - 400 (r = 0.999, n = 12) for MeOH, and y = 15488x - 277 (r = 0.999, n = 12) for MEK. 3) The detection limits calculated by IUPAC formula were 0.0092 mg/l for Ac, 0.11 mg/l for MeOH and 0.0063 mg/l for MEK. These results indicated that this method is superior to other methods because the pretreatment is very simple, specificity is excellent, analysis by standard curves is possible, and this method is not affected by specific gravity of the urine.

Acetone↗
PubMed1992

[Biogenesis of peroxisome--targeting signal and peroxisome assembly factor].

Peroxisome, an ubiquitous subcellular organelle in eukaryotes, functions in many crucial pathways in metabolisms such as catabolism by beta-oxidation of very long chain fatty acids, biosynthesis of etherglycerolipids, and metabolism of cholesterol. To address the question how peroxisomes are assembled in eukaryotic cells, we discuss here two topics undertaken in our laboratory. Peroxisomes are formed by posttranslational assembly mechanism; peroxisomal proteins are synthesized on free polysomes in the cytosol, mostly at their final sizes. This implies that topogenic signal(s) for import of newly synthesized polypeptides into peroxisomes reside in the internal sequence of proteins. Peroxisome-targeting signal has been noted in vivo and in vitro for enzymes such as luciferase and acyl-CoA oxidase (AOX). The topogenic signal resides at the extreme C-terminus and comprises tripeptide-Ser-Lys-Leu-COOH (SKL). Further experiments have strongly suggested that the SKL motif, Ser/Ala-Lys/Arg/His-Leu-COOH commonly found at C-termini of many peroxisomal proteins, functions as a peroxisome-targeting signal. Among several human genetic peroxisomal disorders, cerebrohepatorenal syndrome (Zellweger syndrome) is a typical, severe disease with absence of peroxisome, where a peroxisome assembly is likely to be defective. We isolated three mutants (Z24, Z65, and ZP92), recessive to wild-type cell and mutually complementary, of Chinese hamster ovary (CHO) cells that resemble the fibroblasts from Zellweger patients. To investigate molecular mechanism of peroxisome assembly and primary defects of human peroxisome-deficient disorders, we searched for the genes encoding factors that complement dysfunctions of CHO cell mutants. The mutants transfected with a pcD2-rat liver cDNA library were selected in the presence of G418.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Amino-terminal presequence of the precursor of peroxisomal 3-ketoacyl-CoA thiolase is a cleavable signal peptide for peroxisomal targeting.

To examine the function of the amino-terminal presequence of rat peroxisomal 3-ketoacyl-CoA thiolase precursor, fusion proteins of various amino-terminal regions of the precursor with non-peroxisomal enzymes were expressed in cultured mammalian cells. On immunofluorescence microscopy, all constructs carrying the presequence part exhibited punctate patterns of distribution, identical with that of catalase, a peroxisomal marker. Proteins lacking all or a part of the prepiece were found in the cytosol. These results indicate that the presequence of the thiolase has sufficient information for peroxisomal targeting.

Acetyl-CoA C-Acyltransferase↗
PubMed1991

Molecular cloning and deduced amino acid sequence of nonspecific lipid transfer protein (sterol carrier protein 2) of rat liver: a higher molecular mass (60 kDa) protein contains the primary sequence of nonspecific lipid transfer protein as its C-terminal part.

Two types of cDNA for nonspecific lipid transfer protein (nsLTP), identical to sterol carrier protein 2, of rat liver were cloned; one was 787 base pairs (bp) long containing a 429-bp open reading frame of 143 amino acids, with a mass of 15,303 Da (15-kDa protein). The cDNA from the other type was 1966 bp long, including a 1641-bp open reading frame of 547 amino acids, giving a mass of 59,002 Da (60-kDa protein). The deduced primary sequence for the 15-kDa protein was exactly the same as the published sequence of purified nsLTP, except for an extra N-terminal sequence of 20 amino acids, consistent with the finding that nsLTP is synthesized as a larger precursor and processed to a mature form. The sequence for the 60-kDa protein contained, at the 3' end, the full sequence of the 15-kDa protein, a larger precursor to nsLTP. The 15- and 60-kDa proteins, synthesized in vitro from the respective cDNAs, were both immunoprecipitated by rabbit anti-rat liver nsLTP antibody and comigrated in SDS/PAGE with the proteins made in vitro from total liver RNA. These results shed new light on the dispute among several groups of investigators about the crossreactivity of anti-nsLTP antibody with a higher molecular mass, 60-kDa protein. In Northern blot analysis, two major RNA bands, 0.85 and 2.2 kilobases (kb) long, were detected together with two minor bands of 1.6 and 2.9 kb. The 0.85- and 2.2-kb RNAs most likely encode the 15-and 60-kDa proteins, respectively.

Amino Acid Sequence↗
PubMed1991

Blood pressure during sleep: antihypertensive medication.

To investigate whether excessive reduction of blood pressure (BP) by antihypertensive medications correlates with myocardial infarction, especially during sleep in elderly patients, we used telemetry and cuvette dye-dilution methods to assess the direct BP and the hemodynamics of 68 inpatients with essential hypertension during wakefulness and sleep. There were 25 patients greater than or equal to 60 years old (OH-group) and 43 were less than or equal to 59 years old (YH-group). Of the OH-group, 36% showed high BP during the day, with marked decreases (minimum BP less than 110/70 mm Hg) during sleep. Average cardiac index (CI) of the OH-group was low during wakefulness and extremely low during slow-wave sleep. Changes of mean BP in the OH-group correlated with changes in total peripheral vascular resistance index (TPRI) during sleep, but this correlation was not observed in the YH-group. The antihypertensive effects on nocturnal BP of the various medications was: central adrenergic inhibitors less than or equal to beta blockers with intrinsic sympathomimetic activity less than or equal to alpha (alpha beta) blockers less than or equal to angiotensin-converting enzyme inhibitors less than or equal to calcium antagonists. Because BP and CI were found to be very low and TPRI seems to play an important role in BP regulation in sleeping elderly patients, excessive antihypertensive medication may be harmful to this subgroup. However, because the effects on nocturnal BP differ among various antihypertensive treatments, further research is required on the relation between antihypertensive medication and the hemodynamics of sleeping elderly hypertensive patients.

Adult↗
PubMed1991

Restoration by a 35K membrane protein of peroxisome assembly in a peroxisome-deficient mammalian cell mutant.

Peroxisomes are among the intracellular organelles of eukaryotic cells that contain specialized sets of enzymes with specific functions. Little is known of membranous components involved in assembly of the intracellular compartments. We isolated two peroxisome-deficient and mutually complementary, Chinese hamster ovary cell mutants, Z65 and Z24, which closely resembled fibroblasts from patients with autosomal recessive, peroxisome-defective disorders such as Zellweger syndrome. These patients show characteristic dysmorphism, severe hypotonia, psychomotor retardation, and peroxisomal dysfunctions and rarely survive early childhood. Here we report what seems to be the first direct cloning and characterization of a complementary DNA encoding a peroxisomal membrane protein of relative molecular mass 35,000 (Mr35K) that restores the biogenesis of peroxisomes and complements the defect of peroxisomal functions in the mutant Z65.

Amino Acid Sequence↗
PubMed1991

The individual and combined effects of X-irradiation and hyperthermia on early somite mouse embryos in culture.

The effects of 1) X-irradiation and 2) hyperthermia at a temperature of 43 degrees C individually and in combination have been investigated using cultured 8-day mouse embryos. B6C3F1 embryos were exposed to 0.3-2.0 Gy of X-rays, 5-20 min of heating, or 5 min of heating and irradiation at 0.3, 0.6, and 0.9 Gy. Irradiation alone at 0.3 Gy showed no apparent effect on embryonic development, but irradiation at 0.6-2.0 Gy caused a dose-dependent increase in malformed embryos. Heating alone for 5 min produced no malformed embryos, while heating for 10-20 min caused malformations as a function of heating time. Combined treatments produced higher frequencies (22.2-100%) of malformations than those of the sum of the separate treatments (0-41.7%). Malformations observed were primarily microphthalmia, microcephaly, and open neural tubes. The results indicate that in cultured mouse embryos irradiation combined with a "nonteratogenic dose" of hyperthermia directly exerts an additive effect on formation of the malformed embryos. In addition, a single occurrence of left-sided tail was produced by hyperthermia alone, while four occurrences were produced in combination with radiation.

Abnormalities, Radiation-Induced↗
PubMed1991

A study of the effects of delapril, a new angiotensin converting enzyme inhibitor, on the diurnal variation of arterial pressure in patients with essential hypertension using indirect and direct arterial pressure monitoring methods.

In order to investigate the effect of delapril, a new angiotensin converting enzyme inhibitor, on the diurnal variation of arterial pressure in patients with essential hypertension, this study examined 24 h arterial pressure using an indirect or a direct monitoring system. When the effect of twice-a-day administration of delapril at daily doses of 30 to 90 mg was examined using the indirect monitoring system in 12 outpatients, delapril decreased systolic and diastolic arterial pressures significantly only at limited points during the day. However, each of the averaged 24 h daytime and nighttime arterial pressures showed significant reductions. The 24 h intraarterial pressure monitoring demonstrated that delapril decreased systolic and diastolic arterial pressure at most of the measurement points. The arterial pressure reductions during daytime and nighttime were not significantly different, that is, there was no excessive reduction in nighttime arterial pressure. Heart rate and its variability were virtually unaffected by the delapril treatment in either monitoring study. No adverse reactions were observed in the indirect or direct monitoring studies. Thus, it is concluded that twice-a-day administration of delapril at daily doses of 30 to 90 mg brings about a safe and stable antihypertensive effect, without affecting the diurnal variation of arterial pressure.

Administration, Oral↗
PubMed1991

[Determination of trichloroacetic acid and trichloroethanol by head-space gas chromatography (HS.GC)].

Simultaneous determination of trichloroacetic acid (TCA) and trichloroethanol (TCE) in urine was made using head-space gas chromatography (HSGC). TCA was analyzed after methyl esterification by methanol, and TCE was measured with decomposition of conjugation adding sulfuric acid. (1) As preliminary treatment, 0.1 ml of urine and 0.6 ml of esterizer (pure water: sulfuric acid: methanol = 6:5:1, V/V/V) were mixed in a sample vial, which was sealed a septum. This was analyzed in HSGC. (2) By this method, the recovery, standard deviation and coefficient of variation of TCA were 95.7-104.3%, 0.001-0.783 mg/l and 0.8-4.0%, respectively, while those of TCE were 98.6-102.5%, 0.024-1.603 mg/l and 0.8-4.0%, respectively. (3) Calibration curves were linear up to 30 mg/l for TCA (y = 1.838x + 0.023, r = 0.999, n = 8) and 60 mg/l for TCE (y = 0.963x + 0.072, r = 0.999, n = 8). (4) A high correlation between HSGC and alkaline pyridine spectrophotometry was found for both TTC (TCA + TCE = TTC), (y = 0.917x - 3.08, r = 0.980, n = 100, p less than 0.001) and TCA (y = 0.891x - 2.36, r = 0.928, n = 100, p less than 0.001). The values for TCA and TCE obtained with HSGC were lower than those obtained with spectrophotometry. (5) The limits of the detection obtained with this method were 0.002 mg/l for TCA and 0.005 mg/l for TCE according to the formula recommended by International Union of Pure and Applied Chemistry (IUPAC). These results indicate that this simple method is accurate and useful in simultaneous detection of TCA and TCE.

Chromatography, Gas↗
PubMed1991

Sterol carrier protein2 (SCP2)-like protein in rat aorta.

Immunoblot analysis using affinity-purified antibody against sterol carrier protein2 (SCP2) showed that SCP2-like protein exists in 105,000 x g supernatant of rat aorta. Analysis of subcellular distribution of SCP2-like protein in rat aorta was determined with enzyme immunoassay (EIA). The highest level of SCP2-like protein was observed in cytosolic fraction, while the lowest level was in nuclear fraction. Analysis of marker enzymes in subcellular fractions showed that catalase, a marker enzyme of peroxisomes, leaked to cytosolic fraction to a significant extent during subcellular preparation, suggesting that SCP2-like protein in cytosolic fraction of rat aorta might be partially originated from peroxisomes. In vitro addition of homogenous SCP2 purified from rat liver dose-dependently stimulated the formation of [14C]cholesteryl esters from exogenously added [14C]cholesterol by acyl-CoA:cholesterol acyltransferase (ACAT) in microsomal preparation of rat aorta. However the addition of cytosolic fraction did not enhance cholesterol esterification by ACAT, most likely due to a markedly low level of SCP2-like protein in this fraction. The role of SCP2 in the formation of cholesteryl esters by ACAT in rat aorta was discussed.

Animals↗
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↗