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1.
Strahlenther Onkol ; 200(2): 123-127, 2024 Feb.
Article in English | MEDLINE | ID: mdl-37264179

ABSTRACT

PURPOSE: The purpose of this study was to investigate the feasibility and effectiveness of high-dose planned adaptive intensity-modulated radiation therapy (IMRT) with helical tomotherapy (HT) for cutaneous angiosarcoma (cAS) of the scalp. METHODS: This retrospective cross-sectional included a total of 12 consecutive patients with cAS of the scalp who underwent high-dose planned adaptive IMRT with HT. Prescribed doses were 72.5-74 Gy in 35-37 fractions for the gross tumor volume plus a 1-2 mm margin (PTV1), 58-60 Gy in 29-30 fractions for the clinical target volume plus a 2-3 mm margin (PTV2), and 46 Gy in 23 fractions for the clinical target volume plus a 5-15 mm margin (PTV3) over periods of 7.5, 6 and 4.5 weeks, respectively. RESULTS: The estimated 1­year and 2­year overall survival rate were 65.6% and 27.3%, respectively, and the local progression-free survival at 2 years was 74.1%. All local recurrences were either in or marginal to the PTV2 or PTV3. No local recurrence was observed in the PTV1. All patients tolerated the treatment without grade 3 or higher adverse events during the radiotherapy period. No late adverse events were observed during the follow-up period. CONCLUSION: Planned adaptive high-dose IMRT with HT has the potential to improve local control rate without increasing adverse events.


Subject(s)
Hemangiosarcoma , Radiotherapy, Intensity-Modulated , Humans , Radiotherapy Dosage , Hemangiosarcoma/radiotherapy , Radiotherapy Planning, Computer-Assisted , Scalp , Cross-Sectional Studies , Retrospective Studies
2.
J Cell Sci ; 123(Pt 10): 1761-74, 2010 May 15.
Article in English | MEDLINE | ID: mdl-20427315

ABSTRACT

Nuclear transcribed genes produce mRNA transcripts destined to travel from the site of transcription to the cytoplasm for protein translation. Certain transcripts can be further localized to specific cytoplasmic regions. We examined the life cycle of a transcribed beta-actin mRNA throughout gene expression and localization, in a cell system that allows the in vivo detection of the gene locus, the transcribed mRNAs and the cytoplasmic beta-actin protein that integrates into the actin cytoskeleton. Quantification showed that RNA polymerase II elongation progressed at a rate of 3.3 kb/minute and that transactivator binding to the promoter was transient (40 seconds), and demonstrated the unique spatial structure of the coding and non-coding regions of the integrated gene within the transcription site. The rates of gene induction were measured during interphase and after mitosis, demonstrating that daughter cells were not synchronized in respect to transcription initiation of the studied gene. Comparison of the spatial and temporal kinetics of nucleoplasmic and cytoplasmic mRNA transport showed that the beta-actin-localization response initiates from the existing cytoplasmic mRNA pool and not from the newly synthesized transcripts arising after gene induction. It was also demonstrated that mechanisms of random movement were predominant in mediating the efficient translocation of mRNA in the eukaryotic cell.


Subject(s)
Actins/biosynthesis , RNA, Messenger/metabolism , Actins/genetics , Cell Line, Tumor , Cloning, Molecular , Cytoplasm/metabolism , Humans , Lac Operon/genetics , Microscopy, Fluorescence , Transcription, Genetic , Transcriptional Activation
3.
Biochem Biophys Res Commun ; 351(4): 1072-7, 2006 Dec 29.
Article in English | MEDLINE | ID: mdl-17097613

ABSTRACT

To elucidate the roles of DEC1 and DEC2, basic helix-loop-helix transcription factors, in the circadian clock of photosensitive zebrafish peripheral cells, zebrafish Dec1 and Dec2 (zDec1 and zDec2) were cloned and their functions and expression patterns were examined in BRF41, a zebrafish cell line. zDEC1 and zDEC2 have high sequence similarity to mammalian counterparts and the molecular phylogenetic analysis of the zDEC1 and zDEC2 sequences reflected the predicted pattern of species classification. zDEC1 and zDEC2 inhibited zCLOCK1:zBMAL3 mediated transcription as CRY1a. zDec1 and zDec2 mRNA showed robust circadian oscillation in BRF41 cells. However, zDec1 and zDec2 mRNA was not strongly induced by exposure to light. These results indicate that zDec1 and zDec2 are involved in the circadian clock mechanism in photosensitive zebrafish peripheral cells by suppressing CLOCK/BMAL-induced gene expression and that the feedback loops of zDEC1 and zDEC2 may be interlocked with the PER/CRY core circadian feedback loops.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/physiology , Circadian Rhythm/genetics , Gene Expression Regulation , Zebrafish Proteins/physiology , Zebrafish/physiology , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , CLOCK Proteins , Circadian Rhythm/radiation effects , Cloning, Molecular , Light , Phylogeny , Suppression, Genetic , Trans-Activators/genetics , Transcription, Genetic/radiation effects , Zebrafish/genetics , Zebrafish Proteins/genetics
4.
Mol Cell Biol ; 26(11): 4028-40, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16705157

ABSTRACT

A role for the RNA interference (RNAi) pathway in the establishment of heterochromatin is now well accepted for various organisms. Less is known about its relevance and precise role in mammalian cells. We previously showed that tandem insertion of a 1,000-copy inducible transgene into the genome of baby hamster kidney (BHK) cells initiated the formation of an extremely condensed chromatin locus. Here, we characterized the inactive transgenic locus as heterochromatin, since it was associated with heterochromatin protein 1 (HP1), histone H3 trimethylated at lysine 9, and cytosine methylation in CpG dinucleotides. Northern blot analysis did not detect any transgene-derived small RNAs. RNAi-mediated Dicer knockdown did not disrupt the heterochromatic transgenic locus or up-regulate transgene expression. Moreover, neither Dicer knockdown nor overexpression of transgene-directed small interfering RNAs altered the bidirectional transition of the transgenic locus between the heterochromatic and euchromatic states. Interestingly, tethering of HP1 to the transgenic locus effectively induced transgene silencing and chromatin condensation in a Dicer-independent manner, suggesting a role for HP1 in maintaining the heterochromatic locus. Our results suggest that the RNAi pathway is not required for the assembly and maintenance of noncentromeric heterochromatin initiated by tandem transgene repeats in mammalian cells.


Subject(s)
Chromatin Assembly and Disassembly/genetics , Heterochromatin/genetics , RNA Interference , Tandem Repeat Sequences/genetics , Transgenes/genetics , 5' Untranslated Regions/genetics , Animals , Cells, Cultured , Chromobox Protein Homolog 5 , Chromosomal Proteins, Non-Histone/genetics , CpG Islands/genetics , Cricetinae , DNA Methylation , Euchromatin/genetics , Gene Silencing , Green Fluorescent Proteins/metabolism , Humans , Lysine/metabolism , Methyltransferases/metabolism , Promoter Regions, Genetic/genetics , Protein Transport , RNA, Small Interfering , Repressor Proteins/metabolism , Ribonuclease III/deficiency , Transcription, Genetic , Up-Regulation/genetics
5.
Brain Dev ; 28(5): 287-92, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16376506

ABSTRACT

Peroxisomes are ubiquitous organelles in eukaryotic cells and surrounded by a single membrane, and undergo considerable changes in size, shape and number. Peroxisomal disorders are classified into two categories: peroxisome biogenesis disorders (PBDs) and single-enzyme deficiencies (SEDs). Morphologically aberrant peroxisomes called 'peroxisomal ghosts' in PBDs are well known, however, a morphological approach to the study of peroxisomes in SEDs has been rarely reported. Here, we investigated the morphology of peroxisomes in cultured fibroblasts from patients lacking peroxisomal beta-oxidation enzymes, including acyl-CoA oxidase (AOX) or D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein (D-BP). Morphological analysis by immunofluorescence examination using an antibody against catalase revealed a smaller number of large peroxisomes in fibroblasts from these patients. Moreover, immunoelectron microscopy using an antibody against the 70-kDa peroxisomal membrane protein (PMP70) showed large peroxisomes with various horseshoe-shaped membrane structures. These results give an important clue to elucidating the division of peroxisomes and how peroxisomes change in size, shape, number and position within cells, which are subjects for future study.


Subject(s)
Fibroblasts/cytology , Peroxisomal Disorders/enzymology , Peroxisomal Disorders/pathology , Peroxisomes/enzymology , Peroxisomes/pathology , 3-Hydroxyacyl CoA Dehydrogenases/deficiency , Acyl-CoA Oxidase/deficiency , Adult , Cells, Cultured , Child, Preschool , DNA Mutational Analysis/methods , Enoyl-CoA Hydratase/deficiency , Fluorescent Antibody Technique/methods , Humans , Hydro-Lyases/deficiency , Infant , Isomerases/deficiency , Microscopy, Electron, Transmission/methods , Multienzyme Complexes/deficiency , Peroxisomal Bifunctional Enzyme , Peroxisomes/ultrastructure
6.
Biochimie ; 88(3-4): 329-40, 2006.
Article in English | MEDLINE | ID: mdl-16300873

ABSTRACT

The turnip mosaic virus (TuMV) genome-linked protein (VPg) and Arabidopsis thaliana translation initiation factors were expressed and purified in order to investigate their binding properties and kinetics. Affinity chromatography on m(7)GTP-sepharose showed that bound A. thaliana eIF(iso)4E was eluted with crude TuMV VPg. Further column studies with purified VPg and other A. thaliana eIF4E isoforms showed that VPg preferentially bound eIF(iso)4E. Structural data implicate Trp-46 and Trp-92 in eIF(iso)4E in cap recognition. When Trp-46 or Trp-92 were changed to Leu, eIF(iso)4E lost the ability to form a complex with both VPg and m(7)GTP-sepharose. This suggests that the VPg-binding site is located in or near the cap-recognition pocket on eIF(iso)4E. Affinity constants for the interactions with eIF(iso)4E of VPg and capped RNA oligomer were determined using surface plasmon resonance (SPR). The K(D) values showed that the binging affinity of VPg for eIF(iso)4E is stronger than that of capped RNA. This suggests that viral VPg can interfere with formation of a translational initiation complex on host plant cellular mRNA by sequestering eIF(iso)4E. Further experiments with affinity chromatography showed that VPg forms a ternary complex with eIF(iso)4E and eIF(iso)4G. Thus, VPg may participate in viral translational initiation by functioning as an alternative cap-like structure.


Subject(s)
Arabidopsis Proteins/metabolism , Eukaryotic Initiation Factor-4E/metabolism , Eukaryotic Initiation Factors/metabolism , Tymovirus , Viral Proteins/metabolism , Amino Acid Sequence , Animals , Arabidopsis Proteins/genetics , Chromatography, Affinity , Crystallography, X-Ray , Eukaryotic Initiation Factor-4E/genetics , Eukaryotic Initiation Factors/genetics , Gene Expression Regulation, Plant , Genome, Plant , Guanosine Triphosphate/analogs & derivatives , Guanosine Triphosphate/metabolism , Kinetics , Mice , Molecular Sequence Data , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sepharose/analogs & derivatives , Sepharose/metabolism , Sequence Alignment , Surface Plasmon Resonance , Time Factors , Viral Proteins/genetics
7.
Endocrinology ; 147(3): 1067-73, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16293658

ABSTRACT

The photoperiodic response of the gonads requires T3, which is generated photoperiodically from T4 by type 2 iodothyronine deiodinase in the hypothalamus. Although thyroid hormones were long thought to traverse the plasma membrane by passive diffusion due to their lipophilic nature, it is now known that several organic anion transporting polypeptides (Oatp) transport thyroid hormones into target cells. In this study, we have used database searches to isolate DNA sequences encoding members of the chicken Oatp family and constructed a molecular phylogenetic tree. Comprehensive expression analyses using in situ hybridization revealed strong expression of cOatp1c1 and weak expression of cOatp1b1 in the ventro-lateral walls of basal tuberal hypothalamus, whereas expression of four genes (cOatp1a1, cOatp1b1, cOatp1c1, and cOatp3a2) was observed in the choroid plexus. Expression levels of all these genes in both regions were not different between short-day and long-day conditions. Functional expression of cOatp1c1 in Chinese hamster ovary cells revealed that cOatp1c1 is a highly specific transporter for T4 with an apparent Km of 6.8 nm and a Vmax of 1.50 pmol per milligram of protein per minute. These results suggest that cOatp1c1 could be involved in the thyroxine transport necessary for the avian photoperiodic response of the gonads.


Subject(s)
Membrane Proteins/biosynthesis , Membrane Proteins/physiology , Organic Anion Transporters/biosynthesis , Organic Anion Transporters/physiology , Animals , Biological Transport , Birds , Brain/metabolism , CHO Cells , Chickens , Cloning, Molecular , Computational Biology , Coturnix , Cricetinae , DNA/metabolism , DNA Primers/chemistry , Databases, Genetic , Gonads/metabolism , Hypothalamus/metabolism , In Situ Hybridization , Kinetics , Light , Male , Models, Biological , Photoperiod , Phylogeny , Thyroid Hormones/metabolism , Thyroxine/metabolism , Time Factors
8.
Gen Comp Endocrinol ; 141(1): 84-92, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15707606

ABSTRACT

To expand our knowledge on the evolution of prepro-thyrotropin-releasing hormone (ppTRH) from fish to tetrapods, sequences of ppTRH cDNAs from two cyprinid teleosts, the common carp Cyprinus carpio and goldfish Carassius auratus, were determined. Degenerate primers were designed based on the conserved regions between the zebrafish ppTRH sequence identified from the zebrafish EST database and the sockeye salmon ppTRH sequence, and PCR amplification was performed. Full-length ppTRHs were confirmed from ppTRH cDNAs obtained by 5'- and 3'-rapid amplification of cDNA ends. The common carp ppTRH cDNA encodes 187 amino acids including 6 copies of the TRH progenitor sequence (Lys/Arg-Arg-Gln-His-Pro-Gly-Lys/Arg-Arg), whereas the goldfish ppTRH cDNA encodes 231 amino acids including 8 copies of the TRH progenitor sequence. The molecular phylogenetic analysis of the ppTRH sequences reflected the predicted pattern of species classification. The common carp, goldfish, and zebrafish ppTRHs have some unique characteristics. The common carp and zebrafish ppTRHs are smaller than that of the goldfish mainly due to the absence of 29 and 17 consecutive amino acids, respectively. The deleted region includes one or two TRH progenitor sequences flanked by some glutamate residues, similar to the glutamate-rich regions of human ppTRH. Hydropathy profiles showed that the presence of a TRH progenitor sequence in the C-terminal hydrophilic region is a characteristic of teleosts and human ppTRHs. These observations may provide clues to a better understanding of the molecular evolution of ppTRH.


Subject(s)
Carps/genetics , DNA, Complementary/genetics , Goldfish/genetics , Protein Precursors/genetics , Thyrotropin-Releasing Hormone/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Evolution, Molecular , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Analysis, DNA
9.
Gene ; 340(2): 275-82, 2004 Oct 13.
Article in English | MEDLINE | ID: mdl-15475169

ABSTRACT

Estrogen receptor-related receptors (ERRs) constitute a subfamily of the nuclear hormone receptor superfamily. ERRs are closely related to estrogen receptors (ERs), but apparently lack ligand dependence. In this study, we cloned rat ERRgamma as an interacting partner of an orphan nuclear receptor, small heterodimer partner (SHP). ERRgamma exhibited significant binding affinities with a wide spectrum of sequences: inverted and direct repeat motifs composed of AGGTCA half-sites with various spacings, as well as a monovalent motif of the same sequence carrying extra T(C/G)A trinucleotides on the 5' side. On the other hand, inverted repeat spaced by three nucleotides was dominantly efficient for the binding of ERalpha. These results were mostly consistent with those of gene reporter assays. ERRgamma bound as a homodimer to all binding sequences tested, including a monovalent binding site, and ERRgamma did not heterodimerize with ERalpha. Taken together, ERRgamma recognizes a tremendously broad range of sequences as a homodimer. Finally, we found that SHP efficiently represses the transcriptional activity of ERRgamma, even at a far lower concentration than that of ERRgamma.


Subject(s)
DNA/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Estrogen/genetics , Animals , Base Sequence , Binding Sites/genetics , Binding, Competitive , Cloning, Molecular , DNA/genetics , DNA, Complementary/chemistry , DNA, Complementary/genetics , Dimerization , Electrophoretic Mobility Shift Assay , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Gene Expression Regulation , HeLa Cells , Humans , Molecular Sequence Data , Oligonucleotides/genetics , Oligonucleotides/metabolism , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/metabolism , Rats , Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Cytoplasmic and Nuclear/metabolism , Response Elements/genetics , Sequence Analysis, DNA , Transcription, Genetic/genetics , Transcriptional Activation/genetics , Two-Hybrid System Techniques
10.
Hum Mutat ; 23(6): 552-8, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15146459

ABSTRACT

Peroxisome biogenesis disorders (PBD) are lethal hereditary diseases caused by abnormalities in the biogenesis of peroxisomes. At present, 12 different complementation groups have been identified and to date, all genes responsible for each of these complementation groups have been identified. The peroxisomal membrane protein PEX14 is a key component of the peroxisomal import machinery and may be the initial docking site for the two import receptors PEX5 and PEX7. Although PEX14 mutants have been identified in yeasts and CHO-cells, human PEX14 deficiency has apparently not been documented. We now report the identification of a new complementation group of the peroxisome biogenesis disorders with PEX14 as the defective gene. Indeed, human PEX14 rescues the import of a PTS1-dependent as well as a PTS2-dependent protein into the peroxisomes in fibroblasts from a patient with Zellweger syndrome belonging to the new complementation group. This patient was homozygous for a nonsense mutation in a putative coiled-coil region of PEX14, c.553C>T (p.Q185X). Furthermore, we showed that the patient's fibroblasts lacked PEX14 as determined by immunocytochemical analysis. These findings indicate that there are 13 genotypes in PBD and that the role of PEX14 is also essential in humans.


Subject(s)
Mutation , Peroxisomal Disorders/genetics , Animals , Cells, Cultured , Fibroblasts , Genetic Complementation Test , Humans , Infant, Newborn , Peroxisomal Targeting Signal 2 Receptor , Peroxisome-Targeting Signal 1 Receptor , Rats , Receptors, Cytoplasmic and Nuclear/metabolism , Zellweger Syndrome/genetics
11.
Cell ; 116(5): 683-98, 2004 Mar 05.
Article in English | MEDLINE | ID: mdl-15006351

ABSTRACT

We have developed an inducible system to visualize gene expression at the levels of DNA, RNA and protein in living cells. The system is composed of a 200 copy transgene array integrated into a euchromatic region of chromosome 1 in human U2OS cells. The condensed array is heterochromatic as it is associated with HP1, histone H3 methylated at lysine 9, and several histone methyltransferases. Upon transcriptional induction, HP1alpha is depleted from the locus and the histone variant H3.3 is deposited suggesting that histone exchange is a mechanism through which heterochromatin is transformed into a transcriptionally active state. RNA levels at the transcription site increase immediately after the induction of transcription and the rate of synthesis slows over time. Using this system, we are able to correlate changes in chromatin structure with the progression of transcriptional activation allowing us to obtain a real-time integrative view of gene expression.


Subject(s)
Chromosomal Proteins, Non-Histone , Gene Expression Regulation , Gene Silencing , Immunohistochemistry/methods , Animals , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Line , Chromatin/chemistry , Chromatin/metabolism , Chromobox Protein Homolog 5 , DNA/metabolism , Heterochromatin/genetics , Heterochromatin/metabolism , Histones/metabolism , Humans , In Situ Hybridization, Fluorescence , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transcription, Genetic , Transgenes
12.
Biochem Biophys Res Commun ; 316(2): 512-7, 2004 Apr 02.
Article in English | MEDLINE | ID: mdl-15020246

ABSTRACT

SKIP has been described as a transcriptional coregulator as well as a spliceosome component, but the relationship between these functions is not clear. We found that SKIP activated reporter gene expression from the basal promoters of viral origin. SKIP exhibited more prominent effect on the promoters with stronger activities, in an experiment employing a series of reporter constructs carrying different numbers of GC boxes. We also found that SKIP suppressed aberrant splicing at a cryptic splice donor site in the luciferase reporter gene. In addition, SKIP suppressed splicing of an extra intron created by a beta-thalassemia mutation in the human beta-globin gene. In the transfection experiment, an intronless reporter exhibited a higher level of expression, but was less significantly activated by SKIP, than the intron-containing reporter. These results indicate that SKIP affects gene expression by both transcriptional activation and regulation of pre-mRNA splicing.


Subject(s)
Nuclear Proteins/physiology , RNA Splicing , Trans-Activators/physiology , Transcriptional Activation , Animals , HeLa Cells , Humans , Introns , Nuclear Receptor Coactivators , Promoter Regions, Genetic , RNA Precursors/metabolism , Transcription Factors
13.
Mol Cell Biol ; 24(3): 1313-23, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14729975

ABSTRACT

Most cis-acting regulatory elements have generally been assumed to activate a single nearby gene. However, many genes are clustered together, raising the possibility that they are regulated through a common element. We show here that a single peroxisome proliferator response element (PPRE), located between the mouse PEX11 alpha and perilipin genes, confers on both genes activation by peroxisome proliferator-activated receptor alpha (PPAR alpha) and PPAR gamma. A functional PPRE 8.4 kb downstream of the promoter of PEX11 alpha, a PPAR alpha target gene, was identified by a gene transfection study. This PPRE was positioned 1.9 kb upstream of the perilipin gene and also functioned with the perilipin promoter. In addition, this PPRE, when combined with the natural promoters of the PEX11 alpha and perilipin genes, conferred subtype-selective activation by PPAR alpha and PPAR gamma 2. The PPRE sequence specifically bound to the heterodimer of RXR alpha and PPAR alpha or PPAR gamma 2, as assessed by electrophoretic gel mobility shift assays. Furthermore, tissue-selective binding of PPAR alpha and PPAR gamma to the PPRE was demonstrated in hepatocytes and adipocytes, respectively, by chromatin immunoprecipitation assay. Hence, the expression of these genes is induced through the same PPRE in the liver and adipose tissue, where the two PPAR subtypes are specifically expressed.


Subject(s)
Genes, Regulator , Membrane Proteins/metabolism , Phosphoproteins/metabolism , Animals , Base Sequence , Carrier Proteins , Conserved Sequence , Dimerization , Genes, Reporter , Humans , Mice , Molecular Sequence Data , Organ Specificity , Perilipin-1 , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Retinoic Acid/metabolism , Retinoid X Receptors , Transcription Factors/metabolism , Transfection
14.
Mol Biol Cell ; 13(2): 711-22, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11854424

ABSTRACT

Pex6p belongs to the AAA family of ATPases. Its CHO mutant, ZP92, lacks normal peroxisomes but contains peroxisomal membrane remnants, so called peroxisomal ghosts, which are detected with anti-70-kDa peroxisomal membrane protein (PMP70) antibody. No peroxisomal matrix proteins were detected inside the ghosts, but exogenously expressed green fluorescent protein (GFP) fused to peroxisome targeting signal-1 (PTS-1) accumulated in the areas adjacent to the ghosts. Electron microscopic examination revealed that PMP70-positive ghosts in ZP92 were complex membrane structures, rather than peroxisomes with reduced matrix protein import ability. In a typical case, a set of one central spherical body and two layers of double-membraned loops were observed, with endoplasmic reticulum present alongside the outer loop. In the early stage of complementation by PEX6 cDNA, catalase and acyl-CoA oxidase accumulated in the lumen of the double-membraned loops. Biochemical analysis revealed that almost all the peroxisomal ghosts were converted into peroxisomes upon complementation. Our results indicate that 1) Peroxisomal ghosts are complex membrane structures; and 2) The complex membrane structures become import competent and are converted into peroxisomes upon complementation with PEX6.


Subject(s)
ATP-Binding Cassette Transporters , Adenosine Triphosphatases/genetics , Peroxisomes/physiology , ATPases Associated with Diverse Cellular Activities , Adenosine Triphosphatases/deficiency , Animals , CHO Cells , Cricetinae , Fibroblasts/physiology , Fibroblasts/ultrastructure , Genetic Complementation Test , Humans , Membrane Proteins/physiology , Peroxisomes/ultrastructure
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