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M Fransen

Publications and source records attributed to M Fransen.

At least 37 records · Page 2Linked to original sources
PubMed1998

Identification and characterization of human PMP34, a protein closely related to the peroxisomal integral membrane protein PMP47 of Candida boidinii.

The cDNA of a human protein displaying 33% identity to the peroxisomal integral membrane protein PMP47 of Candida boidinii (CbPMP47) was cloned. It encodes a protein of 307 amino acids, with a calculated molecular mass of 34,566 Da. The protein, which we name HsPMP34, contains six membrane spans and belongs, like PMP47, to the family of mitochondrial solute carriers. Based on overlapping available expressed sequence tags (ESTs) combined with the sequencing of one EST, the amino acid sequence of the mouse counterpart (molecular mass of 34,422 Da) could also be deduced. By means of reverse-transcription PCR, the presence of PMP34 mRNA was revealed in liver of man and in liver and other tissues of mouse and rat. The human gene is localized to chromosome 22q13. Given the similarity of HsPMP34 not only to CbPMP47 but also to known or putative mitochondrial transporters, its subcellular localization was investigated. Fluorescence microscopy of HepG2 cells or mouse fibroblasts transfected with a plasmid encoding a HsPMP34/green fluorescent fusion protein revealed a punctate pattern, reminiscent of a peroxisomal distribution. By indirect immunofluorescence, the HsPMP34/green fluorescent fusion protein clearly colocalized with the dimeric peroxisomal thiolase. Upon transfection of mouse fibroblasts lacking functional peroxisomes due the knock-out of PEX5 [Baes, M., Gressens, P., Baumgart, E., Carmeliet, P., Casteels, M., Fransen, M., Evrard, P., Fahimi, D., Declercq, P. E., Collen, D., Van Veldhoven, P. P. & Mannaerts, G. P. (1997) Nat. Genet. 17, 49-53], fluorescence was associated with larger and less-abundant structures. Taken together, the data indicate that HsPMP34 is a peroxisomal membrane protein and is either the human counterpart of CbPMP47 or closely related to it. According to its structure, the protein is most probably involved in transport.

Acetyl-CoA C-Acetyltransferase↗
PubMed1998

Identification of a human PTS1 receptor docking protein directly required for peroxisomal protein import.

The discovery of many fatal human disorders resulting from impaired peroxisomal protein import makes the functional characterization of human peroxins critical. As part of our attempt to identify novel human genes and gene products involved in the import of peroxisomal proteins, we raised antisera against peroxisomal membrane proteins. One such antiserum inhibited peroxisomal protein import in semipermeabilized mammalian cells. This "import inhibiting" antiserum, ab-MF3, specifically recognized a 57-kDa protein. Immunoblot analysis of rat liver subcellular fractions demonstrated that this protein was present exclusively in peroxisomal membranes. Functional analysis revealed that this 57-kDa molecule bound the PTS1 receptor, Pex5p, in ligand blots, suggesting it is a docking site on the peroxisomal membrane. Previous studies have identified two yeast proteins, Pex14p and Pex13p, as Pex5p-binding proteins. To facilitate the biochemical analysis of peroxisomal membrane docking proteins, we cloned and expressed the previously unidentified human Pex14p, as well as a human Pex13p that is 39 aa longer than previously reported. Recombinant Pex14p was specifically recognized by the "import inhibiting" ab-MF3 and bound Pex5p and the Src homology 3 (SH3) domain of Pex13p in ligand blots. These studies demonstrate that the ab-MF3-immunoreactive, 57-kDa peroxisomal membrane protein is Pex14p. Furthermore, this peroxin interacts with Pex5p and Pex13p(SH3) and is directly required for peroxisomal protein import.

Amino Acid Sequence↗
PubMed1997

Off-the-shelf orthopedic footwear for people with rheumatoid arthritis.

OBJECTIVES: To assess the effectiveness of off-the-shelf orthopedic footwear for people with rheumatoid arthritis (RA) reporting chronic foot pain, in terms of self-reported pain and physical function, as well as objectively measured gait variables using an electric footswitch walkway. METHODS: A small, randomized, controlled trial followed by a larger repeated measures analysis was used. RESULTS: The control group (n = 15) demonstrated no significant changes over a 2-month period in pain, physical function, or gait scores. In contrast, after supply of the footwear both the original footwear group (n = 15) and the control group demonstrated significant improvements, with small to large effect sizes, in weight-bearing pain scores, physical function, gait velocity, and gait stride length without increases in use of arthritis medications or walking aids. CONCLUSION: These data suggest that off-the-shelf orthopedic footwear is beneficial for people with RA even when subjects were unselected on basis of age, sex, disease duration, or disability as measured by the Stanford Health Assessment Questionnaire.

Activities of Daily Living↗
PubMed1997

A revised group exercise program for osteoarthritis of the knee.

The purpose of this study was to determine the effectiveness of a group exercise program for subjects with osteoarthritis of the knee referred for physiotherapy in terms of pain, physical function and gait. This study was initiated after an initial group exercise program at this centre failed to demonstrate significant changes in pain, physical function or gait. The study was based on repeated measures with a two month follow up, carried out in the outpatients department of a large public hospital. Pain levels in various functional situations were assessed on visual analogue scales, physical function with the Stanford Health Assessment Questionnaire, and quantitative gait variables at a normal and a fast self-selected speed with an electric foot switch walkway. On completion of the program (n = 40) more than 90% of the pain and physical function scores demonstrated significant improvements without increases in medication, use of walking aids or fatigue. The gait variables of velocity, cadence and stride length at both self-selected speeds demonstrated significant increases at this assessment without deterioration of gait symmetry. All improvements were maintained at the two month follow up assessments. This study suggests that a clinically realistic group exercise program supplemented with a home program can reduce pain, increase physical function and objective gait measures in subjects with osteoarthritis of the knee.

Aged↗
PubMed1997

A mouse model for Zellweger syndrome.

The cerebro-hepato-renal syndrome of Zellweger is a fatal inherited disease caused by deficient import of peroxisomal matrix proteins. The pathogenic mechanisms leading to extreme hypotonia, severe mental retardation and early death are unknown. We generated a Zellweger animal model through inactivation of the murine Pxr1 gene (formally known as Pex5) that encodes the import receptor for most peroxisomal matrix proteins. Pxr1-/- mice lacked morphologically identifiable peroxisomes and exhibited the typical biochemical abnormalities of Zellweger patients. They displayed intrauterine growth retardation, were severely hypotonic at birth and died within 72 hours. Analysis of the neocortex revealed impaired neuronal migration and maturation and extensive apoptotic death of neurons.

Animals↗
PubMed1997

Reliability of gait measurements in people with osteoarthritis of the knee.

BACKGROUND AND PURPOSE: The purpose of this study was to assess the intrasession and intersession reliability of measurements of quantitative gait variables at two self-selected walking speeds. SUBJECTS: Forty-one patients with osteoarthritis in one or both knees who were referred for physical therapy participated. METHODS: Three measurements were made at 1-week intervals. The quantitative gait variables of walking speed, cadence, and stride length were measured using an 8-m electronic footswitch walkway. At each measurement, subjects were asked to walk five times at a self-selected pace that they considered to be normal and five times at a self-selected pace that they considered to be fast. RESULTS: At the normal walking speed, although intraclass correlations were consistently high for all gait variables, an additive factor within and across the first two measurements was evident even when a mean of several trials was used. At the fast walking speed, the intraclass correlations were again consistently high for all gait variables, but there were no changes within or across the measurements. CONCLUSION AND DISCUSSION: The data suggest that quantitative gait analysis is a practical objective assessment tool for persons with osteoarthritis of the knee. Gait at the fast walking speed, however, will provide the more reliable stable measure on which to evaluate the effect of therapy when compared with gait at the normal walking speed. The comparative responsiveness to change between the two walking speeds still needs to be determined.

Aged↗
PubMed1996

Molecular characterization of the human peroxisomal branched-chain acyl-CoA oxidase: cDNA cloning, chromosomal assignment, tissue distribution, and evidence for the absence of the protein in Zellweger syndrome.

Peroxisomes in human liver contain two distinct acyl-CoA oxidases with different substrate specificities: (i) palmitoyl-CoA oxidase, oxidizing very long straight-chain fatty acids and eicosanoids, and (ii) a branched-chain acyl-CoA oxidase (hBRCACox), involved in the degradation of long branched fatty acids and bile acid intermediates. The accumulation of branched fatty acids and bile acid intermediates leads to severe mental retardation and death of the diseased children. In this study, we report the molecular characterization of the hBRCACox, a prerequisite for studying mutations in patients with a single enzyme deficiency. The composite cDNA sequence of hBRCACox, derived from overlapping clones isolated via immunoscreening and hybridization of human liver cDNA expression libraries, consisted of 2225 bases and contained an open reading frame of 2046 bases, encoding a protein of 681 amino acids with a calculated molecular mass of 76,739 Da. The C-terminal tripeptide of the protein is SKL, a known peroxisome targeting signal. Sequence comparison with the other acyl-CoA oxidases and evolutionary analysis revealed that, despite its broader substrate specificity, the hBRCACox is the human homolog of rat trihydroxycoprostanoyl-CoA oxidase (rTHCCox) and that separate gene duplication events led to the occurrence in mammals of acyl-CoA oxidases with different substrate specificities. Northern blot analysis demonstrated that--in contrast to the rTHCCox gene--the hBRCACox gene is transcribed also in extrahepatic tissues such as heart, kidney, skeletal muscle, and pancreas. The highest levels of the 2.6-kb mRNA were found in heart, followed by liver. The enzyme is encoded by a single-copy gene, which was assigned to chromosome 3p14.3 by fluorescent in situ hybridization. It was absent from livers of Zellweger patients as shown by immunoblot analysis and immunocytochemistry.

Amino Acid Sequence↗
PubMed1996

Molecular cloning and further characterization of rat peroxisomal trihydroxycoprostanoyl-CoA oxidase.

The composite trihydroxycoprostanoyl-CoA oxidase cDNA sequence, derived from overlapping clones isolated via screening of two different rat liver expression libraries, consisted of 2509 bases and contained an open reading frame of 2046 bases, encoding a protein of 681 amino acids with a calculated molecular mass of 76711 Da. The reading frame and identity of the trihydroxycoprostanoyl-CoA oxidase cDNA were confirmed by the location of various tryptic peptides, obtained from the purified enzyme, in the deduced amino acid sequence. The C-terminus (His-Lys-Met) of trihydroxycoprostanoyl-CoA oxidase did not seem to interact with the C-terminal peroxisomal targeting signal 1 (PTS1) import receptor, although the tripeptide fits the rule of conserved PTS1 variants for targeting of proteins to glycosomes of Trypanosomatidae. At the protein level, trihydroxycoprostanoyl-CoA oxidase showed 45% identical amino acids with rat palmitoyl-CoA oxidase, whereas the identity with pristanoyl-CoA oxidase was much lower (22%). Northern analysis of multiple rat tissues revealed a signal (approx. 2.6 kb) only in liver and (although much weaker) in kidney. Dot-blot analysis of total liver RNA revealed that the mRNA for trihydroxy-coprostanoyl-CoA oxidase is not induced after treatment of rats with structurally unrelated peroxisome proliferators and indicates that highly similar mRNAs are present in other mammals, including man. Immunocytochemistry showed a decrease in trihydroxycoprostanoyl-CoA oxidase protein in individual liver peroxisomes ('diluting-out effect') after treatment of rats with bezafibrate, whereas the palmitoyl-CoA oxidase labelling was significantly increased.

Amino Acid Sequence↗
PubMed1996

The visualization of peroxisomal proteins containing a C-terminal targeting sequence on western blot by using the biotinylated PTS1-receptor.

A procedure to visualize proteins containing a C-terminal peroxisomal targeting signal (PTS1) in complex protein mixtures was developed using a bacterially expressed, biotinylated form of the human PTS1-receptor. The binding of this fusion product to purified PTS1-containing proteins that were separated by SDS-PAGE and blotted onto nitrocellulose was detected by means of streptavidin-alkaline phosphatase and shown to be both saturable and specific. When applied to total tissue extracts, in addition to PTS1-containing proteins various endogenous biotinylated proteins were visualized. Therefore, a two-step staining procedure was optimized whereby the endogenous biotinylated proteins were shielded with a blue precipitate, followed by incubation with the biotinylated receptor and detection of the resulting PTS1-receptor/PTS1-protein complexes with a phosphatase reaction coupled to the formation of a red-colored precipitate. This relatively inexpensive, simple, and fast technique enabled us to visualize a variety of PTS1-containing proteins. In addition, the information presented in this study can be used to facilitate the identification and characterization of receptor-ligand interaction in general and to eliminate interference by endogenous biotinylated proteins intrinsic to the streptavidin-biotin detection system.

Alkaline Phosphatase↗
PubMed1996

Further characterization of the peroxisomal 3-hydroxyacyl-CoA dehydrogenases from rat liver. Relationship between the different dehydrogenases and evidence that fatty acids and the C27 bile acids di- and tri-hydroxycoprostanic acids are metabolized by separate multifunctional proteins.

Recently, we purified five 3-hydroxyacyl-CoA dehydrogenases from isolated rat liver peroxisomal fractions. The enzymes were designated I-V according to their order of elution from the first column used in the purification procedure. Determination of the substrate (L- or D-hydroxyacyl-CoA) stereo-specificity and (de)hydratase measurements with the different 3-hydroxyacyl-CoA stereoisomers of straight-chain fatty acids and the bile acid intermediate trihydroxycoprostanic acid, immunoblotting analysis with antibodies raised against the different enzymes and peptide sequencing, all performed on enzymes I-V and molecular cloning of enzyme III revealed the following picture. Rat liver peroxisomes contain two multifunctional beta-oxidation proteins: (a) multifunctional protein 1 (the classical multifunctional protein; MFP-1) displaying 2-enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase and delta 3, delta 2-enoyl-CoA isomerase activity (enzyme IV) and (b) multifunctional protein 2 (MFP-2) displaying 2-enoyl-CoA hydratase and D-3-hydroxyacyl-CoA dehydrogenase activity (enzyme III). Because of their substrate stereospecificity and because of the stereochemical configuration of the naturally occurring beta-oxidation intermediates, MFP-1 and MFP-2 appear to be involved in the beta-oxidation of fatty acids and bile acids intermediates, respectively. The deduced amino acid sequence of the cloned MFP-2 cDNA is highly similar to that of the recently described porcine endometrial estradiol 17 beta-dehydrogenase [Leenders, F., Adamski, J., Husen, B., Thole, H. H. & Jungblut, P. W. (1994) Eur. J. Biochem. 222, 221-227]. In agreement, MFP-2 also displayed estradiol 17 beta-dehydrogenase activity, indicating that MFP-2 and the steroid dehydrogenase are identical enzymes. MFP-2 is partially cleaved, most probably in vivo, in a estradiol 17 beta-dehydrogenase/D-3-hydroxyacyl-CoA dehydrogenase that forms a dimeric complex (enzyme I) and a hydratase. The physiological significance of enzyme I in bile acid synthesis (and steroid metabolism) remains to be determined. MFP-1 (enzyme IV) is artefactually cleaved during purification giving rise to 3-hydroxyacyl-CoA dehydrogenase V. 3-Hydroxyacyl-CoA dehydrogenase II is a mitochondrial contaminant similar to porcine and murine mitochondrial 3-hydroxyacyl-CoA dehydrogenase.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1996

Rat pristanoyl-CoA oxidase. cDNA cloning and recognition of its C-terminal (SQL) by the peroxisomal-targeting signal 1 receptor.

The composite pristanoyl-CoA oxidase cDNA sequence, derived from two overlapping clones from a rat liver cDNA library and a 5'-RACE (rapid amplification of cDNA ends) PCR fragment, consisted of 2600 bases and contained an open reading frame of 2100 bases, encoding a protein of 700 amino acids with a calculated molecular mass of 78445 Da. This value is somewhat larger than the reported molecular mass of 70 kDa as determined earlier by SDS-gel electrophoresis. The amino acid identity with rat palmitoyl-CoA oxidase was rather low (28%) and barely higher than that with the yeast acyl-CoA oxidases (20%), suggesting that the palmitoyl-CoA oxidase/pristanoyl-CoA oxidase duplication occurred early in evolution. The carboxy-terminal tripeptide of pristanoyl-CoA oxidase was SQL. In vitro studies with the bacterially expressed human peroxisomal-targeting signal-1 import receptor indicated that SQL functions as a peroxisome-targeting signal. Northern analysis of tissues from control and clofibrate treated rats demonstrated that the pristanoyl-CoA oxidase gene is transcribed in liver and extrahepatic tissues and that transcription is not enhanced by treatment of rats with peroxisome proliferators. No mRNA could be detected by northern analysis of human tissues, suggesting that the human pristanoyl-CoA oxidase gene, if present, is only poorly or not transcribed.

Amino Acid Sequence↗
PubMed1995

The gene for the peroxisomal targeting signal import receptor (PXR1) is located on human chromosome 12p13, flanked by TPI1 and D12S1089.

Mutations in the PXR1 gene can cause a generalized peroxisome biogenesis disorder (complementation group 2). Fluorescence in situ hybridization with the PXR1 cDNA and two cosmids containing the PXR1 gene was used for the cytogenetic assignment of the gene to chromosome 12p13. Analysis of a radiation hybrid panel for chromosome 12 with a PXR1 gene-based sequence-tagged site (STS) mapped the PXR1 gene between TPI1 and D12S1089. The STS also detects an unusual, highly polymorphic, short tandem repeat (heterozygosity, 0.82). This repeat shows one set of short alleles containing a pentanucleotide repeat and one set of long alleles containing a complex array of different pentanucleotides.

Alleles↗
PubMed1995

Identification and characterization of the putative human peroxisomal C-terminal targeting signal import receptor.

To identify proteins interacting with the C-terminal peroxisomal targeting signal (PTS1), we screened a human liver cDNA library by means of a Saccharomyces cerevisiae genetic system, known as the two-hybrid system. We isolated a cDNA encoding a protein that specifically bound the PTS1 topogenic signal in the intact yeast cell but also in vitro after bacterial expression and purification. Sequence analysis of the full-length cDNA revealed the presence of an open reading frame encoding a 70-kDa polypeptide that belongs to the tetratricopeptide repeat family and that is homologous to the PAS8 and PAS10 gene products, which are required for the formation of normal peroxisomes in yeast. Subcellular fractionation of human liver and immunofluorescence studies on HepG2 cells demonstrated that this PTS1-binding protein is present exclusively in peroxisomes and that the PTS1-binding domain is located to the cytosolic side of the peroxisomal membrane. All available evidence indicates that the PTS1-binding protein is part of the peroxisomal protein import machinery and most probably is the long sought after human PTS1 import receptor.

Amino Acid Sequence↗
PubMed1994

The 80 kDa cytosolic protein that binds the C-terminal part of rat acyl-CoA oxidase is not a peroxisomal import receptor but a prolyl-endopeptidase.

In an attempt to identify putative peroxisomal import receptors, we investigated the cross-linking of a radioiodinated peptide consisting of the 13 last amino acids of acyl-CoA oxidase and comprising the carboxy-terminal SKL-peroxisomal targeting motive, to proteins present in different subcellular fractions from rat liver. The radiolabeled peptide could be cross-linked to an 80 kDa protein present in the cytosol but not to proteins present in other subcellular fractions including highly purified peroxisomes. Binding was reversible, saturable and dependent on the presence of Mg2+ and ATP or GTP but hydrolysis of the nucleotides was not required. Binding was abolished by pretreatment of the cytosol--but not of the peptide--with N-ethylmaleimide. Binding was not specific for peptides containing the carboxy-terminal SKL-motive, since binding was competed for by the SKL-peptide from which the SKL-motive had been deleted, by the SKL-peptide with reversed sequence and by the SV40 T-antigen nuclear localisation signal peptide, but not by other peptides tested. The 80 kDa binding protein cross-reacted with a monoclonal antibody against hsp90. Purification and internal peptide sequencing of the binding protein revealed its identity as prolyl-endopeptidase. In retrospect, we realized that the SKL-peptide and all competing peptides contained a proline residue, which was not present in the non-competing peptides. In recent experiments in yeast McNew et al. (McNew, J.A., Sykes, K. and Goodman, J.M. (1993) Mol. Biol. Cell 4, 223-232) cross-linked a peroxisomal targeting peptide to a 20 kDa cytosolic protein that was identified as proline isomerase despite the fact that the peptide did not contain proline. The experiments by McNew et al. in yeast and our experiments in the rat suggest that the (peroxisomal) targeting sequence cross-linking approach may not be suited for the identification of (peroxisomal) import receptors.

Acyl-CoA Oxidase↗