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R R Frants

Publications and source records attributed to R R Frants.

At least 163 records · Page 9Linked to original sources
PubMed1993

Low-density lipoproteins are degraded in HepG2 cells with low efficiency.

In previous studies we have shown that in HepG2 cells, as compared with fibroblasts, the low-density lipoprotein (LDL) receptor is only weakly down-regulated upon incubation of the cells with LDL. To explain this difference in down-regulation of the LDL-receptor activity, we studied simultaneously the intracellular processing of 125I-labelled LDL in both cell lines. Upon incubation of HepG2 cells with 125I-LDL, the appearance of degradation products started at 90 min, whereas in fibroblasts this lag time was only 30 min. The degradation efficiency (representing the ratio degradation/cell association of LDL) in HepG2 was less than 50% of that in fibroblasts up to 5h of incubation at 37 degrees C. The longer lag time and low efficiency of the degradation of LDL in HepG2 cells were independent of the cell density. Pulse-chase experiments indicated that the internalization rate of surface-bound LDL in HepG2 cells is similar to that of fibroblasts. Endosomal loading of 125I-LDL by incubation at 18 degrees C for 4.5 h, followed by a shift to 37 degrees C, resulted in degradation of LDL within 30 min in fibroblasts, whereas in HepG2 cells the lag time of the degradation was 90 min. In parallel experiments using subcellular fractionation by Percoll-gradient centrifugation of homogenized cells and 125I-tyramine-cellobiose-labelled LDL, we observed that in both cell types LDL is equally rapidly shifted from a low- to a high-density compartment (within 15 min), representing the endosomal and the late-endosomal plus lysosomal compartment respectively. We conclude that in HepG2 cells the cell-bound LDL, upon internalization, goes through the intracellular itinerary at the same rate as in fibroblasts, but that either the fusion between late endosomes and lysosomes or the lysosomal degradation itself is proceeding at a lower efficiency. A low degradation rate of LDL may contribute to explain the relatively weak down-regulation of the LDL-receptor activity in HepG2 cells by LDL.

Biological Transport↗
PubMed1993

Evolutionary conservation of the mouse apolipoprotein e-c1-c2 gene cluster: structure and genetic variability in inbred mice.

The human apolipoprotein E (APOE), APOC1, pseudo APOC1 (APOC1'), and APOC2 genes are clustered within 48 kb on the long arm of chromosome 19. A mouse Apoe cDNA probe was used to isolate overlapping cosmid clones from a cosmid library of the C57BL/Rij inbred mouse strain. These clones were investigated for the presence of the Apoc1 and Apoc2 genes by heterologous hybridization. Our results show that the Apoe-c1-c2 gene cluster is conserved in the mouse. In line with evolutionary data, the mouse lacks the equivalent of APOC1'. These data were confirmed using a mouse Apoc2 cDNA clone, and surprisingly the cDNA clone isolated here was 965 bp in size, which is on average 450 bp longer than other APOC2 cDNAs described so far. Correspondingly, the Apoc2 gene occupies an unusually large genomic region, due to an extended 5' end. Interestingly, a variable number of tandem repeat (VNTR) in the third intron of the human APOC2 gene shows a high sequence homology and is located at the identical position in the mouse gene. Despite the high copy number of this VNTR (27 or 34 copies) only two variants were found among 11 different inbred strains. With the aid of six restriction fragment length variations in this gene cluster only two different haplotypes could be deduced, indicating that the Apoe-c1-c2 gene cluster is highly conserved in the inbred strains that were studied.

Animals↗
PubMed1993

Structure and expression of the mouse apolipoprotein C2 gene.

Three cDNA clones containing the mouse apolipoprotein C2 (Apoc2) gene were isolated from a mouse liver cDNA library. The inserts from two cDNA clones were 500 bp in size while the insert from the third clone was unexpectedly large, 962 bp. All three clones contained a single open reading frame encoding apoC2. The exon-intron structure of the mouse Apoc2 gene was determined by sequence analysis. Northern blotting and primer extension analysis of mouse RNA showed that the major liver transcript is 500 bp in size and is encoded by four exons. Transcripts for Apoc2 were found in fetal liver, adult liver, intestine, and peritoneal macrophages. The largest cDNA clone, mAPOC2c4, contained an additional 440 bp at the 5' end that are evolutionary conserved between man and mouse. These additional sequences are encoded by two exons located 5' to the major liver start site. Although the larger transcript could not be detected by Northern blot analysis, products resulting from an upstream transcription initiation site were detected in the liver using RT-PCR analysis. The sizes of the RT-PCR products are consistent with alternative splicing.

Amino Acid Sequence↗
PubMed1993

The mouse apolipoprotein C1 gene: structure and expression.

We have isolated and characterized cDNA and genomic clones containing the mouse apolipoprotein C1 (Apoc1) gene. The Apoc1 gene is part of the Apoe-c1-c2 gene cluster and is located 3.4 kb 3' of the Apoe gene. The mouse Apoc1 gene spans a region of approximately 3.3 kb and consists of four exons. The exon-intron structure is similar to those of human and baboon genes, although in mouse introns 2 and 3 are smaller. Significant sequence homology is found between man and mouse in the promoter and exonic regions (80 and 67%, respectively). Northern blotting and primer extension analysis of mouse RNA showed that a major transcript 409 bp in size is expressed primarily in fetal and adult liver. The mouse Apoc1 cDNA contains an open reading frame encoding a protein of 88 amino acids, including a signal peptide of 26 amino acid residues. Comparisons of the deduced amino acid sequence of mouse apoC1 with the human, baboon, rat, and dog sequences showed discrete regions with a high degree of conservation. The delineation of the sequence and structural organization of the mouse Apoc1 gene is an essential step in enhancing the use of mouse models to study the function of apoC1 in the lipoprotein metabolism.

Amino Acid Sequence↗
PubMed1993

Identification of a splice-site mutation in the low density lipoprotein receptor gene by denaturing gradient gel electrophoresis.

We have applied the denaturing gradient gel electrophoresis (DGGE) technique to detect sequence variations in exon 9 of the low density lipoprotein receptor (LDLR) gene in individuals with heterozygous familial hypercholesterolemia (FH). A fragment containing exon 9 and 25 base pairs (bp) of the intron boundary sequence at either side was amplified. To this fragment a 40-bp GC-clamp was attached by the polymerase chain reaction (PCR). We have analyzed a total of 165 DNA samples of FH patients and have detected a mutation in three cases. Two patients were found to have the previously described "South African" G to A transition in codon 408. In a third patient, we observed a different banding pattern of the DNA fragments on DGGE indicating a different mutation. The mutant homoduplex band of this sample was purified from the gel, cloned in an AT-vector and sequenced. Sequence analysis demonstrated a G to A transition of the consensus G-nucleotide at the intron 9 splice donor site. Cosegregation between this mutation and elevated plasma cholesterol levels was observed in family members of this FH patient. This mutation probably prevents normal splicing of the mRNA and represents the first identified splice-site mutation in the LDLR gene. We conclude that the use of DGGE of GC-clamped PCR-amplified exon sequences offers a general strategy for the detection of disease-producing mutations in the LDLR gene.

Base Sequence↗
PubMed1993

The effect of the apolipoprotein E phenotype on plasma lipids is not influenced by environmental variability: results of a Dutch twin study.

We tested the influence of the apolipoprotein E (apoE) polymorphism on the intrapair differences in the levels of plasma cholesterol, plasma triglycerides, low density lipoprotein-cholesterol, apoB and apoE in monozygotic (MZ) twins, and estimated whether or not there was a interaction between the apoE polymorphism and environmental factors. In 65 MZ twin pairs, the intrapair differences in the measured lipoprotein parameters were similar in the different apoE phenotype classes. This indicates that the effect of the apoE polymorphism is not influenced by environmental variability between the MZ pair members and accordingly identifies the APOE gene as a "level" gene.

Adolescent↗
PubMed1993

The human skeletal muscle adenine nucleotide translocator gene maps to chromosome 4q35 in the region of the facioscapulohumeral muscular dystrophy locus.

Facioscapulohumeral muscular dystrophy (FSHD) is a relatively common autosomal dominant neuromuscular disorder. The gene for FSHD has recently been assigned to chromosome 4q35. Although abnormal mitochondrial and biochemical changes have been observed in FSHD, the molecular defect is unknown. In addition to the FSHD gene, the human muscle adenine nucleotide translocator gene (ANT1) is located on chromosome 4. Interestingly, biochemical studies recently showed a possible defect of ANT1. In order to evaluate the potential role of ANT1 in the etiology of FSHD, the human ANT1 gene was isolated by cosmid cloning and localized to 4q35, in the region containing the FSHD gene. However, in situ hybridization and physical mapping of somatic cell hybrids localized the ANT1 gene proximal to the FSHD gene. In addition, a polymorphic CA-repeat 5 kb upstream of the ANT1 gene was used as a marker in FSHD and Centre d'Etude du Polymorphisme Humain families to perform linkage analysis. These data together exclude ANT1 as the primary candidate gene for FSHD. The most likely order of the loci on chromosome 4q35 is cen-ANT1-D4S171-F11-D4S187-D4S163-D4S139-+ ++FSHD-tel.

Animals↗
PubMed1993

An acceptor splice site mutation in intron 16 of the low density lipoprotein receptor gene leads to an elongated, internalization defective receptor.

In this report, we describe the characterization of a mutation in the low density lipoprotein (LDL) receptor gene of a true homozygous familial hypercholesterolemic (FH) patient. The combined use of denaturing gradient gel electrophoresis (DGGE) and DNA sequence analysis revealed a unique A to G transition in the penultimate 3'-nucleotide of intron 16 of the LDL receptor gene, which disrupts the acceptor splice site. cDNA sequence analysis indicated that a cryptic splice site was activated in intron 16, upstream from the original splice site, leading to the inclusion of 62 nucleotides and a reading frame-shift. The resulting new translation product contains a stretch of 154 amino acids at the carboxy-terminal that have no resemblance to the normal receptor protein. To elucidate the biological effects of the mutation, the structural and functional properties of the mutated LDL receptor protein were studied. Immunoprecipitation of the newly synthesized LDL receptors showed that an aberrant precursor form of the LDL receptor protein was synthesized, about 10 kDa larger than normal, which is not further processed to the mature form. Some 50% of the normal LDL binding activity was found on the cell surface of the patient's fibroblasts, whereas internalization and degradation of LDL were abolished.

Adult↗
PubMed1993

Treatment of E2E2 homozygous familial dysbetalipoproteinemic subjects with gemfibrozil does not enhance the binding of their d < 1.019 lipoprotein fraction to the low-density lipoprotein receptor.

Six E2E2 homozygous familial dysbetalipoproteinemic (FD) patients were treated with gemfibrozil (2 x 600 mg/d) for a period of 4 weeks. For all subjects, normalization of serum cholesterol concentrations with treatment did not result in a significant change in the cholesterol/triglyceride ratio of the d < 1.019 lipoprotein fraction. In addition, the binding efficiency of this lipoprotein fraction to the low-density lipoprotein (LDL) receptor on HepG2 cells did not change consistently with treatment. We conclude that normalization of serum cholesterol concentrations in FD patients by treatment with gemfibrozil is the result of an effect of gemfibrozil on the synthesis of d < 1.019 lipoproteins rather than an effect on the receptor-mediated clearance of these particles.

Apolipoproteins A↗
PubMed1993

Hereditary spastic paraparesis. Clinical and genetic data from a large Dutch family.

A large Dutch family is presented with pure hereditary spastic paraparesis. Pedigree analysis indicates autosomal dominant inheritance with complete penetrance and possibly anticipation in successive generations. These observations may have implications for genetic studies. Linkage studies in this family have excluded more than 40% of the autosomal genome.

Central Nervous System Diseases↗
PubMed1993

Facioscapulohumeral muscular dystrophy: the impact of genetic research.

Recent developments in genetic research have led to the localization and identification of the causative gene defect in a large number of neurological diseases. This paper describes some of the basic principles of molecular genetics and the strategies that have been followed in the search for the gene for facioscapulohumeral muscular dystrophy (FSHD), beginning with the recent localization to chromosome 4q. Many questions remain concerning the pathogenesis and possible genetic heterogeneity of this autosomal dominant myopathy. Hitherto, most evidence favours a genetically homogeneous disorder, but only the isolation and detailed characterization of the FSHD gene will resolve these issues completely.

Chromosome Aberrations↗
PubMed1993

PCR-based microsatellite polymorphisms in the detection of loss of heterozygosity in fresh and archival tumour tissue.

PCR-based microsatellite polymorphisms have proved their power in genetic linkage analysis and other identification methods, due to their high information content and even distribution over the chromosomes. In the present study we applied microsatellite polymorphisms to detect loss of heterozygosity in fresh (snap-frozen) and in archival ovarian tumour tissue. Clear allele losses were found in fresh and paraffin embedded tumour samples. Conventional Southern analysis of flanking markers on the same tumour DNA samples confirmed the observed losses detected by microsatellite polymorphisms. Titration experiments suggest that loss of heterozygosity remains detectable in tumour samples despite 60% contamination with normal DNA. This technique provides a fast and reproducible alternative to conventional Southern blotting in the detection of loss of heterozygosity, with the crucial additional advantages of minimal sample requirements, making archival material available for genetic investigation.

Adenocarcinoma↗
PubMed1993

Physical mapping and YAC-cloning connects four genetically distinct 4qter loci (D4S163, D4S139, D4F35S1 and D4F104S1) in the FSHD gene-region.

We have constructed a long-range restriction map of the region on chromosome 4q that contains the gene for facioscapulohumeral muscular dystrophy (FSHD). This region contains the linkage group cen ... D4S163-D4S139-D4F35S1-D4F104S1-FSHD ... 4qter, which spans a genetic distance of about 5 cM. Pulse field gel electrophoresis (PFGE) mapping indicated that these loci span a region not more than 1 Mb. STSs were developed for several of these loci, which served to isolate four overlapping yeast artificial chromosomes (YACs). These YACs confirmed the PFGE map and have allowed us to generate a more detailed restriction map using cosmid contig mapping. The physical distances were smaller than was expected on the basis of the genetic map. Two potential HTF islands have been detected within the cloned region. One HTF island maps about 100 kb centromeric from the tandem repeats involved in the FSHD mutation, whereas the other maps within these tandem repeats.

Base Sequence↗
PubMed1993

Fine mapping of the FSHD gene region orientates the rearranged fragment detected by the probe p13E-11.

We have produced a fine restriction map around the locus D4F104S1 (previously designated D4S810); a probe to this locus, p13E-11, identifies a polymorphic EcoRI fragment containing 3.2kb tandem repeats and detects DNA rearrangements associated with facioscapulohumeral muscular dystrophy (FSHD). We developed an STS (D4F106S1) which maps 2kb proximal to D4F104S1, and used this to isolate a 470kb YAC (y25C2E) from the ICI YAC library and a 930kb YAC (y956A11) from the CEPH megabase library. Both YACs contain the loci D4S139, D4F35S1 and D4F104S1. A cosmid library was produced from YAC y25C2E and two cosmid contigs constructed; a 115kb contig encompassing D4S139, and one of 135kb linking D4F35S1 and D4F104S1 and extending distal to the EcoRI fragment detected by p13E-11. A fine restriction map of both these contigs has been generated, allowing the orientation of the EcoRI fragment rearranged in FSHD to be determined. YAC y956A11 was used to confirm the integrity of y25C2E and the map of this region. 9B6A, a probe to the homeobox region of the tandem repeat D4Z4, identified a cross-hybridising sequence proximal to D4F104S1, however, p13E-11 does not detect this additional locus. CpG islands were identified between D4S139 and D4F35S1 and within each copy of the tandem repeat. The probe 9B6A detected each copy of the repeat motif, suggesting there is homeobox present in every copy of the 3.2kb repeat.

Base Sequence↗
PubMed1993

FSHD associated DNA rearrangements are due to deletions of integral copies of a 3.2 kb tandemly repeated unit.

Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disorder characterized by progressive weakness of the facial, shoulder and upper arm muscles. The disease is associated with DNA rearrangements which are detectable using probe p13E-11 (D4F104S1) in DNA digested with EcoRI or other restriction enzymes. We have cloned and characterized the rearranged EcoRI fragment of four unrelated FSHD patients. Restriction fragment mapping and DNA sequence analysis showed that the proximal and distal parts of the EcoRI fragment, which flank a region of tandemly repeated 3.2 kb units, are identical in normal and rearranged EcoRI fragments. These results strongly support the hypothesis that the FSHD associated rearrangements are due to deletions of integral copies of the 3.2 kb repeated unit. Since these repeated units are likely to form part of the FSHD transcription unit, the variation in repeat unit number might affect the function of the gene product. Hence, our data confine the FSHD gene region and thus provide a starting point for cloning the FSHD gene.

Arm↗
PubMed1993

Linkage analysis in Dutch familial atypical multiple mole-melanoma (FAMMM) syndrome families. Effect of naevus count.

Familial atypical multiple mole-melanoma (FAMMM) syndrome is characterized by the familial occurrence of malignant melanoma of the skin in combination with multiple atypical precursor naevi. In the present study we performed linkage analysis in seven Dutch FAMMM families to define the relationship between the ultimate phenotype melanoma and the postulated precursors, atypical (dysplastic) naevi. Various models were defined, varying from melanoma only to various combinations of melanoma and atypical naevi, reflecting the FAMMM phenotype. Using 124 microsatellite markers spread across all autosomes, hints for linkage were obtained between several chromosome 9p markers and a melanoma locus (D9S171; odds for linkage, 275:1). In a model including melanoma and a florid manifestation of atypical naevi a considerably higher lod score was obtained with D9S171 (odds for linkage, 4365:1); models including milder manifestations yielded less support. We conclude that, also in the Dutch FAMMM families, a melanoma gene is located on the short arm of chromosome 9 and that multiple atypical naevi, at least in certain cases, seems to be a component of the FAMMM phenotype.

Adult↗