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L Bolund

Publications and source records attributed to L Bolund.

At least 37 records · Page 2Linked to original sources
PubMed2000

Human and mouse mitochondrial orthologs of bacterial ClpX.

We have determined the cDNA sequence and exon/intron structure of the human CLPX gene encoding a human ortholog of the E. coli ClpX chaperone and protease subunit. The CLPX gene comprises 14 exons and encodes a 633-amino acid-long precursor polypeptide. The polypeptide contains an N-terminal putative mitochondrial transit peptide, and expression of a full-length ClpX cDNA tagged at its C-terminus (Myc-His) shows that the polypeptide is transported into mitochondria. FISH analysis localized the CLPX gene to human Chromosome (Chr) 15q22.1-22.32. This localization was refined by radiation hybrid mapping placing the CLPX gene 4.6 cR distal to D15S159. Murine ClpX cDNA was sequenced, and the mouse Clpx locus was mapped to a position between 31 and 42 cM offset from the centromere on mouse Chr 9. Experimental observations indicate the presence of a pseudogene in the mouse genome and sequence variability between mouse ClpX cDNAs from different strains. Alignment of the human and mouse ClpX amino acid sequences with ClpX sequences from other organisms shows that they display the typical modular organization of domains with one AAA(+) domain common to a large group of ATPases and several other domains conserved in ClpX orthologs linked by non-conserved sequences. Notably, a C-4 zinc finger type motif is recognized in human and mouse ClpX. This motif of so far unknown function is present only in a subset of the known ClpX sequences.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2000

Transgene expression in human epidermal keratinocytes: cell cycle arrest of productively transfected cells.

We have analysed the consequences of liposome mediated gene transfer into human primary epidermal keratinocytes and compared non-Epstein-Barr Virus (EBV) and EBV based expression vectors that carry the genes encoding human Growth Hormone (hGH) or Enhanced Green Fluorescent Protein (EGFP). Different kinetics between the non-EBV and EBV based vectors were revealed upon subcultivation of hGH transfected keratinocytes. The keratinocytes transfected with non-EBV based vector showed a rapid reduction in hGH production. Although the EBV based vector resulted in more stable expression, this was also reduced over time. Chromatin inactivation by deacetylation was investigated by treatment with sodium butyrate and found not to be the reason for the decreasing expression. Keratinocytes divided into subpopulations enriched for either stem cells or transit amplifying cells, based on beta1-integrin expression and function, do not differ significantly with respect to susceptibility to productive transfection. However, when the keratinocytes were transfected with the EGFP gene and sorted live by FACS into EGFP negative and positive populations, only the negative cells were capable of forming significant numbers of colonies. This is consistent with the observation that the ability to incorporate BrdU was dramatically reduced in the EGFP expressing population within 24-48 h post transfection indicating an almost complete cell cycle arrest. p53 levels were unaffected by the procedures, and the keratinocyte cell line HaCat, mutated in both p53 alleles, also shows a marked reduction in clonogenic potency upon transfection. There was a slight increase of TUNEL positive apoptotic nuclei in the positive population at early time points. However, the apoptotic index was still very low. When we measured the frequency of involucrin expressing cells, we found an increase in the productively transfected population over time indicating an initiation of terminal differentiation. In contrast to the transfected cultures, keratinocytes that were transduced using a retroviral vector showed no decrease in colony forming efficiency. In conclusion we find that transgene expressing cells from transfected cultures of epidermal keratinocytes undergo cell cycle arrest and initiate terminal differentiation by mechanisms which are independent of p53 levels.

Cell Adhesion↗
PubMed2000

Strain-specific variations in the development of dendritic cells in murine bone-marrow cultures.

The dendritic cell (DC) is a professional antigen-presenting cell of central importance in immunity. In this paper, we examined DCs generated by 11-day culture of bone-marrow cells from the four mouse strains C57BL/6J, BALB/cA, C3H/HeN and B10.PL-H2u (73NS)/Sn with respect to cell yield as well as surface-marker phenotype and morphology. We also investigated the phenotypic changes and the T-cell stimulatory activity of the DCs induced by bacterial lipopolysaccharide (LPS). Morphologically, we observed low levels (5-10%) of granulocyte contamination of the cultures after a culture period of 11 days. Considerable strain-specific differences were found in the expression levels of the surface markers in addition to the differences in the ratio of the immature to mature DCs in the cultures that were not stimulated with LPS. Furthermore, we found that LPS strongly induces maturation of DCs in all strains investigated with the exception of the B10.PL strain.

Animals↗
PubMed2000

Characterization of murine dendritic cells derived from adherent blood mononuclear cells in vitro.

The therapeutic potential of dendritic cells loaded with tumour antigens for the induction of effective immune responses against cancer is currently being tested in numerous clinical trials. In most cases, the dendritic cells are generated in vitro from peripheral blood monocytes. Many aspects of dendritic cell-based vaccination have not yet been examined in detail, and homologous mouse model systems may prove very valuable for optimizing clinical procedures. In the murine system, however, dendritic cells are usually isolated from either lymphoid tissues or bone marrow cultures. To date, murine monocyte-derived dendritic cells have been described only sporadically. Here, we describe a culture system for the generation of murine dendritic cells from adherent peripheral blood mononuclear cells by culturing in the presence of granulocyte-macrophage colony stimulating factor and interleukin-4. After 7 days of culture the nonadherent cells were harvested from the cultures. Most of these cells exhibited well-accepted characteristics of mature dendritic cells (e.g. veiled appearance, high expression of major histocompatibility complex class II and CD86) and stimulated vigorous proliferation of allogeneic T cells in a primary mixed leucocyte reaction following stimulation with bacterial lipopolysaccharide. Interestingly, staining the cells for expression of the putative antigen-uptake receptor DEC-205 revealed a distinct bimodal distribution.

Animals↗
PubMed1999

Localization of a human nucleoporin 155 gene (NUP155) to the 5p13 region and cloning of its cDNA.

Nucleoporins are the main components of nuclear pore complexes (NPCs) involved in nucleo-cytoplasmic transport. Starting with an expressed DNA fragment retrieved by exon trapping from pooled human BAC clones mapped to the short arm of chromosome 5, we identified a human nucleoporin cDNA sequence by PCR from a human testis cDNA library. The coding sequence showed high homology to that of the rat nucleoporin 155 (Nup155) cDNA. FISH analysis with the human BAC clone as probe localized the human NUP155 gene to chromosome band 5p13. Northern analysis showed that the human NUP155 gene was expressed at different levels in all tissues tested. Two species of transcripts were observed with estimated lengths of 5.4 and 4.7 kb, respectively, in concordance with the finding of two alternative polyadenylation sites in the cDNA. The genomic location of the human NUP155 gene suggests a possible role in the mental and developmental retardation associated with hemizygous deletions of the 5p13 region.

Amino Acid Sequence↗
PubMed1999

Protein misfolding and degradation in genetic diseases.

Investigations of genetic diseases such as cystic fibrosis, alpha-1-antitrypsin deficiency, phenylketonuria, mitochondrial acyl-CoA dehydrogenase deficiencies, and many others have shown that enhanced proteolytic degradation of mutant proteins is a common molecular pathological mechanism. Detailed studies of the fate of mutant proteins in some of these diseases have revealed that impaired or aberrant folding of mutant polypeptides typically results in prolonged interaction with molecular chaperones and degradation by intracellular proteases before the functional conformation is acquired. This appears to be the case for many missense mutations and short in-frame deletions or insertions that represent a major fraction of the mutations detected in genetic diseases. In some diseases, or under some circumstances, the degradation system is not efficient. Instead, aberrant folding leads to accumulation of protein aggregates that damage the cell. Mechanisms by which misfolded proteins are selected for degradation have first been delineated for the endoplasmatic reticulum; this process has been termed "protein quality control." Similar mechanisms appear to be operative in all cellular compartments in which proteins fold. Within the context of genetic diseases, we review knowledge on the molecular processes underlying protein quality control in the various subcellular compartments. The important impact of such systems for variability of the expression of genetic deficiencies is emphasised.

Cell Compartmentation↗
PubMed1999

Normolipidemia and hypercholesterolemia in persons heterozygous for the same 1592 + 5G --> A splice site mutation in the low-density lipoprotein receptor gene.

In the present study, we have characterized a unique splice donor G to A substitution in the moderately conserved + 5 position in intron 10 of the low-density lipoprotein (LDL) receptor gene. In two Danish families, carriers of the 1592 + 5G --> A mutation display a clinical phenotype ranging from healthy normocholesterolemic persons to classical heterozygous familial hypercholesterolemia (FH) patients. Reverse transcription-polymerase chain reaction (RT-PCR) of RNA from Epstein Barr virus (EBV)-transformed lymphoblasts obtained from members of both families demonstrated abnormal splicing generating two aberrant mRNAs due to either alternative splicing and skipping of exon 10 or activation of a cryptic splice site in intron 10 inserting 66 intronic base pairs. These abnormally spliced mRNAs were predicted to encode two abnormal receptor proteins containing an in-frame deletion of 75 amino acids and an insertion of 22 novel amino acids, respectively. Results obtained by immunofluorescence staining, flow cytometry, and confocal microscopy of transfected Chang and COS-7 cells expressing normal and mutant LDL receptors were compatible with nearly complete retention of the mutant proteins in the endoplasmic reticulum. Quantitative measurements of LDL receptor mRNAs from EBV-transformed lymphoblasts, however, did not reveal any significant differences in variant mRNA contents between mutation carriers in the families that could be related to degree of hypercholesterolemia.

Adult↗
PubMed1999

Identification of novel and known mutations in the genes for keratin 5 and 14 in Danish patients with epidermolysis bullosa simplex: correlation between genotype and phenotype.

Epidermolysis bullosa simplex (EBS) is a group of autosomal dominant inherited skin diseases caused by mutations in either the keratin 5 (K5) or the keratin 14 (K14) genes and characterized by development of intraepidermal skin blisters. The three major subtypes of EBS are Weber-Cockayne, Koebner, and Dowling-Meara, of which the Dowling-Meara form is the most severe. We have investigated five large Danish families with EBS and two sporadic patients with the Dowling-Meara form of EBS. In the sporadic Dowling-Meara EBS patients, a novel K14 mutation (N123S) and a previously published K5 mutation (N176S) were identified, respectively. A novel K14 mutation (K116N) was found in three seemingly unrelated families, whereas another family harbored a different novel K14 mutation (L143P). The last family harbored a novel K5 mutation (L325P). The identified mutations were not present in more than 100 normal chromosomes. Six polymorphisms were identified in the K14 gene and their frequencies were determined in normal controls. These polymorphisms were used to show that the K14 K116N mutation was located in chromosomes with the same haplotype in all three families, suggesting a common ancestor. We observed a strict genotype-phenotype correlation in the investigated patients as the same mutation always resulted in a similar phenotype in all individuals with the mutation, but our results also show that it is not possible to predict the EBS phenotype merely by the location (i.e., head, rod, or linker domains) of a mutation. The nature of the amino acid substitution must also be taken into account.

Denmark↗
PubMed1999

Comparison of three methods of microsatellite detection.

Examination of microsatellites is frequent in the diagnosis of cancer. Microsatellites are repeat DNA sequences scattered throughout the human genome. These repeat regions are very frequent and highly polymorphic elements. In this study we focus on dinucleotide repeats. We compared three different methods for the detection of microsatellites: use of the ABI Prism 377 fluorescence sequencer, autoradiography and silver-stained gels. DNA was extracted from various clinical samples and amplified by different polymerase chain reaction (PCR) protocols. DNA from normal and tumor tissues was analysed using each method. The fluorescence method was more sensitive than the two other methods; however, this technology is very expensive. It seems possible, when examining microsatellites on a low budget, to avoid radioactivity by using silver-stained gels as an alternative. In conclusion, we observed identical results when comparing autoradiography with the fluorescence technique. However, we observed variability in the results when interpreting a single locus comparing silver staining with autoradiography and the fluorescence technique. Classification of the tumors based on several microsatellite loci was always identical.

Autoradiography↗
PubMed1999

Clear correlation of genotype with disease phenotype in very-long-chain acyl-CoA dehydrogenase deficiency.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the initial rate-limiting step in mitochondrial fatty acid beta-oxidation. VLCAD deficiency is clinically heterogenous, with three major phenotypes: a severe childhood form, with early onset, high mortality, and high incidence of cardiomyopathy; a milder childhood form, with later onset, usually with hypoketotic hypoglycemia as the main presenting feature, low mortality, and rare cardiomyopathy; and an adult form, with isolated skeletal muscle involvement, rhabdomyolysis, and myoglobinuria, usually triggered by exercise or fasting. To examine whether these different phenotypes are due to differences in the VLCAD genotype, we investigated 58 different mutations in 55 unrelated patients representing all known clinical phenotypes and correlated the mutation type with the clinical phenotype. Our results show a clear relationship between the nature of the mutation and the severity of disease. Patients with the severe childhood phenotype have mutations that result in no residual enzyme activity, whereas patients with the milder childhood and adult phenotypes have mutations that may result in residual enzyme activity. This clear genotype-phenotype relationship is in sharp contrast to what has been observed in medium-chain acyl-CoA dehydrogenase deficiency, in which no correlation between genotype and phenotype can be established.

Acyl-CoA Dehydrogenase, Long-Chain↗
PubMed1999

Clinical and molecular evidence of abnormal processing and trafficking of the vasopressin preprohormone in a large kindred with familial neurohypophyseal diabetes insipidus due to a signal peptide mutation.

The autosomal dominant form of familial neurohypophyseal diabetes insipidus (adFNDI) is a rare disease characterized by postnatal onset of polyuria and a deficient neurosecretion of the antidiuretic hormone, arginine vasopressin (AVP). Since 1991, adFNDI has been linked to 31 different mutations of the gene that codes for the vasopressin-neurophysin II (AVP-NPII) precursor. The aims of the present study were to relate the clinical phenotype to the specific genotype and to the molecular genetic effects of the most frequently reported adFNDI mutation located at the cleavage site of the signal peptide of AVP-NPII [Ala(-1)Thr]. Genetic analysis and clinical studies of AVP secretion, urinary AVP, and urine output were performed in 16 affected and 16 unaffected family members and 11 spouses of a Danish adFNDI kindred carrying the Ala(-1)Thr mutation. Mutant complementary DNA carrying the same mutation was expressed in a neurogenic cell line (Neuro2A), and the cellular effects were studied by Western blotting, immunocytochemistry, and AVP measurements. The clinical studies showed a severe progressive deficiency of plasma and urinary AVP that manifested during childhood. The expression studies demonstrated that the Ala(- 1)Thr mutant cells produced 8-fold less AVP than wild-type cells and accumulated excessive amounts of 23-kDa NPII protein corresponding to uncleaved prepro-AVP-NPII. Furthermore, a substantial portion of the intracellular AVP-NPII precursor appeared to be colocalized with an endoplasmic reticulum antigen (Grp78). These results provide independent confirmation that this Ala(-1)Thr mutation produces adFNDI by directing the production of a mutant preprohormone that accumulates in the endoplasmic reticulum, because it cannot be cleaved from the signal peptide and transported to neurosecretory vesicles for further processing and secretion.

Adolescent↗
PubMed1998

Rapid degradation of short-chain acyl-CoA dehydrogenase variants with temperature-sensitive folding defects occurs after import into mitochondria.

Most disease-causing missense mutations in short-chain acyl-CoA dehydrogenase (SCAD) and medium-chain acyl-CoA dehydrogenase are thought to compromise the mitochondrial folding and/or stability of the mutant proteins. To address this question, we studied the biogenesis of SCAD proteins in COS-7 cells transfected with cDNA corresponding to two SCAD missense mutations, R22W (identified in a patient with SCAD deficiency) or R22C (homologous to a disease-associated R28C mutation in medium-chain acyl-CoA dehydrogenase deficiency). After cultivation at 37 degreesC the steady-state amounts of SCAD antigen and activity in extracts from cells transfected with mutant SCAD cDNAs were negligible compared with those of cells transfected with SCAD wild type cDNA, documenting the deleterious effect of the two mutations. Analysis of metabolically labeled and immunoprecipitated SCAD wild type and mutant proteins showed that the two mutant proteins were synthesized as the 44-kDa precursor form, imported into mitochondria and processed to the mature 41.7-kDa form in a normal fashion. However, the intramitochondrial level of matured mutant SCAD proteins decreased rapidly to very low levels, indicating a rapid degradation of the mutant proteins at 37 degreesC. A rapid initial elimination phase was also observed following cultivation at 26 degreesC; however, significantly higher amounts of metabolically labeled and immunoprecipitated mature mutant SCAD proteins remained detectable. This corresponds well with the appreciable steady-state levels of SCAD mutant enzyme activity observed at 26 degreesC. In addition, confocal laser scanning microscopy of immunostained cells showed that the SCAD mutant proteins were localized intramitochondrially. Together, these results show that newly synthesized SCAD R22W and R22C mutant proteins are imported and processed in the mitochondrial matrix, but that a fraction of the proteins is rapidly eliminated by a temperature-dependent degradation mechanism. Thermal stability profiles of wild type and mutant enzymes revealed no difference between the two mutants and the wild type protein. Furthermore, the turnover of the SCAD mutant enzymes in intact cells was comparable to that of the wild type, indicating that the rapid degradation of the mutant SCAD proteins is not due to lability of the correctly folded tetrameric structure but rather to elimination of partly folded or misfolded proteins along the folding pathway.

Acyl-CoA Dehydrogenase↗
PubMed1998

A human homologue of Escherichia coli ClpP caseinolytic protease: recombinant expression, intracellular processing and subcellular localization.

We have recently cloned a human cDNA (hClpP) with significant sequence similarity to the ATP-dependent Escherichia coli ClpP protease [Bross, Andresen, Knudsen, Kruse and Gregersen (1995) FEBS Lett. 377, 249-252]. In the present study, synthesis, intracellular processing and subcellular localization of hClpP have been analysed in intact cells and in a cell-free system. Using pulse-labelling/immunoprecipitation of Chang cells transfected with the hClpP cDNA, we observed two major bands with apparent molecular masses of approx. 39 and 37 kDa. A pulse-chase experiment showed that these bands were converted into one mature-enzyme band with a molecular mass of approx. 32 kDa that was stable for at least 24 h. The 37 kDa band co-migrated with a band produced upon expression of full-length hClpP in E. coli, and the 32 kDa band co-migrated with the product of E. coli-expressed hClpP in which the 56 N-terminal residues had been deleted, indicating that the 37 kDa moiety represents the precursor and that approx. 56 residues are cleaved off during maturation. The processing of hClpP in intact cells was dependent on mitochondrial membrane potential. These results were confirmed in an import assay system using in vitro transcription and translation directed by the hClpP cDNA and isolated rat liver mitochondria. No protease activity towards a series of fluorogenic peptides could be observed in extracts of Chang cells overexpressing hClpP, indicating that the protease may not be active without co-factors. Immunofluorescence studies using confocal-laser-scanning microscopy showed co-localization of the hClpP and the mitochondrially located Hsp60 (heat-shock protein 60). Taken together, the results reported here show that hClpP is localized inside mitochondria and that the trafficking and processing of hClpP resembles the typical biogenesis pathway for nuclear-encoded mitochondrial proteins.

Adenosine Triphosphatases↗
PubMed1997

Two mutations in the same low-density lipoprotein receptor allele act in synergy to reduce receptor function in heterozygous familial hypercholesterolemia.

Mutations in genes are not necessarily pathogenic. Expression of mutant genes in cells can therefore be required to demonstrate that mutations in fact disturb protein function. This applies especially to missense mutations, which cause an amino acid to be replaced by another amino acid. In the present study of two families with familial hypercholesterolemia in the heterozygous form, we found two mutations in the same allele of the low-density lipoprotein (LDL) receptor gene: a missense Asn543. His mutation (N543H) in exon 11, and an in-frame 9-bp deletion (2393del9) in exon 17. The two mutations were identified in heterozygous FH index patients in whom no other pathogenic mutations were detected by SSCP analysis of the remaining 16 exons and the promoter region. Both mutations cosegregated with hypercholesterolemia within the families. Each of these mutations had little or no effect on receptor function in transfected COS cells, but when both mutations were present simultaneously, receptor function, as assessed by flow cytometric measurement of fluorescent LDL uptake in cells, was reduced by 75%. Immunostainable receptors on the cell surface were decreased by 80% as measured by flow cytometry. The two mutations therefore acted in synergy to affect receptor function, possibly during intracellular receptor transport, since Northern blot analysis suggested that mRNA levels were unaffected. Without screening of the entire coding regions of the gene, the synergistic action of these two LDL receptor mutations would not have been detected.

Alleles↗
PubMed1997

A de nevo complex t(7;13;8) translocation with a deletion in the TRPS gene region.

Molecular cytogenetic analyses have resolved the pathogenetic aberration of an 8-year-old girl with tricho-rhino-phalangeal syndrome type I (TRPS I), normal intelligence, and a karyotype originally described as 46,XX,t(8;13)(q24;q21). R- and Q-banding and high resolution R-banding analyses have also disclosed a seemingly mosaic abnormality of the distal short arm of chromosome 7 but have not fully characterized this abnormality. Combined primed in situ labelling and chromosome painting, and three-colour chromosome painting have revealed a complex, apparently balanced translocation t(7;13;8). Fluorescence in situ hybridization with yeast artificial chromosome and cosmid clones from 8q24.1 has shown an interstitial deletion of at least 3 Mb covering most of the TRPS I critical region.

Child↗
PubMed1997

A common W556S mutation in the LDL receptor gene of Danish patients with familial hypercholesterolemia encodes a transport-defective protein.

In a group of unrelated Danish patients with familial hypercholesterolemia (FH) we recently reported two common low-density lipoprotein (LDL) receptor mutations, W23X and W66G, accounting for 30% of the cases. In this study, we describe another common LDL receptor mutation, a G to C transition at cDNA position 1730 in exon 12, causing a tryptophan to serine substitution in amino acid position 556 (W556S). In the Danish patients, the W556S mutation was present in 12% of 65 possible mutant alleles. The pathogenicity of the W556S mutation, which is located in one of the five conserved motifs Tyr-Trp-Thr-Asp in the epidermal growth factor homology region, was studied in transfected COS-7 cells expressing normal and mutant LDL receptor cDNAs. Results obtained by immunofluorescence flow cytometry and confocal microscopy, as well as by immunoprecipitation, were compatible with complete retention of the mutant protein in the endoplasmic reticulum. The transport-defective W556S mutation and the W23X and W66G mutations seem to account for about 40% of the LDL receptor defects in Danish families with FH.

Amino Acid Sequence↗
PubMed1997

The molecular basis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency in compound heterozygous patients: is there correlation between genotype and phenotype?

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most commonly recognized defect of mitochondrial beta-oxidation. It is potentially fatal, but shows a wide clinical spectrum. The aim of the present study was to investigate whether any correlation exists between MCAD genotype and disease phenotype. We determined the prevalence of the 14 known and seven previously unknown non-G985 mutations in 52 families with MCAD deficiency not caused by homozygosity for the prevalent G985 mutation. This showed that none of the non-G985 mutations are prevalent, and led to the identification of both disease-causing mutations in 14 families in whom both mutations had not previously been reported. We then evaluated the severity of the mutations identified in these 14 families. Using expression of mutant MCAD in Escherichia coli with or without co-overexpression of the molecular chaperonins GroESL we showed that five of the missense mutations affect the folding and/or stability of the protein, and that the residual enzyme activity of some of them could be modulated to a different extent depending on the amounts of available chaperonins. Thus, some of the missense mutations may result in relatively high levels of residual enzyme activity, whereas the mutations leading to premature stop codons will result in no residual enzyme activity. By correlating the observed types of mutations identified to the clinical/biochemical data in the 14 patients in whom we identified both disease-causing mutations, we show that a genotype/phenotype correlation in MCAD deficiency is not straightforward. Different mutations may contribute with different susceptibilities for disease precipitation, when the patient is subjected to metabolic stress, but other genetic and environmental factors may play an equally important role.

Acyl-CoA Dehydrogenase↗