PubMed Health⌕ Search

Biomedical subjects

J van der Vlag

Publications and source records attributed to J van der Vlag.

At least 19 recordsLinked to original sources
PubMed2006

Increased expression of heparanase in overt diabetic nephropathy.

In overt diabetic nephropathy (DNP), an increase in the permeability of the glomerular basement membrane (GBM) has been associated with a loss of negatively charged heparan sulfates (HS) in the GBM. Heparanase (HPSE), an endo-beta(1-4)-D-glucuronidase, can cleave HS and could be a potential candidate for the degradation of glomerular HS, leading to the development of proteinuria. We analyzed whether changes in HS expression are associated with HPSE expression in overt DNP. Immunofluorescence staining was performed to analyze HS, HPSE, and agrin core protein expression in kidney biopsies from patients with overt DNP and from rats and mice with streptozotocin (STZ)-induced diabetes. We also investigated the effect of transgenic HPSE overexpression in mice on glomerular HS and agrin expression. We demonstrate that the loss of GBM HS (-50%) and tubular HS (-60%) is associated with a four-fold increased HPSE expression in overt DNP. In addition, glomerular HPSE expression is upregulated in rats (messenger RNA (mRNA) 2.5-fold, protein three-fold) and mice (mRNA seven-fold, protein 1.5-fold) with STZ-induced diabetes. Furthermore, transgenic HPSE overexpression results in disappearance of HS, whereas expression of the core protein agrin remains unaltered. Our observations suggest that HPSE is involved in the pathogenesis of proteinuria in overt DNP by degradation of HS.

Aged↗
PubMed2006

Transmission of modified nucleosomes from the mouse male germline to the zygote and subsequent remodeling of paternal chromatin.

Rapidly after gamete fusion, the sperm nucleus loses its specific chromatin conformation and the DNA is repopulated with maternally derived nucleosomes. We evaluated the nature of paternally derived nucleosomes and the dynamics of sperm chromatin remodeling in the zygote directly after gamete fusion. We observed histone H4 acetylated at K8 or K12 already prior to full decondensation of the sperm nucleus, suggesting that these marks are transmitted by the spermatozoon. Tracking down the origin of H4K8ac and H4K12ac during spermiogenesis revealed the retention of nucleosomes with these modifications in the chromocenter of elongating spermatids. We show that sperm constitutive heterochromatin is enriched for nucleosomes carrying specific histone modifications which are transmitted to the zygote. Our results suggest an epigenetic mechanism for inheritance of chromosomal architecture. Furthermore, up to pronucleus formation, histone acetylation and phosphorylation build up in a cascade-like fashion in the paternal chromatin. After formation of the pronucleus, a subset of these marks is removed from the heterochromatin, which suggests a reestablishment of the euchromatin-heterochromatin partition.

Acetylation↗
PubMed2006

Selection and characterization of a unique phage display-derived antibody against dermatan sulfate.

Dermatan sulfate (DS) is a member of the glycosaminoglycan (GAG) family and is primarily located in the extracellular matrix. Using a modified phage display procedure, we selected 2 different antibodies against DS of which one antibody, LKN1, was specific for DS. LKN1 was especially reactive with 4/2,4-di-O-sulfated DS, and did not react with other classes of GAGs including chondroitin sulfate and heparan sulfate. Immunohistochemical analysis of kidney, skin and tendon showed a typical fibrillar staining pattern, co-localizing with type I collagen. Staining was abolished by specific enzymatic digestion of DS. Immunoelectron microscopy confirmed the association of the DS epitope with collagen fibrils. The location of DS did not follow the main banding period of collagen, which is in line with the current concept that the core protein rather than the DS moiety of DS-proteoglycans specifically binds to collagen fibrils. This unique anti-DS antibody and the availability of its coding DNA may be instrumental in studies of the structure and function of DS.

Animals↗
PubMed2006

The binding of lupus-derived autoantibodies to the C-terminal peptide (83-119) of the major SmD1 autoantigen can be mediated by double-stranded DNA and nucleosomes.

OBJECTIVES: To evaluate the binding of lupus-derived autoantibodies, double-stranded DNA and nucleosomes to the positively charged C-terminal SmD1(residues 83-119) peptide and the full-length SmD protein. METHODS: The binding of lupus-derived monoclonal antibodies, sera from patients with systemic lupus erythematosus, rheumatoid arthritis and systemic sclerosis, dsDNA and nucleosomes to the SmD1(83-119) peptide or the full-length SmD protein was determined using different ELISA methods. RESULTS: Monoclonal anti-dsDNA antibodies and the serum of patients with systemic lupus erythematosus that are positive for anti-dsDNA antibodies react with the SmD1(83-119) peptide in ELISA. However, DNaseI treatment of the blocking reagents leads to a decreased reactivity. Purified dsDNA and nucleosomes bind to the SmD1 peptide but not to the full-length SmD protein. CONCLUSIONS: The SmD1(83-119) peptide is able to bind dsDNA and nucleosomes, and dsDNA or nucleosomes in applied reagents lead to an apparent reactivity of anti-dsDNA, anti-histone or nucleosome-specific antibodies with the SmD1(83-119) peptide in ELISA.

Animals↗
PubMed2006

Reactive oxygen species deglycosilate glomerular alpha-dystroglycan.

In the kidney, dystroglycan (DG) has been shown to cover the basolateral and apical membranes of the podocyte. alpha-DG is heavily glycosilated, which is important for its binding to laminin and agrin in the glomerular basement membrane. Furthermore, alpha-DG is negatively charged, which maintains the filtration slit open. Reactive oxygen species (ROS) are known to degrade and depolymerize carbohydrates, and to play a role in several glomerular diseases. Therefore, we evaluated the effect of ROS on the glycosilation of glomerular alpha-DG. By using specific antibodies directed against the core protein or glyco-epitopes of alpha-DG, this was studied in a solid-phase assay, in situ on kidney sections, and in vivo in adriamycin nephropathy. A ligand overlay assay was used to study binding of alpha-DG to its ligands. Exposure to ROS leads to a loss of carbohydrate epitopes on alpha-DG both in vitro and on kidney sections. In the in vitro assays, a decreased binding of deglycosilated alpha-DG to laminin and agrin was found. In adriamycin nephropathy, where radicals play a role, we observed a loss of alpha-DG carbohydrate epitopes. We conclude that deglycosilation of glomerular alpha-DG by ROS leads to disruption of the agrin-DG complex, which in vivo may lead to the detachment of podocytes. Furthermore, loss of negative charge in the filtration slit may lead to foot process effacement of podocytes.

Agrin↗
PubMed2004

[Podocyte dysfunction and proteinuria].

Podocytes play a central role in the pathogenesis of several glomerular diseases. In recent years, this has been revealed by molecular analysis of a number of rare hereditary renal diseases. Podocytes contain three domains: the domain bound to the glomerular basement membrane (GBM), the domain of the slit diaphragms and the apical domain. The slit diaphragms are situated basolaterally between the pedicles and form together with the GBM a mechanism for the selective filtration of blood to primary urine. The apical cell membrane forms a negatively charged layer which prevents adhesion to the adjacent cell membranes, thus keeping the slit diaphragms and urinary space open. Many podocyte diseases are characterised by foot process effacement, which causes the loss of slit diaphragms, and could lead to podocyte loss. Specific abnormalities have been discovered in the three domains of the podocyte to which a number of glomerular diseases can be attributed.

Humans↗
PubMed2002

Triggers for anti-chromatin autoantibody production in SLE.

The formation of autoantibodies against chromatin is the main feature of systemic lupus erythematosis (SLE), an autoimmune disease, which is T-cell dependent and autoantigen-driven. Historically, antibodies against dsDNA, one of the components of chromatin, are considered as a hallmark of SLE. However, dsDNA is poorly immunogenic. Nucleosome-specific T helper cells have been identified. These T cells propagate not only nucleosome-specific antibodies, but also anti-dsDNA antibodies. Nucleosomes are formed during apoptosis by cleavage of chromatin, and evidence of disturbed apoptosis has been found especially in certain murine models of lupus. In addition to an increased rate of apoptosis, autoimmunity against chromatin might also result from an impaired phagocytosis of apoptotic material, for which strong evidence has been provided by studies in certain knock-out mice (C1q, SAP, Dnase I). The induction of an immune response to nucleosomes could be enhanced by modifications of histones or DNA during apoptosis, altered presentation by antigen presenting cells or a viral infection. The release of nucleosomes and the formation of anti-chromatin autoantibodies result in formation of complexes, which bind to the glomerular basement membrane via heparan sulfate. This deposition incites glomerulonephritis, the most serious manifestation of SLE.

Autoantibodies↗
PubMed2000

Transcriptional repression mediated by polycomb group proteins and other chromatin-associated repressors is selectively blocked by insulators.

Polycomb group (PcG) proteins repress gene activity over a considerable distance, possibly by spreading along the chromatin fiber. Insulators or boundary elements, genetic elements within the chromatin, may serve to terminate the repressing action of PcG proteins. We studied the ability of insulators to block the action of chromatin-associated repressors such as PcG proteins, HP1, and MeCP2. We found that the Drosophila special chromatin structure insulator completely blocks transcriptional repression mediated by all of the repressors we tested. The Drosophila gypsy insulator was able to block the repression mediated by the PcG proteins Su(z)2 and RING1, as well as mHP1, but not the repression mediated by MeCP2 and the PcG protein HPC2. The 5'-located DNase I-hypersensitive site in the chicken beta-globin locus displayed a limited ability to block repression, and a matrix or scaffold attachment region element was entirely unable to block repression mediated by any repressor tested. Our results indicate that insulators can block repression mediated by PcG proteins and other chromatin-associated repressors, but with a high level of selectivity. This high degree of specificity may provide a useful assay to define and characterize distinct classes of insulators.

Animals↗
PubMed1999

Transcriptional repression mediated by the human polycomb-group protein EED involves histone deacetylation.

Polycomb-group (PcG) proteins form multimeric protein complexes, which are involved in maintaining the transcriptional repressive state of genes over successive cell generations. Components of PcG complexes and their mutual interactions have been identified and analysed through extensive genetic and biochemical analyses. Molecular mechanisms underlying PcG-mediated repression of gene activity, however, have remained largely unknown. Previously we reported the existence of two distinct human PcG protein complexes. The EED/EZH protein complex contains the embryonic ectoderm development (EED) and enhancer of zeste 2 (EZH2; refs 9,10) PcG proteins. The HPC/HPH PcG complex contains the human polycomb 2 (HPC2; ref. 11), human polyhomeotic (HPH), BMI1 (ref. 13 ) and RING1 (refs 14, 15) proteins. Here we show that EED (refs 4, 5, 6, 7, 8) interacts, both in vitro and in vivo, with histone deacetylase (HDAC) proteins. This interaction is highly specific because the HDAC proteins do not interact with other vertebrate PcG proteins. We further find that histone deacetylation activity co-immunoprecipitates with the EED protein. Finally, the histone deacetylase inhibitor trichostatin A (ref. 17) relieves transcriptional repression mediated by EED, but not by HPC2, a human homologue of polycomb. Our data indicate that PcG-mediated repression of gene activity involves histone deacetylation. This mechanistic link between two distinct, global gene repression systems is accomplished through the interaction of HDAC proteins with a particular PcG protein, EED.

Acetylation↗
PubMed1999

C-Terminal binding protein is a transcriptional repressor that interacts with a specific class of vertebrate Polycomb proteins.

Polycomb (Pc) is part of a Pc group (PcG) protein complex that is involved in repression of gene activity during Drosophila and vertebrate development. To identify proteins that interact with vertebrate Pc homologs, we performed two-hybrid screens with Xenopus Pc (XPc) and human Pc 2 (HPC2). We find that the C-terminal binding protein (CtBP) interacts with XPc and HPC2, that CtBP and HPC2 coimmunoprecipitate, and that CtBP and HPC2 partially colocalize in large PcG domains in interphase nuclei. CtBP is a protein with unknown function that binds to a conserved 6-amino-acid motif in the C terminus of the adenovirus E1A protein. Also, the Drosophila CtBP homolog interacts, through this conserved amino acid motif, with several segmentation proteins that act as repressors. Similarly, we find that CtBP binds with HPC2 and XPc through the conserved 6-amino-acid motif. Importantly, CtBP does not interact with another vertebrate Pc homolog, M33, which lacks this amino acid motif, indicating specificity among vertebrate Pc homologs. Finally, we show that CtBP is a transcriptional repressor. The results are discussed in terms of a model that brings together PcG-mediated repression and repression systems that require corepressors such as CtBP.

Alcohol Oxidoreductases↗
PubMed1998

Characterization of interactions between the mammalian polycomb-group proteins Enx1/EZH2 and EED suggests the existence of different mammalian polycomb-group protein complexes.

In Drosophila melanogaster, the Polycomb-group (PcG) and trithorax-group (trxG) genes have been identified as repressors and activators, respectively, of gene expression. Both groups of genes are required for the stable transmission of gene expression patterns to progeny cells throughout development. Several lines of evidence suggest a functional interaction between the PcG and trxG proteins. For example, genetic evidence indicates that the enhancer of zeste [E(z)] gene can be considered both a PcG and a trxG gene. To better understand the molecular interactions in which the E(z) protein is involved, we performed a two-hybrid screen with Enx1/EZH2, a mammalian homolog of E(z), as the target. We report the identification of the human EED protein, which interacts with Enx1/EZH2. EED is the human homolog of eed, a murine PcG gene which has extensive homology with the Drosophila PcG gene extra sex combs (esc). Enx1/EZH2 and EED coimmunoprecipitate, indicating that they also interact in vivo. However, Enx1/EZH2 and EED do not coimmunoprecipitate with other human PcG proteins, such as HPC2 and BMI1. Furthermore, unlike HPC2 and BMI1, which colocalize in nuclear domains of U-2 OS osteosarcoma cells, Enx1/EZH2 and EED do not colocalize with HPC2 or BMI1. Our findings indicate that Enx1/EZH2 and EED are members of a class of PcG proteins that is distinct from previously described human PcG proteins.

Amino Acid Sequence↗
PubMed1997

Interference with the expression of a novel human polycomb protein, hPc2, results in cellular transformation and apoptosis.

Polycomb (Pc) is involved in the stable and heritable repression of homeotic gene activity during Drosophila development. Here, we report the identification of a novel human Pc homolog, hPc2. This gene is more closely related to a Xenopus Pc homolog, XPc, than to a previously described human Pc homolog, CBX2 (hPc1). However, the hPc2 and CBX2/hPc1 proteins colocalize in interphase nuclei of human U-2 OS osteosarcoma cells, suggesting that the proteins are part of a common protein complex. To study the functions of the novel human Pc homolog, we generated a mutant protein, delta hPc2, which lacks an evolutionarily conserved C-terminal domain. This C-terminal domain is important for hPc2 function, since the delta hPc2 mutant protein which lacks the C-terminal domain is unable to repress gene activity. Expression of the delta hPc2 protein, but not of the wild-type hPc2 protein, results in cellular transformation of mammalian cell lines as judged by phenotypic changes, altered marker gene expression, and anchorage-independent growth. Specifically in delta hPc2-transformed cells, the expression of the c-myc proto-oncogene is strongly enhanced and serum deprivation results in apoptosis. In contrast, overexpression of the wild-type hPc2 protein results in decreased c-myc expression. Our data suggest that hPc2 is a repressor of proto-oncogene activity and that interference with hPc2 function can lead to derepression of proto-oncogene transcription and subsequently to cellular transformation.

3T3 Cells↗
PubMed1997

RING1 is associated with the polycomb group protein complex and acts as a transcriptional repressor.

The Polycomb (Pc) protein is a component of a multimeric, chromatin-associated Polycomb group (PcG) protein complex, which is involved in stable repression of gene activity. The identities of components of the PcG protein complex are largely unknown. In a two-hybrid screen with a vertebrate Pc homolog as a target, we identify the human RING1 protein as interacting with Pc. RING1 is a protein that contains the RING finger motif, a specific zinc-binding domain, which is found in many regulatory proteins. So far, the function of the RING1 protein has remained enigmatic. Here, we show that RING1 coimmunoprecipitates with a human Pc homolog, the vertebrate PcG protein BMI1, and HPH1, a human homolog of the PcG protein Polyhomeotic (Ph). Also, RING1 colocalizes with these vertebrate PcG proteins in nuclear domains of SW480 human colorectal adenocarcinoma and Saos-2 human osteosarcoma cells. Finally, we show that RING1, like Pc, is able to repress gene activity when targeted to a reporter gene. Our findings indicate that RING1 is associated with the human PcG protein complex and that RING1, like PcG proteins, can act as a transcriptional repressor.

Amino Acid Sequence↗
PubMed1995

Regulation of glycerol and maltose uptake by the IIAGlc-like domain of IINag of the phosphotransferase system in Salmonella typhimurium LT2.

In Enterobacteriaceae the nonphosphorylated form of IIAGlc of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) can inhibit the uptake and subsequent metabolism of glycerol and maltose by binding to, and inhibiting, glycerol kinase and the MalK protein of the maltose transport system, respectively. In this report we show that the IIAGlc-like domain of the membrane-bound IIN-acetylglucosamine (IINag) of the PTS can replace IIAGlc in a Salmonella typhimurium crr mutant strain that lacks all soluble IIAGlc. The inhibition was most severe in cells which were partially induced for the glycerol or maltose uptake systems. The Streptococcus thermophilus lactose transporter LacS, which also contains a IIAGlc-like domain, could not replace IIAGlc. Neither IINag nor LacS could replace IIAGlc in activation of adenylate cyclase.

Acetylglucosamine↗
PubMed1995

Control of glucose metabolism by the enzymes of the glucose phosphotransferase system in Salmonella typhimurium.

The quantitative role of the phosphoenolpyruvate:glucose phosphotransferase system (glucose phosphotransferase system) in glucose uptake and metabolism, and phosphotransferase-system-mediated regulation of glycerol uptake, was studied in vivo in Salmonella typhimurium. Expression plasmids were constructed which contained the genes encoding enzyme I (ptsI), HP (ptsH), IIAGlc (crr), and IICBGlc (ptsG) of the glucose phosphotransferase system behind inducible promoters. These plasmids allowed the controlled expression of each of the glucose phosphotransferase system proteins from about 30% to about 300% of its wild-type level. When enzyme I, HPr or IIAGlc were modulated between 30% and 300% of their wild-type value, hardly any effects on the growth rate on glucose, the glucose oxidation rate, the rate of methyl alpha-D-glucopyranoside (a glucose analog) uptake or the phosphotransferase-system-mediated inhibition of glycerol uptake by methyl alpha-D-glucopyranoside were observed. Employing the method of metabolic control analysis, it was shown that the enzyme flux control coefficients of these phosphotransferase system components on the different measured processes were close to zero. The enzyme flux control coefficient of IICBGlc on growth on glucose or glucose oxidation was also close to zero. In contrast, the enzyme flux control coefficient of IICBGlc on the flux through the glucose phosphotransferase system (transport and phosphorylation) was 0.72. The experimentally determined enzyme flux control coefficients allowed us to calculate the flux control coefficients of the phosphoenolpyruvate/pyruvate and methyl alpha-D-glucopyranoside/methyl alpha-D-glucopyranoside 6-phosphate couples and the process control coefficients of the phosphotransfer reactions of the glucose phosphotransferase system. We discuss the implications of these values and the possible control points in the glucose phosphotransferase system.

Bacterial Proteins↗
PubMed1994

Quantification of the regulation of glycerol and maltose metabolism by IIAGlc of the phosphoenolpyruvate-dependent glucose phosphotransferase system in Salmonella typhimurium.

The amount of IIAGlc, one of the proteins of the phosphoenolpyruvate:glucose phosphotransferase system (PTS), was modulated over a broad range with the help of inducible expression plasmids in Salmonella typhimurium. The in vivo effects of different levels of IIAGlc on glycerol and maltose metabolism were studied. The inhibition of glycerol uptake, by the addition of a PTS sugar, was sigmoidally related to the amount of IIAGlc. For complete inhibition of glycerol uptake, a minimal ratio of about 3.6 mol of IIAGlc to 1 mol of glycerol kinase (tetramer) was required. Varying the level of IIAGlc (from 0 to 1,000% of the wild-type level) did not affect the growth rate on glycerol, the rate of glycerol uptake, or the synthesis of glycerol kinase. In contrast, the growth rate on maltose, the rate of maltose uptake, and the synthesis of the maltose-binding protein increased two- to fivefold with increasing levels of IIAGlc. In the presence of cyclic AMP, the maximal levels were obtained at all IIAGlc concentrations. The synthesis of the MalK protein, the target of IIAGlc, was not affected by varying the levels of IIAGlc. The inhibition of maltose uptake was sigmoidally related to the amount of IIAGlc. For complete inhibition of maltose uptake by a PTS sugar, a ratio of about 18 mol of IIAGlc to 1 mol of MalK protein (taken as a dimer) was required.

ATP-Binding Cassette Transporters↗
PubMed1994

Enzymes of glucose and methanol metabolism in the actinomycete Amycolatopsis methanolica.

The actinomycete Amycolatopsis methanolica was found to employ the normal bacterial set of glycolytic and pentose phosphate pathway enzymes, except for the presence of a PPi-dependent phosphofructokinase (PPi-PFK) and a 3-phosphoglycerate mutase that is stimulated by 2,3-bisphosphoglycerate. Screening of a number of actinomycetes revealed PPi-PFK activity only in members of the family Pseudonocardiaceae. The A. methanolica PPi-PFK and 3-phosphoglycerate mutase enzymes were purified to homogeneity. PPi-PFK appeared to be insensitive to the typical effectors of ATP-dependent PFK enzymes. Nevertheless, strong N-terminal amino acid sequence homology was found with ATP-PFK enzymes from other bacteria. The A. methanolica pyruvate kinase was purified over 250-fold and characterized as an allosteric enzyme, sensitive to inhibition by P(i) and ATP but stimulated by AMP. By using mutants, evidence was obtained for the presence of transketolase isoenzymes functioning in the pentose phosphate pathway and ribulose monophosphate cycle during growth on glucose and methanol, respectively.

Actinobacteria↗