Biomedical subjects

J H Veerkamp

Publications and source records attributed to J H Veerkamp.

At least 19 recordsLinked to original sources

PubMed1999

Functional and conformational characterization of new mutants of heart fatty acid-binding protein.

In this study we investigated the possible involvement of several amino acids (not located in the ligand-binding centre) in fatty acid binding and conformational stability of heart fatty acid-binding protein (H-FABP). We prepared recombinant human H-FABP proteins with mutations in the hydrophobic patch (Phe(4), Trp(8) and Phe(64)), portal region (Phe(16)), hinge region (Leu(66), Gly(67)), second portal region (Glu(72)) and at the protein surface (Lys(21)) respectively. Oleic acid-binding affinity and conformational stability of human H-FABP are significantly decreased or completely lost by mutation of Trp(8) or Phe(16). NMR spectra confirmed that these residues are important for the stability of the protein fold. Substitution of Phe(4) or Phe(64) resulted in less stability, but oleic acid-binding affinity was not affected. Mutation of Lys(21) had no effect on either structural integrity or fatty acid-binding affinity. Replacement of Leu(66) or Gly(67) did not affect fatty acid binding, but protein stability was reduced. Finally, mutation of Glu(72) to Ser caused no change of affinity, but NMR spectra and urea-denaturation curves showed the extremely poor stability of this mutant. In conclusion, no relationship was observed between fatty acid-binding affinity and conformational stability.

Carrier Proteins↗

PubMed1999

The fatty acid-binding heterocomplex FA-p34 formed by S100A8 and S100A9 is the major fatty acid carrier in neutrophils and translocates from the cytosol to the membrane upon stimulation.

Since no data are available concerning fatty acid (FA) transport in neutrophils we studied the presence of possible FA carriers. The kFA-p34 complex, composed of S100A8 and S100A9, has been implicated in the intracellular transport of arachidonic acid and its precursors in human keratinocytes. Here, we show that FA-p34 is the major FA carrier in human neutrophils (nFA-p34). The complex is highly expressed in resting neutrophils (2.65% of cytosolic proteins) and translocates to the membrane fraction upon stimulation with opsonized zymosan. Comparison of purified nFA-p34 with kFA-p34 shows that both complexes are composed of nearly the same subunits and possess similar binding properties for oleic acid. Densitometrical analyses of 2D gels show that n and kFA-p34 contain twice as much S100A8 and S100A9 suggesting an estimated stoichiometry of (S100A8)2S100A9. A method is described allowing to distinguish n and kFA-p34 from S100A8/S100A9 homo- and heteromer complexes that are devoid of FA-binding properties. After solvent extraction, we find by GC analysis linoleic acid as major endogenous ligand of purified kFA-p34. Our results suggest that nFA-p34, might be involved in the shuttling of unsaturated FA between the cytosol and the plasma membrane of neutrophils.

Animals↗

PubMed1999

Differentiation markers of mouse C2C12 and rat L6 myogenic cell lines and the effect of the differentiation medium.

The differentiation grade of cells in culture is dependent on the composition of the culture medium. Two commonly used myogenic cell lines, mouse C2C12 and rat L6, usually differentiate at a low concentration of horse serum. In this study we compared the effect of horse serum with a medium containing a low percentage of Ultroser G and rat brain extract. The maturation grade was evaluated on the basis of various biochemical, (immuno)histochemical and cell-physiological parameters. Substitution of horse serum by Ultroser G and rat brain extract during the differentiation phase resulted in a higher maturation grade of the myotubes of both cell lines, on the basis of creatine kinase activity and the diameter of the myotubes. In addition, the C2C12 myotubes display cross-striation, contain a higher percentage of creatine kinase muscle-specific isoenzyme MM, show a ninefold increase in acetylcholine receptor (AChR) clusters, form a continuous basement membrane, and have a lower resting cytosolic Ca2+ concentration. L6 myotubes show a fivefold increase in AChR clusters and a twofold increase in the expression of the mRNA of the epsilon-subunit of AChR.C2C12 cells show spontaneous contraction and response of cytosolic Ca2+ to various stimulants in contrast to L6 cells which do not. These studies established that the Ultroser G/brain extract medium leads to a higher differentiation grade of both cell lines, but parameters appropriate for use as differentiation markers appear to differ between both cell lines.

Animals↗

PubMed1999

Preparation and characterization of porous crosslinked collagenous matrices containing bioavailable chondroitin sulphate.

Porous collagen matrices with defined physical, chemical and biological characteristics are interesting materials for tissue engineering. Attachment of glycosaminoglycans (GAGs) may add to these characteristics and valorize collagen. In this study, porous type I collagen matrices were crosslinked using dehydrothermal (DHT) treatment and/or 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC), in the presence and absence of chondroitin sulphate (CS). EDC covalently attaches CS to collagen. DHT crosslinking preserved a porous matrix structure. However, attachment of CS to DHT-treated matrices using EDC, resulted in collapsed surfaces, CS located only at the matrix exterior. EDC crosslinking resulted in a partial matrix collapse. This could be prevented if crosslinking was carried out in the presence of ethanol. Matrix porosity was then preserved. The presence of CS during EDC crosslinking resulted in covalent immobilization of CS throughout the matrix. The amount of CS incorporated was increased if crosslinking was performed in the presence of ethanol. EDC-crosslinked matrices, with and without CS, had increased denaturation temperatures and decreased free amine group contents. The susceptibility of these matrices towards degradation by proteolytic enzymes was diminished. Immobilized CS increased the water-binding capacity and decreased the denaturation temperature and tensile strength. Immobilized CS bound anti-CS antibodies and was susceptible to chondroitinase ABC digestion, demonstrating its bioavailability. The modified matrices were not cytotoxic as was established using human myoblast and fibroblast culture systems. It is concluded that the use of ethanol during EDC crosslinking, offers an elegant means for the preparation of defined porous collagenous matrices containing bioavailable, covalently attached CS.

Biocompatible Materials↗

PubMed1999

Isolation, amino acid sequence determination and binding properties of two fatty-acid-binding proteins from axolotl (Ambistoma mexicanum) liver. Evolutionary relationship.

Up until now, the primary structure of fatty-acid-binding proteins (FABPs) from the livers of four mammalian (rat, human, cow and pig) and three nonmammalian (chicken, catfish and iguana) species has been determined. Based on amino acid sequence comparisons, it has been suggested that mammalian and nonmammalian liver FABPs may be paralogous proteins that originated by gene duplication, rather than as a consequence of mutations of the same gene. In this paper we report the isolation and amino acid sequence determination of two FABPs from axolotl (Ambistoma mexicanum) liver. One of them is similar to mammalian liver FABPs (L-FABPs) and the other to chicken, catfish and iguana liver FABPs (Lb-FABPs). The finding of both L-FABP and Lb-FABP in a single species, as reported here, indicates that they are paralogous proteins. The time of divergence of these two liver FABP types is estimated to be of approximately 694 million years ago. The ligand-binding properties of axolotl liver FABPs were studied by means of parinaric-acid-binding and parinaric-acid-displacement assays. L-FABP binds two fatty acids per molecule but Lb-FABP displays a fatty-acid-conformation-dependent binding stoichiometry; L-FABP shows a higher affinity for fatty acids, especially oleic acid, while Lb-FABP has a higher affinity for other hydrophobic ligands, especially retinoic acid. In addition, the tissue-expression pattern is different, L-FABP is present in liver and intestinal mucosa while the expression of Lb-FABP is restricted to liver. Data indicate distinct functional properties of both liver FABP types.

Ambystoma↗

PubMed1999

Recent insights into the structure and functions of heparan sulfate proteoglycans in the human glomerular basement membrane.

As the first barrier to be crossed on the way to urinary space, the glomerular basement membrane (GBM) plays a key role in renal function. The permeability of the GBM for a given molecule is highly dependent on its size, shape and charge. As early as 1980, the charge-selective permeability was demonstrated to relate to the electrostatic properties of covalently bound heparan sulfates (HS) within the GBM. Since the identification of perlecan as a heparan sulfate proteoglycan (HSPG) of basement membranes, the hypothesis that perlecan could be a crucial determinant of GBM permselectivity received considerable attention. In addition to perlecan, the GBM also contains other HSPG species, one of which was identified as agrin. The high local expression of agrin in the GBM, together with the presence of agrin receptors at the cell matrix interface, suggests that this HSPG contributes to glomerular function in multiple ways. Here, we review the current knowledge regarding the structure and functions of HSPGs in the GBM, and discuss how these molecules could be involved in various glomerular diseases. Possible directions for future investigation are suggested.

Animals↗

PubMed1999

Hemoglobin and myoglobin content in muscles of broiler chickens.

The two main heme proteins, hemoglobin and myoglobin, are important factors determining meat quality aspects such as color and hemorrhage. The extent of hemorrhage in muscle tissue can probably be determined by measuring the hemoglobin content. The objective of this study was twofold: 1) to develop a specific and reproducible method to quantify the hemoglobin and myoglobin content in muscle tissue of broiler chickens, and 2) to study the effect of hemorrhage on the hemoglobin content in muscle tissue. We tested several methods to determine the total heme, hemoglobin, and myoglobin content in broiler chicken muscles on their specificity, sensitivity, and reproducibility. Methods based on immunological techniques appeared to be very specific and sensitive. The results obtained applying these methods on muscle tissue extracts were, however, not reproducible due to concentration effects. A combination of spectrophotometric analysis of the total heme protein concentration and measurement of the myoglobin concentration, applying size exclusion chromatography, proved to be a reliable and reproducible method to determine the hemoglobin and myoglobin content in chicken muscles. The total heme, hemoglobin, and myoglobin contents were related to muscle type. Extensive hemorrhage increased the hemoglobin content.

Animals↗

PubMed1999

Markers of capacity to utilize fatty acids in human skeletal muscle: relation to insulin resistance and obesity and effects of weight loss.

A number of biochemical defects have been identified in glucose metabolism within skeletal muscle in obesity, and positive effects of weight loss on insulin resistance are also well established. Less is known about the capacity of skeletal muscle for the metabolism of fatty acids in obesity-related insulin resistance and of the effects of weight loss, though it is evident that muscle contains increased triglyceride. The current study was therefore undertaken to profile markers of human skeletal muscle for fatty acid metabolism in relation to obesity, in relation to the phenotype of insulin-resistant glucose metabolism, and to examine the effects of weight loss. Fifty-five men and women, lean and obese, with normal glucose tolerance underwent percutaneous biopsy of vastus lateralis skeletal muscle for determination of HADH, CPT, heparin-releasable (Hr) and tissue-extractable (Ext) LPL, CS, COX, PFK, and GAPDH enzyme activities, and content of cytosolic and plasma membrane FABP. Insulin sensitivity was measured using the euglycemic clamp method. DEXA was used to measure FM and FFM. In skeletal muscle of obese individuals, CPT, CS, and COX activities were lower while, conversely, they had a higher or similar content of FABP(C) and FABP(PM) than in lean individuals. Hr and Ext LPL activities were similar in both groups. In multivariate and simple regression analyses, there were significant correlations between insulin resistance and several markers of FA metabolism, notably, CPT and FABP(PM). These data suggest that in obesity-related insulin resistance, the metabolic capacity of skeletal muscle appears to be organized toward fat esterification rather than oxidation and that dietary-induced weight loss does not correct this disposition.

Body Composition↗

PubMed1999

Effect of genetic variants of the heart fatty acid-binding protein gene on intramuscular fat and performance traits in pigs.

In order to find genetic markers to improve the meat quality of pigs by breeding we studied the relationship between variation in the heart fatty acid-binding protein (H-FABP) gene (FABP3) and intramuscular fat (IMF) content. To estimate the effect of H-FABP, pigs from two Duroc populations were selectively mated in such a way that at least two genotypes were present in each litter. In total, data from 983 pigs and pedigree information from three preceding generations were analyzed. Offspring were tested for IMF content as well as backfat thickness (BFT), BW, and drip loss of the meat (DRIP). All pigs were assigned to H-FABP RFLP genotype classes either by the assessed genotype (75%) or based on a probability score determined according to genotypic information of their relatives (25%). Contrasts were detected between homozygous H-FABP RFLP genotype classes for IMF content (.4%, P < .05), BFT (.6 mm, P < .01), and BW (2.4 kg, P < .10). No significant contrasts were detected for DRIP. Results for IMF content, BFT, and BW were confirmed when only genotyped animals were analyzed. Variation in BFT partially explained the effect on IMF content. Although other closely linked genes on porcine chromosome 6 might be responsible for the observed effect, interference of the halothane gene was excluded because all parental animals were noncarriers. In conclusion, H-FABP RFLP can be used as markers to select for increased IMF content and growth in breeding programs.

Adipose Tissue↗

PubMed1999

Structural and functional studies on different human FABP types.

Interaction of various ligands with recombinant proteins of 5 human FABP types was studied by radiochemical and fluorescence procedures. Liver, heart, intestinal and myelin FABP showed a higher affinity for oleic acid than adipocyte FABP. Intestinal and adipocyte FABP had a relatively high Kd value for arachidonic acid. Liver and intestinal FABP showed high affinity for DAUDA in contrast to the other FABP types. ANS was only well bound by liver and adipocyte FABP. Retinol was not bound by any FABP type, retinoic acid only by adipocyte FABP. Data indicate the importance of both electrostatic and hydrophobic interaction for the ligand-FABP binding. The immunological crossreactivity between six human FABP types including epidermal FABP and their respective antibodies raised in rabbit, chicken and mouse appeared to be low and may suggest heterogeneity of protein surface.

Amino Acid Sequence↗

PubMed1998

Members of the fatty acid-binding protein family inhibit cell-free protein synthesis.

Fatty acid-binding proteins (FABPs) are 15-kDa cytosolic proteins which are involved in the intracellular binding and targeting of fatty acids. Some members have been implicated in the regulation of cell growth and differentiation. In this study we investigated the effect of a series of FABPs and heart FABP (H-FABP) mutants on cell-free protein synthesis. Human myelin, intestinal, heart and brain FABP showed a dose-dependent inhibition of in vitro mRNA translation. Adipocyte, liver and epidermal types had no effect. The inhibition was not influenced by delipidation and for H-FABP mutants not related to their affinity for fatty acids. Our results indicate that some FABPs may modulate cell growth and/or differentiation by inhibition of protein synthesis.

Animals↗

PubMed1998

Generation and application of type-specific anti-heparan sulfate antibodies using phage display technology. Further evidence for heparan sulfate heterogeneity in the kidney.

Detailed analysis of various heparan sulfate (HS) species is seriously hampered by a lack of appropriate tools, such as antibodies. We adopted phage display technology to generate anti-HS antibodies. A "single pot" semisynthetic human antibody phage display library was subjected to four rounds of selection on HS from bovine kidney using panning methodology. Three different phage clones expressing anti-HS single chain variable fragment antibodies (HS4C3, HS4D10, and HS3G8) were isolated, with an amino acid sequence of the complementarity-determining region 3 of GRRLKD (VH3 gene, DP-38), SLRMNGCGAHQ (VH3 gene, DP-42), and YYHYKVN (VH1 gene, DP-8), respectively. The antibodies react with HS and heparin, but not with DNA or other glycosaminoglycans. Kd values for HS are about 0.1 microM. The three antibodies react differently toward various HS preparations and show different staining patterns on rat kidney sections, indicating recognition of different HS molecules. This also holds for two described mouse anti-HS IgMs (JM403 and 10E4; both generated by conventional hybridoma technique) and indicates the presence of at least 5 different HS species in the kidney. O- and N-sulfation are important for binding of HS to HS4C3 and HS3G8. The three single chain antibodies, but not JM403, block a basic fibroblast growth factor binding site of HS. It is concluded that phage display technology presents a powerful technique to generate antibodies specific for HS epitopes. This is the first time this technique has been successfully applied to obtain directly antibodies to (poly)saccharides.

Amino Acid Sequence↗

PubMed1998

Primary structure and high expression of human agrin in basement membranes of adult lung and kidney.

Agrin is a heparan sulfate proteoglycan involved in the development of the neuromuscular junction during embryogenesis. In addition to this well-characterized function, agrin may have additional functions in other tissues and during other stages in development. In this study we present the cDNA sequence of human agrin, and demonstrate a high agrin content in adult basement membranes. The N-terminal domain of human agrin is highly similar to that of chick agrin, suggesting a similar function in laminin binding. The presence of three SGXG sequences supports serine-linked glycosylation of the core protein, two sites being particularly favorable for heparan sulfate attachment. Comparison of levels of agrin mRNA in fetal and adult human tissues showed a remarkable upregulation in adult kidney and lung. In both tissues truncated agrin transcripts were detected, lacking the region that encodes the laminin-binding domain. The high transcription levels in lung and kidney corresponded with the accumulation of agrin in the alveolar and glomerular basement membranes, suggesting a filtration-associated function. These data provide new directions for investigating the role of agrin in its different physiological environments, including the basement membranes of the neuromuscular junction, kidney and lung.

Age Factors↗

PubMed1998

Transfection of L6 myoblasts with adipocyte fatty acid-binding protein cDNA does not affect fatty acid uptake but disturbs lipid metabolism and fusion.

We studied the involvement of fatty acid-binding protein (FABP) in growth, differentiation and fatty acid metabolism of muscle cells by lipofection of rat L6 myoblasts with rat heart (H) FABP cDNA or with rat adipocyte (A) FABP cDNA in a eukaryotic expression vector which contained a puromycin acetyltransferase cassette. Stable transfectants showed integration into the genome for all constructs and type-specific overexpression at the mRNA and protein level for the clones with H-FABP and A-FABP cDNA constructs. The rate of proliferation of myoblasts transfected with rat A-FABP cDNA was 2-fold higher compared with all other transfected cells. In addition, these myoblasts showed disturbed fusion and differentiation, as assessed by morphological examination and creatine kinase activity. Uptake rates of palmitate were equal for all clone types, in spite of different FABP content and composition. Palmitate oxidation over a 3 h period was similar in all clones from growth medium. After being cultured in differentiation medium, mock- and H-FABP-cDNA-transfected cells showed a lower fatty acid-oxidation rate, in contrast with A-FABP-cDNA-transfected clones. The ratio of [14C]palmitic acid incorporation into phosphatidylcholine and phosphatidylethanolamine of A-FABP-cDNA-transfected clones changed in the opposite direction in differentiation medium from that of mock- and H-FABP-cDNA-transfected clones. In conclusion, transfection of L6 myoblasts with A-FABP cDNA does not affect H-FABP content and fatty acid uptake, but changes fatty acid metabolism. The latter changes may be related to the observed fusion defect.

Adipocytes↗

PubMed1998

The adipocyte fatty acid-binding protein locus: characterization and association with intramuscular fat content in pigs.

The porcine A-FABP gene (FABP4) was isolated and sequenced to study the role of A-FABP in the differentiation of intramuscular fat (IMF) accretion in pigs. The coding sequence of the porcine A-FABP gene is highly conserved across human, mouse, and rat. Moreover, all the functionally important amino acids are conserved. This high similarity extends into the first 270 bp of the 5' upstream region. Within this region, a 56-bp nucleotide sequence was completely identical with the corresponding sequence in the mouse A-FABP gene, which contains the transcription factor binding sites for C/EBP and AP-1, and is implicated in the differentiation-dependent regulation of A-FABP. The A-FABP gene was assigned to porcine Chromosome (Chr) 4 by a porcine sequence-specific PCR on a cell hybrid panel, fully consistent with comparative mapping data with human and mouse. In the first intron of the porcine A-FABP gene, a microsatellite sequence was detected that was polymorphic for all six pig breeds tested. This genetic variation within the A-FABP gene was associated with differences in IMF content and possibly growth in a Duroc population, whereas no effect on backfat thickness and drip loss of the meat were detected. A considerable and significant contrast of approximately 1% IMF was observed between certain genotype classes. We conclude that the A-FABP locus is involved in the regulation of intramuscular fat accretion in Duroc pigs.

Adipose Tissue↗

PubMed1998

Intravenous immunoglobulin preparation increases myoplasmic calcium concentration by activating the dihydropyridine-ryanodine receptor complex.

A human intravenous immunoglobulin preparation (IVIg) released Ca2+ from the sarcoplasmic reticulum of cultured human skeletal muscle cells in a dose-dependent manner. Blocking the dihydropyridine-ryanodine receptor complex abrogated the IVIg-mediated Ca2+ response, whereas inhibition of the voltage-operated Na+-channels or acetylcholine receptors did not. This effect of IVIg was not mediated by its main component, the IgG molecules, and differed between preparations from different manufacturers. Heating destroyed the activity. Data shows that an unidentified serum protein present in IVIg can influence human muscle cells by an effect on the dihydropyridine receptor. This phenomenon may be important in interpreting the (side) effects of IVIg in neuromuscular diseases.

Calcium↗

PubMed1998

The use of nonhomologous scatchard analysis in the evaluation of ligand-protein interactions.

Scatchard plots are widely used for the graphical presentation of receptor-ligand binding data. When a combination of labelled and unlabelled ligand molecules is used in a binding assay, equations for Scatchard plots are readily available if the labelled and unlabelled ligands have similar binding affinities. In this article, Everardus van Zoelen, Roel Kramer, Herman van Moerkerk and Jacques Veerkamp present mathematical equations to obtain the binding characteristics of an unlabelled ligand in a Scatchard plot, which has a dissociation equilibrium constant different from that of the labelled ligand used.

Drug Interactions↗

PubMed1998

Contribution of mitochondria and peroxisomes to palmitate oxidation in rat and bovine tissues.

Total and peroxisomal palmitate oxidation capacities and mitochondrial enzyme activities were compared in tissues from growing rats, preruminant calves and 15-month-old bulls. Total palmitate oxidation rates were 1.9-5.2-fold higher in rat than in bovine tissues and 1.7-fold higher in the heart and muscles from calves than from growing bulls. The peroxisomal contribution to palmitate oxidation was similar between rats and bovines (i.e. calves and bulls) in liver (35-51%), heart (26%) but not in muscles (14 +/- 3% in rats vs 33 +/- 4.5% in bovines, P < 0.05). Mitochondrial enzyme activities were 1.8-4.8-fold higher in rat than in bovine tissues but the citrate synthase to cytochrome-c oxidase ratio was the highest in the liver (17-38), intermediate in the heart and muscles from calves and rats (6-10) and the lowest in heart and muscles from bulls (2-3, P < 0.05). In all tissues and animal groups, palmitate oxidation rates were similar per unit cytochrome-c oxidase activity, but not always per unit citrate synthase activity. Therefore, differences in mitochondrial contents (as between rats and bovines) or in mitochondrial characteristics (as between liver and muscles) relate to the differences in palmitate oxidation capacity.

Animals↗