Biomedical subjects

T H van Kuppevelt

Publications and source records attributed to T H van Kuppevelt.

At least 37 records · Page 2Linked to original sources

PubMed2000

Restoration by vacuum inflation of original alveolar dimensions in small human lung specimens.

Resection of lung specimens results in deflated and distorted lung structures. If no major airway is present (as in the case of small lung specimens from biopsies), lung dimensions cannot be restored by inflation under 25 cmH20. This impedes morphological analysis of the tissue. This report describes a simple and easy procedure to restore alveolar dimensions in deflated small lung specimens. Small human lung samples were inflated using moderate vacuum conditions which are provided by a common water stream-driven vacuum device. Restoration of alveolar dimensions and morphology was evaluated for paraffin-embedded as well as frozen tissue, using morphometric and immunohistological analysis. Vacuum inflation results in restoration of original lung dimensions as judged by light and scanning electron microscopy, and by analysis of the mean linear intercept, and the average length, width, perimeter and surface area. It also results in markedly improved cutting characteristics, allowing reliable sectioning of 2 microm cryosections and achieving high resolution images in immunofluorescence. Vacuum inflation is a simple and easy procedure to restore lung architecture of small human lung specimens/biopsies with a concomitant improvement of cutting characteristics. It allows for correct histological analysis of small specimens which cannot be inflated otherwise.

Adult↗

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

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

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

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

Susceptibility of broiler chickens to hemorrhages in muscles: the effect of stock and rearing temperature regimen.

In this study, the effect of genetic constitution (stock) and rearing temperature on the occurrence of hemorrhages in thighs and breasts of water bath stunned broilers was investigated. Particular attention was given to the relation between the susceptibility for hemorrhages, body composition, and adaptations in blood variables induced by low rearing temperatures. A factorial experiment was performed with five parental stocks, differing with respect to growth rate, feed conversion, and body composition, and two temperature regimens (thermoneutral and below the zone of thermoneutrality). Hemorrhage scores in thighs were dependent on rearing temperature, not on stock, and were highest in broilers reared at low temperatures. In all stocks, high scores in the thighs were accompanied by an increased carbon dioxide pressure and bicarbonate and triiodothyronine concentration of the venous blood, and by an increased relative heart weight, hematocrit, and blood loss at slaughter. Hemorrhage scores in breasts were dependent on stock, confounded with day of scoring. An effect of rearing temperature was present only for the leanest, slow-growing stock having the lowest feed conversion ratio at thermoneutral rearing conditions. Scores in breasts were related neither to body composition nor to metabolic and hemodynamic adaptations to low rearing temperatures. It is concluded that high hemorrhage scores in thighs are related to hemodynamic and metabolic adaptations to an increased need for energy and oxygen caused by low rearing temperatures. Hemorrhage scores are not related to stock-dependent differences.

Adaptation, Physiological↗

PubMed1997

Induction of emphysematous lesions in rat lung by beta-D-xyloside, an inhibitor of proteoglycan synthesis.

The possible involvement of proteoglycans in the pathogenesis of emphysema was studied in rats by a single intratracheal instillation of p-nitrophenyl-beta-D-xylopyranoside (beta-D-xyloside), an inhibitor of proteoglycan synthesis. The first 3 days after instillation are characterized by mild hemorrhages, some infiltration of inflammatory cells, and edema. After 1 wk, lung morphology is normal again. Forty days after instillation, considerable parenchymal destruction has occurred as determined by the mean linear intercept (81 +/- 12 microns versus 57 +/- 5 microns for control [P < 0.001]). Pulmonary fibrosis is not observed. Instillation with p-nitrophenyl-alpha-D-xylopyranoside and p-nitrophenol do not induce parenchymal destruction, indicating the specificity of beta-D-xyloside action. Urinary glycosaminoglycan (GAG) content of the beta-D-xyloside-treated rats is increased 15-fold during the first day after instillation, mainly due to elevated levels of chondroitin sulfate and dermatan sulfate. The increase is correlated to the extent of parenchymal destruction after 40 days (r = 0.68; P < 0.002). At day 2 and thereafter, levels are normal again. A short-term increase in dermatan and chondroitin sulfate content is also observed in serum, bronchoalveolar lavage (BAL) fluid, and lung tissue. Heparan sulfate content is decreased in BAL fluid and lung tissue. Instillation with p-nitrophenyl-alpha-D-xylopyranoside and p-nitrophenol do not induce elevated GAG concentration in urine. We suggest that a disturbance in proteoglycan synthesis accompanied by an increase of (beta-D-xyloside-primed) free GAGs results in loss of stability and integrity of the alveolar wall, leading to parenchymal destruction and emphysematous lesions. beta-D-xyloside treatment may be an alternative experimental method for inducing emphysema.

Animals↗

PubMed1996

Altered composition of urinary heparan sulfate in patients with COPD.

In patients with emphysema the integrity of the extracellular matrix (connective tissue skeleton) is compromised. In this study we analyzed glycosaminoglycans, which are main constituents of this matrix, in urines from patients with chronic obstructive pulmonary disease (COPD)/emphysema. Glycosaminoglycans (GAGs) were purified by anion exchange chromatography and quantified using the 1,9-dimethylmethylene blue assay. Heparan sulfate (HS) was assayed using three different chemical methods: digestion with heparitinase or with nitrous acid and by use of an adapted 1,9-dimethylmethylene blue assay. A specific epitope on the HS molecule, defined by the monoclonal antibody JM403, was determined using an inhibition enzyme immunoassay. In patients with COPD total urinary glycosaminoglycan and HS content were not altered. The JM403 epitope of HS, however, was greatly decreased in patients (0.6 versus 4.1 units/mg creatinine for control subjects, p < 0.0001). A similar pattern was observed when patients with bronchial carcinoma with and without emphysema were compared (0.4 versus 2.4 units/mg creatinine respectively, p < 0.0005). Patients with sarcoidosis did not show a decreased epitope content. These results indicate a structural change or an altered processing of the HS molecule in patients with emphysema. Taking into consideration the importance of HS for the stability of the alveolar extracellular matrix, this change may be associated with the pathogenesis of emphysema.

Adult↗

PubMed1995

Immunoquantification of type I, III, IV and V collagen in small samples of human lung parenchyma.

The involvement of collagen in pathological conditions underscores the need for sensitive, collagen type-specific assays. A method for the quantification of different types of collagen in lung has been developed. Human lung parenchyma is digested with cyanogen bromide which results in almost complete solubilization of collagen to type-specific collagen peptides. The peptides are quantified using inhibition enzyme immunoassays with type-specific antibodies. The 50% inhibition value for the type I collagen assay is 1 microgram type I collagen peptides, for the type III collagen assay 350 ng type III collagen peptides, for the type IV collagen 200 ng type IV collagen peptides, and for the type V collagen assay 50 ng type V collagen peptides. Using less than 1 mg lyophilized human parenchymal lung tissue it was established that the amount of collagen/mg dry tissue (+/- SD, n = 10) is 84.6 +/- 16.1 micrograms for type I collagen, 26.6 +/- 10.3 micrograms for type III collagen, 9.6 +/- 2.0 micrograms for type IV collagen and 1.8 +/- 0.3 micrograms for type V collagen. The procedure is useful for the quantification of different types of collagen, including minor collagens, and requires only minimal sample preparation.

Aged↗

PubMed1995

Digestion of proteoglycans in porcine pancreatic elastase-induced emphysema in rats.

Pulmonary emphysema was induced in rats by a single intratracheal instillation of pancreatic elastase. The short-term effects of elastase instillation on basement membrane components were evaluated using immunohistochemical and biochemical methods. Lung alveoli showed a decrease in heparan sulphate proteoglycan content (especially of its heparan sulphate chains) 3 h to 7 days after induction. Type IV collagen, laminin and fibronectin were not affected. The glycosaminoglycan content of the lung was decreased during the first 3 days after induction, while the glycosaminoglycan concentration in urine was increased during the first 4 days by an increase of heparan sulphate and dermatan sulphate. The increase in urinary glycosaminoglycan content was positively correlated with the extent of emphysema developed after 40 days. We conclude that proteoglycans are target molecules for elastase, and may be involved in the pathogenesis of emphysema.

Animals↗

PubMed1995

ImageCalc: a Microsoft Windows application for quantitative image analysis and comparison.

In this report we describe a Microsoft Windows-based computer program (ImageCalc) for the analysis of gels and autoradiograms and for computation of stand-alone images and images that are related to each other (i.e., images with the same image parameters). The program is able to subtract, add, multiply and divide constant values or full images from another image. It measures the intensity of (part of) an image in two ways: (i) by calculation of the total intensity and the average intensity/pixel and (ii) by line scanning. The program allows importation of all images that are stored in an 8-bit uncompressed format and saves them in a Microsoft Windows bitmap format. Densitometric analysis of gels and autoradiograms, digitized using an ordinary optical scanner, is illustrated for glycosaminoglycans separated by electrophoresis on agarose gels and stained with Azure A silver, and for [32P]phosphatidic acid, separated by thin-layer chromatography. Another application of the program is the analysis of multiple related images that are resolved in time or for which different emission or excitation wavelengths are used (fluorescence microscopy). As an example, the change of cytosolic [Ca2+] is demonstrated in cultured human skeletal muscle cells after stimulation with acetylcholine.

Autoradiography↗

PubMed1994

A spectrophotometric method for the determination of heparan sulfate.

A simple and reliable spectrophotometric method for the determination of heparan sulfate is described. The method is based on the 1,9-dimethylmethylene blue assay for sulfated glycosaminoglycans. Addition of bovine serum albumin, together with a specific NaCl concentration and pH, results in a specific decrease of heparan sulfate-based absorbance. The amount of heparan sulfate can be calculated by subtracting the values obtained in the presence of albumin from those obtained in its absence. The sensitivity is 0.5 microgram heparan sulfate. Two applications are given: the quantification of heparan sulfate in urine, including urine from patients with mucopolysaccharidosis, and the evaluation of fractions from gel filtration and ion exchange column chromatography for isolation of heparan sulfate proteoglycans.

Glycosaminoglycans↗

PubMed1994

Application of cationic probes for the ultrastructural localization of proteoglycans in basement membranes.

The application of cationic probes for the ultrastructural detection of proteoglycans in basement membranes is reviewed. Proteoglycans are highly negatively charged macromolecules due to their glycosaminoglycan side chains. The interaction of cationic probes with proteoglycans is of an electrostatic nature. Methods are discussed to increase the specificity of probes for proteoglycans. The use of phthalocyanin-like dyes such as Cuprolinic blue, according to the critical electrolyte concentration method, results in a selective staining of proteoglycans. Enzymatic or chemical digestions, however, should be done to validate the proteoglycan nature of the dye-positive granules/filaments, and to establish the class of proteoglycan. The value of cationic probes in basement membrane research on development and pathology is discussed. The potential for deducting molecular information from the ultrastructural appearance of stained proteoglycans is indicated.

Animals↗

PubMed1994

Quantification and characterization of glycosaminoglycans at the nanogram level by a combined azure A-silver staining in agarose gels.

A rapid, sensitive, and nonradioactive method has been developed for the quantification and characterization of glycosaminoglycans. The method is based on the separation of different types of glycosaminoglycans in agarose gel and subsequent fixation and staining with the cationic dye azure A, followed by silver enhancement. Densitometric analysis of the silver deposition gives a linear response between 1 and 20 ng for chondroitin and dermatan sulfate and between 2 and 40 ng for heparan sulfate. The detection limit is about 250 pg. The staining procedure was applied for the quantification and characterization of glycosaminoglycans in human serum, urine, and lung and in mouse kidney glomeruli. It requires only 10 microliters serum, 2 microliters urine, and only a single cryosection in case of tissue. The method is at least as sensitive as staining with radioactive markers and about 200 times more sensitive than conventional glycosaminoglycan-staining methods.

Animals↗

PubMed1994

Immunolocalization of AMP-deaminase isozymes in human skeletal muscle and cultured muscle cells: concentration of isoform M at the neuromuscular junction.

The three major isoforms of AMP-deaminase (AMPda) were localized in human skeletal muscle and cultured muscle cells by immunocytochemistry. The M isoform was mainly located in Type II muscle fibers and showed a clear cross-striation. Particularly strong staining was present at the neuromuscular junction. Capillaries were also immunoreactive. The L isoform was predominantly observed in nerve bundles and to a minor extent in smooth muscle cells and endothelial cells. The E isoform was predominantly present in smooth muscle cells, and to a lesser extent in Type I muscle fibers and nerve bundles. In quadriceps muscle of patients with myoadenylate deaminase deficiency, no immunostaining for the M isozyme was observed, whereas reactivity for the L and E isoforms was unaltered. In human muscle cell cultures, mononuclear cells, including myoblasts, were immunoreactive for the L isoform and to a lesser extent the E isoform, whereas the M isoform was absent. In myotubes, diffuse or fibrillar staining was present for all three isoforms, but only the M isoform showed a clear cross-striation pattern in highly differentiated myotubes.

AMP Deaminase↗

PubMed1993

Deficiency of Na+/K(+)-ATPase and sarcoplasmic reticulum Ca(2+)-ATPase in skeletal muscle and cultured muscle cells of myotonic dystrophy patients.

Since defective regulation of ion transport could initiate or contribute to the abnormal cellular function in myotonic dystrophy (MyD), Na+/K(+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+)-ATPase were examined in skeletal muscle and cultured skeletal muscle cells of controls and MyD patients. Na+/K(+)-ATPase was investigated by measuring ouabain binding and the activities of Na+/K(+)-ATPase and K(+)-dependent 3-O-methylfluorescein phosphate (3-O-MFPase). SR Ca(2+)-ATPase was analysed by e.l.i.s.a., Ca(2+)-dependent phosphorylation and its activities with ATP and 3-O-methylfluorescein phosphatase (3-O-MFP). In MyD muscle the K(+)-dependent 3-O-MFPase activity and the activity and concentration of SR Ca(2+)-ATPase were decreased by 40%. In cultured muscle cells from MyD patients the activities as well as the concentration of both Na+/K(+)-ATPase and SR Ca(2+)-ATPase were reduced by about 30-40%. The ouabain-binding constant and the molecular activities, i.e. catalytic-centre activities with ATP or 3-O-MFP, of Na+/K(+)-ATPase and SR Ca(2+)-ATPase were similar in muscle as well as in cultured cells from both controls and MyD patients. Thus the decreased activity of both ATPases in MyD muscle is caused by a reduction in the number of their molecules. To check whether the deficiency of ATP-dependent ion pumps is a general feature of the pathology of MyD, we examined erythrocytes from the same patients. In these cells the Ca2+ uptake rate and the Ca(2+)-ATPase activity were lower than in controls, but the Ca(2+)-ATPase concentration was normal. Thus the reduced Ca(2+)-ATPase activity is caused by a decrease in the molecular activity of the ion pump. The Na+/K(+)-ATPase activity is also lower in erythrocytes of MyD patients. It is concluded that the observed alterations in ion pumps may contribute to the pathological phenomena in the muscle and other tissues in patients with MyD.

Adult↗