H Galjaard's research while affiliated with Erasmus University Rotterdam and other places
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Publications (144)
Human fibroblasts or amniotic fluid cells can be cultivated in special dishes with a bottom of thin transparent plastic foil. After quick freezing and freeze-drying small groups of cultured cells or single cells can be isolated by dissecting small pieces of plastic foil under the stereomicroscope. These can be incubated in (sub)microlitre volumes o...
In a pregnancy at risk for Fabry's disease, a prenatal diagnosis could be established 11 days after amniocentesis in the 15th week of pregnancy. This was possible by microchemical analysis of the galactosidase activity in isolated groups of 100–200 freeze-dried, cultured amniotic fluid cells. The results of this microassay were confirmed by (micro)...
Since the completion of the human genome map, genomics, proteomics and pharmacogenomics have become popular headings. In this review some 40 years of development in research and laboratory diagnosis of inborn errors of metabolism are summarized. It is shown that collaborative approaches of clinicians, geneticists, pathologists, biochemists and mole...
Since the completion of the human genome map, genomics, proteomics and pharmacogenomics have become popular headings. In this review some 40 years of development in research and laboratory diagnosis of inborn errors of metabolism are summarized. It is shown that collaborative approaches of clinicians, geneticists, pathologists, biochemists and mole...
Fragile X syndrome is caused by the absence of the fragile X mental-retardation protein (FMRP). FMRP and the fragile X-related proteins 1 and 2 (FXR1P and FXR2P) form a gene family with functional similarities, such as RNA binding, polyribosomal association and nucleocytoplasmic shuttling. In a previous study, we found that FMRP and FXR1P shuttle b...
Fragile X syndrome is caused by the absence of the fragile X mental retardation protein (FMRP). FMRP and its structural homologues
FXR1P and FXR2P form a family of RNA-binding proteins (FXR proteins). The three proteins associate with polyribosomes as cyto-plasmic
mRNP particles. Here we show that small amounts of FMRP, FXR1P and FXR2P shuttle betw...
Enzyme assays for the diagnosis of lysosomal storage diseases have been underway in China only in a few laboratories, and the specimens for enzyme assay must not be inactivated by environmental factors. To solve the problem, the stability of freeze-drying lysosomal enzymes was tested.
Three pools for samples were set up: control leucocytes pool and...
Lack of expression of the fragile X mental retardation protein (FMRP) results in mental retardation and macroorchidism, seen as the major pathological symptoms in fragile X patients. FMRP is a cytoplasmic RNA-binding protein which cosediments with the 60S ribosomal subunit. Recently, two proteins homologous to FMRP were discovered: FXR1 and FXR2. T...
Fragile X syndrome is caused by mutations in the FMR1 gene and is one of the most frequent forms of inherited mental retardation in males. Postnatal and prenatal diagnosis of fragile X syndrome is feasible by direct DNA analysis. A new approach to prenatal diagnosis of fragile X syndrome in amniotic fluid cells is described, using a rapid and simpl...
About 10% of the human genome has now been mapped and for more than 1000 diseases closely linked DNA polymorphisms or the responsible genes have been identified. These developments have extended the scope of (prenatal) diagnosis and carrier detection which already was possible by biochemical analysis of protein defects in some 400 Mendelian disorde...
Deficiency of lysosomal protective protein/cathepsin A in humans is the primary cause of galactosialidosis, a lysosomal storage disease characterized by combined deficiency of beta-galactosidase and neuraminidase. We have investigated 20 galactosialidosis patients and nine of their obligate heterozygous parents. A group of 12 patients with the earl...
The FMR1 transcript is alternatively spliced and generates different splice variants coding for FMR1 proteins (FMRP) with a predicted molecular mass of 70-80 kDa. FMRP is widely expressed and localized in the cytoplasm. To study a possible interaction with other cellular components, FMRP was isolated and characterized under non-denaturing condition...
In most wealthy industrialized countries, socioeconomic circumstances, hygiene and medical care have improved to such an extent that perinatal complications and congenital disorders have become the major causes of infant mortality and of chronic handicap in children [1].
In the past four decades, we have made impressive advances understandig the cy...
Medical genetics made during the last decades immense advances and new findings from the sphere of fundamental research are increasingly penetrating into different clinical disciplines. The greatest attention was concentrated on investigations devoted to the molecular etiology of malignant diseases and the molecular mechanism of embryonic developme...
FMR1 protein expression was studied in different tissues. In human, monkey and murine tissues, high molecular mass FMR1 proteins (67-80 kDa) are found, as shown in lymphoblastoid cells lines. The identity of these proteins was confirmed by their absence in tissues from patients with the fragile X syndrome and a FMR1 knock-out mouse. An Ile367Asn su...
Fragile X syndrome is the most common known cause of inherited mental retardation. Identification of patients and carriers of fragile X syndrome is usually done with a DNA test system but we have developed a rapid antibody to identify fragile X patients. This non-invasive test requires only 1 or 2 drops of blood and can be used for screening large...
Recently a striking elevation of the activity of chitotriosidase, an endo beta-glucosaminidase distinct from lysozyme, was found in plasma from patients with Gaucher type I disease (McKusick 230800). Plasma chitotriosidase originates from activated macrophages and this elevation is secondary to the basic defect in Gaucher disease. To investigate th...
Hereditary multiple exostoses (EXT) is an autosomal dominant disorder of enchondral bone formation characterized by multiple bony outgrowths (exostoses), with progression to osteosarcoma in a minority of cases. The exclusive involvement of skeletal abnormalities distinguishes EXT from the clinically more complex Langer-Giedion syndrome (LGS), which...
The lysosomal carrier for the acidic monosaccharides sialic acid and glucuronic acid was solubilized from rat liver lysosomal membranes and reconstituted into phospholipid vesicles. Membrane proteins were extracted from lysosomal membranes with Triton X-100. Upon removal of the detergent by absorption on Amberlite XAD-2 beads, the solubilized prote...
The lysosomal disorder galactosialidosis is caused by deficiency of the protective protein in the absence of which the activities of the enzymes beta-galactosidase and neuraminidase are reduced. Aside from its protective function towards the two glycosidases, this protein has cathepsin A-like activity. A point mutation in the protective protein gen...
The recent identification of the cystic fibrosis (CF) gene and its putative protein product, the CF transmembrane conductance regulator (CFTR), enabled us to synthesize oligopeptides corresponding with a predicted extracellular domain (position 103-117; peptide A) and a cytoplasmic domain (position 501-515; peptide B) constituting the phenylalanine...
The Cystic Fibrosis antigen (CFA) is a 14 kDa. Ca2(+)-binding protein known to be expressed in cells of myeloid origin during normal cell differentiation. CFA serum levels are elevated in Cystic Fibrosis (CF) patients and heterozygotes. We examined the expression of CFA in different cultured epithelial cells from controls and patients with CF. The...
The localization of several GTP-binding regulatory proteins in teh apical membrane of intestinal epithelial cells has prompted us to investigate a possible role for G-proteins as modulators of apical Cl- channels. In membrane vesicles isolated from rat small intestine or human HT29-cl.19A colon carcinoma cells, the entrapment of guanosine 5'-O-(3-t...
At present some 4500 disorders in man are known or assumed to be of Mendelian inheritance. Most of the genetic disorders have initially been defined by clinicians on the basis of clinical and pathological manifestations [1–3]. The development of chromatographic methods in the 1950s stimulated the use of chemical analyses in the diagnosis of genetic...
In the Netherlands the clinical application of laboratory methods in cytogenetics and biochemical analysis started in departments involved in basic research and in hospital departments with a special interest in early diagnosis of congenital and genetic disorders. During the 1960s and early 1970s the various activities that later were concentrated...
4-Methylumbelliferyl-beta-D-galactopyranoside-6-sulphate was synthesized and used for the determination of galactose-6-sulphate sulphatase activity. Fibroblasts and leucocytes from 12 different Morquio A patients, showed 0.0-2.7% of mean normal galactose-6-sulphate sulphatase activity. Heterozygotes showed intermediate activities. The enzymatic lib...
Highly purified lysosomal membrane vesicles, obtained from rat liver lysosomes, were used to study characteristics of NeuAc transport across the lysosomal membrane. Uptake of [14C]NeuAc was found to be strongly influenced by a pH gradient across the membrane. When a proton gradient (pHin greater than pHout) was generated by impermeable buffers, Neu...
Highly purified lysosomal membrane vesicles, obtained from rat liver lysosomes, were used to study characteristics of NeuAc transport across the lysosomal membrane. Uptake of [¹⁴C]NeuAc was found to be strongly influenced by a pH gradient across the membrane. When a proton gradient (pHin > pHout) was generated by impermeable buffers, NeuAc uptake a...
Lysosomal neuraminidase from human placenta has been obtained in its active form by association of an inactive neuraminidase polypeptide with beta-galactosidase and the protective protein. Using a specific antiserum, we have now identified a 66-kDa protein as the inactive neuraminidase polypeptide. It is specifically recognized on immunoblots only...
From the placenta of a human fetus with galactosialidosis, detected by prenatal diagnosis, sialyloligosaccharides were isolated by successively gel-permeation chromatography on Bio-Gel P-6, anion-exchange chromatography on Mono Q and high-performance liquid chromatography on Lichrosorb-NH2. 16 sialic acid-containing N-glycosidic N-acetyllactosamine...
We describe the results and follow-up of chorionic villus studies in 1,034 pregnancies at risk for chromosome or metabolic disorders. Direct chromosome studies were successful in 99.7% and yielded results within a few days. Fifty pregnancies at risk for an unbalanced translocation, inherited from parents with many small reciprocal translocations, w...
A new lysosomal storage disease with autosomal recessive inheritance is described in two male siblings of 5 1/2 and 4 years of age. Clinical manifestations started after 9 months of age with neurological symptoms, followed by progressive psychomotor deterioration. Urinary oligosaccharide excretion was abnormal and showed a characteristic pattern on...
Nearly 4000 different human disorders are supposed or known to be due to a single gene mutation. In about 10%, an early diagnosis is possible by the demonstration of specific abnormalities of metabolites in blood and/or urine, an abnormal structure/function of a particular (enzyme) protein or by the (in)direct demonstration of the gene mutation usi...
The molecular basis of clinical diversity in glycogenosis type II (Pompe's disease) was investigated by comparing the nature of acid alpha-glucosidase deficiency in cultured fibroblasts from 30 patients. Biosynthetic forms of acid alpha-glucosidase with different molecular mass were separated electrophoretically and identified by immunoblotting. Im...
This study represents the first example of immunological localization of lysosomal acid phosphatase. The intracellular localization of lysosomal acid phosphatase was investigated with immunocytochemical methods at the light and electron microscopical level in cultured fibroblasts obtained from normal subjects and from a patient with I-cell disease....
Human lysosomal beta-galactosidase and neuraminidase exist in a complex together with a 32-kilodalton (kd) glycoprotein. The latter protein was found to have a dual function: it is required for the aggregation of monomeric 64-kd beta-galactosidase into high molecular weight (600-700 kd) multimers and it is an essential subunit of neuraminidase toge...
To study the biochemical defect underlying N-acetylneuraminic acid (NANA) storage disorders (NSD), a tritium-labeled NANA-methylester was prepared and its metabolism was studied in normal and mutant human fibroblasts. The uptake of methylester, its conversion into free NANA, and the release of free NANA was studied in lysosome-enriched fractions. I...
Cultured fibroblasts from different variants of GM1-gangliosidosis synthesize normal amounts of 88-kDa beta-galactosidase precursor. Yet the amount of the mature 64-kDa form is reduced to 5-15% of normal values. In this communication it is shown that the mutation in the infantile and adult form of GM1-gangliosidosis interferes with the phosphorylat...
The uptake and catabolism of [3H-ceramide]-GM1 was followed in living fibroblasts from patient with different forms of beta-galactosidase deficiency. Gangliosides are identified according to the nomenclature of Svennerholm (1963). A total inability to metabolize the ingested substrate was found in infantile GM1-gangliosidosis whereas cells from an...
Immunoelectron microscopy was performed to study the biosynthesis of lysosomal beta-galactosidase (beta-gal) in normal and mutant human fibroblasts. Using polyclonal and monoclonal antibodies we show in normal cells precursor forms of beta-gal in the rough endoplasmic reticulum (RER) and in the Golgi apparatus throughout the stack of cisternae. In...
The lysosomal storage disorder galactosialidosis has been recognized as a distinct genetic and biochemical entity, associated with a combined beta-galactosidase and neuraminidase deficiency that is due to the lack of a 32-kilodalton (kDa) glycoprotein. The molecular basis of different clinical variants of galactosialidosis has been investigated. In...
The role of cytochemistry in human genetics is reviewed. In basic research, autoradiography and cytochemical staining procedures for DNA, RNA, proteins and other cell constituants have contributed to the understanding of the way DNA is localized, duplicated and translated. The development of new “banding techniques” for the identification of human...
The results of 350 first trimester fetal diagnoses are reported. Before the time of chorion sampling in the 10th week of pregnancy 15% of the appointments had to be cancelled because of a spontaneous abortion. After successful chorion aspiration the results of cytogenetic and (micor)biochemical analysis were available in all instances within 1-2 da...
A cytochemical fluorescence method is described that makes possible simple, rapid, and specific demonstration and measurement of the activities of a wide variety of lysosomal enzymes in single cells using 4-methylumbelliferyl derivatives as substrates. The validity of the method and a number of applications using normal and mutant human cells are p...
A series of man-Chinese hamster and man-mouse somatic cell hybrids was investigated to study the localization of the genes coding for the human lysosomal enzyme β-galactosidase (EC 3.2.1.23) and for its protective protein. Using a monoclonal antibody, raised against human placental β-galactosidase, it was observed that the structural locus for the...
Cell hybridizations between fibroblasts of four variants (B, O, AB, and B1) of infantile GM2 gangliosidosis were performed. Cocultivated as well as hybrid cells were analyzed for their capability to degrade exogenously added [3H]-GM2. Hybridization of variant AB fibroblasts with fibroblasts of variant O, variant B, or variant B1 resulted in an enha...
The intestinal microvillar enzyme complex sucrase-isomaltase has been studied in cystic fibrosis and control ileum. A number of biochemical parameters of the enzyme in ileum homogenates have been determined. Both solubilized as well as membrane-bound sucrase-isomaltase were analyzed with respect to their reaction with monoclonal antibodies against...
First trimester fetal chromosome analysis has been successfully carried out for 180 patients in our center. Cytogenetic studies of chorionic villi have been greatly facilitated by the observation of Brambati and Simoni (1983) that direct preparations after sampling are possible. This method is a great improvement for parents at high risk of produci...
Early diagnosis of congenital disease in patients, genetic counselling, and prenatal monitoring for fetal abnormalities has generated increasing interest during the last decade. At present, nearly 3500 disorders of Mendelian inheritance are known, and in about 10% of them, the responsible (enzyme) protein defect has been elucidated (McKusick 1983;...
We report the diagnosis of Krabbe's disease in a fetus in the ninth week of pregnancy of a mother who had previously lost a one-year-old son with this autosomal recessive disorder. A sample of 15 mg of chorionic villi was obtained by ultrasound-directed aspiration. Under microscopical control, the villi were washed in saline and homogenized in 75 μ...
The effects of the sequential application of specific glycosidases on surfaces of living mammalian cells were studied with respect to their ability to bind the beta-galactoside-specific lectin, Ricinus communis agglutinin (RCA). Sialidase and beta-galactosidases from different sources were tested for their actions on two strains of mouse lymphoma c...
The acid hydrolases α-glucosidase, β-galactosidase,N-acetyl-β-d-hexosaminidase, β-glucocerebrosidase and cathepsin D were studied immunocytochemically in normal and mutant human cells using monoclonal and affinity-purified polyclonal antibodies. For light microscopy, Rhodamine or Fluorescein-labelled conjugates were used, and for electron microscop...
The nature of the molecular defect resulting in the beta-galactosidase deficiency in different forms of GM1-gangliosidosis and mucopolysaccharidosis IV B (Morquio B syndrome) was investigated. Normal and mutant cultured skin fibroblasts were labeled in vivo with [3H]leucine and immunoprecipitation studies with human anti-beta-galactosidase antiseru...
The current approach to the chromosomal localization of genes coding for lysosomal enzymes has been the correlation of enzymatic and karyotypic analyses of human-rodent somatic cell hybrids. The feasibility of regional mapping depends on the availability of human cells with informative chromosomal rearrangements. In this communication we report the...
Human leucocytes contain two different MU-NANA neuraminidases, which can be distinguished by Concanavalin A binding. The Con A binding form is predominant in lymphocytes (more than 80%) and the non-binding form predominates in granulocytes. The pH optima of both these neuraminidases as well as their subcellular localization as determined by Percoll...
Cultured skin fibroblasts from patients with the lysosomal storage disease galactosialidosis lack a 54-kDa protein which is a precursor of 32-kDa and 20-kDa proteins, which immunoprecipitate with human anti-beta-galactosidase antiserum. The lack of a 32-kDa "protective protein" results in a combined deficiency of beta-galactosidase and sialidase. T...
A series of man-Chinese hamster somatic cell hybrids with a variable content of human chromosomes was used to study the localization of the human gene coding for the lysosomal enzyme beta-glucocerebrosidase (EC 3.2.1.45). In lysates made from hybrid cells, the human enzyme was specifically recognized by a mouse monoclonal antibody raised against hu...
In normal human fibroblasts, an enzymically active 85,000-dalton precursor form of beta-galactosidase is processed, via a number of intermediates, into a mature 64,000-dalton form. In addition there is an enzymically inactive 32,000-dalton component and its 54,000-dalton precursor. In fibroblasts from patients with a combined deficiency of beta-gal...
The endocytosis of β-galactosidase purified from bovine and monkey testis was studied in cultured fibroblasts from patients with GM1-gangliosidosis, mucolipidosis I and combined β-galactosidase and neuraminidase deficiency (β-gal−/neur−). The uptake of 3H-labelled β-galactosidase was of the same order of magnitude in all cell strains. Polyacrylamid...
The turnover of lysosomal beta-galactosidase was studied in fibroblast cultures from patients with Gm1-gangliosidosis and combined beta-galactosidase and neuraminidase deficiency, which had 5-10% residual beta-galactosidase activity. beta-Galactosidase was specifically inactivated with the suicide substrate beta-D-galactopyranosylmethyl-p-nitro-phe...
Complementation analysis by somatic cell hybridization to produce heterokaryons has shown that at least three complementation groups exist within the disorders in which the enzyme sialidase is deficient. We have confirmed these results by electrophoretic analysis of two glycoprotein enzymes, adenosine deaminase and acid phosphatase, which show aber...
Red- and green-fluorescing polystyrene beads were used to label different populations of cultured human fibroblasts. After enucleation of the green-fluorescing population, the cytoplasts were fused with red-fluorescing cells. Twenty-four hours after cell fusion the population of red-green heterofluorescent cells was isolated with a FACS II cell sor...
For the study of intra- and intercellular interactions between cultured human fibroblasts, balanced fluorescent cell markers were developed which allow flow-cytophotometric identification and flow sorting of cell populations. Using these aspecific cell labels, it was possible to isolate heterofluorescent heterokaryons by two-colour flow sorting (FA...
Human fibroblasts with a genetic deficiency of a single lysosomal enzyme and fibroblasts from a patient with ‘I-cell’ disease with a multiple deficiency of lysosomal hydrolases were used as recipient cells in studies on recognition and uptake of β-N-acetylhexosaminidase (hexosaminidase), β-glucuronidase and β-galactosidase. Normal human fibroblasts...
Special preparation procedures and miniaturization of biochemical analyses enable enzyme studies to be performed on small numbers of culture cells or even a single cell. Ultramicrochemical techniques have already been successfully applied to the rapid prenatal diagnosis of genetic disease, co-cultivation studies and complementation analyses after s...
I-cell fibroblasts with a multiple intracellular lysosomal enzyme deficiency were hybridized with cells from patients with different types of single lysosomal enzyme defects. Fusion with G(M2) gangliosidosis, type 2, (Sandhoff disease) fibroblasts resulted in a restoration of the hexosaminidase activity, in a normalization of the electrophoretic mo...
Fusions have been carried out between fibroblasts from patients with “I-cell” disease and enucleated human fibroblasts with a single lysosomal enzyme deficiency derived from patients with GM1-gangliosidosis, Sandhoff disease and mannosidosis. Pure cytoplasts were obtained using cytochalasin B treatment followed by fluorescence activated cell sortin...
There is a deficiency of human alpha-N-acetylneuraminidase in several inherited diseases. In patients with mucolipidosis I (refs 1,2) and in adults with a variant form with out bony abnormalities and mental retardation, both also classified as sialidoses, it is the only deficient enzyme. In mucolipidosis II ('I-cell' disease) neuraminidase is one o...
The intracellular and extracellular acidic alpha-mannosidase in cultures of fibroblasts from mucolipidosis-II patients has normal kinetics. The extracellular activity in cultures of cells from mannosidosis patients is normal, but a mutant enzyme is associated with the cell surface and intracellular fraction. The results support the involvement of m...
1. beta-D-Galactopyranosylmethyl-p-nitrophenyltriazene can specificag detectable changes in cell viability or the activities of other hydrolases. 2. beta-D-Glucopyranosylmethyl-p-nitrophenyltriazene behaves similarly towards lysosomal beta-glucosidase, but also inactivates some alpha-glucosidase. 3. Both beta-galactosidase and beta-glucosidase acti...
A pregnancy at risk for adenosine deaminase deficiency and severe combined immunodeficiency disease has been investigated by assay of adenosine deaminase activity in cultured amniotic fluid cells using a microradioassay. A low-normal level of activity consistent with heterozygote status in the foetus was found and confirmed after birth by assay of...
Cultured skin fibroblasts from patients with different clinical types of Niemann-Pick disease were hybridized and sphingomyelinase activities were measured in the heterokaryon cell population. Both the natural substrate (3H-choline) sphingomyelin and the chromogenic analogue hexadecanoylamino-4-nitrophenylphosphorylcholine were used in the compleme...
In chapter 1 the Hagberg’s conclude that out of four major neurodevelopmental impairments cerebral palsy is most closely related to negative perinatal events. Yet, genetic factors and disturbances during organogenesis and in later pregnancy have also been mentioned as a possible etiology (1). These observations need not be contradictory since many...
Recently a combined deficiency of neuraminidase and β-galactosidase was reported in two patients who had earlier been described as having variant types of GM 1-gangliosidosis. The authors have found similar deficiencies in the second of two children who both died at birth with severe hydrops fetalis. The parents are consanguineous, of Turkish origi...
Acid β-galactosidase from human liver consists, after gel filtration at pH 7.0, of a monomeric isoenzyme, β-galactosidase A and small amounts of a multimer, β-galactosidase B (Norden, A.G.W., Tennant, L.; and O'Brien, J.S. (1974) J. Biol. Chem. 249, 7969–7976).Our studies showed identical gel filtration patterns for β-galactosidase from human liver...
Both N-acetyl-β-D-glucosaminidase A and B (EC 3.2.1.30) are continuously secreted by normal cultured fibroblasts and can be taken up by deficient Sandhoff cells without cellular contact. The absence of intercellular transfer of β-galactosidase (EC 3.2.1.23) and acid α-glucosidase (EC 3.2.1.20) in cocultivations of normal and deficient fibroblasts i...
Somatic cell hybridization of β-galactosidase fibroblasts derived from patients with the infantile type 1 and the adult type 4 GM1-gangliosidosis results in restoration of the β-galactosidase activity. The kinetic properties of the enzyme activity in heterokaryons were found to be similar as in controls. Genetic complementation did not occur after...
Fibroblasts from patients with the adult, juvenile, and infantile form of glycogenosis type II (Pompe disease) were cultured under standardized conditions, and the activity of acid alpha-glucosidase (E.C.3.2.1.20) towards glycogen, maltose, and 4-methylumbelliferyl-alpha-D-glucopyranoside was measured. Glycogen levels in muscle biopsies and in cult...