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Biomedical subjects

H Galjaard

Publications and source records attributed to H Galjaard.

At least 145 records · Page 8Linked to original sources
PubMed1977

Autoradiographic detection of mucopolysaccharide accumulation in single fibroblasts.

A method is described for localizing acid mucopolysaccharides autoradiographically in cultured cells. Normal fibroblasts and fibroblasts, from patients suffering from Mucopolysaccharidosis II disease (MPS II), were cultured for six days in the presence of 35SO4 and one day in unlabelled medium. The cultured cells were transferred to plastic film dish and, after settling, they were rapidly quenched, freeze-dried, fixed in osmium tetroxide vapour and embedded in Epon. Grain counting after autoradiography in 2 mum sections revealed a significant difference (P greater than 0.001) in 35SO4 incorporation in the perinuclear cytoplasm of MPS II cells and control cells grown under the same conditions. Autoradiography was also performed after mixing MPS II cells and control fibroblasts in a ratio 1:1-8 prior to freezing and the same ratio was found between labelled and unlabelled fibroblasts. These results demonstrate the feasibility of the present autoradiographic technique for the detection of the acid mucopolysaccharide storage at the single cell level.

Autoradiography↗
PubMed1977

Electron-microscopic autoradiography of tritiated testosterone in rat testis.

The feasibility of a technique for autoradiography of diffusible substances has been further tested by analysing the localization of steroids in rat testes with the light-and electron-microscope. Testes of rats were perfused with tritiated testosterone (3 min) followed by 15-min perfusion with buffer containing a 100-fold of unlabelled testosterone. Tissue samples were frozen, freeze dried, fixed in osmium vapour and embedded in Epon. To exclude extraction of steroids, contact with water and other solvents was prevented during cutting of thin sections on an ultracryotome and further treatments for autoradiography. Light- and electron-microscopic observations indicate that the highest concentration of labelled testosterone was present within the basal parts of the Sertoli cell cytoplasm and in lipid inclusions of Sertoli cells within the seminiferous tubules. This is the first account of autoradiography of steroids at the electron-microscope level.

Autoradiography↗
PubMed1977

Influence of experimental bypass on cellular kinetics and maturation of small intestinal epithelium in the rat.

The effect of experimental bypass on the intestinal epithelium of the rat was investigated from 2 to 360 days after creating a Thiry-Vella fistula. Within 7 to 14 days the number of cells per villus column and, to a lesser degree, the number of cells per crypt column decreased and subsequently these values remained constant up to 1 year. Radioautography did not show any changes in the percentage of labelled cells per crypt column, after incorporation of [3H]thymidine. In addition, the relative size of the proliferative cell compartment in the crypt, the number of crypts per unit length of small intestine and the crypt-villus ratio remained unchanged. Scintillation counting of isolated crypts from the Thiry-Vella fistulas showed that the number of cells per crypt was markedly reduced. Scanning electron microscopy revealed that the three-dimensional size of the villi was also reduced. The activities of nonspecific esterases and neutral alpha-glucosidase remained unchanged in crypt cells and were above control values in villus cells. The development of these biochemical characteristics of the villus cells seems to be independent of the luminal contents. It was concluded that deprivation of food passage and gastric, pancreatic, and liver secretions from a small intestinal segment primarily causes a reduction in the number of cells per crypt, which leads to a lower cell production. In turn, this is responsible for the reduction of the functional villus cell compartment.

Animals↗
PubMed1976

The relation between cell proliferation, differentiation and ultrastructural development in rat intestinal epithelium.

The ultrastructural development of the principal cells in rat small intestine was studied by morphometric analyses in relation to the exact cell position along crypt and villus. From the bottom to the tip of the crypt, a gradual increase occurred in absolute size of the total cell, the cytoplasm, the terminal web and of nearly all cell organelles. Also, the relative size of the cytoplasm, mitochondria, microvilli and endoplasmic reticulum increased during crypt cell differentiation. No sudden changes in ultrastructure were observed in the so-called "critical decision zone", normally located halfway up the crypt where the proliferative activity ceases. At the crypt-villous junction a 1.4-3 fold increase in cell size, cytoplasm, terminal web and of most organelles was noted. Expansion of the proliferative cell compartment over the total length of the crypt as occurs during recovery after a low X-irradiation dose (72 h after 400 R) does not affect the normal development of cellular ultrastructure. These findings are discussed in relation to biochemical and cell kinetic data.

Animals↗
PubMed1976

[Genetic heterogeneity of congenital metabolic disorders. A bridge between the physician and the basic science researcher].

Earlier diagnosis of genetic diseases was based on clinical symptoms. Today, molecular biology and biochemistry have led to a better understanding of genetic principles. The exact diagnosis of more than 100 metabolic diseases rests on biochemical investigations which in general are carried out by specialized groups outside the sphere of direct patient care. The study of genetic heterogeneity of inherited metabolic disorders may be a bridge between the doctor and the basic researcher. The doctor will principally concentrate on the relation between gene mutation, enzyme deficiency and the resulting clinical peculiarities. The investigation of the genetic heterogeneity may provide important information on the various aspects of molecular genetics and cell metabolism.

Amniotic Fluid↗
PubMed1976

Prenatal diagnosis of genetic disorders.

Three hundred and fifty pregnancies were monitored by transabdominal amniocentesis in the fourteenth to sixteenth week of gestation followed by karyotyping or biochemica assays of cultured amniotic fluid cells and analysis of alpha-fetoprotein in the amniotic fluid supernatant. The pregnancy was interrupted in 36 cases (10%) either becasue of a fetal abnormality or the presence of a male fetus in pregnancies at risk for an X-linked disease. Four chromosomal aberrations were found in 87 pregnancies tested because of advanced maternal age. In 101 pregnancies with a recurrence risk of Down's syndrome, 2 fetuses with an abnormal karyotype were detected. In 11 cases, in which 1 parent was a carrier of a balanced translocation, 2 unbalanced fetal karyotypes were found. Fetal chromosome studies in 43 pregancies at risk for an X-linked disease indicated the presence of a male fetus in 21 cases. Prenatal diagnosis of 11 different metabolic diseases was performed in a total of 34 cases. Microchemical techniques were used to allow completion of the diagnosis of seven different enzyme deficiencies within 9 to 22 days after amniocentesis. Alpha-fetoprotein assay in the amniotic fluid supernatant of 47 pregnancies at risk for an open neural tube defect resulted in the detection of 3 anencephalic fetuses during the second half of pregnancy. The safety and reliability of amniocentesis and the possible effects on the outcome of pregnancy are evaluated. Prenatal diagnosis offers a promising alternative for parents who are at risk of having a child with a genetic disease which can be detected in amniotic fluid or in cultured amniotic fluid cells.

Amniocentesis↗
PubMed1976

European experience with prenatal diagnosis of congenital disease: a survey of 6121 cases.

The results of 6121 prenatal diagnoses established by 46 centers in eight West-European countries are reported. The percentage of "spontaneous" abortions following early amniocentesis (1.4%) does not seem to exceed the normal rate of spontaneous abortion in this period of pregnancy. A total of nearly 300 affected fetuses was detected, and in all but 5 instances the parents asked for interruption of the pregnancy. The majority of prenatal monitoring (75%) was carried out because of an increased risk of chromosomal aberration. In the group with higher maternal age 2.8% of the fetuses were affected; this figure was 4.6% for mothers older than 38 years. Monitoring because of an earlier child with trisomy 21 showed 1.3% fetuses with a chromosomal aberration, and 1.7% abnormal karyotypes were detected in the group where amniocentesis was performed for "miscellaneous reasons." In pregnancies where a parental translocation has been demonstrated, 6.7% of the fetuses showed an unbalanced karyotype and 25% were balanced carriers. Also discussed are results and organizatioal problems of prenatal monitoring for inherited metabolic disorders (206 analyses for 23 different enzyme defects) and for neural tube defects (about 2700 cases).

Abortion, Spontaneous↗
PubMed1976

Methods for analysis of acid alpha-1,4-glucosidase activity in single hybrid cells.

Two methods are described which allow the quantitative assay of the lysosomal enzyme alpha-1,4-glucosidase in single fibroblasts. In the first procedure the substrate was maltose, and liberated glucose was measured with an enzymatic cycling procedure for reduced nicotinamide adenine dinucleotide phosphate. Single cultured fibroblasts were found to have enzyme activities in the range of 0.5-10 X 10(-13) moles glucose/hr. In the second procedure the artificial substrate 4-methylumbelliferyl-alpha-D-glucopyransodie was used. It is hydrolyzed in a single step reaction to the fluorescent product 4-methylumbelliferone (MU). By reducing the incubation volume and by measuring the fluorescence in microdroplets with a microscope fluorometer, a sensitivity of 10(-14) moles MU could be obtained. Activities were found ranging from 0.5-10 X 10(-14) moles MU/hr/cell. Both procedures for single cell analysis proved to be reliable when compared with conventional assays on cell homogenates. Cocultivation and cell fusion studies were performed to demonstrate that these methods can be used to study the metabolic and genetic interaction between normal and enzyme-deficient fibroblasts derived from patients with glycogenosis II.

Fibroblasts↗
PubMed1975

The effect of continuous irradiation on cell proliferation and maturation in small intestinal epithelium.

Autoradiographic studies and scintillation counting of crypt material after pulse labelling with 3H-thymidine showed that during continuous irradiation with 290 rads/day a reduced proliferative activity is present in the crypts of rat small intestine after 1 day of irradiation and of normal activity during the remaining period (5 days) irradiation. After cessation of irradiation an increase in proliferative activity can be observed after 1 day of recovery. From the time (36-48 hr after starting of the irradiation) that the number of villus cells is reduced an expansion of the proliferation zone in the crypt was observed. Both effects last until 1 day of recovery after cessation of irradiation. The process of crypt cell maturation and of villus cell function has also been studied during and after continuous irradiation by micro-chemical enzyme analyses in isolated crypts and villi. It was found that the expansion of the proliferation zone in the crypt is accompanied by a decrease in activity of only those enzymes (i.e. non-specific esterases) which normally become active during crypt cell maturation. The activity of enzymes normally present mainly in the functional villus cells remained relatively unaffected by changes in crypt cell kinetics. A hypothesis of different regulation mechanisms of the proliferative activity in the intestinal crypt and a possible explanation of the different behaviour of various enzyme activities as a result of changes in crypt cell proliferation is discussed.

Alkaline Phosphatase↗
PubMed1975

Prenatal diagnosis of type II glycogenosis (Pompe's disease) using microchemical analyses.

1. In order to reduce the time interval between amniocentesis and prenatal diagnosis of Pompe's disease microchemical techniques were used for assay of acid alpha-1,4-glucosidase activities in cultured amniotic fluid cells. 2. Microtechniques used on homogenates of cultured amniotic fluid cells enabled the waiting period to be reduced to 2-3 weeks. 3. When dissected lyophilized groups of 200-300 cultured cells were analyzed, a prenatal diagnosis was possible at about 10 days after amniocentesis. 4. The acid alpha-1,4-glucosidase activity in the amniotic fluid supernatant is not informative in prenatal diagnosis of Pompe's disease. 5. Conditions of cell cultivation such as length of time in culture were found to influence markedly the acid alpha-1,4-glucosidase activity in cultured amniotic fluid cells. 6. For a reliable prenatal diagnosis of metabolic disorders primary cultures of control amniotic fluid cells should be used and the analytical results from the pregnancy at risk should be compared with primary cultures of control amniotic fluid cells and with those in cultured fibroblasts from heterozygous carriers, and an affected sibling from the particular family.

Amniocentesis↗