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M I Cleton-Soeteman

Publications and source records attributed to M I Cleton-Soeteman.

14 recordsLinked to original sources
PubMed1999

Accumulation and release of iron in polarly and non-polarly cultured trophoblast cells isolated from human term placentas.

We investigated the usefulness of membrane grown human term trophoblast cells in transferrin-mediated iron transfer studies. We showed that diferric transferrin is taken up both at the microvillous and at the basal membrane by means of receptor-mediated endocytosis. Uptake from the microvillous side is predominant. This corresponded with a much higher expression of transferrin receptors at the microvillous membrane as compared to the basal one. Iron appeared to accumulate in the cell. Accumulation was higher when transferrin was supplied at the microvillous side. Transfer of iron could not be assessed because uptake of transferrin by the cells was much less than passive diffusion of transferrin through the cell-free filter. The observation of iron accumulation was unexpected for a transfer epithelium. Could it be that part of the iron taken up by the cells is rapidly released whereas the remaining part accumulates? In this case the rate of iron uptake should be higher than the rate of iron accumulation. This question was assessed with non-polarly cultured trophoblast cells. We showed that like in polar cells iron accumulated in ferritin. A new experimental design enabled us to demonstrate that indeed the rate of transferrin-mediated iron is in excess over iron accumulation. We thus provide evidence for a mechanism that enables rapid transfer of iron across the syncytiotrophoblast cell layer.

Biological Transport↗
PubMed1998

Iron uptake in blood-brain barrier endothelial cells cultured in iron-depleted and iron-enriched media.

Iron is essential in the cellular metabolism of all mammalian tissues, including the brain. Intracerebral iron concentrations vary with age and in several (neurological) diseases. Although it is evident that endothelial cells lining the capillaries in the brain are of importance, factors governing the regulation of intracerebral iron concentration are unknown. To investigate the role of blood-brain barrier endothelial cells in cerebral iron regulation, primary cultures of porcine blood-brain barrier endothelial cells were grown in either iron-enriched or iron-depleted medium. Iron-enriched cells showed a reduction in surface-bound and total transferrin receptor numbers compared with iron-depleted cells. Transferrin receptor kinetics showed that the transferrin receptor internalization rate in iron-enriched cultures was higher, whereas the transferrin receptor externalization rate in iron-enriched cultures was lower than the rate in iron-depleted cultures. Moreover, blood-brain barrier endothelial cells cultured in iron-enriched medium were able to accumulate more iron intracellularly, which underlines our kinetic data on transferrin receptors. Our results agree with histopathological studies on brain tissue of patients with hemochromatosis, suggesting that at high peripheral iron concentrations, the rate of iron transport across the blood-brain barrier endothelial cells is to some extent proportional to the peripheral iron concentration.

Animals↗
PubMed1997

Transcytosis of 6.6-nm gold-labeled transferrin: an ultrastructural study in cultured porcine blood-brain barrier endothelial cells.

The mechanism and regulation of iron transport to the brain are largely unknown. The large surface area of the blood-brain barrier capillaries and the presence of transferrin receptors on the luminal plasma membranes of the blood-brain barrier endothelial cells (BBB-ECs) suggest that these cells actively participate in the transport of iron into the brain. In this paper, we describe the ultrastructural morphology of primary and first-passage cultures of BBB-ECs grown on different types of porous membranes. To investigate the mechanism of iron transport into and across the BBB-ECs, porous membrane grown first-passage cells were incubated with 6.6-nm gold-labeled transferrin and studied with electron microscopy. Results are suggestive for a transcytosis of transferrin through the BBB-ECs.

Animals↗
PubMed1997

New developments and applications in quantitative electron spectroscopic imaging of iron in human liver biopsies.

Reliable iron concentration data can be obtained by quantitative analyses of image sequences, acquired by electron spectroscopic imaging. A number of requirements are formulated for the successful application of this recently developed in situ quantitative type of analysis. A demonstration of the procedures is given. By application of the technique it is established that there are no significant differences in the average iron loading of structures analysed in liver parenchymal cells of a patient with an iron storage disease, before and after phlebotomy. This supports the hypothesis that the process of iron unloading is an organelle specific process. Measurement of the binary morphology, represented by the area and contour ratio of the iron containing objects revealed no information about differences between the objects. This finding contradicts the visual suggestion that ferritin clusters are more irregularly shaped than the other iron objects. Also, no differences could be found in this sense between the situations before and after phlebotomy. With respect to the density appearance, objects that have an inhomogeneous iron loading averagely contain more iron. This observation does correspond well with the visual impression of the increasingly irregular appearance of more well-loaded structures.

Biopsy↗
PubMed1996

Isolation and partial characterization of two porcine spleen ferritin fractions with different electrophoretic mobility.

Ferritin isolated from porcine spleen could routinely be separated in two fractions on nondenaturating gradient gels. Both fractions could be isolated with a purity of 96% when applied to two serially linked columns, each 200 cm in length, packed respectively with Sepharose 4B and Sepharose 6B. Both fractions were similar as judged by electron microscopy. Assessed biochemically fractions were equal with respect to subunit composition, iron and phosphorus content, as well as amino acid composition (with the exception of N-acetylglucosamine). Carbohydrate analysis showed that the fraction with an apparent mass of 440 kDa (= FFL) contained 1.8% (w/w) glycans, whereas the fraction with an apparent mass of 670 kDa (= FFH) contained nearly five times as much (neutral) sugar residues (8.9%, w/w) and 10 times as much sialic acid. This difference in amount of carbohydrate side chains might explain the dissimilarity in electrophoretic mobility of the two fractions.

Amino Acids↗
PubMed1996

Quantitative electron spectroscopic imaging in bio-medicine: evaluation and application.

Electron spectroscopic imaging (ESI) with the energy-filtering transmission electron microscope enables the investigation of chemical elements in ultrathin biological sections. An analysis technique has been developed to calculate elemental maps and quantitative distributions from ESI sequences. Extensive experience has been obtained with a practical implementation of this technique. A procedure for more robust element detection has been investigated and optimized. With the use of Fe-loaded Chelex beads, the measurement system has been evaluated with respect to the linearity of the element concentration scale, the reproducibility of the measurements and the visual usage of image results. In liver specimens of a patient with an iron storage disease the detectability of iron was tested and we tried to characterize iron-containing components. The concentration measurement scale is approximately linear up to a relative section thickness of approximately equal to 0.5. Monitoring of this parameter is therefore considered to be important. The reproducibility was measured in an experiment with Fe-Chelex. The iron concentration differed by 6.4% between two serial measurements. Element distributions are in many applications interpreted visually. For this purpose the frequently used net-intensity distributions are regarded as unsuitable. For the quantification and visual interpretation of concentration differences mass thickness correction has to be performed. By contrast, for the detection of elements the signal-to-noise ratio is the appropriate criterion. Application of ESI analysis demonstrated the quantitative chemical capabilities of this technique in the investigation of iron storage diseases. Based on an assumed ferritin iron loading in vivo, different iron components can be discerned in liver parenchymal cells of an iron-overloaded patient.

Ferritins↗
PubMed1995

Quantification of different transferrin receptor pools in primary cultures of porcine blood-brain barrier endothelial cells.

Distribution of iron in the brain varies with region, cell type, and age. Furthermore, some neurological diseases are accompanied by an abnormal accumulation of iron in specific areas of the CNS. These findings implicate a mobile intracerebral iron pool; however, transport of iron across the blood-brain barrier and its regulation are largely unknown. In an extensive series of experiments in primary cultures of porcine blood-brain barrier endothelial cells, we separately quantified surface-bound and total cellular transferrin receptor pools. Although 90% of all transferrin receptors were located inside the cell, only 10% of these intracellular receptors actively took part in the endocytic cycle. This large "inactive" intracellular transferrin receptor pool could either function as a storage site for spare receptors or be activated by the cell to increase its capacity for iron transport. Data were corrected for nonspecific binding by a separate biochemical assessment using a 100-fold excess of unlabeled ligand. Data were also analyzed in a nonlinear curve-fit program. This resulted in a less elaborate and less biased estimate of nonspecific binding.

Animals↗
PubMed1994

Energy-filtering transmission electron microscopy as a tool for structural and compositional analysis of isolated ferritin particles.

Structural and compositional analysis of isolated horse-spleen ferritin particles was performed by energy filtering transmission electron microscopy (EFTEM). Ferritin particles were collected in ultrathin (2 nm thick) chromium films and analyzed without any additional stain by electron energy-loss spectroscopy (EELS) for iron and carbon and by electron-spectroscopic imaging (ESI) for carbon. The ultrastructure of the proteinaceous shell of the ferritin particle, as obtained by the carbon net-intensity electron spectroscopical and carbon concentration-distribution images, was qualitatively compared to the structure as acquired by a negative-staining procedure. Quantitative analysis of the number of carbon atoms in the ferritin-shell proteins was carried out through an ESI-acquisition protocol and processing procedure with calibrated attenuation filters in the optical path to the TV camera. This procedure included images acquired with calibrated attenuation filters for the compensation of shading and the non-linear performance of the TV camera used in the analytical part of the procedure. A new ¿ESI-Spectra¿ program is proposed that allows element-related spectra to be generated at any place and with any frame size in a contrast-sensitive or other type of image present on the computer monitor screen.

Animals↗
PubMed1993

Ferritin accumulation and uroporphyrin crystal formation in hepatocytes of C57BL/10 mice: a time-course study.

To establish the time-sequence relationship between ferritin accumulation and uroporphyrin crystal formation in livers of C57BL/10 mice, a biochemical, morphological and morphometrical study was performed. Uroporphyria was induced by the intraperitoneal administration of hexachlorobenzene plus iron dextran and of iron dextran alone. Uroporphyrin crystal formation started in hepatocytes of mice treated with hexachlorobenzene plus iron dextran at 2 weeks and in mice treated with iron dextran alone a 9 weeks. In the course of time, uroporphyrin crystals gradually increased in size. Uroporphyrin crystals were initially formed in hepatocytes in the periportal areas of the liver, in which also ferric iron staining was first detected. The amount and the distribution of the main storage form of iron in hepatocytes, ferritin, did not differ between the two treatment groups. Ferritin accumulation preceded the formation of uroporphyrin crystals in hepatocytes in both treatment groups. Moreover, uroporphyrin crystals were nearly always found close to ferritin iron. We conclude that uroporphyrin crystals are only formed in hepatocytes in which also iron (ferritin) accumulates. Hexachlorobenzene accelerates the effects of iron in porphyrin metabolism, but does not influence the accumulation of iron into the liver.

Animals↗
PubMed1992

The role of iron in experimental porphyria and porphyria cutanea tarda.

Porphyria cutanea tarda (PCT) and experimental porphyria are characterized by a decreased activity of the enzyme uroporphyrinogen decarboxylase, and accumulation of uroporphyrins and heptacarboxylporphyrins in the liver. Iron (Fe) plays an important role in PCT and experimental porphyria. Biochemically and electron microscopically, we examined the relationship between Fe and porphyrins in liver tissue of C57BL/10 mice made porphyric by administration of iron dextran as Imferon (IMF), and in liver biopsies of patients with symptomatic PCT. Accumulation of uroporphyrins and heptacarboxylporphyrins, and an increased amount of Fe were observed in livers of mice treated with IMF and in liver biopsies of patients with PCT. In mice treated with IMF, the activity of uroporphyrinogen decarboxylase was decreased. Both in livers of mice treated with IMF and in livers of patients with PCT, needle-like structures, representing uroporphyrin crystals, were observed by electron microscopy. Uroporphyrin crystals and Fe (as ferritin) were observed in the same hepatocyte. Moreover, there was a striking morphological correlation between uroporphyrin crystals and ferritin-Fe, suggesting a role for (ferritin-)Fe in the pathogenesis of porphyria.

Animals↗
PubMed1990

Quantitative energy-filtered image analysis in cytochemistry. II. Morphometric analysis of element-distribution images.

A combination of energy-filtered electron microscopy (EFEM) and an image-analysis system (IBAS/2000) is used for a morphometric analysis of chemical reaction products in cells. Electron energy-loss spectroscopic element-distribution images are acquired from cytochemical reaction products in a variety of cellular objects: (1) colloidal thorium particles in extra-cellular coat material, (2) iron-containing ferritin particles in liver parenchymal cells, (3) barium-containing reaction products in endoplasmic reticulum stacks, (4) elements present in lysosomal cerium- and barium-containing precipitates connected with acid phosphatase (AcPase) or aryl sulphatase (AS) enzyme activity. Areas or area fractions are determined from such element-distribution images by application of an objective image segmentation method. By superposition of two or more element-distribution images, mutual element relations are qualitatively established in lysosomal cerium- and barium-containing precipitates connected with acid phosphatase (AcPase) or aryl sulphatase (AS) enzyme activity. By comparing electron spectroscopic images (ESI) with element-distribution images, the mutual contrast per element relations are quantitatively investigated. The obtained gain in resolution in such electron energy-loss spectroscopic element-distribution images will be explained and discussed.

Animals↗
PubMed1987

Image analysis and X-ray microanalysis in cytochemistry.

When cytochemical reaction products are homogeneously distributed within an organelle, point analyses suffice for the quantitative approach. However, quantitative analysis becomes tedious, when the elements in the reaction product are inhomogeneously distributed. Problems arise when elements from two reaction products have to be related to each other, or to endogenous cytological products (ferritin, haemosiderin, calcium, electron dense markers), either topographically or in concentration. When analyzing inhomogeneous/heteromorphical reaction product-containing organelles special attention has to be paid to measure and relate both volume and concentration. In this paper a relative simple structure (eosinophil granules) is chosen to demonstrate that the acquisition of the requested morphometrical plus chemical information and their integration is possible. The following points will be covered to acquire the morphometrical and chemical information: a). How to estimate the total cell cross-sectioned area. b). How to estimate the total cross-sectioned area of all reaction product-containing particles inside that cell. The ratio of these two areas will provide the requested information about the particle volume fraction. By using the X-ray detector in addition: c). How to acquire the chemical information at the requested resolution, within a reasonable total acquisition time d). How to integrate the morphometrical and chemical data per organelle, by matrix analysis in a reduced scan area. e). How to acquire quantitative chemical information, by the use of cross-sectioned standards. f). How to make this acquisition method independent from changes in the instrumental conditions during the acquisition.

Animals↗
PubMed1985

Application of Chelex standard beads in integrated morphometrical and X-ray microanalysis.

Chelex ion exchange beads loaded with a known amount (18.3% weight percentage (w/w)) of platinum, have been co-embedded with a mouse peritoneal cell population. To establish the influence of the various deconvolution methods applied, upon the platinum concentration in cytoplasmic granules and erythrocytes these cross-sectioned beads are used as a standard. It is concluded that irrespective of the deconvolution method chosen 1) the Pt concentration inside the particles is identical when the particles and the co-embedded Chelex Pt standard, are analysed strictly under the same instrumental conditions 2) the Pt concentration outside the particle is zero, or virtually zero when that element is absent there (erythrocyte surrounded by Epon) 3) the Pt concentration outside the particle in the surrounding cytoplasm was identical (when the element Pt was present there). The information about the elemental concentration obtained by point analysis in the STEM-mode in homogeneous objects was compared with the mean value obtained by the reduced raster method. The ratio between these values were constant. As an example of a heteromorphic, heterogeneous cell organelle population, the application of the method of integrated morphometrical and chemical (X-ray) analysis is demonstrated on lysosomes within a single human liver parenchymal cell, containing iron and cerium. It was shown that the cerium concentration (from the cytochemical reaction to detect acid phosphatase activity in lysosomes) was rather homogeneously distributed over the small population and in the individual lysosomes. The iron distribution was very inhomogeneous, both in its distribution over the lysosomal area, and among the lysosomes in the population.

Animals↗