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Effect of weak bases on the secretion of lysomal enzymes by Caco-2 cells
Abstracts of The Netherlands Society of Electron Microscopy
EFFECT OF WEAK BASES OF LYSOSOMAL ENZYMES
ON THE SECRETION BY CACO-2 CELLS
J. Klumperman, Z.A.M. Fransen, J.C. Boekesteijn, H.P. Hauei, J.M. Teger and L.A. Ginsel Laboratory for Electron Microscopy, Rijnsburqerweq 10, 2333 AA Leiden, The Netherlands
Previously, we have shown that aprecursor form of the lysosomal enzyme a-glucosidase is present in the brush border of Caco-2 cells, and that 70-80% of the total amount of secreted a-glucosidase precursor is found in the apical medium. Therefore we investigated whether the mannose 6-phosphate receptor is involved in the transport and secretion of a-glucosidace. Immunocytochemistry of the 215 kDa mannose 6-phosphate receptor showed its presence in the brush border of the Caco-2 cells. However, the intensity of labeling of the brush border varied considerably between the cells, and sometimes label was completely absent Besides the brush border, labeling was observed over the Golgi region, dense vesicular and tubular structures dispersed through the cytoplasm, multivesicular bodies and, although weakly, over the basolateral membrane. Receptor-mediated transport can be disturbed by the addition of weak bases to the culture medium leading to a secretion of precursors of lysosomal enzymes. Here we investigated the effect of NH4Cl on the secretion of a-glucosidase and some other lysosomal enzymes. No effect of NH4Cl was observed on the secretion of a-giucosidase, except maybe a slight reduction of the apical secretion. In contrast, NH4Cl led to a significant increase in the basolateral secretion of all other lysosomal enzymes studied. We therefore conclude that the transport of a-glucosidase to the brush border is not receptor mediated, although mannose 6-phosphate receptors are present in the brush border membrane.
INFLAMMATORY PHOSPHATES
ACTIVITY
VP' CALCIUM
H.K. Koerten, C.A. van Blittersvijk, S.C. Hesseling, R.A. Terpstra, J.P.W. Vogelaar, K. de Groot and J.J. Grote Biomaterials Research Group, Laboratory for Electron Microscopy, University of Leiden, Leiden, The Netherlands
473
Most biomaterials encounter inflammation, either as a consequence of a wound reaction which coincides with the impiantation, or as a result of a forThe aim of the eign body reaction. present study was to investigate the inflammatory process associated with five calcium phosphate ceramics in vivo and in vitro, the latter omittingeffects of a wound reaction. Suspensions containing the calcium phosphates were injected intraperitoneally, and added to peritoneal macrophages in culture. Control groups received a physiological solution only. Isolation of the peritoneal cell suspensions was performed at 4, 16, 24, 48, 72 h, 1 wk, 1, 2 and 6 mo. The cultures were studied at2wkandl mo. The material of both experiments was studied by LM, TEM, SEM and X-ray microanalysis (XRMA). In the in vivo experiments the proportions of peritoneal cells changed as a reaction to the introduction of the materials, and phagocytosis of the materials was observed. All materials showed particle concentrations after 4 h already, which developed into granulomas at the longer intervals. XRMA performed on inclusion bodies in the macrophaqes of both the in vivo and in vitro experiments demonstrated the oresence of Ca and P. derived from the originally injected materials. In addition, elements like Si, Fe, Cr and Ni were demonstrated.
PRACTICAL AUTOTUNING ELECTRON MICROSCOPE
OF A TRANSMISSION
A. J. Koster Tietz Video and Image Processing GmbH, Herbstr. 7, D-8035 Gautinq Munich), Fed. Repub. Germany
Systems (near
The quality and reliability of the TEM as an analytical tool is becoming ever more dependent on the design and integration of image processing equipment and computer control facilities. For low-dose and high-resolution electron microscopy, the automatic correction of optical misalignment, defocus and astigmatism (autotuning) is highly desirable. An autotuning method, based on measur. ing image displacements when the illuminating beam is tilted, has been tested on a Philips EM 420 TEM with a Gatan622 video camera and a TVIPS image processing system connected. The method works
EFFECT OF WEAK BASES OF LYSOSOMAL ENZYMES
ON THE SECRETION BY CACO-2 CELLS
J. Klumperman, Z.A.M. Fransen, J.C. Boekesteijn, H.P. Hauei, J.M. Teger and L.A. Ginsel Laboratory for Electron Microscopy, Rijnsburqerweq 10, 2333 AA Leiden, The Netherlands
Previously, we have shown that aprecursor form of the lysosomal enzyme a-glucosidase is present in the brush border of Caco-2 cells, and that 70-80% of the total amount of secreted a-glucosidase precursor is found in the apical medium. Therefore we investigated whether the mannose 6-phosphate receptor is involved in the transport and secretion of a-glucosidace. Immunocytochemistry of the 215 kDa mannose 6-phosphate receptor showed its presence in the brush border of the Caco-2 cells. However, the intensity of labeling of the brush border varied considerably between the cells, and sometimes label was completely absent Besides the brush border, labeling was observed over the Golgi region, dense vesicular and tubular structures dispersed through the cytoplasm, multivesicular bodies and, although weakly, over the basolateral membrane. Receptor-mediated transport can be disturbed by the addition of weak bases to the culture medium leading to a secretion of precursors of lysosomal enzymes. Here we investigated the effect of NH4Cl on the secretion of a-glucosidase and some other lysosomal enzymes. No effect of NH4Cl was observed on the secretion of a-giucosidase, except maybe a slight reduction of the apical secretion. In contrast, NH4Cl led to a significant increase in the basolateral secretion of all other lysosomal enzymes studied. We therefore conclude that the transport of a-glucosidase to the brush border is not receptor mediated, although mannose 6-phosphate receptors are present in the brush border membrane.
INFLAMMATORY PHOSPHATES
ACTIVITY
VP' CALCIUM
H.K. Koerten, C.A. van Blittersvijk, S.C. Hesseling, R.A. Terpstra, J.P.W. Vogelaar, K. de Groot and J.J. Grote Biomaterials Research Group, Laboratory for Electron Microscopy, University of Leiden, Leiden, The Netherlands
473
Most biomaterials encounter inflammation, either as a consequence of a wound reaction which coincides with the impiantation, or as a result of a forThe aim of the eign body reaction. present study was to investigate the inflammatory process associated with five calcium phosphate ceramics in vivo and in vitro, the latter omittingeffects of a wound reaction. Suspensions containing the calcium phosphates were injected intraperitoneally, and added to peritoneal macrophages in culture. Control groups received a physiological solution only. Isolation of the peritoneal cell suspensions was performed at 4, 16, 24, 48, 72 h, 1 wk, 1, 2 and 6 mo. The cultures were studied at2wkandl mo. The material of both experiments was studied by LM, TEM, SEM and X-ray microanalysis (XRMA). In the in vivo experiments the proportions of peritoneal cells changed as a reaction to the introduction of the materials, and phagocytosis of the materials was observed. All materials showed particle concentrations after 4 h already, which developed into granulomas at the longer intervals. XRMA performed on inclusion bodies in the macrophaqes of both the in vivo and in vitro experiments demonstrated the oresence of Ca and P. derived from the originally injected materials. In addition, elements like Si, Fe, Cr and Ni were demonstrated.
PRACTICAL AUTOTUNING ELECTRON MICROSCOPE
OF A TRANSMISSION
A. J. Koster Tietz Video and Image Processing GmbH, Herbstr. 7, D-8035 Gautinq Munich), Fed. Repub. Germany
Systems (near
The quality and reliability of the TEM as an analytical tool is becoming ever more dependent on the design and integration of image processing equipment and computer control facilities. For low-dose and high-resolution electron microscopy, the automatic correction of optical misalignment, defocus and astigmatism (autotuning) is highly desirable. An autotuning method, based on measur. ing image displacements when the illuminating beam is tilted, has been tested on a Philips EM 420 TEM with a Gatan622 video camera and a TVIPS image processing system connected. The method works