Wim Voorhout - Academia.edu (original) (raw)
Papers by Wim Voorhout
Lung Surfactant: Basic Research in the Pathogenesis of Lung Disorders
Journal of Biological Chemistry, 2001
Proteolytic processing of surfactant protein C (SP-C) proprotein in multivesicular bodies of alve... more Proteolytic processing of surfactant protein C (SP-C) proprotein in multivesicular bodies of alveolar type II cells results in a 35-residue mature peptide, consisting of a transmembrane domain and a 10-residue extramembrane domain. SP-C mature peptide is stored in lamellar bodies (a lysosomal-like organelle) and secreted with surfactant phopholipids into the alveolar space. This study was designed to identify the peptide domain of SP-C required for sorting and secretion of this integral membrane peptide. Deletion analyses in transiently transfected PC12 cells and isolated mouse type II cells suggested the extramembrane domain of mature SP-C was cytosolic and sufficient for sorting to the regulated secretory pathway. Intratracheal injection of adenovirus encoding SP-C mature peptide resulted in secretion into the alveolar space of wild type mice but not SP-C (؊/؊) mice. SP-C secretion in null mice was restored by the addition of the N-terminal propeptide. The cytosolic domain, consisting of the Nterminal propeptide and extramembrane domain of mature SP-C peptide, supported secretion of the transmembrane domain of platelet-derived growth factor receptor. Collectively, these studies indicate that the N-terminal propeptide of SP-C is required for intracellular sorting and secretion of SP-C.
D electron tomography is becoming a rou- tine research tool, thus warranting a streamlined workfl... more D electron tomography is becoming a rou- tine research tool, thus warranting a streamlined workflow optimization. We describe the quality factors in the various stages (acquisition, alignment and recon- struction) of electron tomography and how they can be optimized using an integrated suite of software programs. We show that the main time bottleneck is the (iterative) reconstruction process. We subsequently present a solution for this bottleneck in the form of GPU acceleration of the calcula- tions, speeding up the reconstruction by a factor of about 60.
Scanning microscopy. Supplement, 1991
The success of post-embedding immunocytochemistry depends largely on the preparation methods. The... more The success of post-embedding immunocytochemistry depends largely on the preparation methods. The requirements for structural preservation and immunocytochemistry are in some cases contradictory. This is especially the case in the study of lipid-rich structures and the localization of lipid components. Earlier work on freeze-substitution has shown that this method is very promising for the preservation of lipids and the immunocytochemical localization of lipids at the electron microscopical level. In this study we show that freeze-substitution in combination with low temperature embedding in Lowicryl HM20 has fulfilled this promise. Lamellar bodies in alveolar type II cells contain about 90% lipids and are very difficult to preserve in ultrathin cryosections. Lowicryl sections of freeze-substituted lung tissue shows excellent preservation of lamellar bodies in combination with immunogold localization of a hydrophobic surfactant protein. With an antibody against the Forssman glycolip...
Microscopy and Microanalysis
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1988
Journal of …, 1996
Peanut agglutinin (PNA) was used to assess the sperm acrosomal status and the acrosome reaction d... more Peanut agglutinin (PNA) was used to assess the sperm acrosomal status and the acrosome reaction during gamete interaction in the equine species. PNA exclusively binds to the outer acrosomal membrane of stallion spermatozoa, as was established by transmission electron microscopy. Fluorescein isothiocyanate-PNA (FITC-PNA) labeling was used to monitor sperm acrosomal changes during a prolonged incubation period of 24 hours and during a 2-hours incubation in the presence of 5 microM calcium ionophore A23187. In addition, after a 4-hours preincubation in SP-TALP medium, sperm samples were incubated with matching hemizonae for 1 minute (onset binding) followed by a 60-minute incubation (1-hour binding) of the sperm-hemizona complexes in sperm-free medium to assess the acrosomal status of the bound spermatozoa. For acrosome assessment, spermatozoa and washed sperm-hemizona complexes were air dried onto microscope slides, fixed, permeabilized in ethanol, stained with FITC-PNA, and counterstained with the DNA dye ethidium homodimer. Both zona-bound and non-bound spermatozoa showed similar staining patterns. Acrosome-intact spermatozoa displayed intensively green fluorescence over the acrosomal cap, whereas reacting spermatozoa showed a patchy disrupted image of fluorescence. Sperm cells that completed the acrosome reaction were principally stained on the equatorial segment or not stained at all. During prolonged incubation and during the calcium ionophore treatment, the proportion of spermatozoa with an acrosomal modification (reacting) and a complete breakdown of the acrosome (reacted) increased noticeably. Significant induction of the acrosome reaction was observed within 60 minutes of sperm-zona contact (P < 0.001). In conclusion, a rapid and reliable assessment of the acrosomal status and the incidence of the acrosome reaction of stallion spermatozoa at the zona surface were demonstrated in this study.
American Journal of Respiratory Cell and Molecular Biology, 2003
Lung surfactant protein A (SP-A) is a collectin produced by alveolar type II cells and Clara cell... more Lung surfactant protein A (SP-A) is a collectin produced by alveolar type II cells and Clara cells. It binds to carbohydrate structures on microorganisms, initiating effector mechanisms of innate immunity and modulating the inflammatory response in the lung. Reverse transcriptase-polymerase chain reaction was performed on a panel of RNAs from human tissues for SPA mRNA expression. The lung was the main site of synthesis, but transcripts were readily amplified from the trachea, prostate, pancreas, and thymus. Weak expression was observed in the colon and salivary gland. SPA sequences derived from lung and thymus mRNA revealed the presence of both SP-A1 and SP-A2, whereas only SP-A2 expression was found in the trachea and prostate. Monoclonal antibodies were raised against SPA and characterized. One of these (HYB 238-4) reacted in Western blotting with both reduced and unreduced SPA , with N-deglycosylated and collagenase-treated SPA , and with both recombinant SP-A1 and SP-A2. This antibody was used to demonstrate SPA in immunohistochemistry of human tissues. Strong SPA immunoreactivity was seen in alveolar type-II cells, Clara cells, and on and within alveolar macrophages, but no extrapulmonary SPA immunoreactivity was observed. In contrast to lung surfactant protein D (SP-D), which is generally expressed on mucosal surfaces, SPA seems to be restricted to the respiratory system. Lung surfactant protein A (SP-A) is a member of the collectin family, a group of oligomeric proteins in which a collagenous region is connected by an ␣-helical neck region to a C-type lectin or carbohydrate-recognition domain (CRD) (1-3). The structural subunit consists of three such polypeptide chains, and the functional protein is made up of six subunits linked by interchain disulfide bridges near the N-terminus (4). Post-translational modifications include complex N-linked glycosylation at Asn 187 in the CRD (4). Two genes encoding SPA transcripts, SP-A1 and SP-A2, are found in humans (2, 3, 5), and several alleles are known for each gene (6). Transcripts of both genes undergo
Abstract. Forssman antigen, a neutral glycosphingolipid carrying five monosaccharides, was locali... more Abstract. Forssman antigen, a neutral glycosphingolipid carrying five monosaccharides, was localized in epithelial MDCK cells by the immunogold technique. Labeling with a well defined mAb and protein A-gold after freeze-substitution and low temperature embedding in Lowicryl HM20 of aldehyde-fixed and cryoprotected cells, resulted in high levels of specific labeling and excellent retention of cellular ultrastructure compared to ultra-thin cryosections. No Forssman glycolipid was lost from the cells during freezesubstitution as measured by radio-immunostaining of lipid extracts. Redistribution of the glycolipid between membranes did not occur. Forssman glycolipid, abundantly expressed on the surface of MDCK II cells, did not move to neighboring cell surfaces in cocultures with Forssman negative MDCK 1 cells, even though
Lung Surfactant: Basic Research in the Pathogenesis of Lung Disorders
Journal of Biological Chemistry, 2001
Proteolytic processing of surfactant protein C (SP-C) proprotein in multivesicular bodies of alve... more Proteolytic processing of surfactant protein C (SP-C) proprotein in multivesicular bodies of alveolar type II cells results in a 35-residue mature peptide, consisting of a transmembrane domain and a 10-residue extramembrane domain. SP-C mature peptide is stored in lamellar bodies (a lysosomal-like organelle) and secreted with surfactant phopholipids into the alveolar space. This study was designed to identify the peptide domain of SP-C required for sorting and secretion of this integral membrane peptide. Deletion analyses in transiently transfected PC12 cells and isolated mouse type II cells suggested the extramembrane domain of mature SP-C was cytosolic and sufficient for sorting to the regulated secretory pathway. Intratracheal injection of adenovirus encoding SP-C mature peptide resulted in secretion into the alveolar space of wild type mice but not SP-C (؊/؊) mice. SP-C secretion in null mice was restored by the addition of the N-terminal propeptide. The cytosolic domain, consisting of the Nterminal propeptide and extramembrane domain of mature SP-C peptide, supported secretion of the transmembrane domain of platelet-derived growth factor receptor. Collectively, these studies indicate that the N-terminal propeptide of SP-C is required for intracellular sorting and secretion of SP-C.
D electron tomography is becoming a rou- tine research tool, thus warranting a streamlined workfl... more D electron tomography is becoming a rou- tine research tool, thus warranting a streamlined workflow optimization. We describe the quality factors in the various stages (acquisition, alignment and recon- struction) of electron tomography and how they can be optimized using an integrated suite of software programs. We show that the main time bottleneck is the (iterative) reconstruction process. We subsequently present a solution for this bottleneck in the form of GPU acceleration of the calcula- tions, speeding up the reconstruction by a factor of about 60.
Scanning microscopy. Supplement, 1991
The success of post-embedding immunocytochemistry depends largely on the preparation methods. The... more The success of post-embedding immunocytochemistry depends largely on the preparation methods. The requirements for structural preservation and immunocytochemistry are in some cases contradictory. This is especially the case in the study of lipid-rich structures and the localization of lipid components. Earlier work on freeze-substitution has shown that this method is very promising for the preservation of lipids and the immunocytochemical localization of lipids at the electron microscopical level. In this study we show that freeze-substitution in combination with low temperature embedding in Lowicryl HM20 has fulfilled this promise. Lamellar bodies in alveolar type II cells contain about 90% lipids and are very difficult to preserve in ultrathin cryosections. Lowicryl sections of freeze-substituted lung tissue shows excellent preservation of lamellar bodies in combination with immunogold localization of a hydrophobic surfactant protein. With an antibody against the Forssman glycolip...
Microscopy and Microanalysis
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1988
Journal of …, 1996
Peanut agglutinin (PNA) was used to assess the sperm acrosomal status and the acrosome reaction d... more Peanut agglutinin (PNA) was used to assess the sperm acrosomal status and the acrosome reaction during gamete interaction in the equine species. PNA exclusively binds to the outer acrosomal membrane of stallion spermatozoa, as was established by transmission electron microscopy. Fluorescein isothiocyanate-PNA (FITC-PNA) labeling was used to monitor sperm acrosomal changes during a prolonged incubation period of 24 hours and during a 2-hours incubation in the presence of 5 microM calcium ionophore A23187. In addition, after a 4-hours preincubation in SP-TALP medium, sperm samples were incubated with matching hemizonae for 1 minute (onset binding) followed by a 60-minute incubation (1-hour binding) of the sperm-hemizona complexes in sperm-free medium to assess the acrosomal status of the bound spermatozoa. For acrosome assessment, spermatozoa and washed sperm-hemizona complexes were air dried onto microscope slides, fixed, permeabilized in ethanol, stained with FITC-PNA, and counterstained with the DNA dye ethidium homodimer. Both zona-bound and non-bound spermatozoa showed similar staining patterns. Acrosome-intact spermatozoa displayed intensively green fluorescence over the acrosomal cap, whereas reacting spermatozoa showed a patchy disrupted image of fluorescence. Sperm cells that completed the acrosome reaction were principally stained on the equatorial segment or not stained at all. During prolonged incubation and during the calcium ionophore treatment, the proportion of spermatozoa with an acrosomal modification (reacting) and a complete breakdown of the acrosome (reacted) increased noticeably. Significant induction of the acrosome reaction was observed within 60 minutes of sperm-zona contact (P < 0.001). In conclusion, a rapid and reliable assessment of the acrosomal status and the incidence of the acrosome reaction of stallion spermatozoa at the zona surface were demonstrated in this study.
American Journal of Respiratory Cell and Molecular Biology, 2003
Lung surfactant protein A (SP-A) is a collectin produced by alveolar type II cells and Clara cell... more Lung surfactant protein A (SP-A) is a collectin produced by alveolar type II cells and Clara cells. It binds to carbohydrate structures on microorganisms, initiating effector mechanisms of innate immunity and modulating the inflammatory response in the lung. Reverse transcriptase-polymerase chain reaction was performed on a panel of RNAs from human tissues for SPA mRNA expression. The lung was the main site of synthesis, but transcripts were readily amplified from the trachea, prostate, pancreas, and thymus. Weak expression was observed in the colon and salivary gland. SPA sequences derived from lung and thymus mRNA revealed the presence of both SP-A1 and SP-A2, whereas only SP-A2 expression was found in the trachea and prostate. Monoclonal antibodies were raised against SPA and characterized. One of these (HYB 238-4) reacted in Western blotting with both reduced and unreduced SPA , with N-deglycosylated and collagenase-treated SPA , and with both recombinant SP-A1 and SP-A2. This antibody was used to demonstrate SPA in immunohistochemistry of human tissues. Strong SPA immunoreactivity was seen in alveolar type-II cells, Clara cells, and on and within alveolar macrophages, but no extrapulmonary SPA immunoreactivity was observed. In contrast to lung surfactant protein D (SP-D), which is generally expressed on mucosal surfaces, SPA seems to be restricted to the respiratory system. Lung surfactant protein A (SP-A) is a member of the collectin family, a group of oligomeric proteins in which a collagenous region is connected by an ␣-helical neck region to a C-type lectin or carbohydrate-recognition domain (CRD) (1-3). The structural subunit consists of three such polypeptide chains, and the functional protein is made up of six subunits linked by interchain disulfide bridges near the N-terminus (4). Post-translational modifications include complex N-linked glycosylation at Asn 187 in the CRD (4). Two genes encoding SPA transcripts, SP-A1 and SP-A2, are found in humans (2, 3, 5), and several alleles are known for each gene (6). Transcripts of both genes undergo
Abstract. Forssman antigen, a neutral glycosphingolipid carrying five monosaccharides, was locali... more Abstract. Forssman antigen, a neutral glycosphingolipid carrying five monosaccharides, was localized in epithelial MDCK cells by the immunogold technique. Labeling with a well defined mAb and protein A-gold after freeze-substitution and low temperature embedding in Lowicryl HM20 of aldehyde-fixed and cryoprotected cells, resulted in high levels of specific labeling and excellent retention of cellular ultrastructure compared to ultra-thin cryosections. No Forssman glycolipid was lost from the cells during freezesubstitution as measured by radio-immunostaining of lipid extracts. Redistribution of the glycolipid between membranes did not occur. Forssman glycolipid, abundantly expressed on the surface of MDCK II cells, did not move to neighboring cell surfaces in cocultures with Forssman negative MDCK 1 cells, even though