Wim F. Voorhout

Immunocytochemical localization of surfactant protein D(SP-D) in type II cells, Clara cells, and alveolar macrophages of rat lung

We investigated the cellular and subcellular distribution of surfactant protein D (SP-D) by immunogold labeling in lungs of adult rats that had been given bovine serum albumin coupled to 5-nm gold (BSAG) for 2 hr to visualize the endocytotic pathway. Specific gold labeling for SP-D was found in alveolar Type II cells, Clara cells, and alveolar macrophages. In Type II cells abundant labeling was observed in the endoplasmic reticulum, whereas the Golgi complex and multivesicular bodies were labeled to a limited extent only. Lamellar bodies did not seem to contain SP-D. Gold labeling in alveolar macrophages was restricted to structures containing endocytosed BSAG. In Clara cells labeling was found in the endoplasmic reticulum, the Golgi complex, and was most prominent in granules present in the apical domain of the cell. Double labeling experiments with anti-surfactant protein A (SP-A) showed that both SP-A and SP-D were present in the same granules. However, SP-A was distributed throughout the granule contents, whereas SP-D was confined to the periphery of the granule. The Clara cell granules are considered secretory granules and not lysosomes, because they were not labeled for the lysosomal markers cathepsin D and LGP120, and they did not contain endocytosed BSAG.

Surfactant protein A is localized at the corners of the pulmonary tubular myelin lattice.

Immunogold labeling on sections of a freeze-substituted tubular myelin-enriched fraction isolated from a bronchoalveolar lavage of rat lung showed that surfactant protein A (SP-A) occurs predominantly at the corners of the tubular myelin lattice. Seventy-nine percent of the gold particles were located within 20 nm from a corner. Extracellular SP-A was detected only in the tubular myelin lattice and not in vesicles or secreted lamellar bodies. Ultra-thin cryosections of rat lung fixed in vivo showed that intracellular SP-A was distributed homogeneously over the stacked membranes of lamellar bodies in alveolar Type II cells. The presence of SP-A at the corners of the tubular myelin lattice suggests an important role of this protein in the formation and/or maintenance of this highly ordered lattice.

Detection of calcium ionophore induced membrane changes in dog sperm as a simple method to predict the cryopreservability of dog semen

The sensitivity of dog sperm cells for extracellular Ca2+/Ca2+‐ionophore challenge was compared to the detrimental effects of an optimized freeze/thawing protocol. Three sperm‐rich fractions of ejaculates from 9 dogs were obtained, and one aliquot of each ejaculate was washed in a modified Tyrodes medium (HBT containing 0.1 mM Ca2+), without (control sample) and with 2.5 μM Ca2+‐ionophore (induced sample) and incubated for 60 min at 38°C in humidified atmosphere. Another aliquot from the same semen fractions was diluted, washed in a Tris buffer, and packed into 0.5‐ml straws with a Tris buffer containing 7.5 vol % glycerol. The samples were stored for 1 week in liquid nitrogen after a computer‐driven three‐step freeze protocol and subsequently thawed for 50 sec in a 37°C water bath and reconstituted into HBT. The acrosome integrity was determined using fluorescein‐conjugated peanut agglutinin (PNA‐FITC) as an acrosomal marker, while the vitality of the sperm cells was simultaneously assessed with the membrane impermeable DNA supravital stain ethidium homodimer 1 (EthD‐1) using fluorescence microscopy and flow cytometry. The motility of frozen/thawed sperm samples was evaluated by microscopic as well as computerized motility analyses. Remarkably, the percentage sperm cells that underwent acrosome reactions induced by Ca2+‐ionophore correlated very positively (r = 0.93) with the amount of acrosome damage observed in cryopreserved sperm samples. Furthermore, the degree of cellular damage induced by Ca2+‐ionophore treatment correlated very negatively (r = −0.99) with the relative amount of sperm cells that remained motile after cryopreservation. Such clear correlations between Ca2+‐ionophore induced acrosome reaction and motility parameters for frozen/thawed dog sperm cells were not found, suggesting that the generation of acrosome leakage and sperm immotility are two independent detrimental processes occurring during cryopreservation. From these results it can be concluded that Ca2+‐ionophore treatment followed by simultaneous determination PNA‐FITC and EthD‐1 staining can be used to predict the cryopreservability of ejaculates from individual dogs used as donors. Mol. Reprod. Dev. 55:289–298, 2000.

Effect of sperm diluents on the acrosome reaction in canine sperm

In this study we investigated the influence of sperm diluting media and temperature on the incidence of the acrosome reaction in dog sperm. Ejaculates were collected from 5 dogs, diluted with six different media and then incubated at 37 degrees C and 20 degrees C. Fluorescein isothiocynate conjugated peanut agglutinin (FITC-PNA) and ethidium homodimer as a vital stain were used in combination to determine the acrosomal status of viable spermatozoa, the technique was validated using electron microscopy. The outer acrosomal membrane of dog spermatozoa was shown to be the specific binding site for FITC-PNA. After 6 h of incubation, ejaculates diluted in media with a high Ca2+ concentration showed a significantly higher percentage (means +/- SD) of acrosome reacted spermatozoa [64 +/- 7 and 58 +/- 9 in sperm capacitation medium with (SP-TALP-1) and without BSA (SP-TALP-2), respectively] than those diluted in media with a low Ca2+ concentration [36 +/- 5, 39 +/- 4, 18 +/- 2 and 20 +/- 4 in Canine Capacitation Medium (CCM), Egg Yolk Tris dog semen extender (EXT-1), Modified Egg Yolk Tris extender (EXT-2) and Modified CCM (MCCM), respectively]. The increase in the percentage of acrosome reaction (AR) was slower at 20 degrees C than at 37 degrees C. In addition, the percentage of viable acrosome reacted spermatozoa increased significantly from 19 +/- 5 and 22 +/- 3 in non-bound sperm to 27 +/- 4 and 30 +/- 6 in zona pellucida bound sperm (diluted in EXT-2 and MCCM, respectively). We conclude that the composition of the spermatozoa diluent has a marked effect on the incidence of the acrosome reaction. Therefore, both the media used to dilute dog sperm and the temperature at which the spermatozoa are handled are important factors to consider when processing spermatozoa for artificial insemination, IVF procedures or preservation.

Aerosolized endotoxin is immediately bound by pulmonary surfactant protein D in vivo

Collectins are carbohydrate binding proteins that are implicated in innate host defense. The lung collectins, surfactant proteins A and D (SP-A and SP-D), bind a variety of pathogens in vitro and influence phagocytosis by alveolar macrophages. In this report we show that SP-D binds endotoxin (lipopolysaccharide, LPS) in vivo in a rat model of acute respiratory distress syndrome (ARDS). Intratracheal aerosolization of LPS in rats resulted in the typical features of human ARDS. Total amounts of SP-D, as well as the carbohydrate binding properties of SP-D were measured in lung lavage as a function of time. The amount of SP-D did not change during 24 h. Interestingly, SP-D in lung lavage isolated from rats during the first 2 h after LPS treatment, was not able to bind to carbohydrate. Further analysis revealed that the carbohydrate binding sites of SP-D were occupied by LPS, suggesting that SP-D is an LPS scavenging molecule in vivo. Electron microscopic analysis indicated that, 1 h after LPS aerosolization, aggregates of SP-D with LPS were found in lysosomal structures in alveolar macrophages. We conclude that the lung collectin SP-D binds inhaled endotoxin in vivo, which may help to protect the lung from endotoxin-induced disease.

Cytofluorescence detection of adriamycin-mitochondria interactions in isolated, perfused rat heart

The major side-effect of the anthracycline anti-tumor drug adriamycin is a specific, dose-dependent cardiotoxicity. Impairment of mitochondrial function has been suggested to play an important role in this toxicity. The present study addresses the question as to whether direct drug-mitochondria interactions occur in the isolated, perfused rat heart. To this aim, cytofluorescence microscopy experiments were performed on thin cryosections. To demonstrate the applicability of this technique it is shown that adriamycin bound to isolated rat liver and heart mitochondria can be visualized through its characteristic fluorescence. Longitudinal sections from heart tissue perfused with 50 microM adriamycin display two distinct cellular sites of drug accumulation, i.e., nuclei which exhibit very bright fluorescence and, in addition, mitochondria which become significantly labeled with the drug. The mitochondrial localization of adriamycin is confirmed independently by quantification of the drug content of the mitochondrial fraction after cell fractionation. These results are discussed in the light of the potential role of adriamycin-nuclei versus adriamycin-mitochondria interactions in the deterioration of heart performance.

Steric hindrance in immunolabelling

In this paper we describe immunocytochemical detection of PhoE pore protein in the outer membrane of E. coli K‐12 cells in dependence of a variety of labelling approaches.

Accumulation of LamB-LacZ Hybrid Proteins in Intracytoplasmic Membrane-like Structures in Escherichia coli K12

The subcellular location of LamB-LacZ hybrid proteins in the Escherichia coli K12 strains pop3234 and pop3299 was investigated by immunocytochemical detection and protease-accessibility experiments. Induction of the synthesis of the hybrid proteins resulted in the appearance of membrane-like structures within the cytoplasm of the cells. Labelling of ultrathin cryosections of the cells with anti-beta-galactosidase or anti-LamB protein serum and protein-A-gold complexes revealed that the hybrid proteins were associated with these membrane-like structures or accumulated within the cytoplasm. Protease-accessibility experiments confirmed this localization. Moreover, when low quantities of hybrid proteins were produced, i.e. in uninduced pop3234 cells or in induced pop3299 cells, the hybrid proteins were accessible to trypsin from the periplasmic side of the inner membrane, leaving protected fragments with an apparent Mr of 83,000. Apparently, these hybrid proteins are partly translocated through the inner membrane, resulting in membrane-spanning forms of the proteins.

Visualization of contact sites between outer and inner envelope membranes in isolated chloroplasts

Abstract Contact sites between outer and inner envelope membranes of isolated chloroplasts have been studied at the ultrastructural level by two different electron microscopical methods, i.e., freeze fracturing and freeze substitution followed by ultrathin sectioning. Both methods demonstrate that approx. 10% of the chloroplast population exhibits blister-like structures. Cross fractures and ultrathin sections of freeze-substituted chloroplasts reveal that the blisters originated from a separation of the outer and inner envelope membranes. Exposure of isolated chloroplasts to hypertonic conditions results in an almost complete separation of the two envelope membranes, except for small regions in which the two membranes are in close contact. These contact sites are clearly recognized in freeze-fracture replicas by ridges of a high density of intramembrane particles. In addition, cross fractures and thin sections of freeze-substituted chloroplasts demonstrate the presence of small vesicles associated with the outer envelope membrane. As indicated by the opacity of the vesicle and demonstrated by immuno-gold labeling, these vesicles, which originate from the inner envelope membrane, contain stroma-derived proteins.

Congenital Alveolar Proteinosis in the Netherlands: A Report of Five Cases with Immunohistochemical and Genetic Studies on Surfactant Apoproteins

Congenital alveolar proteinosis is a recently described cause of lung dysfunction and respiratory distress in term neonates. In several cases a deficiency or insufficiency of surfactant apoprotein B (SP-B) has been caused by a frameshift mutation in the gene encoding SP-B. Five full-term children in three unrelated families from The Netherlands are reported. Immunohistochemistry demonstrated large amounts of surfactant proteins A and C (SP-A and SP-C) and precursors in alveolar cells and in intra-alveolar material. Results were positive for antibovine SP-B antibody but negative for antipig SP-B1 antibody, most probably reflecting differences in the antibody specificity. The findings suggest abnormal SP-B function. In two sibs, no pre-SP-C was demonstrated in the alveoli, although it was found in considerable amounts in alveolar cells. One such case has previously been reported. In two families, the parents were heterozygous for the 121 ins 2 mutation in the SP-B gene. Our findings suggest that congenital alveolar proteinosis may result from abnormalities in one or more of the surfactant proteins.