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Biomedical subjects

D Drenckhahn

Publications and source records attributed to D Drenckhahn.

At least 55 records · Page 3Linked to original sources
PubMed1996

Cytoskeletal markers allowing discrimination between brush cells and other epithelial cells of the gut including enteroendocrine cells.

Brush cells are specialised epithelial cells scattered throughout the simple epithelia of the respiratory and alimentary tracts. These cells have been suggested to serve a still unknown receptive function and use nitric oxide as a gaseous messenger molecule. At the light microscope level, brush cells can be identified by antibodies against the actin filament crosslinking proteins villin and fimbrin that not only stain the apical tuft of microvilli and their rootlets, but also label projections emanating from the basolateral surface of these cells. Since brush cells contain numerous intermediate filaments and microtubules and display a complicated basolateral cell morphology, we tested in this study whether antibodies against cytokeratin, tubulin and components of the membrane cytoskeleton might provide further markers for these cells at the light microscope level. Here we show that brush cells (identified by villin antibodies) can be discriminated from the neighbouring simple epithelium of the stomach, pancreatic duct and duodenum by particularly strong immunoreactivity with antibodies specific for cytokeratin 18. Tubulin antibodies reacted strongly with the upper half of brush cells in a pattern not observed in the other epithelial cells of these tissues, including enteroendocrine cells of the duodenum. Ankyrin, a protein that links the spectrin-based membrane cytoskeleton to integral proteins of the plasma membrane was revealed as a third cytoskeleton-associated protein, prominently expressed in brush cells where ankyrin is restricted to the basolateral membrane domain. The apparently high concentration of cytokeratin 18, tubulin and ankyrin in brush cells suggests that these cytoskeletal proteins might play a role in the mechanical stability and polarised organisation of these putative receptor cells.

Animals↗
PubMed1996

Dystrophin and the dystrophin-associated glycoprotein, beta-dystroglycan, co-localize in photoreceptor synaptic complexes of the human retina.

Mutations in the gene encoding for dystrophin, a membrane-associated cytoskeletal protein of muscle and several non-muscle cells, are the cause of Duchenne muscular dystrophy and Becker muscular dystrophy. Patients suffering from Duchenne muscular dystrophy have recently been shown to display an abnormal b-wave of the electroretinogram, suggesting that dystrophin is important for normal retinal transmission. In the retina, dystrophin has been localized in the outer plexiform layer where dystrophin co-localizes with postsynaptic markers of photoreceptor synaptic complexes. In the present study we addressed the question of whether two major dystrophin-associated integral membrane proteins of the muscular plasma membrane, beta-dystroglycan and adhalin, are also present in photoreceptor synaptic complexes. By double immunostaining and immunoblotting we show here that beta-dystroglycan is expressed in the human retina where it co-localizes with dystrophin in photoreceptor synaptic complexes most likely on the postsynaptic side. Adhalin was not detected in the retina. Since beta-dystroglycan is a member of a transmembrane supramolecular complex thought to be important for differentiation of the neuromuscular junction, it is an attractive hypothesis that dystroglycan (linked to dystrophin) might also play a similar role in differentiation of the photoreceptor synapse. A further outcome of this study is that beta-dystroglycan is not only present in the neuromuscular junction but also associated with a well-defined synaptic complex of the central nervous system. These findings indicate a more general role of this dystrophin-associated membrane protein in synaptic functions.

Adult↗
PubMed1996

Development of pericyte-like cells during angiogenesis in quail chick chimeras as detected by combined Feulgen reaction and immunohistochemistry.

Perivascular fibroblasts have been proposed as possible precursor cells for microvascular pericytes. To investigate the development of pericytes during angiogenesis we examined interspecific grafts between chick and quail embryos. Limb buds of three-day old quail embryos were transferred to the chorioallantoic membrane (CAM) of ten to fourteen day-old chick embryos. Six days after grafting, the limb buds were explanted and histologically examined by combined Feulgen reaction and immunohistochemistry using an antibody to quail endothelial and hemopoietic cells (QH-1). Limb buds were found to be vascularized by a network of capillaries which were partially derived from sprouts of the chick CAM microvasculature. Numerous hybrid capillaries were detected, consisting of host endothelial cells (chick) and graft pericytes (quail). These results provide further support for the idea that microvascular pericytes can evolve from perivascular fibroblasts.

Allantois↗
PubMed1996

Filovirus-induced endothelial leakage triggered by infected monocytes/macrophages.

The pathogenetic mechanisms underlying hemorrhagic fevers are not fully understood, but hemorrhage, activation of coagulation, and shock suggest vascular instability. Here, we demonstrate that Marburg virus (MBG), a filovirus causing a severe form of hemorrhagic fever in humans, replicates in human monocytes/macrophages, resulting in cytolytic infection and release of infectious virus particles. Replication also led to intracellular budding and accumulation of viral particles in vacuoles, thus providing a mechanism by which the virus may escape immune surveillance. Monocytes/macrophages were activated by MBG infection as indicated by tumor necrosis factor alpha (TNF-alpha) release. Supernatants of monocyte/macrophage cultures infected with MBG increased the permeability of cultured human endothelial cell monolayers. The increase in endothelial permeability correlated with the time course of TNF-alpha release and was inhibited by a TNF-alpha specific monoclonal antibody. Furthermore, recombinant TNF-alpha added at concentrations present in supernatants of virus-infected macrophage cultures increased endothelial permeability in the presence of 10 micron H2O2. These results indicate that TNF-alpha plays a critical role in mediating increased permeability, which was identified as a paraendothelial route shown by formation of interendothelial gaps. The combination of viral replication in endothelial cells (H.-J. Schnittler, F. Mahner, D. Drenckhahn, H.-D. Klenk, and H. Feldmann, J. Clin. Invest. 19:1301-1309, 1993) and monocytes/macrophages and the permeability-increasing effect of virus-induced cytokine release provide the first experimental data for a novel concept in the pathogenesis of viral hemorrhagic fever.

Base Sequence↗
PubMed1995

Identification of a binding motif for ankyrin on the alpha-subunit of Na+,K(+)-ATPase.

Cytoskeleton membrane associations are important for a variety of cellular functions. The anion exchanger of erythrocytes (AE1) and Na+,K(+)-ATPase of polarized epithelial cells provide well studied examples of how integral membrane proteins are anchored via the linker molecule ankyrin to the spectrin-based membrane cytoskeleton. In the present study we have generated several recombinant fragments of the large (third) cytoplasmic domain (CD3) of Na+,K(+)-ATPase to define binding sites of ankyrin on CD3 at a molecular level. We provide evidence that a cluster of four amino acids, ALLK, is essential for binding of ankyrin to both recombinant CD3 and to native Na+,K(+)-ATPase. Once bound, conformational changes might uncover further binding sites for ankyrin on Na+,K(+)-ATPase. A motif related to the ALLK cluster is also present in the cytoplasmic domain of AE1 where this sequence (ALLLK) turned out to be also important for ankyrin binding. These motifs are highly conserved during evolution of both Na+,K(+)-ATPase and AE1, further underlining their potential role in cytoskeleton to membrane linkage.

Amino Acid Sequence↗
PubMed1995

A deletional frameshift mutation in protein 4.2 gene (allele 4.2 Lisboa) associated with hereditary hemolytic anemia.

We studied a 26-year-old Portuguese patient with recessively transmitted hereditary hemolytic anemia. Protein 4.2 was absent from red cell ghosts by Western blotting. Although the 4.2 mRNA was not detected in Northern blots, it was shown to be present by a procedure based on nested reverse transcription-polymerase chain reaction (RT-PCR). Partial nucleotide sequencing disclosed a one-nucleotide deletion at nt 264 (or 265): AAG GTG-->AAG TG, in codon 88 (or 89) belonging to exon 2. This change, defining allele 4.2 Lisboa, placed in frame the nonsense triplet that normally overlaps codons 136 and 137 (GTG ACC). This mutation, which abolishes an EcoNI site, was also found in the gene of the proband (homozygous state), her parents, and her brother (heterozygous state). Apart from anemia, the patient was free of clinical manifestations. Platelet membranes were also investigated using Western blots. Antibodies to red cell protein 4.2 showed a doublet (72 and 70 kD) both in the controls and the patient. This finding raises an interesting question concerning the relationship between this doublet and erythroid protein 4.2.

Adult↗
PubMed1995

A novel, microcarrier-based in vitro assay for rapid and reliable quantification of three-dimensional cell migration and angiogenesis.

Angiogenesis in situ occurs within the interstitial extracellular matrix. The complexity of currently used three-dimensional in vitro angiogenesis systems makes it difficult to quantify cellular growth and neovessel formation. To overcome this problem we were interested to develop an angiogenesis system which allows rapid and reliable quantification of three-dimensional neovessel formation in vitro. Endothelial cells were seeded on gelatine-coated microcarriers (MCs). Cell-coated MCs were suspended in a solution of fibrinogen which was then induced to polymerize by addition of thrombin. By this way, MCs were entrapped in a three-dimensional fibrin matrix. Within a few hours, endothelial cells began to leave their supporting microcarriers and to migrate into the fibrin gel. Without addition of stimulators of angiogenesis, endothelial cells showed incoherent migration into the matrix. In contrast, in response to fibronectin, basic fibroblast growth factor (bFGF), or vascular endothelial growth factor (VEGF), respectively, endothelial cells assembled to form multicellular capillary-like structures occasionally exceeding 1000 microns in length. Each MC gave rise to a limited number of capillaries. A single culture dish contained hundreds of MCs, ensuring that a sufficient number of random samples was present for a reliable statistical evaluation. The angiogenic response could be easily quantified by determination of the average number of capillary-like formations per MC (cap/MC). The capillary count for macrovascular endothelial cells from the bovine pulmonary artery was 0.14 cap/MC when no angiogenic stimulators were contained within the fibrin gel. Addition of 200 micrograms/ml fibronectin increased capillary formation to 0.63 cap/MC (P < 0.0001) at Day 6. Already after 3 days, addition of bFGF (30 ng/ml) yielded a capillary count of 1.05 and addition of VEGF (100 ng/ml) resulted in 0.91 cap/MC. In contrast, addition of hyaluronic acid stimulated migration of dispersed endothelial cells into the fibrin matrix without leading to significant capillary formation (0.09 cap/MC). Hydrocortisone alone or in combination with heparin led to a significant inhibition of bFGF-stimulated angiogenesis. We thus have developed a convenient angiogenesis in vitro system which allows reliable quantification of capillary formation in a three-dimensional environment. Based on this assay we conclude that apart from proliferation and migration of endothelial cells, angiogenesis additionally requires the assembly of cells to form multicellular capillaries. This process is strongly induced by the extracellular matrix protein fibronectin. Hyaluronic acid, on the other hand, promotes migration but not capillary formation (assembly).

Animals↗
PubMed1995

Distribution of neurofilament-positive nerve fibres and sensory endings in the human anterior cruciate ligament.

The present immunocytochemical study describes the distribution of neurofilament-containing nerve fibres and corpuscular-like endings in the human anterior cruciate ligament. The entire anterior cruciate ligament of a non-injured knee joint from a child was cut into serial 15-microns-thick frozen sections, which were processed for immunofluorescence staining with a monoclonal antibody against the 68-kDa neurofilament subunit. Numerous neurofilament-positive fibres were found in bundles. These bundles were mostly located near blood vessels in the subsynovial layer and in interfascicular gaps. Only a few single nerve fibres were found independent of blood vessels in interfascicular gaps and between collagen bundles. Neurofilament-containing nerve fibres were preferentially located near the bony attachments of the anterior cruciate ligament. Two types of corpuscular-like endings were found, i.e. "spiral-like" (type I) and "spray-like" (type II) endings. Similarly to nerve fibres, both types of corpuscular-like endings were found mainly near the tibial and femoral attachment sites (15 of 17), whereas only two were found in the middle third of the ligament. Most likely, the type I and type II corpuscular-like endings serve a mechanoreceptive function involved in the sensory control of normal movements and in stress protection.

Anterior Cruciate Ligament↗
PubMed1995

A microcarrier-based cocultivation system for the investigation of factors and cells involved in angiogenesis in three-dimensional fibrin matrices in vitro.

Angiogenesis in situ includes coordinated interactions of various microvascular cell types, i.e., endothelial cells, pericytes and perivascular fibroblasts. To study the cellular interactions of microvascular cells in vitro, we have developed a microcarrier-based cocultivation system. The technical details of this method include seeding of endothelial cells on unstained cytodex-3 microcarriers and seeding of pericytes, fibroblasts or vascular smooth muscle cells on microcarriers which have been labeled by trypan blue staining. A mixture of both unstained and trypan blue-stained microcarriers was subsequently embedded in a three-dimensional fibrin clot. The growth characteristics of each cell type could be conveniently observed since the majority of cells left their supporting microcarriers in a horizontal direction to migrate into the transparent fibrin matrix. As differently stained microcarriers were randomly arranged in the fibrin matrix, the characteristic patterns of the microcarriers allowed location of particular points of interest at different developmental stages, facilitating the observation of cellular growth over the course of time. One further advantage of this microcarrier-based system is the possibility of reliably quantifying capillary growth by determination of average numbers of capillary-like formations per microcarrier. Thus, this model allows convenient evaluation of the effects of non-endothelial cells on angiogenesis in vitro. By using this coculture system, we demonstrate that endothelial capillary-like structures in vitro do not become stabilized by contacting vascular smooth muscle cells or pericytes during the initial stages of capillary formation.

Angiogenesis Inducing Agents↗
PubMed1994

The effect of fibroblasts, vascular smooth muscle cells, and pericytes on sprout formation of endothelial cells in a fibrin gel angiogenesis system.

We have recently shown that during angiogenesis in situ, sprouting and newly formed capillaries appear to be composed of two cell types, endothelial cells and nonendothelial, pericyte-like cells. The effect of pericytes on the process of neovessel formation is largely unknown. To study the influence of nonendothelial cell types on endothelial tubule formation, we have performed coculture experiments in a fibrin-clot angiogenesis system. When seeded below a critical density on the surface of fibrin gels, endothelial cells (from macro- or microvascular origin) did not show spontaneous formation of sprouts. However, in superconfluent cell cultures or after stimulation of endothelial cells with basic fibroblast growth factor (bFGF), endothelial cells frequently acquired an elongated shape. By stimulation of endothelial cells with both bFGF and vascular endothelial growth factor (VEGF), development of short capillary-like structures was induced. When endothelial cells were cocultivated with a cell type of high fibrinolytic potential, i.e., fibroblasts, development of capillary-like formations could not be detected. Cocultivation of endothelial cells with vascular smooth muscle cells or with retinal pericytes also did not increase the number of capillary-like formations in fibrin gels. In contrast, vascular smooth muscle cells on their own could be demonstrated to give rise to branched capillary-like networks in fibrin, which easily could be mistaken for true capillaries. Our results indicate that periendothelial cells contribute to angiogenesis not only by fibrinolysis and proteolytic permeation of the extracellular matrix. Rather, the interactions of endothelial cells and pericyte-like cells, as frequently observed during neovessel formation in situ, appear to be more specific and may require factors hitherto unknown.

Animals↗
PubMed1994

Basolateral localization of anion exchanger 2 (AE2) and actin in acid-secreting (parietal) cells of the human stomach.

Basolateral uptake of chloride by the HCl-secreting parietal cells of the gastric (oxyntic) glands is most likely mediated by a HCO3-/Cl- anion exchange mechanism. Circumstantial evidence indicates that in rodents the anion exchange proceeds through an anion exchanger 2(AE2)-like membrane protein. In the present study, we raised antibodies against a bacterial fusion protein expressing a approximately 26-kDa portion of the human AE2 sequence. These antibodies were used to identify and localize AE2 in the human stomach. Here we report that the mucosa of the human stomach expresses an approximately 160-kDa immunoreactive form of AE2 containing the AE2-specific exoplasmic domain (Z-loop) as identified by polymerase chain reaction. Immunostaining specific for AE2 was restricted to the basolateral membrane domain of parietal cells and was also detected in small epithelial cells localized in the glandular isthmus region. The latter cells most likely represent pre-parietal cells. Parietal cells were identified by simultaneous and sequential labelling with antibodies against the gastric H+,K(+)-ATPase and actin, respectively. Both actin and the H+,K(+)-ATPase were localized along the apical membrane of parietal cells and the membrane of their secretory intracellular canaliculi. In addition, actin was shown to be colocalized with AE2 along the basolateral cell surface. Discontinuous staining for AE2 coincided with infoldings of the basolateral plasma membrane labelled by the actin antibody. These observations indicate that AE2 might be placed at specialized (folded) microdomains of the basolateral cell surface by linkage to the actin-based cytoskeleton.

Actins↗
PubMed1994

Colocalization of cytokeratin 18 and villin in type III alveolar cells (brush cells) of the rat lung.

Alveoli of the rat lung are lined by three different cell types, the flat type I cells and the cuboidal type II and type III cells. Type III cells differ from type II cells by the presence of an apical tuft of microvilli and the absence of lamellar type secretory granules. In the present study we show by double immunolabelling that type III cells of the rat lung can be identified at the light- and electron microscope level by antibodies against both cytokeratin 18 and the actin-crosslinking protein villin. At the ultrastructural level, microvilli and their rootlets in the apical cytoplasm were labelled by the anti-villin antibodies, whereas a monoclonal antibody against cytokeratin 18 (Ks18.04) labelled bundles of intermediate filaments. In conclusion, antibodies against villin and certain monoclonal antibodies specific for cytokeratin 18 can be used as tools for selective visualization of type III cells in the rat lung.

Animals↗
PubMed1994

The fodrin-ankyrin cytoskeleton of choroid plexus preferentially colocalizes with apical Na+K(+)-ATPase rather than with basolateral anion exchanger AE2.

A unique feature of the choroid plexus as a single-layer epithelium is its localization of Na+K(+)-ATPase at its apical (lumenal) surface. In contrast, a band 3 (AE1)-related anion exchanger protein has been localized to the basolateral surface of the choroid plexus. Both Na+K(+)-ATPase and AE1 in other tissues have been shown to bind via ankyrin to the spectrin-actin-based membrane cytoskeleton. Since linkage of integral membrane proteins to the membrane cytoskeleton is important for their restriction to specialized domains of the cell surface, we investigated the polarity of the choroid plexus membrane cytoskeleton. We developed isoform-specific antibodies to confirm the identity of choroid plexus band 3-related polypeptide as AE2. We demonstrated that ankyrin, fodrin/spectrin, actin, myosin, and alpha-actinin are predominantly apical in choroid plexus and preferentially colocalize with apical Na+K(+)-ATPase rather than with basolateral anion exchanger AE2. Colchicine administration did not alter the polarity of apical cytoskeletal and transport proteins or basolateral AE2 in choroid plexus, suggesting that biosynthetic targeting of these proteins is not microtubule dependent. In choroid plexus papilloma, Na+K(+)-ATPase and AE2 were decreased in amount and failed to preserve their polarized distributions.

Animals↗
PubMed1994

Nitric oxide synthase and NADP-linked glucose-6-phosphate dehydrogenase are co-localized in brush cells of rat stomach and pancreas.

The epithelia of the respiratory and gastrointestinal tract and their appendages contain a distinct population of disseminated epithelial cells called brush cells or caveolated cells. On the basis of their structure, it was suggested that brush cells might serve as chemo- or volume receptors that play a role in certain aspects of gastrointestinal and bronchopulmonary secretion or motility. In the present study we provide first clues to a possible function of this widespread epithelial cell type. Brush cells of the rat gastric cardia and major pancreatic duct display strong immunoreactivity for nitric oxide synthase (NOS) and also exhibit high activity of NADPH-diaphorase. This NADPH-oxidizing activity was previously shown to be mediated by a specific domain of the sequence of the NOS. NADPH, in turn, appears to be delivered by glucose-6-phosphate dehydrogenase, which we found in brush cells at particularly high levels. We conclude that brush cells of the stomach and pancreas may represent a specialized population of paracrine cells that use nitric oxide as a messenger molecule to control certain gastrointestinal functions.

Amino Acid Oxidoreductases↗
PubMed1994

Intracellular distribution of kinesin in chromaffin cells.

In this paper we examined the association of the microtubule motor protein kinesin with organelles in chromaffin cells. Approximately 15% of kinesin was associated with membranes as determined by differential and equilibrium centrifugation on sucrose gradients. Kinesin was not enriched in a particular organelle fraction but cofractionated with a variety of organelle markers including markers for early and late endosomes, smooth and rough endoplasmic reticulum (ER) and the Golgi apparatus. Surprisingly, low amounts of kinesin were present in fractions of purified chromaffin granules. The absence of kinesin from the bulk of chromaffin granules was also indicated by immunostaining of tissue sections. A polyclonal antibody that specifically recognized the 120 kDa kinesin heavy chain labeled predominantly a perinuclear region that is typical for most of the kinesin-binding organelles identified by cell fractionation (endosomes, Golgi, ER). Since these organelles are compartments with high membrane turnover, we speculate that kinesin might be involved in certain aspects of trafficking of these membrane systems.

Animals↗
PubMed1993

Li(+)-induced structural changes of synaptic ribbons are related to the phosphoinositide metabolism in photoreceptor synapses.

Synaptic ribbons are specialized cytoskeletal components of the presynaptic exocytotic machinery in photoreceptors. In cone photoreceptors, these structures are highly dynamic disappearing during darkness and reforming in the light phase. In this study we wanted to characterize the second messengers involved in the regulation of these cyclic changes. We show that synaptic ribbons in cone photoreceptors are very sensitive to both Li+ and inositol 1,4,5-trisphosphate suggesting that inositol polyphosphates might play a physiological role in the disassembly of synaptic ribbons. The presence of the phosphoinositide pathway was demonstrated in isolated photoreceptor synapses. The phosphoinositide metabolism in photoreceptor synapses was dark-activated and correlated with the disappearance of synaptic ribbons during dark-adaptation. Since Li+ and inositol 1,4,5-trisphosphate only influenced synaptic ribbons in cones but not in rods the dark-activated phosphoinositide metabolism should be largely ascribed to cone synapses.

Animals↗
PubMed1993

Colocalization of retinal dystrophin and actin in postsynaptic dendrites of rod and cone photoreceptor synapses.

In this paper we demonstrate immunostaining specific for dystrophin in photoreceptor synapses of human, bovine and rat retinas. Cryosections of retinas incubated with dystrophin-specific monoclonal antibodies displayed a punctuate staining pattern in the outer plexiform layer. This pattern resulted from binding of the antibodies to synaptic complexes of both rods and cones, shown by double-labelling with antibodies to either synaptophysin or actin. Confocal laser fluorescence microscopy demonstrated that dystrophin staining colocalized predominantly with actin, which is concentrated in the postsynaptic portions of the synaptic complex. No significant dystrophin immunolabel was seen in the presynaptic terminals labelled with antibodies to synaptophysin, a marker of synaptic vesicles. Immunoblot analysis confirmed the presence of approximately 420 kDa and approximately 360 kDa dystrophin-like polypeptide bands associated with membranes of the bovine retina. We speculate that retinal dystrophin is involved in the linkage of actin filaments to the postsynaptic plasma membrane. Such a linkage may be important for the generation of synaptic microdomains and for certain phenomena of synaptic plasticity. The absence of dystrophin in patients suffering from Duchenne's muscular dystrophy is accompanied by visual problems and abnormalities of the electroretinogram. Therefore it is likely that retinal dystrophin plays a role in certain stages of synaptic transmission between photoreceptors and the postsynaptic dendritic complex formed by horizontal and bipolar cells.

Actins↗