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AbstractTo evaluate the involvement of frizzled receptors (Fzds) in oncogenesis, we investigated mRNA expression levels of several human Fzds in more than 30 different human tumor samples and their corresponding (matched) normal tissue samples, using real-time quantitative PCR. We observed that the mRNA level of Fzd5 was markedly increased in 8 of 11 renal carcinoma samples whilst Fzd8 mRNA was increased in 7 of 11 renal carcinoma samples. Western blot analysis of crude membrane fractions revealed that Fzd5 protein expression in the matched tumor/normal kidney samples correlated with the observed mRNA level. Wnt/β-catenin signaling pathway activation was confirmed by the increased expression of a set of target genes. Using a kidney tumor tissue array, Fzd5 protein expression was investigated in a broader panel of kidney tumor samples. Fzd5 membrane staining was detected in 30% of clear cell carcinomas, and there was a strong correlation with nuclear cyclin D1 staining in the samples. Our data suggested that altered expression of certain members of the Fzd family, and their downstream targets, could provide alternative mechanisms leading to activation of the Wnt signaling pathway in renal carcinogenesis. Fzd family members may have a role as a biomarker.Copyright © 2004 S. Karger AG, Basel

The present study investigates whether aluminium-transferrin (Al-Tf) uptake by Tf receptor-mediated endocytosis induces hypoparathyroidism and thus might contribute to the increasing prevalence of adynamic bone disease (ABD) in the current dialysis population.

The distribution of alcian blue (AB) positivity, and its sensitivity to streptococcal and testicular hyaluronidase, were studied in primitive streak stage chick blastoderms. Accumulation of hyaluronate was observed in deep layer (DL) cells and on laterally migrating middle layer (ML) cells. During the formation of the middle layer, a first stage, namely de-epithelialization of the upper layer cells, is recognized and correlated with the absence of hyaluronate. A second stage, namely migration of the de-epithelialized upper layer cells laterally to the edge of the area pellucida, is correlated with the accumulation of AB-positivity.

Primitive streak stage chick blastoderms were cultured for 30 min on a medium containing tritiated glucosamine. Light microscope autoradiography revealed extracellular labeling, and pretreatment of the sections with testicular hyaluronidase suggested the glycosaminoglycan nature of the labeled products.

The intercellular contacts of the migrating edge of chick and quail blastoderms during gastrulation were studied by transmission electron microscopy of thin sections and of freeze-fracture replicas. Tight junctions and gap junctions as well as desmosomes were found. Tight junctions were organized as single junctional strands or as a complex of numerous junctional strands interposed between the lamellae and the bodies of the cells building up the margin of overgrowth. The function of these intercellular junctions is considered in relation to the locomotion of the margin of overgrowth cells.

Selective permeability of endocardial endothelium has been suggested as a mechanism underlying the modulation of the performance of subjacent myocardium. In this study, we characterized the organization and permeability of junctional complexes in ventricular endocardial endothelium in rat heart. The length of intercellular clefts viewed en face per unit endothelial cell surface area was lower, and intercellular clefts were deeper in endocardial endothelium than in myocardial vascular endothelium, whereas tight junctions had a similar structure in both endothelia. On this basis, endocardia endothelium. might be less permeable than capillary endothelium. However, confocal scanning laser microscopy showed that intravenously injected dextran 10000 coupled to Lucifer Yellow penetrated first the endocardial endothelium and later the myocardial capillary endothelium. Penetration of dextran 10000 in myocardium occurred earlier through subepicardial capillary endothelium than through subendocardial capillary endothelium. Penetration of tracer might thus be influenced by hydrostatic pressure. Dextran of MW 40000 did not diffuse through either endocardial endothelium or capilary endothelium. The ultrastructure of endocardial endothelium may constitute an adaptation to limit diffusion driven by high hydrostatic pressure in the heart. Differences in paracellular diffusion of dextran 10000 between endocardial endothelium and myocardial vessels, may result from differing permeability properties of the endocardium and underlying myocardium.

SEM reveals that the subgerminal yolk surface of the chick and quail embryo during stages X to XIV possesses microvilli and small pits. Threadlike extensions and globular structures are found on the subgerminal yolk surface mainly in the central area. The cellular nature of the subgerminal yolk was confirmed with TEM that showed the presence of a plasma membrane, mitochondria, micro-invaginations and microfilaments. The ventral cells of the germ wall are yolky and can be attached to the subgerminal yolk surface with filopodial extensions during stages X to XII. From stage XIII, the shape of these cells is usually more flattened and they protrude lamellae and filopodia.

The epiblast and the surface of the perigerminal yolk of the just laid quail blastoderm were examined by scanning electron microscopy (SEM). The dorsal surface structures are microvilli, mainly along the cell borders. The scarcity of the dimples does not support that ingression occurs at this stage. Flat or round cells on the epiblast are possibly deep layer cells that failed to incorporate after passing through the epiblast. The majority of the blastoderms have an irregular margin. The large edge cells possess microvilli at their borders only. A few blastoderms, probably the more developed, have a smooth edge with closely packed cells. The margin of these germs shows round cells and lamellae that could be protruded by deep cells. The process of cell rounding and extension of lamellae may be the onset of the formation of the margin of overgrowth. Concentric zones are present on the surface of the perigerminal yolk, on which microvilli and blebs are found near the germ. The presence of cell projections on the perigerminal surface suggests its living nature.

The blastoderm edge migrates by the active locomotion of a multilayer of epithelial cells, the so-called margin of overgrowth (MO), that uses the vitelline membrane as its substratum. The structural unity formed by the margin of overgrowth cells and their rapid migration suggest coordination of locomotion between individual cells. Using transmission electron microscopy of thin sections and freeze-fracture, we attempted to determine if the pattern of junctions of the migrating margin of overgrowth is related to the suggested cell-cell cooperation between individual cells in this region. In the leading edge there are large areas of closely apposed cell membranes. Incipient desmosomes and small gap junctions were observed. Tight junctions consisted of isolated strands or isolated networks of tight-junctional strands. In the proximal part of the margin of overgrowth the size of the gap junctions increased and the desmosomes were fully developed. Tight-junctional strands were either isolated or arranged into an isolated network. A broad belt of tight junctions was observed at the transition between margin of overgrowth and non-marginal cells. The distribution of the junctional elements in the MO suggests that junctions contribute to the maintenance of the structural and functional organization of the margin of overgrowth. Furthermore, the spatial distribution of the junctions might give information about the mechanism of locomotion of the margin of overgrowth.

The ventral surface of the upper layer shows a band of extracellular fibrils around the anterior and lateral border of tfieareapellucida during gastrulation of the chick embryo. Using scanning electron microscopy, we found that this disposition is correlated with the motility of the middle-layer cells of gastrulating chick and quail embryos. Outside the fibrous band, single middle-layer cells and a sheet of mesoblast cells were spread out and possessed lamellae. Single cells on the fibrous band did not form lamellae. The same cell behaviour was obtained with the expiants of deep layer on the fibrous band. The fibrous band is assumed to operate as a barrier that inhibits cell motility during gastrulation.

The cytoskeleton in endocardial endothelium of rat heart was examined by en face confocal scanning laser microscopy. In the ventricular cavity, endocardial endothelial cells had a polygonal shape and F-actin staining was generally restricted to the peripheral junctional actin band. Central F-actin bundles, or stress fibers, in endocardial endothelial cells were found on the tendon end of papillary muscles, especially in the right ventricle, and frequently in the outflow tract of both ventricles; elsewhere, stress fibers were scarce. Many endocardial endothelial cells were elongated in areas of endothelium with stress fibers, but no correlation was found between cell elongation and the number of stress fibers. An inverse correlation was found between the number of stress fibers and the surface area of endocardial endothelial cells. Shear stress as well as mechanical deformation of the surface of the ventricular wall during the cardiac cycle may affect cell shape and the organization of actin filaments in endocardial endothelial cells. Vimentin in endocardial endothelial cells formed a filamentous network with some distinct cytoplasmic and juxtanuclear vimentin bundles. No perinuclear ring of vimentin filaments was observed in endocardial endothelium. Microtubules in endocardial endothelial cells were, in contrast to endothelial cells of rat aorta, not aligned, less closely packed and originated from randomly distributed centriolar regions. The cytoskeleton has been suggested to play an important role in cellular functions of vascular endothelial cells. Accordingly, differences in the cytoskeletal organization between endocardial and vascular endothelial cells may relate to differences in functional properties.

Scanning electron microscopy was used to investigate the surface architecture of the human tympanic membrane. The morphology of the eardrum was compared with the surface structures of preserved tympanic membranes (allografts), fresh air-dried temporalis fascia and preserved calf jugular veins (xenografts). The role of the physical structure and the composition of the extracellular matrix in the regeneration of a tympanic graft is discussed.

Acid mucopolysaccharides in the extracellular compartment of early chick blastoderms (16 h of incubation) were labelled with tritiated glucosamine and/or [35S]sulphate. The incorporation pattern was studied autoradiographically. Treatment with testicular hyaluronidase revealed a testicular hyaluronidase-sensitive fraction, mainly at the periphery of Middle Layer and Deep Layer cells, and a testicular hyaluronidase-resistant fraction, mainly at the ventral side of the Upper Layer. A biochemical analysis, utilizing chondroitinase ABC and nitrous acid, followed by cellulose acetate electrophoresis, demonstrated the synthesis of a non-sulphated fraction, i.e. hyaluronic acid and/or chondroitin, and a sulphated fraction, comprising two undersulphated components, i.e. chondroitin sulphate, and heparan sulphate or heparin. The appearance of different AMPS in specific areas of the early chick blastoderm is regarded as an early specialization of the extracellular compartment.