Spontaneous formation of intercellular bile canaliculi and hybrid biliary-pancreatic canaliculi in co-culture of hepatocytes and exocrine pancreas cells from carp

When cultured together in a primary serum-free hormone-free system, hepatocytes and exocrine pancreas cells from the carp, Cyprinus carpio, spontaneously establish unique morphological structures that do not occur in vivo. These structures include intercellular bile canaliculi between neighbouring hepatocytes and hybrid canaliculi between hepatocytes and pancreas cells. In vivo, carp hepatocytes form only unicellular bile canaliculi; hybrid canaliculi between hepatocytes and exocrine pancreas cells do not exist at all in nature. This study shows that, in an artificial environment, cells are able spontaneously to establish novel morphological structures that are absent in the animal from which the cells have been obtained. Received: 3 January 1996 / Accepted: 17 March 1997     

Pathfinding of peripheral neurons in the central nervous system of an embryonic grasshopper (Chorthippus biguttulus)

The peripheral leg nerves of grasshoppers are initially formed by a set of pioneer neurons and guidepost cells. These cells are used as guiding structures for later-arising axons of sensory neurons. The development of the central projections of the pioneer cells, the guidepost cells and some sensory cells is shown with Lucifer Yellow injection or with DiI application. The axons of the pioneer cells Ti1 enter the central nervous system at 38% of embryonic development. They turn anteriorly close to the midline and ascend with no major branching to the brain. The axons of the guidepost cells Fe1 and Tr1 follow the same path but do not ascend to the brain. Sensory axons of the subgenual organ and the femoral organ probably do not follow the central path pioneered by the former neurons. They end ipsilaterally in the respective thoracic neuromere, as is found in the adult.     

Electron microscopic observations on the cholesterol distributed in the epithelial cells of the gallbladder.

Epithelial cells of eight human gallbladders with cholesterosis were examined. In the supranuclear portion of the epithelial cells of one case, many spicular, circular and plate-like crystalline structures are present. Spicular and circular structures have not a limiting membrane, but plate-like structures are apparently bounded by a limiting membrane that clearly shows trilamellar structure. After digitonin treatment, the spicular and circular crystalline structures become denser. On the other hand, the plate-like structures do not become denser by digitonization. In the epithelial cells that contain no crystalline structure, there also occur many reaction precipitates after digitonization. These findings may suggest that free cholesterol is highly present in the epithelial cells of gallbladder with cholesterosis and that it, in some case, precipitates in the form of spicular or circular structure for the rapid fixation process.     

Heat dissociation and maceration of marine Cnidaria

Summary The effect of increased temperature on the tissue integrity of polyps and medusae ofPodocoryne carnea is described. Animals exposed for 10 to 20 min to a temperature of 35°C are easily dissociated into single cells. These dissociated cells round up, form reaggregates and, depending on their origin, regenerate polyp or medusa structures. However, as the exposure time is increased, the dissociated cells gradually lose the ability to reaggregate or to regenerate defined structures. At incubation times exceeding 50 min, the tissue separates into single cells which retain their normalin vivo shapes but which do not form reaggregates. These are termed macerated cells. The ultrastructure and protein profile of macerated cells demonstrate no major changes from those of untreated cells. Both the dissociation and maceration methods are applicable to other cnidarian species for developmental, histological and biochemical studies.     

The Ascoma Development in Mycoporum elabens (Mycoporaceae, Dothideales)

Abstract: The structure and development of the ascomata in Mycoporum elabens were studied. No pseudoparaphyses or para-physoids are developed but the asci grow into a network formed by stretched ascostromatic tissue which appears similar to pseudoparaphyses at the mature stage. The ascomata do not open regularly but burst open at several points, thus imitating plurilo-cularous stromata. This development agrees with that observed for the Dothideales sensu stricto and so it is proposed to place the Mycoporaceae here. Although algal cells were found in the surroundings of the ascomata, they did not have close contact with the fungal hyphae. Thus, Mycoporum elabens is interpreted as being non-lichenized.     

Filamin-a and rheological properties of cultured melanoma cells

Here we report the rheological properties of cultured hsFLNa (filamin-A)-expressing (FIL+) and hsFLNa-deficient (FIL-) melanoma cells. Using magnetic twisting cytometry over a wide range of probing frequencies, and targeting either cortical or deeper cytoskeletal structures, we found that differences in stiffness of FIL+ versus FIL- cells were remarkably small. When probed through deep cytoskeletal structures, FIL+ cells were, at most, 30% stiffer than FIL- cells, whereas when probed through more peripheral cytoskeletal structures FIL- cells were not different except at very high frequencies. The loss tangent, expressed as an effective cytoskeletal temperature, was systematically greater in FIL- than FIL+ cells, but these differences were small and showed that the FIL+ cells were only slightly closer to a solidlike state. To quantify cytoskeletal remodeling, we measured spontaneous motions of beads bound to cortical cytoskeletal structures and found no difference in FIL+ versus FIL- cells. Although mechanical differences between FIL+ and FIL- cells were evident both in cortical and deeper structures, these differences were far smaller than expected based on measurements of the rheology of purified actin-filamin solutions. These findings do not rule out an important contribution of filamin to the mechanical properties of the cortical cytoskeleton, but suggest that effects of filamin in the cortex are not exerted on the length scale of the probe used here. These findings would appear to rule out any important contribution of filamin to the bulk mechanical properties of the cytoplasm, however. Although filamin is present in the cytoplasm, it may be inactive, its mechanical effects may be small compared with other crosslinkers, or mechanical properties of the matrix may be dominated by an overriding role of cytoskeletal prestress.     

60Ma of legume nodulation. What's new? What's changing?

Current evidence suggests that legumes evolved about 60 million years ago. Genetic material for nodulation was recruited from existing DNA, often following gene duplication. The initial process of infection probably did not involve either root hairs or infection threads. From this initial event, two branched pathways of nodule developmental processes evolved, one involving and one not involving the development of infection threads to 'escort' bacteria to young nodule cells. Extant legumes have a wide range of nodule structures and at least 25% of them do not have infection threads. The latter have uniform infected tissue whereas those that have infection threads have infected cells interspersed with uninfected (interstitial) cells. Each type of nodule may develop indeterminately, with an apical meristem, or show determinate growth. These nodule structures are host determined and are largely congruent with taxonomic position. In addition to variation on the plant side, the last 10 years have seen the recognition of many new types of 'rhizobia', bacteria that can induce nodulation and fix nitrogen. It is not yet possible to fit these into the emerging pattern of nodule evolution.     

F-actin aggregates in transformed cells contain alpha-actinin and fimbrin but apparently lack tropomyosin

Transformation-specific F-actin structures are examined in tumor cells after in vitro tumor cell growth alone or on an untransformed cell monolayer. In transformed cells F-actin aggregates near the ventral plasma membrane in close substrate adhesion areas contain the cytoskeletal proteins alpha-actinin and fimbrin but, unlike microfilament bundles, are not labeled with antibody against tropomyosin. By electron microscopy the dense ventral aggregates in transformed cells resemble stress fiber termini found at the membrane in normal cells. These transformed-cell cytoskeletal structures are not limited solely to substrate adhesion areas; they are also expressed at cell-cell contacts about 48 h after transformed cells are plated on untransformed cells. These specialized F-actin aggregates appear to be implicated in the processes of penetration of these transformed cells between adjoining untransformed cells in vitro.     

Antennal chemoreceptors of the desert burrowing cockroach, Arenivaga sp

The antennae of Arenivaga have six types of chemoreceptor sensilla. Some of these have unusual morphological features which may be adaptations for survival in a dry habitat. The sensory dendrites are well protected by cuticular structures, and in some receptors stimulatory molecules must pass through long channels or through pores filled with strands to reach the sensory cells. Large grooved pegs (possibly pheromone receptors) are numerous on antennae of adult males, and grooved sensilla are described here in detail for the first time. Thin-walled pegs, present in males and females, do not have pore tubules or hollow filaments as observed in many other insects. Rather, they contain structures designated here as pore strands, since they have a dense core rather than a light center as previously described for pore tubules and filaments. These strands do not appear to be evaginations of the dendritec membrane, but are probably formed in association with the cuticular structures of the sensilla.     

Specific attachment of desmin filaments to desmosomal plaques in cardiac myocytes

Intercellular junctions which are similar in ultrastructure and protein composition to typical desmosomes have so far only been found in epithelial cells and in heart tissue, specifically in the intercalated disks of cardiac myocytes and at cell boundaries between Purkinje fiber cells. In epithelial cells the cytoplasmic side of desmosomes, the 'desmosomal plaque', represents a specific attachment structure for the anchorage of intermediate filaments (IF) of the cytokeratin type. Cardiac myocytes do not contain cytokeratin filaments. In primary cultures of rat cardiac myocytes, we have examined by immunofluorescence and electron microscopy, using single and double label techniques, whether other types of IF are attached to the desmosomal plaques of the heart. Antibodies to desmoplakin, the major protein of the desmosomal plaque, have been used to label specifically the desmosomal plaques. It is shown that the desmoplakin-containing structures are often associated with IF stained by antibodies to desmin, i.e., the characteristic type of IF present in these cells. Like cytokeratin filaments in epithelial cells, desmin filaments attach laterally to the desmosomal plaque. They also remain attached to these plaques after endocytotic internalization of desmosomal domains by treatment of the cells with EGTA. These desmin filaments do not appear to attach to junctions of the fascia adherens type and to nexuses (gap junctions). These observations show that anchorage at desmosomal plaques is not restricted to IF of the cytokeratin type and that IF composed of either cytokeratin or desmin, specifically attach, in a lateral fashion, to desmoplakin-containing regions of the plasma membrane. We conclude that special domains exist in these two IF proteins that are involved in binding to the desmosomal plaque.     

The chick oviduct in tissue culture. I. Initial characterization of growing primary oviduct tissue cultures

A simple three-enzyme treatment of collagenase, dispase and hyaluronidase on finely minced chick oviduct yields clumps of 50-150 cells. These cells attach to collagen-treated dishes and survive in culture for at least 2 weeks without subculturing. Oviduct cell cultures can also be induced to grow. Estradiol or epidermal growth factor (EGF) induce a 40% increase in cells in 4 days when cultures are grown in serum levels that do not support growth. Serum from estrogen-stimulated chicks promotes rapid cellular proliferation (doubling times of 1-2 days). Sera from estrogen withdrawn chicks, laying hen or horse do not support as rapid proliferation. The oviduct growth-promoting factors in serum from estrogen-stimulated chicks are not steroids or fibroblast growth factors (FGF). Removal of steroids from these sera by charcoal treatment or delipidization does not decrease the rate of growth. The addition of 1-100 nM estradiol does not increase a serum's ability to promote growth. Purified FGF or platelet-derived growth factor (PDGF) do not induce oviduct proliferation. These results were reproduced in oviduct cell cultures started from estrogen-stimulated and withdrawn chicks as well as laying hens. Thus the factors in serum from estrogen-stimulated chicks that promote rapid oviduct growth are induced by estrogen treatments in vivo, but do not seem to be only steroids.     

Cell junctions in the early chick embryo--a freeze etch study

Cell junctions in the early chick embryo have been examined in freeze-etch specimen. Well developed zonulae occludentes are found in the epiblast as early as stage 4. Large gap junctions are also found in the epiblast at this stage. In those cells which have left the surface to form mesenchymal structures (Hensen's node, juxtanodal mesenchyme, primitive streak mesenchyme), one finds not only gap, but also tight, junctions. These junctions do not form continuous belts, but appear as fragments, often reduced to single strands, of typical tight junctions. They probably correspond to the focal tight junctions described earlier in sectioned material. The origin and possible significance of these contacts is discussed, and it is suggested that they represent remnants of junctions between neighboring cells in the epiblast. These junctional remnants slowly disappear by “dilution,” either through cell division and/or cell movement. The appearance of newly formed gap junctions is also described.     

On the occurrence of twisted fibrillar structures in the cytoplasm of the red algaAlsidium helminthochorton (La Tourette) Kütz., ultrastructural and cytochemical observations

Summary The cytoplasm ofAlsidium cells contains unusual structures showing various fibrillar arrangements, either stacked rows of arcs or parallel fibrils in longitudinal view alternating with fibrils in cross-section. These regions, located exclusively in the cytoplasmic matrix, are highly proteinaceous and do not seem to be complexed with polysaccharides. Tilting observations under the electron microscope show that the appearance of arcs is an illusion and that the pattern fits the explanation given byBouligand and collaborators for various twisted biological structures.     

The differentiation behaviour of MDCK cells grown on matrix components and in collagen gels

MDCK cells are grown on various substrates (Thermanox pure, extracellular matrix (ECM), dried or wet collagen type I or type III), on floating collagen and enclosed in collagen gels, and their differentiation behaviour is investigated electron microscopically. The cells grown on ECM or dried collagen (type I and type III) do not show any changes as compared with the controls (Thermanox). Differentiation processes can only be observed when the cells are grown on wet collagen (type I and type III), especially on floating collagen and enclosed in collagen gels. These differentiation processes comprise changes in the cell shape, an increase in the number of microvilli, an increase in the length of the lateral contact zone with the formation of gap junctions and desmosomes, and an increase in the number and size of the cell organelles. A basement membrane only develops in the form of short segments. Moreover, on floating collagen and in collagen gels three-dimensional, organoid structures develop: cell aggregates with central lumina and tubuli. They are formed by cuboid cells that also exhibit indications of differentiation. Basement membrane fragments occur more often and are longer. It can be concluded from these findings that the chemical structure of the substrate does not play the primary role in the described process. It is rather the physical properties, probably the plasticity, that are of significance. Due to this property the cells change their shape and the contact areas increase in size. The establishment of contacts might be the triggering factor for differentiation. Organoid structures with lumina develop when the apical surface comes into contact with other cells or collagen gels. The pronounced tendency towards polarization necessitates a re-arrangement of three-dimensionally growing cells to structures with lumina. The formation of the basement membrane is the result and not the cause of differentiation.     

Electron microscopical observation of RNA on the surface of Ehrlich ascites tumor cells

The structure of RNA on the surface of Ehrlich ascites tumor cells was studied under an electron microscope using both plasma polymerization replica films and ultrathin sections of the cells. Necklace-like structures were found on the cell surface where anti-RNA antibody was bound in replica film, and particles which resemble cytoplasmic ribosomes in size and density were found distributed sparsely on the cell surface in ultrathin sections. These particles were found to gather at one pole of the cell surface after the cell was incubated at 4 degrees C with anti-RNA antibody and then incubated at 37 degrees C for 10 min in antibody-free medium. On the other hand, L1210 cells which do not bind with anti-RNA antibody showed hardly any such structures on the cell surface. These results suggest that RNA on the surface of Ehrlich ascites tumor cells is present in the form of particles.     

Interaction Between TRPC Channel Subunits in Endothelial Cells

Transient Receptor Potential Canonical (TRPC) proteins have been identified in mammals as a family of plasma membrane calcium-permeable channels activated by different kinds of stimuli in several cell types. We have studied TRPC subunit expression in bovine aortic endothelial (BAE-1) cells, where stimulation with basic fibroblast growth factor (bFGF), a potent angiogenetic factor, induces calcium entry carried at least partially by TRPC1 channels. By means of a RT-PCR approach, we have found that, in addition to TRPC1, only TRPC4 is expressed, both at the mRNA and protein level, as confirmed by immunoblotting and immunocytochemical analysis. Because functional TRPC channels are formed by assembly of four subunits in either homo- or heterotetrameric structures, we have carried out immunoprecipitation experiments and showed that TRPC1 and TRPC4 interact to form heteromers in these cells, independently from culture conditions (high or low percent of fetal calf serum, stimulation with bFGF). Moreover, the data show that TRPC subunits are not tyrosine-phosphorylated after bFGF stimulation and they do not co-immunoprecipitate with the type 1 FGF receptor. These results suggest that BAE-1 cells are a suitable model to study function and regulation of endogenous TRPC1/TRPC4 heteromers.     

Interaction between TRPC channel subunits in endothelial cells

Transient Receptor Potential Canonical (TRPC) proteins have been identified in mammals as a family of plasma membrane calcium-permeable channels activated by different kinds of stimuli in several cell types. We have studied TRPC subunit expression in bovine aortic endothelial (BAE-1) cells, where stimulation with basic fibroblast growth factor (bFGF), a potent angiogenetic factor, induces calcium entry carried at least partially by TRPC1 channels. By means of a RT-PCR approach, we have found that, in addition to TRPC1, only TRPC4 is expressed, both at the mRNA and protein level, as confirmed by immunoblotting and immunocytochemical analysis. Because functional TRPC channels are formed by assembly of four subunits in either homo- or heterotetrameric structures, we have carried out immunoprecipitation experiments and showed that TRPC1 and TRPC4 interact to form heteromers in these cells, independently from culture conditions (high or low percent of fetal calf serum, stimulation with bFGF). Moreover, the data show that TRPC subunits are not tyrosine-phosphorylated after bFGF stimulation and they do not co-immunoprecipitate with the type 1 FGF receptor. These results suggest that BAE-1 cells are a suitable model to study function and regulation of endogenous TRPC1/TRPC4 heteromers.     

Some structural,biochemical and biophysical characteristics of L-929 cells growing in the presence of hyperosmotic sorbitol concentrations

Mouse L-929 cells (a fibroblast-like line) were transferred from normal growth medium to one supplemented with 0.3 M sorbitol, doubling the normal external osmotic pressure. After a short lag phase and minimal cell death, the cells began to grow, and the growth rate reached that of controls after about one week. These chronically grown cells (S) have been compared to those of control cultures (C) with regard to general morphology, ability to reverse when returned to normal condition, water content, volume and selected metabolic parameters. S-cell cultures exhibited considerable heterogeneity but most contained vesicle-like cytoplasmic structures, sometimes in abundance. These structures do not appear to be completely bounded by membranes, but that is uncertain. S cells become larger and contain more water than C cells; however, the ratio of total water to total dry mass is indistinguishable from controls suggesting regulation at that level. S and C cells were found to be remarkably similar, on a per cell basis, with regard to their rate of respiration and the incorporation of glucose into metabolites and macromolecules. These results are interpreted in terms of current views on the composition and organization of the aqueous compartments of animal Cells.     

Epithelium of lips and associated structures of the Indian major carp,Catla catla

Structural organization of the epithelium of the lips and associated structures of the Indian major carp,Catla catla, is described. The upper lip is thin and is associated on its dorsal side with a membranous fold of skin and the rostral cap. In contrast, the lower lip is thick and very conspicuous. It is associated on its ventral side with a fold of skin between it and the ventral head skin. The lower lip is divided into a non-projectile portion, a projectile portion and an intermediate groove region. The projectile portion remains folded covering a part of the ventral head skin when the mouth is closed. Their role in relation to the formation of the characteristic feeding tube is discussed. The epithelium of the lips and associated structures is stratified in nature and is composed of the epithelial cells, mucous cells, club cells, lymphocytes and the taste buds. The mucous cells are small, few or even absent and do not appear to secrete profusely at the surfaces of the upper and the lower lips. This suggests that the lips inCatla catla, which feeds on micro-organisms, do not need extra lubrication for protection against abrasion during feeding. In the epithelium at the folds of skin, the voluminous mucous cells secrete profusely and provide extra lubrication to their surface. This reduces the resistance to surface drag during stretching and enables the jaws to protrude with increasing efficiency and swiftness. The club cells are developed additionally to complement the mucous cells in the rostral cap and the upper lip epithelium. Their primary function appears protective in some way, which needs further confirmation. The taste buds, though few in the lower lip, are located in a good number in the upper lip on the characteristic epithelial papillae-like projections, and are projected at the surface. These have been associated with the acute gustatory sense of the fish. The taste buds are absent on the folds of skin where they may not be of much significance.