A novel antiapoptotic mechanism based on interference of Fas signaling by CD44 variant isoforms.

There is growing evidence that one of the central common characteristics of tumor and inflammatory cells is their resistance to programmed cell death. This feature results in the accumulation of harmful cells, which are mostly refractory to Fas (FAS, APO-1)-mediated apoptosis. A molecule found on these cells is the transmembrane receptor CD44 with its variant isoforms (CD44v). The establishment of transfectants expressing different CD44v isoforms allowed us to demonstrate that the CD44v6 and CD44v9 isoforms exhibit an antiapoptotic effect and can block Fas-mediated apoptosis. Moreover, we observed that CD44v6 and CD44v9 colocalize and interact with Fas. Importantly, an anti-CD44v6 antibody can abolish the antiapoptotic effect of CD44v6. These results are the first to show that CD44v isoforms interfere with Fas signaling. Our findings improve the understanding of the pathogenesis of cancer and autoimmunity and open new strategies to treat such disorders.

Extrahepatic obstructive cholestasis reverses the bile salt secretory polarity of rat hepatocytes.

To elucidate the consequences of extrahepatic cholestasis on the structure and function of hepatocytes, we studied the effects of bile duct ligation on the turnover, surface distribution, and functional activity of the canalicular 100-kD bile salt transport protein (cBSTP). Basolateral (blLPM) and canalicular (cLPM) liver plasma membrane vesicles were purified to the same degree from normal and cholestatic rat livers and the membrane bound cBSTP identified and quantitated using polyclonal anti-cBSTP antibodies. Cholestasis of 50 h resulted in an increased release of cBSTP into bile, thereby decreasing its in vivo half-life from 65 to 25 h. Furthermore, a significant portion of cBSTP accumulated at the basolateral surface and in intracellular vesicles of cholestatic hepatocytes. This redistribution of cBSTP was functionally paralleled by decreased and increased electrogenic taurocholate anion transport in cLPM and blLPM vesicles, respectively. These results demonstrate that biliary obstruction causes a reversal of the bile salt secretory polarity of rat hepatocytes. The resulting increase in basolateral (sinusoidal) bile salt efflux might protect hepatocytes from too high an accumulation of toxic bile salts within the cell interior.

Acylglucosylceramide causes flattening and stacking of liposomes. An analogy for assembly of the epidermal permeability barrier.

When 5% of 1-(3'-O-acyl)-beta-D-glucosyl-N-(omega-hydroxyacyl)sphingosine, isolated from pig epidermis, was added to distearoylphosphatidylcholine and cholesterol (10:1), the lipid mixture formed liposomes in phosphate buffer which were flattened and aggregated like stacks of coins. Other glycolipids from pig epidermis did not cause this phenomenon. This supports the hypothesis that the acylglucosylceramide is responsible for assembly of the lamellar granules found in epidermal cells.

Early development of the human mesonephros.

The mesonephrogenic cord disintegrates into approximately 35-40 provesicular cell masses which are in close contact with the mesonephric (Wolffian) duct (WD) on their lateral side. Here, the epithelium of the WD is columnar and shares a common basal lamina with the provesicular cell masses. This in turn gives rise to a sickle-shaped pseudostratified epithelium. The concavity of the sickle is filled by spherical cells, the transition of which into the surrounding connective tissue is continuous. The sickle is transformed into a distillation flask and becomes separated from the mesonephric duct while the spherical cells maintain a connection to it by a-for the time being-solid outlet pipe. The columnar epithelium of the mesonephric duct becomes a multilayered cone, whose surface is in contact with the outlet tube. Shortly after, a continuous lumen is formed in the cone and the outlet pipe which is delimited by cells becoming columnar and forming a basal lamina. The epithelial anlage of the nephron is clearly separated from the surrounding mesenchyma by these processes. The flask eventually becomes a corpusculum, the outlet pipe a secretory (proximal) as well as collecting tubule, and the cone of the mesonephric duct a mesoureter. The various sections display differentially differentiated epithelia that are clearly distinct from each other. The mesoureter behaves differently during differentiation of epi- and paragenitale: in the epigenitale, it is short and runs into the collecting tubules of the nephrons at the lateral side of the convolved tubules, whereas a long mesoureter crosses the dorsal side of the convolved tubules and joins the corresponding collecting tubules at the far end of the mesonephros in the paragenitale.

The epidermal permeability barrier. Comparison between in vivo and in vitro lipid structures.

The epidermal permeability barrier is established by the lamellar contents of membrane-coating granules which are discharged into the intercellular space of the stratum granulosum and form continuous lipid layers in the stratum corneum. Artificial lipid systems, prepared with a composition similar to that found in stratum granulosum and stratum corneum, were able to form a lamellar phase. These systems show dense line thickness and center-to-center spacing comparable to those found in membrane-coating granules and intercellular layers. The significance of lipid composition in relation to barrier function is discussed and a model showing the molecular arrangement of the lipid structures in the epidermal barrier is proposed.

Epidermal permeability barrier: transformation of lamellar granule-disks into intercellular sheets by a membrane-fusion process, a freeze-fracture study.

Freeze-fracture replication of lamellar granules and intercellular sheets of the horny layer in mouse, chicken, and snake epidermis reveals a pattern of serial fracture faces which is highly suggestive of polar lipids in a bilayer configuration. The occurrence of alternating wide and narrow fracture faces separated by intervening steps supports the view that epidermal barrier bilayers display lipid asymmetry similar to membranes. Within the lamellar granules, bilayers arrange to form disks which in fact are equivalent to flattened unilamellar liposomes. Stacking of the disks in turn gives rise to the lamellar pattern. After exocytosis into the intercellular space, the disks are arranged parallel to the cell membranes. In tangentially fractured specimens, the cleavage plane jumps back and forth from the plasma membrane to a disk-bilayer, thereby giving rise to the known phenomenon of EF-ridges (on the extracellular fracture face) and PF-grooves (in the plasmatic fracture face) which both represent the level of the plasma membrane sur- or subjacent to the aisles between disks. Concomitantly with the upward movement of the keratinocytes, the ridges and grooves become narrower until they fade away by the second or third cell layer of the stratum corneum. This phenomenon is explained by the fusion of adjacent disks at their highly curved brims due to a mechanism similar to the process of membrane fusion which causes the formation of wide, uninterrupted sheets.

Stratum corneum lipid liposomes: calcium-induced transformation into lamellar sheets.

The epidermal water barrier in mammalian stratum corneum is formed of broad lamellar sheets of lipids consisting principally of ceramides (40%), cholesterol (25%), cholesteryl sulfate (10%), and free fatty acids (25%). Such lipid mixtures have been shown to form lipid bilayers in the form of small, unilamellar liposomes when sonicated at 80 degrees C in water containing Tris buffer and 100 mM NaCl. In the present study it is shown that such liposomes are slowly transformed into large unilamellar liposomes and then into broad lamellar sheets after the addition of stoichiometric amounts of calcium chloride. The presence of free fatty acids was a necessary condition for this calcium-induced fusion. These observations may provide a useful analogy for the transformation of flattened liposomes into broad lamellar sheets that occurs during transition of epidermal granular cells into corneocytes.

Preparation of liposomes from stratum corneum lipids.

Mammalian stratum corneum contains multiple intercellular lipid bilayers that constitute the epidermal water barrier. Unlike all other biologic membranes, the epidermal lamellae do not contain phospholipids, as a result of which the ability of the stratum corneum lipid mixture to form bilayers has been questioned. In the present study, a lipid mixture containing only epidermal ceramides (40%), cholesterol (25%), palmitic acid (25%), and cholesteryl sulfate (10%), approximating the composition of stratum corneum lipids, formed stable, unilamellar liposomes when sonicated at 80 degrees C in buffer containing 100 mM NaCl, 5 mM Tris, and 1 mM EDTA at pH 7.5. The size and form of the liposomes were studied by both freeze fracture and negative staining electron microscopy. Lipid mixtures from which either the palmitic acid or the cholesteryl sulfate were omitted were still capable of forming similar liposomes, but a mixture of ceramides and cholesterol, or ceramides alone, were incapable of forming liposomes. The results indicate that lipid mixtures similar to those found in stratum corneum are capable of forming bilayers at physiologic pH.

Expression, distribution, and activity of Na+,K+-ATPase in normal and cholestatic rat liver.

Hepatocellular Na+,K+-ATPase is an important driving force for bile secretion and has been localized to the basolateral plasma membrane domain. Cholestasis or impaired bile flow is known to modulate the expression, domain specificity, and activity of various transport systems involved in bile secretion. This study examined Na+, K+-ATPase after ethinylestradiol (EE) treatment and after bile duct ligation (BDL), two rat models of cholestasis. It applied quantitative immunoblotting, biochemical and cytochemical determination of enzyme activity, and immunocytochemistry to the same livers. The data showed a good correlation among the results of the different methods. Neither EE nor BDL induced alterations in the subcellular distribution of Na+,K+-ATPase, which was found in the basolateral but not in the canalicular (apical) plasma membrane domain. Protein expression and enzyme activity showed a small (approximately 10%) decrease after EE treatment and a similar increase after BDL. These modest changes could not be detected by immunofluorescence, immuno EM, or cytochemistry. The data, therefore, demonstrate that Na+,K+-ATPase is only slightly affected by EE and BDL. They suggest that other components of the bile secretory apparatus that take effect downstream of the primary basolateral driving force may play a more prominent role in the pathogenesis of cholestasis.

The epidermal permeability barrier.

The permeability barrier of the skin which prevents transcutaneous water loss and penetration of harmful drugs from the environment is localized in the horny layer of the epidermis. Multiple lipid bilayers obstructing the intercellular space of the stratum corneum fulfill this function. In contrast to cellular membranes consisting predominantly of phospholipids, these lamellae contain mostly ceramides, cholesterol and free fatty acids. The lamellae are derived from the contents of lamellar granules (LGs) which are synthesized in the viable epidermal layers by the keratinocytes. LGs display stacks of small disks each of which represents a flattened vesicle or liposome. Prior to terminal differentiation, the disks are exocytosed into the intercellular space and fused to form uninterrupted sheetlike lamellae. The singular lipid composition of LG-disks and of stratum corneum-lamellae reflects the multistage process of barrier formation. It also renders these structures well suited to provide for a barrier function.

Bovine endothelial-like cells increase intercellular junctions under treatment with interferon-gamma. An in vitro study.

We have recently shown that a subgroup of endothelial-like cells from the bovine corpus luteum maintains cytokeratin (CK) expression in long-term culture. Treatment with interferon-gamma (IFN-gamma) for three days brought about an increase in the intercellular junctions in CK-positive postconfluent cultures, as shown by immunofluorescence with specific monoclonal antibodies and by electron microscopy. Desmosomes, zonulae adhaerentes and both tight and gap junctions were found. The intercellular junctions (excluding the gap junctions) had increased in amount, while permeability of the cell layer to horseradish peroxidase (HRP) had decreased. Hence, IFN-gamma has reinforced intercellular junctions in our CK-positive endothelial-like cells.

Pharmacological manipulation of L-carnitine transport into L6 cells with stable overexpression of human OCTN2.

The high-affinity Na+-dependent carnitine transporter OCTN2 (SLC22A5) has a high renal expression and reabsorbs most filtered carnitine. To gain more insight into substrate specificity of OCTN2, we overexpressed hOCTN2 in L6 cells and characterized the structural requirements of substances acting as human OCTN2 (hOCTN2) inhibitors. A 1905-bp fragment containing the hOCTN2 complete coding sequence was introduced into the pWpiresGFP vector, and L6 cells were stably transduced using a lentiviral system. The transduced L6 cells revealed increased expression of hOCTN2 on the mRNA, protein and functional levels. Structural requirements for hOCTN2 inhibition were predicted in silico and investigated in vitro. Essential structural requirements for OCTN2 inhibition include a constantly positively charged nitrogen atom and a carboxyl, nitrile or ester group connected by a 2-4-atom linker. Our cell system is suitable for studying in vitro interactions with OCTN2, which can subsequently be investigated in vivo.

Synapses form in skeletal muscles lacking neuregulin receptors.

The formation of the neuromuscular junction (NMJ) is directed by reciprocal interactions between motor neurons and muscle fibers. Neuregulin (NRG) and Agrin from motor nerve terminals are both implicated. Here, we demonstrate that NMJs can form in the absence of the NRG receptors ErbB2 and ErbB4 in mouse muscle. Postsynaptic differentiation is, however, induced by Agrin. We therefore conclude that NRG signaling to muscle is not required for NMJ formation. The effects of NRG signaling to muscle may be mediated indirectly through Schwann cells.

Deconvolution improves colocalization analysis of multiple fluorochromes in 3D confocal data sets more than filtering techniques.

Background and noise impair image quality by affecting resolution and obscuring image detail in the low intensity range. Because background levels in unprocessed confocal images are frequently at about 30% maximum intensity, colocalization analysis, a typical segmentation process, is limited to high intensity signal and prone to noise-induced, false-positive events. This makes suppression or removal of background crucial for this kind of image analysis. This paper examines the effects of median filtering and deconvolution, two image-processing techniques enhancing the signal-to-noise ratio (SNR), on the results of colocalization analysis in confocal data sets of biological specimens. The data show that median filtering can improve the SNR by a factor of 2. The technique eliminates noise-induced colocalization events successfully. However, because filtering recovers voxel values from the local neighbourhood false-negative ('dissipation' of signal intensity below threshold value) as well as false-positive ('fusion' of noise with low intensity signal resulting in above threshold intensities), results can be generated. In addition, filtering involves the convolution of an image with a kernel, a procedure that inherently impairs resolution. Image restoration by deconvolution avoids both of these disadvantages. Such routines calculate a model of the object considering various parameters that impair image formation and are able to suppress background down to very low levels (< 10% maximum intensity, resulting in a SNR improved by a factor 3 as compared to raw images). This makes additional objects in the low intensity but high frequency range available to analysis. In addition, removal of noise and distortions induced by the optical system results in improved resolution, which is of critical importance in cases involving objects of near resolution size. The technique is, however, sensitive to overestimation of the background level. In conclusion, colocalization analysis will be improved by deconvolution more than by filtering. This applies especially to specimens characterized by small object size and/or low intensities.

Cholestasis-induced alterations of the trans- and paracellular pathways in rat hepatocytes.

Bile secretion depends on the vectorial transport of solutes from blood to bile and involves three different pathways: transcellular pathways mediated by transport proteins distributed asymmetrically in the basolateral and canalicular plasma membrane and by transcytotic vesicles, and a paracellular pathway allowing selective diffusion through tight junctions. All three pathways are impaired differentially by extrahepatic (bile duct ligation) or intrahepatic (ethinyloestradiol) cholestasis. Ethinyloestradiol treatment leads to tight junctional defects that are less severe than those induced by bile duct ligation. Junctional impairment is reflected functionally in increased permeability for horseradish peroxidase and structurally by decreased strand numbers and increased junctional length, but not by alterations at the level of the individual strands. The parallelism of physiological and morphological perturbations indicates a structure-function relationship in hepatocellular tight junctions. In addition, impaired functional integrity of tight junctions following bile duct ligation is reflected in a partial loss of hepatocellular surface polarity owing to redistribution of some, but not all, domain-specific plasma membrane antigens, which might mimic the behaviour of transport systems. After ethinyloestradiol treatment no alterations of surface polarity were observed. Thus, immunohistochemistry supports the view that ethinyloestradiol results in less severe impairment of the tight junctions than bile duct ligation. Finally, bile duct ligation, but not ethinyloestradiol, affects the transcytotic vesicular pathway; severe impairment of this is reflected in the absence of a late horseradish peroxidase peak in bile and also in the accumulation of pericanalicular vesicles that are immunopositive for canalicular membrane proteins and accessible for bulk phase endocytic markers.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphological evidence for axonal transport of glycogen in neurons innervating cutaneous receptors in Lacerta sicula (Squamata: Reptilia).

Three types of glycogen-containing cutaneous nerve terminals - two of them hitherto unknown - are described. It is shown that the glycogen is synthesized in the perikaryon and transferred to the terminals by means of slow axoplasmic transport.

Zonulae occludentes in the epidermis of the snake Natrix natrix L.

Zonulae occludentes of a very tight type were identified between the uppermost stratum germinativum cells of the grass snake Natrix natrix L. by means of the lanthanum tracer technique. Since zonulae occludentes alone are not capable of preventing transepithelial water flow, an additional barrier mechanism is postulated, responsible for the low rates of cutaneous water loss measured in squamate reptiles. It is suggested that the observed zonulae occludentes are involved in the sloughing process.

Theoretical calculation of layer geometry in rotary shadowed models of membrane-associated particles.

The geometry of the evaporated metal layer on models of membrane-associated particles has been calculated theoretically and the respective density profiles have been estimated. The models were a hollow cylinder, a hemisphere and a rotary ellipsoid substituting the freeze-fracture appearance of gap junction connexons and globular integral membrane proteins respectively. These calculations may be used as a help for choosing an optimal compromise between shadowing angle and layer thickness. They furthermore provide a basis for assaying the extent of shadowing artefacts such as decoration and lead to a more accurate interpretation of rotary shadowed structures.

Glycogen in epidermal nerve terminals of Lacerta sicula (squamata: reptilia).

Proof is given that the granula occuring in the epidermal discoid nerve terminals of Lacerta sicula consist of glycogen. Staining with PA-PbCi shows 300 A sized alpha-particles and 70 A sized beta-particles. The electron-dense boundary appearing after digestion with alpha-amylase consists of limit-dextrins.