Expression of tight-junction proteins in the inflamed and clinically uninvolved skin in patients with venous leg ulcers.

Skin around venous leg ulcers (VLUs) is often inflamed and prone to contact sensitization. Expression of tight-junction components (ZO-1, occludin, and claudins 1 and 4) was studied by immunofluorescence in inflamed and noninflamed lower leg skin (both uncovered skin and skin occluded under hydrocolloid dressings) in patients with VLUs. No major differences were found in the expression of occludin and claudin-4. ZO-1 protein had stronger and more wide-ranging expression in the inflamed epidermis. Expression of claudin-1 was lost from the basal layer of the inflamed skin and skin under the hydrocolloid dressing. The skin on the lower legs affected by VLU may have altered expression of ZO-1 and claudin-1, similar to that seen in psoriatic plaques.

HMG-CoA reductase is regulated by environmental salinity and its activity is essential for halotolerance in halophilic fungi.

The activity and level of HMG-CoA reductase (HMGR) were addressed in halophilic fungi isolated from solar saltpans. Representative fungi belonging to the orders Dothideales, Eurotiales and Wallemiales have a specific pattern of HMGR regulation, which differs from salt-sensitive and moderately salt-tolerant yeasts. In all of the halophilic fungi studied, HMGR amounts and activities were the lowest at optimal growth salinity and increased under hyposaline and hypersaline conditions. This profile paralleled isoprenylation of cellular proteins in H. werneckii. Inhibition of HMGR in vivo by lovastatin impaired the halotolerant character. HMGR may thus serve as an important molecular marker of halotolerance.

Superficial cell differentiation during embryonic and postnatal development of mouse urothelium.

After drastic urothelial destruction around birth and around postnatal day 6, mouse urothelial renewal starts each time de novo. The differentiation of superficial cells during urothelial restoration was followed for the first time from embryonic day 15 to postnatal day 6 by the detection of differentiation markers: cytokeratins, uroplakins and apical membrane specialization. The differentiation markers of short-lived superficial cells were studied before and after urothelial destruction. Three distinctive types of superficial cells, typical for certain developmental period, were characterised: cells at low differentiation stage with microvilli and cilia, expressing CK7 and CK18, detected on embryonic day 15; cells at advanced differentiation stage with star-like arrangement of prominent membrane ridges, expressing CK7 and CK20, present between the two urothelial destruction events; highly differentiated cells with typically jagged apical surface, expressing CK7 and CK20, found twice during development. This cell type appears for the first time on embryonic day 18 as the terminal stage of embryonic differentiation. It was found again on postnatal day 6 as an initial stage of differentiation, leading toward terminally differentiated cells of the adult urothelium. Our work proves that apical membrane specialization is the most valuable differentiation marker of superficial cells.

Succession of events in desquamation of superficial urothelial cells as a response to stress induced by prolonged constant illumination.

The effect of moderate stress induced by prolonged illumination was analysed on urothelial cells of female mouse urinary bladders at ultrastructural and cytochemical levels. This study demonstrates that the urothelium responds to moderate stress with desquamation which involves two subsequent steps. The first step includes a local detachment of tight junctions and consequently the loss of the permeability barrier leading to expanded intercellular spaces among urothelial cells. During the second step, the disjunction of desmosomes accompanied by exocytosis of lysosomal enzymes (NADPase) in the intercellular space results in exfoliation of superficial cells. It is evident that moderate stress elicits an enhanced desquamation of only superficial cells by a subsequent dysfunction of first tight junctions and after that adherens-type junctions. A rapid restoration of the new tight junctions prevents a long-term malfunction of the blood-urine barrier.

Theoretical analysis of shape transformation of V-79 cells after treatment with cytochalasin B.

We observed that after treatment of V-79 fibroblasts with cytochalasin B the area of cell contact with the substrate is essentially reduced, the microtubules are organized into rodlike structures and the actin filaments are disintegrated. Remnants of the actin cortex become concentrated in the form of discrete patches under the plasma membrane. The described changes in the organization of the cytoskeleton and of the cortical shell are accompanied by the formation of a cell shape resembling the Greek letter phi. We calculated that the phi shape corresponds to the minimum of the stretching energy of the cortical shell at relevant geometrical constraints. In line with this result, if cytochalasin B treatment was followed by colchicine application which disrupted the microtubular rod, the characteristic phi shape completely disappeared. This study suggests that the effect of the microtubular rod on the cell shape can be theoretically well described by taking into account some basic conditions for the mechanical equilibrium of the cell cortical shell and the appropriate geometrical constraints.

Postnatal restoration of the mouse urinary bladder urothelium.

Mouse urothelium is disrupted just before birth, followed by a postnatal restoration process which includes cell proliferation, death and differentiation. We assessed urothelial proliferation by the expression of proliferating cell nuclear antigen (PCNA), desquamation by electron microscopy, and apoptosis by TUNEL staining and urothelial differentiation by the expression of uroplakins and cytokeratin 20 (CK20) as well as the apical plasma membrane maturation. Our results indicated that urothelial proliferation was high from birth until about the 14th postnatal day. A majority of basal cells and even occasional superficial cells were PCNA positive during the first 5 postnatal days. Cell death occurred during the first 9 postnatal days. Between birth and day 5, single cells underwent apoptosis, whereas between days 6 and 9 cells mainly desquamated. CK20 and uroplakins were expressed in all superficial cells in postnatal urothelium. Their subcellular distribution characteristically changed in accordance with the progressive differentiation of superficial cells. During the urothelial postnatal development, proliferation activity slowly decreases to the proliferatively quiescent urothelium of the adult animal. Apoptosis is present in the first 9 postnatal days and within a few days of this period it appears simultaneously with desquamation. Superficial urothelial cells gradually differentiate, which is reflected in the changeable morphology of the apical plasma membrane.

Autophagy-related vacuoles in mouse gallbladder epithelium.

The mouse gallbladder epithelial cells contain very heterogeneous vacuolar population. In an attempt to classify these vacuoles we identified NADPase and TPPase activity as well as the location of HRP which is used as the endocytotic marker. The results of the present study show that the vacuoles can be classified into three categories: (1) the vacuoles predominantly containing loose membrane coils related to the nascent autophagic vacuoles, (2) vacuoles containing densely packed membranes and exhibiting a positive HRP reaction, indicating the convergence of endocytotic and autophagic pathway, and (3) vacuoles composed of degraded membrane structures and containing the reaction product of NADPase activity, showing that the fusion of the lysosomes with the autophagosome-endosome took place. The highly developed cis, medial and trans Golgi compartments reflect the biosynthetic and endocytotic activity of the gallbladder epithelium.

The response of junctional complexes to induced desquamation in mouse bladder urothelium.

During desquamation, the cells of mouse urinary bladder epithelium undergo detachment. In this process we examined the disconnection of cell adhesion molecules. Two proteins of cell junctions were studied: ZO1 of tight junctions and desmoplakin of desmosomes. Desquamation was induced by intravesical injection of LPS, constant illumination of mouse for 96 h, application of a combination of stress hormones hydrocortisone and norepinephrine or by removal of calcium with EGTA. All the inducers caused penetration of lanthanum tracer through the tight junctions, indicating paracellular permeability. Dilatation of extracellular spaces between neighboring cells was seen whenever desquamation was induced in bladders containing urine. Desquamation of single cells as well as groups of cells was observed. Contrary to obvious disconnection of cell junctions, as a precondition for desquamation, the distribution of junctional proteins did not change either in urothelial tissue or in desquamated cells. This study demonstrates that all the inducers of desquamation cause first an extensive dysfunction of a blood urine barrier and after that an occasional mechanical disconnection of adhesive junctions which consequently leads to desquamation.

Urothelial cell detachment and differentiation in urinary bladder.

In developing and in repairing bladder, proliferation of the transitional urothelium is followed by cell detachment--desquamation or apoptosis. Proliferation results in formation of terminally differentiated superficial cells and this process may be followed by checking the cells on the presence of differentiation markers. The formation of an asymmetric unit membrane (AUM) structure (plaque) on the cell surface is in correlation with urothelial differentiation. Thus, the microstructure of the luminal surface of the urinary bladder provides a very convenient differentiation biomarker. The surface of immature cells showed a pattern of microvilli. The progress of differentiation was associated with microvili arranged in rows finally forming the characteristic pattern of ridges in terminally differentiated cells. These results demonstrate that the characteristic surface pattern and the AUM plaque formation in the apical plasma membrane of superficial urothelial cells are associated with specific morphology, and patterns and thus help detect differentiation level of cell.

Exogenously added growth factors have no effect on formation of cell junctions and cytoskeleton in urothelial cells in culture.

To elucidate the effects of epidermal growth factor-EGF and transforming growth factor-TGFbeta1 on cellular structure, especially on cell junctions and cytoskeleton, the distribution of ZO1, E-cadherin and desmoplakin as well as the organization of actin and keratin filaments have been examined immunohistochemically. In EGF-treated cultures as well as in TGFbeta1-treated cultures, the distribution of adhesion proteins looked similar. On the sites where cells made contacts, the presence of ZO1, E-cadherin and desmoplakin was revealed seen as a continuous line around cells. EGF as well as TGFbeta1 treatment induced no difference in the presence and distribution of cytokeratin 20; this marker of terminal differentiation was limited to superficial urothelial cells only. Also, the distribution of actin filaments was not significantly altered by any of the growth factors used. This indicates that neither cell junctions nor cytoskeleton of urothelial cells were affected by exogenously added growth factors. This may result from the influence of stroma on the formation of urothelium during the first days of culture of urinary bladder explants and the production of growth factors in the culture itself.

Actin filaments during terminal differentiation of urothelial cells in the rat urinary bladder.

The localisation of actin filaments was studied in rat urothelial cells during differentiation which accompanied regeneration after cell damage induced by cyclophosphamide (CP). By immunofluorescence it was established that actin filaments equally stained along the cell circumference in basal and intermediate cells, while basolateral cell membrane expression was found in terminally differentiated superficial cells. During regeneration, after CP treatment, simple urothelial hyperplasia developed with smaller cuboidal superficial cells, in which actin filaments were equally distributed under the apical and basolateral plasma membranes. As demonstrated by immunoelectron microscopy, the apical surface of these superficial cells was covered with microvilli containing bundles of actin filaments. Within 1 week, the urothelium reverted to its normal three-layer thickness. Superficial cells became larger and flattened and the unthickened apical plasma membrane matured into a thick asymmetric unit membrane. Concomitantly actin filaments disappeared from apical areas of superficial cells while remaining abundant at basolateral areas. Our results indicate that in the urothelium subcellular distribution of actin filaments can be considered as a marker of cell differentiation.

Activation of Ste20 by Nef from human immunodeficiency virus induces cytoskeletal rearrangements and downstream effector functions in Saccharomyces cerevisiae.

The negative factor (Nef) from human and simian immunodeficiency viruses is important for the pathogenesis of acquired immune deficiency syndrome. Among other targets, it activates the Nef-associated kinase, which is related to the p21-activated kinase. In this study, we demonstrate that Nef activates Ste20, the homolog of p21-activated kinase in Saccharomyces cerevisiae. Nef binds to the adaptor proteins Bem1 and Ste20 via its proline-rich (PXXP) and diarginine (RR) motifs, respectively. These interactions induce the mitogen-activated protein kinase and increase the rates of budding, sizes of cells, and patterns of mating projections. These effects of Nef depend on the small GTPase Cdc42 and guanine nucleotide exchange factor Cdc24. Thus, studies in S. cerevisiae identified specific interactions between Nef and cellular proteins and their associated signaling cascade.

A mini organ culture as a model for studying the gallbladder epithelium of mouse.

A mini organ culture of mouse gallbladder was developed as an alternative to primary cultures of epithelial cells of this organ. Small pieces of tissue were prepared and maintained in minimum essential Eagle medium with 10% foetal calf serum, for as long as 7 days. Qualitative and quantitative ultrastructural studies have been performed using electron microscopy. The viability of cells was evaluated by stereological quantification of endocytotic vesicles containing horseradish peroxidase and labelling of exocytotic glycoproteins with tannic acid. The morphology of tissue pieces during the 1st h of culturing and tissue isolated directly from animals exhibited no significant differences. However, after 4 h in culture degradative changes became evident in many cells. At that time, endo- and exocytosis were both dramatically reduced. After 24 h, the morphology, as well as endo- and exocytosis recovered and were comparable to the parameters of the tissue in vivo or after 1 h in culture. The endocytotic activity remained unchanged from day 1 to 7 of culturing, while the number of exocytotic vesicles gradually decreased after 2 days in culture. Our results prove that mini organ culture of gallbladder is morphologically and functionally comparable with the tissue in vivo and for studies of epithelium in culture it is more convenient than primary cultures.

The separation of exocytosis from endocytosis in rat melanotroph membrane capacitance records.

1. Using the patch-clamp technique, we have monitored the secretory activity of single rat melanotrophs. Changes in membrane capacitance (Cm) were measured to detect small discrete femtofarad steps. These are believed to be due to interactions between single secretory organelles (granules) and plasmalemma. 2. A new approach was introduced to measure the amplitude of discrete steps in Cm. Records of Cm were converted into time derivatives, where discrete steps appeared as transients. A transient due to a 2 fF discrete step in Cm was easily distinguished from random noise, since the probability of such a transient being due to random noise was less than 0.01. To distinguish apparent steps from noise the computer-based analysis employed a threshold of 3 times the standard deviation of the noise time derivative (dCm/dt). A phase diagram was created by plotting dCm/dt versus Cm, from which the magnitude and direction of transients were determined. Transients due to 2 fF steps (equivalent to a signal-to-noise ratio of 1) were detected with a reliability of 100%, whereas steps of 1 fF were detected with a reliability of more than 60%. The amplitude of false steps detected by the program was less than 1 fF, and the frequency of false detections of 0.075 S-1 was equal for exocytotic and endocytotic events. 3. Electron microscopy was used to measure secretory organelle size and an immunogold technique was used to label the electron micrographs with an anti-adrenocorticotrophin (ACTH) antibody. Secretory organelles in cultured and non-cultured cells were of similar diameter. All sizes of secretory granules appear to contain ACTH, since secretory organelles of similar diameter stained positively with the anti-ACTH antibodies. 4. Small discrete steps in Cm, recorded with the whole-cell configuration and loosely buffered cytosolic calcium, were similar to the estimated Cm of secretory organelles from morphological data. Thus, measured discrete steps in Cm reflect interactions between single organelle size and plasma membrane. Exocytotic and endocytotic steps were found to be of similar size. 5. To separate exocytosis from endocytosis in Cm records, we assumed that the rates of exocytosis and endocytosis were related to the respective frequencies of discrete steps in Cm. A relationship between the frequency of exocytotic, but not endocytotic events, and the rate of change in Cm was observed. Thus, under our experimental conditions, an increase in Cm could be explained by an increased rate of exocytosis in rat melanotrophs.