Rok Romih
University of Ljubljana, Institute of Cell Biology, Faculty Member
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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... more
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.
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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... more
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.
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The urothelium forms the blood-urine barrier, which depends on the complex organization of transmembrane proteins, uroplakins, in the apical plasma membrane of umbrella cells. Uroplakins compose 16 nm intramembrane particles, which are... more
The urothelium forms the blood-urine barrier, which depends on the complex organization of transmembrane proteins, uroplakins, in the apical plasma membrane of umbrella cells. Uroplakins compose 16 nm intramembrane particles, which are assembled into urothelial plaques. Here we present an integrated survey on the molecular ultrastructure of urothelial plaques in normal umbrella cells with advanced microscopic techniques. We analyzed the ultrastructure and performed measurements of urothelial plaques in the normal mouse urothelium. We used field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), transmission electron microscopy (TEM) on immunolabeled ultrathin sections (immuno-TEM), and freeze-fracture replicas (FRIL). We performed immunolabeling of uroplakins for scanning electron microscopy (immuno-FESEM). All microscopic techniques revealed a variability of urothelial plaque diameters ranging from 332 to 1179 nm. All immunolabeling techniques confirmed the presence of uroplakins in urothelial plaques. FRIL showed the association of uroplakins with 16 nm intramembrane particles and their organization into plaques. Using different microscopic techniques and applied qualitative and quantitative evaluation, new insights into the urothelial apical surface molecular ultrastructure have emerged and may hopefully provide a timely impulse for many ongoing studies. The combination of various microscopic techniques used in this study shows how these techniques complement one another. The described advantages and disadvantages of each technique should be considered for future studies of molecular and structural membrane specializations in other cells and tissues.
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The expression, subcellular localization, and supramolecular organization of structural and differentiation-related proteins were studied in differentiating superficial cells during regeneration of the rat urothelium. Cytokeratin 17 and... more
The expression, subcellular localization, and supramolecular organization of structural and differentiation-related proteins were studied in differentiating superficial cells during regeneration of the rat urothelium. Cytokeratin 17 and epidermal growth factor receptor were expressed in undifferentiated cells, whereas in differentiated superficial cells, this expression ceased. beta-Actin was expressed in undifferentiated and in differentiated cells, but subcellular localization markedly changed by its withdrawal from apical region during differentiation. In differentiating cells, cytokeratin 20 was first observed as diffusely arranged spots distributed throughout the cytoplasm; later, it became organized as a network in the apical part of the cells. Urothelium specific proteins, uroplakins, were weakly expressed at early states of differentiation, but only in terminally differentiated cells did they organize into asymmetric unit membrane plaques. These findings show that in some cases, expression is a sufficient marker of cell differentiation. In other cases, however, the specific subcellular localization and supramolecular organization of proteins should be considered. It is evident that choosing the appropriate methods is crucial for precise evaluation of the urothelial differentiation states.
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In normal urothelium, superficial umbrella cells express four major integral membrane proteins, uroplakins UPIa, UPIb, UPII, and UPIIIa, which compose urothelial plaques. In the apical plasma membrane, urothelial plaques form microridges.... more
In normal urothelium, superficial umbrella cells express four major integral membrane proteins, uroplakins UPIa, UPIb, UPII, and UPIIIa, which compose urothelial plaques. In the apical plasma membrane, urothelial plaques form microridges. During neoplastic changes, microridges are replaced by microvilli, while uroplakin expression is retained. We correlated individual uroplakin expression with apical plasma membrane structure, cytokeratin 20 expression, and urothelial cell proliferation (Ki-67). Male Wistar rats were treated with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in drinking water, which caused flat hyperplasia with mild dysplasia, low-grade papillary urothelial carcinoma, invasive low- and high-grade papillary urothelial carcinoma and invasive squamous cell carcinoma with extensive keratinization, grade 2. During urothelial carcinogenesis, UPII expression was the most decreased in all urothelial lesions, while UPIa, UPIb, and UPIIIa expression was differently altered in different types of lesions. Superficial cells were covered with microvilli and ropy ridges, while microridges were disappearing. The expression of cytokeratin 20 was decreased and limited to superficial urothelial cells. Proliferation indices were increased, except for invasive squamous cell carcinoma with extensive keratinization. Our results indicate that during urothelial carcinogenesis the expression of UPII is diminished, suggesting that UPIb/UPIIIa heterodimer can still be formed, while heterodimer UPIa/UPII formation is disrupted. Correlation between decreased level of UPII expression and changed apical plasma membrane structure suggests that diminished expression of UPII hinders the urothelial plaque formation.
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Although the epithelial lining of much of the mammalian urinary tract is known simply as the urothelium, this epithelium can be divided into at least three lineages of renal pelvis/ureter, bladder/trigone, and proximal urethra based on... more
Although the epithelial lining of much of the mammalian urinary tract is known simply as the urothelium, this epithelium can be divided into at least three lineages of renal pelvis/ureter, bladder/trigone, and proximal urethra based on their embryonic origin, uroplakin content, keratin expression pattern, in vitro growth potential, and propensity to keratinize during vitamin A deficiency. Moreover, these cells remain phenotypically distinct even after they have been serially passaged under identical culture conditions, thus ruling out local mesenchymal influence as the sole cause of their in vivo differences. During vitamin A deficiency, mouse urothelium form multiple keratinized foci in proximal urethra probably originating from scattered K14-positive basal cells, and the keratinized epithelium expands horizontally to replace the surrounding normal urothelium. These data suggest that the urothelium consists of multiple cell lineages, that trigone urothelium is closely related to th...
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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... more
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.
Research Interests:
The apical surface of mammalian bladder urothelium is covered by large (500–1000 nm) two-dimensional (2D) crystals of hexagonally packed 16-nm uroplakin particles (urothelial plaques), which play a role in permeability barrier function... more
The apical surface of mammalian bladder urothelium is covered by large (500–1000 nm) two-dimensional (2D) crystals of hexagonally packed 16-nm uroplakin particles (urothelial plaques), which play a role in permeability barrier function and uropathogenic bacterial binding. How the uroplakin proteins are delivered to the luminal surface is unknown. We show here that myelin-and-lymphocyte protein (MAL), a 17-kDa tetraspan protein suggested to be important for the apical sorting of membrane proteins, is coexpressed with uroplakins in differentiated urothelial cell layers. MAL depletion in Madin–Darby canine kidney cells did not affect, however, the apical sorting of uroplakins, but it decreased the rate by which uroplakins were inserted into the apical surface. Moreover, MAL knockout in vivo led to the accumulation of fusiform vesicles in mouse urothelial superficial umbrella cells, whereas MAL transgenic overexpression in vivo led to enhanced exocytosis and compensatory endocytosis, re...
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ABSTRACT We report on the nanoparticle uptake into MCF10A neoT and PC-3 cells using flow cytometry, confocal microscopy, SQUID magnetometry, and transmission electron microscopy. The aim was to evaluate the influence of the nanoparticles’... more
ABSTRACT We report on the nanoparticle uptake into MCF10A neoT and PC-3 cells using flow cytometry, confocal microscopy, SQUID magnetometry, and transmission electron microscopy. The aim was to evaluate the influence of the nanoparticles’ surface charge on the uptake efficiency. The surface of the superparamagnetic, silica-coated, maghemite nanoparticles was modified using amino functionalization for the positive surface charge (CNPs), and carboxyl functionalization for the negative surface charge (ANPs). The CNPs and ANPs exhibited no significant cytotoxicity in concentrations up to 500 μg/cm3 in 24 h. The CNPs, bound to a plasma membrane, were intensely phagocytosed, while the ANPs entered cells through fluid-phase endocytosis in a lower internalization degree. The ANPs and CNPs were shown to be co-localized with a specific lysosomal marker, thus confirming their presence in lysosomes. We showed that tailoring the surface charge of the nanoparticles has a great impact on their internalization.
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Research Interests: Immunology, Immune response, Biology, Medicine, Dendritic Cells, and 15 moreCell line, Cell Differentiation, Humans, Antigen Presentation, Dendritic cell, Monocytes, cystatin C, Exocytosis, Biological markers, Lymph Node, Golgi Apparatus, Plasma Membrane, Interleukin, Lymph nodes, and Cysteine Protease
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A sodium saccharin (NaSac) diet was used to induce cell damage and regeneration in the urothelium of the male rat urinary bladder. Foci of terminally differentiated superficial cell exfoliation were detected after 5 weeks and their number... more
A sodium saccharin (NaSac) diet was used to induce cell damage and regeneration in the urothelium of the male rat urinary bladder. Foci of terminally differentiated superficial cell exfoliation were detected after 5 weeks and their number increased after 10 and 15 weeks of the diet. At the sites of superficial cell loss, regenerative simple hyperplasia developed. Within 5 weeks of NaSac removal, regeneration re-established normal differentiated urothelium. In order to follow urothelial differentiation during regeneration we studied the expression of uroplakins and cytokeratins by means of immunocytochemistry and immunohistochemistry, respectively. Normal urothelium was characterised by terminally differentiated superficial cells which expressed uroplakins in their luminal plasma membrane and cytokeratin 20 (CK20) in the cytoplasm. Basal and intermediate cells were CK20 negative and cytokeratin 17 (CK17) positive. In hyperplastic urothelium all cells synthesised CK17, but not CK20. Differentiation of the superficial layer was reflected in three successive cell types: cells with microvilli, cells with rounded microridges and those with a rigid-looking plasma membrane on the luminal surface. The cells with microvilli did not stain with anti-uroplakin antibody. When the synthesis of uroplakins was detected rounded microridges were formed. With the elevated expression of uroplakins the luminal plasma membrane becomes rigid-looking which is characteristic of asymmetric unit membrane of terminally differentiated cells. During differentiation, synthesis of CK17 ceased in superficial cells while the synthesis of CK20 started. These results indicate that during urothelial regeneration after NaSac treatment, specific superficial cell types develop in which the switch to uroplakin synthesis and transition from CK17 to CK20 synthesis are crucial events for terminal differentiation.
Research Interests: Regeneration, Biology, Membrane Proteins, Immunohistochemistry, Medicine, and 10 moreAnimals, Male, Carcinogens, Rats, Wistar Rats, Saccharin, Plasma Membrane, Urinary Bladder, Keratins, and Male rat(Animals, Male, Carcinogens, Rats, Wistar Rats, Saccharin, Plasma Membrane, Urinary Bladder, Keratins, and Male rat)
(Animals, Male, Carcinogens, Rats, Wistar Rats, Saccharin, Plasma Membrane, Urinary Bladder, Keratins, and Male rat)
In superficial umbrella cells of normal urothelium, uroplakins (UPs) are assembled into urothelial plaques, which form fusiform vesicles (FVs) and microridges of the apical cell surface. Altered urothelial differentiation causes changes... more
In superficial umbrella cells of normal urothelium, uroplakins (UPs) are assembled into urothelial plaques, which form fusiform vesicles (FVs) and microridges of the apical cell surface. Altered urothelial differentiation causes changes in the cell surface structure. Here, we investigated ultrastructural localization of UPIa, UPIb, UPII and UPIIIa in normal and cyclophosphamide-induced preneoplastic mouse urothelium. In normal urothelium, terminally differentiated umbrella cells expressed all four UPs, which were localized to the large urothelial plaques covering mature FVs and the apical plasma membrane. The preneoplastic urothelium contained two types of superficial cells with altered differentiation: (1) poorly differentiated cells with microvilli and small, round vesicles that were uroplakin-negative; no urothelial plaques were observed in these cells; (2) partially differentiated cells with ropy ridges contained uroplakin-positive immature fusiform vesicles and the apical plasma membrane. Freeze-fracturing showed small urothelial plaques in these cells. We concluded that in normal urothelium, all four UPs colocalize in urothelial plaques. However, in preneoplastic urothelium, the growth of the uroplakin plaques was hindered in the partially differentiated cells, leading to the formation of immature FVs and ropy ridges instead of mature FVs and microridges. Our study demonstrates that despite a lower level of expression, UPIa, UPIb, UPII and UPIIIa maintain their plaque association in urothelial preneoplastic lesions.
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Cytokeratins, uroplakins and the asymmetric unit membrane are biochemical and morphological markers of urothelial differentiation. The aim of our study was to follow the synthesis, subcellular distribution and supramolecular organization... more
Cytokeratins, uroplakins and the asymmetric unit membrane are biochemical and morphological markers of urothelial differentiation. The aim of our study was to follow the synthesis, subcellular distribution and supramolecular organization of differentiation markers, cytokeratins and uroplakins, during differentiation of umbrella cells of mouse bladder urothelium. Regenerating urothelium after destruction with cyclophosphamide was used to simulate de-novo differentiation of cells, which was followed from day 1 to day 14 after cyclophosphamide injection. Cytokeratin 7 and uroplakins co-localized in the subapical cytoplasm of superficial cells from the early stage of differentiation on. At early stages of superficial cell differentiation cytokeratin 7 was filamentary organized, and rare uroplakins were found on the membranes of relatively small cytoplasmic vesicles, which were grouped in clusters under the apical membrane. Later, cytokeratin 7 gradually reorganized into a continuous trajectorial network, and uroplakins became organized into plaques of asymmetric unit membrane, which formed fusiform vesicles. After insertion of fusiform vesicles into the apical plasma membrane, the surface acquired microridged appearance of umbrella cells. Cytokeratin 20 appeared as the last differentiation marker of umbrella cells. Cytokeratin 20 was incorporated into the pre-existing trajectorial cytokeratin network. These results indicate that differentiation of urothelial cells starts with the synthesis of differentiation-related proteins i.e., cytokeratins and uroplakins, and later with their specific organization. We consider that the umbrella cell has reached its final stage of differentiation when uroplakins form plaques of asymmetric unit membrane that are inserted into the apical plasma membrane and when cytokeratin 20 becomes included in a trajectorial cytokeratin network in the subapical area of cytoplasm.
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Processes leading to the recovery of a normal three-layered urothelium from a hyperplastic urothelium induced by cyclophosphamide (CP) treatment in rats have been investigated. A single intraperitoneal (ip) dose of CP caused extensive... more
Processes leading to the recovery of a normal three-layered urothelium from a hyperplastic urothelium induced by cyclophosphamide (CP) treatment in rats have been investigated. A single intraperitoneal (ip) dose of CP caused extensive loss of cells from urothelium, but the remaining cells started to express epidermal growth factor receptor (EGFR) in their plasma membranes. On day 2 after CP injection, proliferating cell nuclear antigen (PCNA) immunohistochemistry showed a rapid increase in positively stained nuclei, from which a hyperplastic urothelium developed, composed of undifferentiated cells expressing EGFR over the entire plasma membrane. Subsequently, EGFR gradually disappeared from the apical plasma membrane but remained in the basolateral membranes. After day 6, PCNA-positive nuclei in all cell layers decreased, except in basal cells. Apoptotic cells were detectable by the TUNEL assay at day 2, and increased in number in all layers of the hyperplastic urothelium until day 10, returning to the control levels by day 14. Electron microscopic evidence showed that apoptotic cells were either pinched off into the bladder lumen or phagocytosed by the neighbouring urothelial cells. Thus, the urothelium responds to the damage by intense proliferation for a week, resulting in an undifferentiated hyperplastic state. Differentiation of superficial cells then begins and damaged cells are gradually removed by apoptosis until the three-layered urothelium is fully restored by two weeks following CP treatment.
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The purpose of this study was to establish an in vitro culture model that closely resembles whole mouse urothelial tissue. Primary explant cultures of mouse bladder were established on porous membrane supports and explant outgrowths were... more
The purpose of this study was to establish an in vitro culture model that closely resembles whole mouse urothelial tissue. Primary explant cultures of mouse bladder were established on porous membrane supports and explant outgrowths were analysed for morphology and the presence of antigenic and ultrastructural markers associated with urothelial cytodifferentiation. When examined at the ultrastructural level, the cultured urothelium was polarized and organized as a multilayered epithelium. Differentiation was found to increase from the porous membrane towards the surface and from the explant towards the periphery of the culture. Scanning and transmission electron microscopical analysis of the most superficially-located cells revealed four successive differentiation stages: cells with microvilli, cells with ropy microridges, cells with rounded microridges, and highly-differentiated cells with asymmetric unit membrane (AUM) plaques forming rigid microridges and fusiform vesicles. The more highly-differentiated cells were numerous at the periphery of the culture, but rare close to the explant. Epithelial organization was stabilized by well developed cell junctions. Immunolabeling demonstrated that superficial urothelial cells in culture: (1) develop tight junctions, E-cadherin adherens junctions and abundant desmosomes and (2) express uroplakins and cytokeratin 20 (CK 20). Using a culture model of primary explant outgrowth we have shown that non-differentiated mouse urothelial cells growing on a porous membrane show a high level of de novo differentiation.
Research Interests: Cell Biology, Cell Differentiation, Mice, Animals, Male, and 15 moreLanthanum, Keratin, Differentiation, Tetraspanins, Time Factors, In vitro culture, Transmission Electron Microscope, Tight Junction, Desmoplakins, Biochemistry and cell biology, Tight Junctions, Phosphate Buffer Saline, Cell Membrane, Cadherins, and Adherens Junction
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Research Interests: Scanning Electron Microscopy, Immunohistochemistry, Cell Differentiation, Mice, Female, and 12 moreAnimals, Complex network, Male, Medical Physiology, Fluorescent Antibody Technique, Urinary Tract, Tetraspanins, Cell Surface Markers, Epithelial Cell Biology, Plasma Membrane, Urinary Bladder, and Keratins
The differentiation of urothelial cells is characterized by the synthesis of uroplakins and their assembly into the asymmetric unit membrane. The Golgi apparatus (GA) has been proposed to play a central role in asymmetric unit membrane... more
The differentiation of urothelial cells is characterized by the synthesis of uroplakins and their assembly into the asymmetric unit membrane. The Golgi apparatus (GA) has been proposed to play a central role in asymmetric unit membrane formation. We have studied the distribution and organization of the GA in normal mouse urothelial cells and in the superficial urothelial cells that undergo differentiation following cyclophosphamide-induced regeneration, in correlation with urothelial cell differentiation. In normal urothelium, immature basal cells have a simple GA, which is small and distributed close to the nucleus. In intermediate cells, the GA starts to expand into the cytoplasm, whereas the GA of terminally differentiated umbrella cells is complex, being large and spread over the whole basal half of the cytoplasm. During early stages of regeneration after cyclophosphamide treatment, the GA of superficial cells is simple and no markers of urothelial differentiation (uroplakins or asymmetric unit membranes, discoidal or fusiform vesicles, apical surface covered with microvilli) are expressed. At a later stage, the GA expands and, in the final stage of regeneration, when cells express all markers of terminal urothelial differentiation, the GA become complex once again. Our results show that: (1) GA distribution and organization in urothelial cells is differentiation-dependent; (2) the GA matures from a simple form in partially differentiated cells to a complex form in terminally differentiated superficial cells; (3) major rearrangements of GA distribution and organization correlate with the beginning of asymmetric unit membrane production. Thus, GA maturation seems to be crucial for asymmetric unit membrane formation.
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To assess the expression and distribution of uroplakins, protein subunits of the asymmetric unit membrane (AUM), and inducible nitric-oxide synthase (iNOS) in the urinary bladder urothelium of patients with bladder outlet obstruction... more
To assess the expression and distribution of uroplakins, protein subunits of the asymmetric unit membrane (AUM), and inducible nitric-oxide synthase (iNOS) in the urinary bladder urothelium of patients with bladder outlet obstruction (BOO) caused by benign prostatic hyperplasia (BPH). Urinary bladder urothelium samples from 15 men (mean age 69 years) with BOO secondary to BPH were processed for light and electron immunocytochemistry. Uroplakins and iNOS were detected, and areas of apical surface covered with AUM were compared with those of iNOS-positive urothelial cells. Areas of superficial urothelial cells with no AUM were found in all obstructed bladder samples. The immuno-electron microscopy showed that the uroplakin-positive cells had the characteristic appearance of terminally differentiated umbrella cells, whereas cells from the uroplakin-negative regions were undifferentiated, typically showing microvilli on their apical surface. iNOS was not detected in areas with continuous AUM staining, but was readily detected in the uroplakin-negative areas. There was an inverse correlation between the intensity of uroplakin and iNOS staining. In patients with BOO associated with BPH, some superficial urothelial cells lacked the AUM, suggesting focal compromise of the blood-urine permeability barrier. In such relatively undifferentiated urothelial zones there was an accompanying increase in the expression of iNOS, which marks perturbed urothelial differentiation and may modulate bladder response to the outlet obstruction.
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In urothelium, nitric oxide (NO) produced by NO synthase (NOS) plays various roles during disease processes. Because diseases influence the expression of uroplakins, the main urothelial differentiation-related proteins, we compared their... more
In urothelium, nitric oxide (NO) produced by NO synthase (NOS) plays various roles during disease processes. Because diseases influence the expression of uroplakins, the main urothelial differentiation-related proteins, we compared their expression and localization with that of inducible NOS (iNOS) in bladder outlet obstruction caused by benign prostatic hyperplasia and in noninvasive urothelial neoplasms (papilloma, low-grade, and high-grade papillary carcinoma). In all cases, we found areas with terminal and areas with partial cell differentiation. Terminally differentiated urothelium was uroplakin positive and iNOS negative. Areas of partial differentiation contained superficial cells with altered apical surface morphology and with no or weak uroplakin staining. These areas showed elevated iNOS staining. By immunoelectron microscopy, it was demonstrated for the first time that iNOS was localized in mitochondria of urothelial cells that show partial differentiation. These results suggest that various urinary bladder lesions alter the normal differentiation pathway of urothelial superficial cells, which induces the expression of NOS in mitochondria of partially differentiated cells.
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Delivery of therapeutic proteins into tissues and across the blood-brain barrier (BBB) is limited by the size and biochemical properties of the proteins. Efficient delivery across BBB is generally restricted to small, highly lipophilic... more
Delivery of therapeutic proteins into tissues and across the blood-brain barrier (BBB) is limited by the size and biochemical properties of the proteins. Efficient delivery across BBB is generally restricted to small, highly lipophilic molecules. However, in the last decades, several peptides that can pass cell membranes have been identified. It has been shown that these peptides are also capable of delivering large hydrophilic cargoes into cells and are therefore a powerful biological tool for transporting drugs across cell membranes and even into the brain. We designed and prepared a single-chain antibody fragment (scFvs), specific for the pathological form of the prion protein (PrP(Sc)), where a cell-penetrating peptide (CPP) was used as a linker between the two variable domains of the scFv. The intravenously administered recombinant scFv-CPP was successfully targeted to and delivered into mouse brain cells. Our single-chain antibody fragments are of special interest in view of possible therapeutic reagents design not only for prion diseases but also for other neurodegenerative diseases.