The small GTPase Rab13 regulates assembly of functional tight junctions in epithelial cells.

Junctional complexes such as tight junctions (TJ) and adherens junctions are required for maintaining cell surface asymmetry and polarized transport in epithelial cells. We have shown that Rab13 is recruited to junctional complexes from a cytosolic pool after cell-cell contact formation. In this study, we investigate the role of Rab13 in modulating TJ structure and functions in epithelial MDCK cells. We generate stable MDCK cell lines expressing inactive (T22N mutant) and constitutively active (Q67L mutant) Rab13 as GFP-Rab13 chimeras. Expression of GFP-Rab13Q67L delayed the formation of electrically tight epithelial monolayers as monitored by transepithelial electrical resistance (TER) and induced the leakage of small nonionic tracers from the apical domain. It also disrupted the TJ fence diffusion barrier. Freeze-fracture EM analysis revealed that tight junctional structures did not form a continuous belt but rather a discontinuous series of stranded clusters. Immunofluorescence studies showed that the expression of Rab13Q67L delayed the localization of the TJ transmembrane protein, claudin1, at the cell surface. In contrast, the inactive Rab13T22N mutant did not disrupt TJ functions, TJ strand architecture nor claudin1 localization. Our data revealed that Rab13 plays an important role in regulating both the structure and function of tight junctions.

Lens connexins alpha3Cx46 and alpha8Cx50 interact with zonula occludens protein-1 (ZO-1).

Connexin alpha1Cx43 has previously been shown to bind to the PDZ domain-containing protein ZO-1. The similarity of the carboxyl termini of this connexin and the lens fiber connexins alpha3Cx46 and alpha8Cx50 suggested that these connexins may also interact with ZO-1. ZO-1 was shown to be highly expressed in mouse lenses. Colocalization of ZO-1 with alpha3Cx46 and alpha8Cx50 connexins in fiber cells was demonstrated by immunofluorescence and by fracture-labeling electron microscopy but showed regional variations throughout the lens. ZO-1 was found to coimmunoprecipitate with alpha3Cx46 and alpha8Cx50, and pull-down experiments showed that the second PDZ domain of ZO-1 was involved in this interaction. Transiently expressed alpha3Cx46 and alpha8Cx50 connexins lacking the COOH-terminal residues did not bind to the second PDZ domain but still formed structures resembling gap junctions by immunofluorescence. These results indicate that ZO-1 interacts with lens fiber connexins alpha3Cx46 and alpha8Cx50 in a manner similar to that previously described for alpha1Cx43. The spatial variation in the interaction of ZO-1 with lens gap junctions is intriguing and is suggestive of multiple dynamic roles for this association.

Chronic hemodynamic overload of the atria is an important factor for gap junction remodeling in human and rat hearts.

OBJECTIVES: The expression and distribution of connexins is abnormal in a number of cardiac diseases, including atrial fibrillation, and is believed to favor conduction slowing and arrhythmia. Here, we studied the role of atrial structural remodeling in the disorganization of gap junctions and whether redistributed connexins can form new functional junction channels. METHODS: Expression of connexin-43 (Cx43) was characterized by immunoblotting and immunohistochemistry in human right atrial specimens and in rat atria after myocardial infarction (MI). Gap junctions were studied by electron and 3-D microscopy, and myocyte-myocyte coupling was determined by Lucifer yellow dye transfer. RESULTS: In both chronically hemodynamically overloaded human atria in sinus rhythm and in dilated atria from MI-rats, Cx43 were dephosphorylated and redistributed from the intercalated disc to the lateral cell membranes as observed during atrial fibrillation. In MI-rats, the gap junctions at the intercalated disc were smaller (20% decrease) and contained very little Cx43 (0 or 1 gold particle vs. 42 to 98 in sham-operated rats). In the lateral membranes of myocytes, numerous connexon aggregates comprising non-phosphorylated Cx43 were observed. These connexon aggregates were in no case assembled into gap junction plaque-like structures. However, N-cadherin was well organized in the intercalated disc. There was very little myocyte-myocyte coupling in MI-rat atria and no myocyte-fibroblast coupling. Regression of the atrial remodeling was associated with the normalization of Cx43 localization. CONCLUSION: Structural alteration of the atrial myocardium is an important factor in the disorganization of connexins and gap junction. Moreover, redistributed Cx43 do not form junction channels.

Viruses of parasites as actors in the parasite-host relationship: A "ménage à trois".

The complex parasite-host relationship involves multiple mechanisms. Moreover, parasites infected by viruses modify this relationship adding more complexity to the system that now comprises three partners. Viruses infecting parasites were described several decades ago. However, until recently little was known about the viruses involved and their impact on the resulting disease caused to the hosts. To clarify this situation, we have concentrated on parasitic diseases caused to humans and on how virus-infected parasites could alter the symptoms inflicted on the human host. It is clear that the effect caused to the human host depends on the virus and on the parasite it has infected. Consequently, the review is divided as follows: Viruses with a possible effect on the virulence of the parasite. This section reviews pertinent articles showing that infection of parasites by viruses might increase the detrimental effect of the tandem virus-parasite on the human host (hypervirulence) or decrease virulence of the parasite (hypovirulence). Parasites as vectors affecting the transmission of viruses. In some cases, the virus-infected parasite might facilitate the transfer of the virus to the human host. Parasites harboring viruses with unidentified effects on their host. In spite of recently renewed interest in parasites in connection with their viruses, there still remains a number of cases in which the effect of the virus of a given parasite on the human host remains ambiguous. The triangular relationship between the virus, the parasite and the host, and the modulation of the pathogenicity and virulence of the parasites by viruses should be taken into account in the rationale of fighting against parasites.

Post-translational amino acid racemization in the frog skin peptide deltorphin I in the secretion granules of cutaneous serous glands.

The dermal glands of the South American hylid frog Phyllomedusa bicolor synthesize and expel huge amounts of cationic, alpha-helical, 24- to 33-residue antimicrobial peptides, the dermaseptins B. These glands also produce a wide array of peptides that are similar to mammalian hormones and neuropeptides, including a heptapeptide opioid containing a D-amino acid, deltorphin I (Tyr-DAla-Phe-Asp-Val-Val-Gly NH2). Its biological activity is due to the racemization of L-Ala2 to D-Ala. The dermaseptins B and deltorphins are all derived from a single family of precursor polypeptides that have an N-terminal preprosequence that is remarkably well conserved, although the progenitor sequences giving rise to mature opioid or antimicrobial peptides are markedly different. Monoclonal and polyclonal antibodies were used to examine the cellular and ultrastructural distributions of deltorphin I and dermaseptin B in the serous glands by immunofluoresence confocal microscopy and immunogold-electron microscopy. Preprodeltorphin I and preprodermaseptins B are sorted into the regulated pathway of secretion, where they are processed to give the mature products. Deltorphin I, [l-Ala2]-deltorphin I and dermaseptin B are all stored together in secretion granules which accumulate in the cytoplasm of all serous glands. We conclude that the L- to D-amino acid isomerization of the deltorphin I occurs in the secretory granules as a post-translational event. Thus the specificity of isomerization depends on the presence of structural and/or conformational determinants in the peptide N-terminus surrounding the isomerization site.

Structural and immunocytochemical alterations in eye lens fiber cells from Cx46 and Cx50 knockout mice.

In the current study we describe the changes of overall organization of lens fiber cells in connexin 46 (Cx46) and connexin 50 (Cx50) knockout mice. Morphometric analyses and the application of immunocytochemical techniques revealed that in Cx46 knockout lens (Cx46 -/-), where Cx50 is expressed alone, the postnatal differentiation of secondary fiber cells proceeds faster and is characterized by an increased number of smaller fiber cells. Conversely, in Cx50 knockout mice (Cx50 -/-), the lenticular mass is considerably reduced and characterized by a small number of fiber cells added during the postnatal period. The process of terminal differentiation was impaired and generated larger fiber cells still possessing cytoplasmic organelles. Freeze-fracture and fracture labeling revealed that the junctional assembly, packing organization and topographic interactions between connexons and MP26 differed when Cx46 and Cx50 were co-assembled in the wild-type or expressed separately in the two distinct knockout phenotypes. Filipin cytochemistry provided indirect evidence that Cx46 and Cx50 expressed alone are recruited into different lipid environments. Our results represent the structural proof that interaction of connexins and MP26 contributes to the overall organization of the fiber cells.

Persistent, noncytolytic infection of neurons by Borna disease virus interferes with ERK 1/2 signaling and abrogates BDNF-induced synaptogenesis.

Infection of the central nervous system by Borna disease virus (BDV) provides a unique model to study the mechanisms whereby a persistent viral infection can impair neuronal function and cause behavioral diseases reminiscent of mood disorders, schizophrenia, or autism in humans. In the present work, we studied the effect of BDV infection on the response of hippocampal neurons, the main target for this virus, to the neurotrophin BDNF. We showed that persistent infection did not affect neuronal survival or morphology. However, it blocked BDNF-induced ERK 1/2 phosphorylation, despite normal expression of the TrkB BDNF receptor. In addition, BDNF-induced expression of synaptic vesicle proteins was abrogated, which resulted in severely impaired synaptogenesis and defects in synaptic organization. Thus, we provide the first evidence that a virus can interfere specifically with neurotrophin-regulated neuroplasticity, thereby hampering proper neuronal connectivity. These results may help to understand the behavioral disorders associated with BDV infection.

A new nidovirus (NamDinh virus NDiV): Its ultrastructural characterization in the C6/36 mosquito cell line.

We describe the ultrastructure of the NamDinh virus (NDiV), a new member of the order Nidovirales grown in the C6/36 mosquito cell line. Uninfected and NDiV-infected cells were investigated by electron microscopy 24-48 h after infection. The results show that the viral nucleocapsid-like particles form clusters concentrated in the vacuoles, the endoplasmic reticulum, and are scattered in the cytoplasm. Mature virions of NDiV were released as budding particles on the cell surface where viral components appear to lie beneath and along the plasma membrane. Free homogeneous virus particles were obtained by ultracentrifugation on sucrose gradients of culture fluids. The size of the round-shaped particles with a complete internal structure was 80 nm in diameter. This is the first study to provide information on the morphogenesis and ultrastructure of the first insect nidovirus NDiV, a missing evolutionary link in the emergence of the viruses with the largest RNA genomes.

Laparoscopy in ureteral engineering: a feasibility study.

OBJECTIVE: We recently bioengineered a ureter substitute from a seeded scaffold implanted by open surgery in the omentum. In view of the development of laparoscopy in the treatment of benign conditions of the ureter, obtaining a ureter substitute by minimally invasive techniques would be a desirable objective. However, conflicting results about the biological impact of carbon dioxide insufflation on the microcirculation of intra-abdominal organs prompted us to investigate first whether the results obtained by open surgery, in terms of vascular supply and maturation, could be reproduced laparoscopically. MATERIALS AND METHODS: Bladder full-thickness tissue was harvested laparoscopically from three pigs for urothelial and smooth muscle cell primary cultures subsequently used to seed a small intestinal submucosa (SIS) matrix. After 2 wk, the in vitro seeded constructs were shaped around silicone drains and transferred laparoscopically into the abdomen for omental maturation. Three weeks later, the constructs were harvested for histological, immunohistochemical, and electron microscopic analysis. RESULTS: The laparoscopic procedures were performed successfully in all animals. After omental maturation, the constructs were vascularized and comprised of a well-differentiated multilayered urothelium with umbrella cells, over connective tissue and smooth muscle cells, with no evidence of fibrosis or inflammation. Electron microscopic analysis showed characteristics of a terminally differentiated urothelium. CONCLUSION: As shown by conventional microscopy, immunochemistry, and electron microscopy, carbon dioxide insufflation does not impact cell growth and differentiation. These findings validate the laparoscopic approach for omental maturation of ureter substitutes.

Absence of alpha3 (Cx46) and alpha8 (Cx50) connexins leads to cataracts by affecting lens inner fiber cells.

Lens development and transparency have been hypothesized to depend on intercellular gap junction channels, consisting of alpha3 (Cx46) and alpha8 (Cx50) connexin subunits, to transport metabolites, secondary messages and ions between lens cells. To evaluate this hypothesis, we have generated alpha3(-/-) alpha8(-/-) double knockout mice and characterized their lens phenotypes. Without gap junctions between lens fiber cells, alpha3(-/-) alpha8(-/-) lenses displayed severe cataracts resulting from cell swelling and degeneration of inner fibers while normal peripheral fiber cells continued to form throughout life. Neither an increase of degraded crystallins nor an increase of water-insoluble crystallins was found in alpha3(-/-) alpha8(-/-) lenses. However, a substantial reduction of gamma-crystallin proteins, but not alpha- and beta-crystallins, was detected. These results suggest that gap junction communication is important for maintaining lens homeostasis of inner fiber cells and that a loss of gap junctions leads to cataract formation as well as reductions of gamma-crystallin proteins and transcripts.

Caveolins and flotillin-2 are present in the blood stages of Plasmodium vivax.

Blood stages of Plasmodium vivax induce the development of caveolae and caveola-vesicle complexes (CVC) in the membrane of their host erythrocyte. Caveolae are found in almost all types of cells and are involved in endogenous processes as calcium and cholesterol homeostasis, cell signalling, transporting, ligand internalization and transcytosis of serum components. Major structural components of caveolae are the proteins caveolins and flotillins. The functional role of caveolae in the P. vivax-infected erythrocyte is not properly understood. As these organelles have been shown to contain malaria antigens, it has been suggested that they are involved in the transport and release of specific parasite antigens from the infected erythrocyte and in the uptake of plasma proteins. Using specific antibodies to classical caveolae proteins and an immunolocalization approach, we found caveolin-2, caveolin-3, and flotillin-2 in the vesicle profiles and some CVC of P. vivax-infected erythrocytes. Caveolin-1-3 were not found in uninfected erythrocytes. This is the first report of identification and localization of caveolins in the CVC present in erythrocytes infected with P. vivax, thereby providing evidence of the role of this particular organelle in the protein-trafficking pathway that connect parasite-encoded proteins with the erythrocyte cytoplasm and the cell surface throughout the asexual blood cycle of vivax malaria parasite.

Traffic pathways of Plasmodium vivax antigens during intraerythrocytic parasite development.

We investigated the secretory traffic of a Plasmodium vivax antigen (Pv-148) synthesised by the parasite during the blood cycle, exported into the host cell cytosol and then transported to the surface membrane of the infected erythrocyte. Studies of the ultrastructure of erythrocytes infected with P. vivax showed that intracellular schizogony is accompanied by the generation of parasite-induced membrane profiles in the erythrocyte cytoplasm. These structures are detectable soon after the parasite invades the erythrocyte and develop an elaborate organisation, leading to a tubovesicular membrane (TVM) network, in erythrocytes infected with mature trophozoites. Interestingly, the clefts formed stacked, flattened cisternae resembling a classical Golgi apparatus. The TVM network stained with the fluorescent Golgi marker Bodipy-ceramide. Specific immunolabelling showed that Pv-148 was transferred from the parasite to the erythrocyte surface membrane via the clefts and the TVM network. These findings suggest that the TVM network is part of the secretory pathways involved in parasite protein transport across the Plasmodium-infected erythrocyte and that Pv- 148 may represent a marker that links the parasite with the host cell cytoplasm and, in turn, with the extracellular milieu.

Disruption of Gja8 (alpha8 connexin) in mice leads to microphthalmia associated with retardation of lens growth and lens fiber maturation.

The development of the vertebrate lens utilizes a sophisticated cell-cell communication network via gap junction channels, which are made up of at least three connexin isoforms, alpha8 (Cx50), alpha3 (Cx46) and alpha1 (Cx43), and which are encoded by three different genes. In a previous study, we reported that, with a disruption of Gja3 (alpha3 connexin), mice developed nuclear cataracts with a normal sized lens. We show that Gja8tm1 (alpha8-/-) mice develop microphthalmia with small lenses and nuclear cataracts, while the alpha8 heterozygous (+/-) mice have relatively normal eyes and lenses. A comparative study of these alpha3 and alpha8 knockout mice showed that the protein levels of both alpha3 and alpha8 were independently regulated and there was no compensation for either the alpha3 or alpha8 protein from the wild-type allele when the other allele was disrupted. More interestingly, western blotting data indicated that the presence of alpha8 in the lens nucleus is dependent on alpha3 connexin, but not vice versa. The staining of the knock-in lacZ reporter gene showed the promoter activity of alpha8 connexin is much higher than that of alpha3 connexin in embryonic lenses and in adult lens epithelium. More importantly, a delayed denucleation process was observed in the interior fibers of the alpha8-/- lenses. Therefore, alpha8 connexin is required for proper fiber cell maturation and control of lens size.