Amino acid substitutions in σ1 and μ1 outer capsid proteins are selected during mammalian reovirus adaptation to Vero cells

Establishment of viral persistence in cell culture has previously led to the selection of mammalian reovirus mutants, although very few of those have been characterized in details. In the present study, reovirus was adapted to Vero cells that, in contrast to classically-used L929 cells, are inefficient in supporting the early steps of reovirus uncoating and are also unable to produce interferon as an antiviral response once infection occurs. The Vero cell-adapted reovirus exhibits amino acids substitutions in both the σ1 and μ1 proteins. This contrasts with uncoating mutants from persistently-infected L929 cells, and various other cell types, that generally harbor amino acids substitutions in the σ3 outer capsid protein. The Vero cell-adapted virus remained sensitive to an inhibitor of lysosomal proteases; furthermore, in the absence of selective pressure for its maintenance, t he virus has partially lost its ability to resist interferon. The positions of the amino acids substitutions on the known protein structures suggest an effect on binding of the viral σ1 protein to the cell surface and on μ1 disassembly from the outer capsid.

Putative outer membrane proteins of Leptospira interrogans stimulate human umbilical vein endothelial cells (HUVECS) and express during infection

Cell adhesion molecules (CAMs) are surface receptors present in eukaryotic cells that mediate cell-cell or cell-extracellular matrix interactions. Vascular endothelium stimulation in vitro that lead to the upregulation of CAMs was reported for the pathogenic spirochaetes, including rLIC10365 of Leptospira interrogans. In this study, we report the cloning of LIC10507, LIC10508, LIC10509 genes of L interrogans using Escherichia coli as a host system. The rational for selecting these sequences is due to their location in L. interrogans serovar Copenhageni genome that has a potential involvement in pathogenesis. The genes encode for predicted lipoproteins with no assigned functions. The purified recombinant proteins were capable to promote the upregulation of intercellular adhesion molecule 1 (ICAM-1) and E-selectin on monolayers of human umbilical vein endothelial cells (HUVECS). In addition, the coding sequences are expressed in the renal tubules of animal during bacterial experimental infection. The proteins are probably located at the outer membrane of the bacteria since they are detected in detergent-phase of L interrogans Triton X-114 extract. Altogether our data suggest a possible involvement of these proteins during bacterial infection and provide new insights into the role of this region in the pathogenesis of Leptospira. (C) 2008 Elsevier Ltd. All rights reserved.

Detection of cytokeratins in ghost cells of calcifying cystic odontogenic tumor indicates an altered keratinization and hair follicle differentiation for their development

Calcifying cystic odontogenic tumors (CCOTs) are benign cystic lesions of odontogenic origin characterized by an ameloblastoma-like epithelium and the presence of a group of cells named ghost cells. The pattern of cytokeratin (Ck) expression on these lesions remains unclear and needs to be clarified. To this end, the expression of Ck6, Ck13, Ck14, Ck18, and Ck19 in the epithelium lining of 7 cases of CCOTs was evaluated by immunohistochemistry. For this, the epithelium lining was divided into 3 distinct regions: basal layer, suprabasal layer, and the compartment composed of ghost cells. In this study, 6 cases (85.7%) were classified as type 1 and 1 (14.3%) as type 4. All cases were negative for Ck13 and Ck18, despite the epithelial layer, as well as in the ghost cells. Ck6 was only positive in the ghost cells. Positivity for Ck14 and Ck19 was found in the basal and suprabasal layers, including the ghost cells. The results showing positivity for Ck14 and Ck19 in all of the analyzed cases reinforce CCOT as being of odontogenic origin, and the restricted expression of Ck6 in the ghost cells may be indicative that these cells suffer an altered differentiation into hair follicles in CCOTs. © 2013 Elsevier Inc. All rights reserved.

Mitochondrial Swelling and Incipient Outer Membrane Rupture in Preapoptotic and Apoptotic Cells

Outer mitochondrial membrane (OMM) rupture was first noted in isolated mitochondria in which the inner mitochondrial membrane (IMM) had lost its selective permeability. This phenomenon referred to as mitochondrial permeability transition (MPT) refers to a permeabilized inner membrane that originates a large swelling in the mitochondrial matrix, which distends the outer membrane until it ruptures. Here, we have expanded previous electron microscopic observations that in apoptotic cells, OMM rupture is not caused by a membrane stretching promoted by a markedly swollen matrix. It is shown that the widths of the ruptured regions of the OMM vary from 6 to 250 nm. Independent of the perforation size, herniation of the mitochondrial matrix appeared to have resulted in pushing the IMM through the perforation. A large, long focal herniation of the mitochondrial matrix, covered with the IMM, was associated with a rupture of the OMM that was as small as 6 nm. Contextually, the collapse of the selective permeability of the IMM may precede or follow the release of the mitochondrial proteins of the intermembrane space into the cytoplasm. When the MPT is a late event, exit of the intermembrane space proteins to the cytoplasm is unimpeded and occurs through channels that transverse the outer membrane, because so far, the inner membrane is impermeable. No channel within the outer membrane can expose to the cytoplasm a permeable inner membrane, because it would serve as a conduit for local herniation of the mitochondrial matrix. Anat Rec, 2012. (c) 2012 Wiley Periodicals, Inc.

Human melanocytes can be isolated, propagated and expanded from plucked anagen hair follicles

Herein, we report a technically simple method for isolation and culture of human follicular melanocytes based on explant cultures of epilated hair follicles. This technique does not require any surgical intervention and allows the isolation and cultivation of follicular melanocytes from a comparatively small amount of raw material. Generally, 30-60 human anagen hair follicles have been plucked from the scalp of healthy donors and cultivated under low oxygen pressure (5%). After a short period of time cells of various types were growing out from the outer root sheath (ORS) of the hair follicles. Under the selected culture conditions, most of the cells other than melanocytes have been eliminated and a nearly 100% pure population of melanocytes has been achieved, as confirmed by immunohistochemical analyses for melanocyte-specific markers, for example, Tyrosinase-1, S-100 and premelanosomal antigens. These melanocytes derived from the ORS were proliferating for up to 2 months.

Outer membrane protein UspA1 and lipooligosaccharide are involved in invasion of human epithelial cells by Moraxella catarrhalis

Invasion of non-professional phagocytes is a strategy employed by several mucosal pathogens, but has not been investigated in detail for Moraxella catarrhalis, a major cause of human respiratory tract infections. We investigated the role of outer membrane protein (OMP) UspA1 and lipooligosaccharide (LOS) in M. catarrhalis invasion into epithelial cells. An isogenic mutant of strain O35E, which lacked expression of the UspA1 adhesin, demonstrated not only severely impaired adherence (86%) to but also reduced invasion (77%) into Chang conjunctival cells in comparison with the wild-type strain. The isogenic, LOS-deficient mutant strain O35E.lpxA was attenuated in adherence (93%) and its capacity to invade was severely reduced (95%), but not abolished. Inhibition assays using sucrose and cytochalasin D, respectively, demonstrated that clathrin and actin polymerization contribute to internalization of M. catarrhalis by Chang cells. Furthermore, inhibition of UspA1-mediated binding to cell-associated fibronectin and alpha5beta1 integrin decreased invasion of M. catarrhalis strain O35E (72% and 41%, respectively). These data indicate that OMP UspA1 and LOS profoundly affect the capacity of M. catarrhalis to invade epithelial cells.

Repigmentation by outer-root-sheath-derived melanocytes: proof of concept in vitiligo and leucoderma

BACKGROUND: Treatment of depigmented skin is an unmet medical need. OBJECTIVE: Melanocytes or stem cells thereof residing in the outer root sheath (ORS) of hair follicles might be used to repigment skin. METHODS: After de-epidermisation, autologous ORS cell solutions were applied to 5 patients with vitiligo and 1 with leucoderma. RESULTS: Stable repigmentation in a variable percentage was documented in all the patients. CONCLUSION: Applying ORS-derived melanocytes is a promising technology to improve autologous melanocyte transplantation.

Phagocytosis of rod outer segments by retinal pigment epithelial cells requires αvβ5 integrin for binding but not for internalization

Phagocytosis of shed photoreceptor rod outer segments (ROS) by the retinal pigment epithelium (RPE) is essential for retinal function. Here, we demonstrate that this process requires αvβ5 integrin, rather than αvβ3 integrin utilized by systemic macrophages. Although adult rat RPE expressed both αvβ3 and αvβ5 integrins, only αvβ3 was expressed at birth, when the retina is immature and phagocytosis is absent. Expression of αvβ5 was first detected in RPE at PN7 and reached adult levels at PN11, just before onset of phagocytic activity. Interestingly, αvβ5 localized in vivo to the apical plasma membrane, facing the photoreceptors, and to intracellular vesicles, whereas αvβ3 was expressed basolaterally. Using quantitative fluorimaging to assess in vitro uptake of fluorescent particles by human (ARPE-19) and rat (RPE-J) cell lines, αvβ5 function-blocking antibodies were shown to reduce phagocytosis by drastically decreasing (85%) binding of ROS but not of latex beads. In agreement with a role for αvβ5 in phagocytosis, immunofluorescence experiments demonstrated codistribution of αvβ5 integrin with internalized ROS. Control experiments showed that blocking αvβ3 function with antibodies did not inhibit ROS phagocytosis and that αvβ3 did not colocalize with phagocytosed ROS. Taken together, our results indicate that the RPE requires the integrin receptor αvβ5 specifically for the binding of ROS and that phagocytosis involves internalization of a ROS-αvβ5 complex. αvβ5 integrin does not participate in phagocytosis by other phagocytic cells and is the first of the RPE receptors involved in ROS phagocytosis that may be specific for this process.

Saccharomyces cerevisiae Cells with Defective Spindle Pole Body Outer Plaques Accomplish Nuclear Migration via Half-Bridge–organized Microtubules

Cnm67p, a novel yeast protein, localizes to the microtubule organizing center, the spindle pole body (SPB). Deletion of CNM67 (YNL225c) frequently results in spindle misorientation and impaired nuclear migration, leading to the generation of bi- and multinucleated cells (40%). Electron microscopy indicated that CNM67 is required for proper formation of the SPB outer plaque, a structure that nucleates cytoplasmic (astral) microtubules. Interestingly, cytoplasmic microtubules that are essential for spindle orientation and nuclear migration are still present in cnm67Δ1 cells that lack a detectable outer plaque. These microtubules are attached to the SPB half- bridge throughout the cell cycle. This interaction presumably allows for low-efficiency nuclear migration and thus provides a rescue mechanism in the absence of a functional outer plaque. Although CNM67 is not strictly required for mitosis, it is essential for sporulation. Time-lapse microscopy of cnm67Δ1 cells with green fluorescent protein (GFP)-labeled nuclei indicated that CNM67 is dispensable for nuclear migration (congression) and nuclear fusion during conjugation. This is in agreement with previous data, indicating that cytoplasmic microtubules are organized by the half-bridge during mating.

Detection of synchrony in the activity of auditory nerve fibers by octopus cells of the mammalian cochlear nucleus

The anatomical and biophysical specializations of octopus cells allow them to detect the coincident firing of groups of auditory nerve fibers and to convey the precise timing of that coincidence to their targets. Octopus cells occupy a sharply defined region of the most caudal and dorsal part of the mammalian ventral cochlear nucleus. The dendrites of octopus cells cross the bundle of auditory nerve fibers just proximal to where the fibers leave the ventral and enter the dorsal cochlear nucleus, each octopus cell spanning about one-third of the tonotopic array. Octopus cells are excited by auditory nerve fibers through the activation of rapid, calcium-permeable, α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptors. Synaptic responses are shaped by the unusual biophysical characteristics of octopus cells. Octopus cells have very low input resistances (about 7 MΩ), and short time constants (about 200 μsec) as a consequence of the activation at rest of a hyperpolarization-activated mixed-cation conductance and a low-threshold, depolarization-activated potassium conductance. The low input resistance causes rapid synaptic currents to generate rapid and small synaptic potentials. Summation of small synaptic potentials from many fibers is required to bring an octopus cell to threshold. Not only does the low input resistance make individual excitatory postsynaptic potentials brief so that they must be generated within 1 msec to sum but also the voltage-sensitive conductances of octopus cells prevent firing if the activation of auditory nerve inputs is not sufficiently synchronous and depolarization is not sufficiently rapid. In vivo in cats, octopus cells can fire rapidly and respond with exceptionally well-timed action potentials to periodic, broadband sounds such as clicks. Thus both the anatomical specializations and the biophysical specializations make octopus cells detectors of the coincident firing of their auditory nerve fiber inputs.

A recombinant Chlamydia trachomatis major outer membrane protein binds to heparan sulfate receptors on epithelial cells.

Chlamydial attachment to columnar conjunctival or urogenital epithelial cells is an initial and critical step in the pathogenesis of chlamydial mucosal infections. The chlamydial major outer membrane protein (MOMP) has been implicated as a putative chlamydial cytoadhesin; however, direct evidence supporting this hypothesis has not been reported. The function of MOMP as a cytoadhesin was directly investigated by expressing the protein as a fusion with the Escherichia coli maltose binding protein (MBP-MOMP) and studying its interaction with human epithelial cells. The recombinant MBP-MOMP bound specifically to HeLa cells at 4 degrees C but was not internalized after shifting the temperature to 37 degrees C. The MBP-MOMP competitively inhibited the infectivity of viable chlamydiae for epithelial cells, indicating that the MOMP and intact chlamydiae bind the same host receptor. Heparan sulfate markedly reduced binding of the MBP-MOMP to cells, whereas chondroitin sulfate had no effect on binding. Enzymatic treatment of cells with heparitinase but not chondroitinase inhibited the binding of MBP-MOMP. These same treatments were also shown to reduce the infectivity of chlamydiae for epithelial cells. Mutant cell lines defective in heparan sulfate synthesis but not chondroitin sulfate synthesis showed a marked reduction in the binding of MBP-MOMP and were also less susceptible to infection by chlamydiae. Collectively, these findings provide strong evidence that the MOMP functions as a chlamydial cytoadhesin and that heparan sulfate proteoglycans are the host-cell receptors to which the MOMP binds.

Phagocytosis of Photoreceptor Outer Segments by Transplanted Human Neural Stem Cells as a Neuroprotective Mechanism in Retinal Degeneration

Purpose. Transplantation of human central nervous system stem cells (HuCNS-SC) into the subretinal space of Royal College of Surgeons (RCS) rats preserves photoreceptors and visual function. To explore possible mechanism(s) of action underlying this neuroprotective effect, we performed a detailed morphologic and ultrastructure analysis of HuCNS-SC transplanted retinas. Methods. The HuCNS-SC were transplanted into the subretinal space of RCS rats. Histologic examination of the transplanted retinas was performed by light and electron microscopy. Areas of the retina adjacent to HuCNS-SC graft (treated regions) were analyzed and compared to control sections obtained from the same retina, but distant from the transplant site (untreated regions). Results. The HuCNS-SC were detected as a layer of STEM 121 immunopositive cells in the subretinal space. In treated regions, preserved photoreceptor nuclei, as well as inner and outer segments were identified readily. In contrast, classic signs of degeneration were observed in the untreated regions. Interestingly, detailed ultrastructure analysis revealed a striking preservation of the photoreceptor–bipolar–horizontal cell synaptic contacts in the outer plexiform layer (OPL) of treated areas, in stark contrast with untreated areas. Finally, the presence of phagosomes and vesicles exhibiting the lamellar structure of outer segments also was detected within the cytosol of HuCNS-SC, indicating that these cells have phagocytic capacity in vivo. Conclusions. This study reveals the novel finding that preservation of specialized synaptic contacts between photoreceptors and second order neurons, as well as phagocytosis of photoreceptor outer segments, are potential mechanism(s) of HuCNS-SC transplantation, mediating functional rescue in retinal degeneration.