p53 immunostaining is correlated with reduced survival and is not correlated with gene mutations in resected pulmonary large cell carcinomas

Malignancy of pulmonary large cell carcinomas (LCC) increases from classic LCC through LCC with neuroendocrine morphology (LCCNM) to large cell neuroendocrine carcinomas (LCNEC). However, the histological classification has sometimes proved to be difficult. Because the malignancy of LCC is highly dependent on proteins with functions in the cell cycle, DNA repair, and apoptosis, p53 has been targeted as a potentially useful biological marker. p53 mutations in lung cancers have been shown to result in expression and protein expression also occurs in the absence of mutations. To validate the importance of both p53 protein expression (by immunostaining) and p53 gene mutations in lung LCC (by PCR-single strand conformational polymorphism analysis of exons 5, 6, 7, and 8) and to study their relationships with clinical factors and sub-classification we investigated the correlation of p53 abnormalities in 15 patients with LCC (5 classic LCC, 5 LCNEC, and 5 LCCNM) who had undergone resection with curative intent. Of these patients, 5/15 expressed p53 and none had mutant p53 sequences. There was a negative survival correlation with positive p53 immunostaining (P = 0.05). After adjustment for stage, age, gender, chemotherapy, radiotherapy, and histological subtypes by multivariate analysis, p53 expression had an independent impact on survival. The present study indicates that p53 assessment may provide an objective marker for the prognosis of LCC irrespective of morphological variants and suggests that p53 expression is important for outcome prediction in patients with the early stages of LCC. The results reported here should be considered to be initial results because tumors from only 15 patients were studied: 5 each from LCC, LCNEC and LCCNM. This was due to the rarity of these specific diseases.

Stem cell factor protects against neuronal apoptosis by activating AKT/ERK in diabetic mice

Neuronal apoptosis occurs in the diabetic brain due to insulin deficiency or insulin resistance, both of which reduce the expression of stem cell factor (SCF). We investigated the possible involvement of the activation of the MAPK/ERK and/or AKT pathways in neuroprotection by SCF in diabetes. Male C57/B6 mice (20-25 g) were randomly divided into four groups of 10 animals each. The morphology of the diabetic brain in mice treated or not with insulin or SCF was evaluated by H&E staining and TUNEL. SCF, ERK1/2 and AKT were measured by Western blotting. In diabetic mice treated with insulin or SCF, there was fewer structural change and apoptosis in the cortex compared to untreated mice. The apoptosis rate of the normal group, the diabetic group receiving vehicle, the diabetic group treated with insulin, and the diabetic group treated with SCF was 0.54 ± 0.077%, 2.83 ± 0.156%, 1.86 ± 0.094%, and 1.78 ± 0.095% (mean ± SEM), respectively. SCF expression was lower in the diabetic cortex than in the normal cortex; however, insulin increased the expression of SCF in the diabetic cortex. Furthermore, expression of phosphorylated ERK1/2 and AKT was decreased in the diabetic cortex compared to the normal cortex. However, insulin or SCF could activate the phosphorylation of ERK1/2 and AKT in the diabetic cortex. The results suggest that SCF may protect the brain from apoptosis in diabetes and that the mechanism of this protection may, at least in part, involve activation of the ERK1/2 and AKT pathways. These results provide insight into the mechanisms by which SCF and insulin exert their neuroprotective effects in the diabetic brain.

Relationships between dendritic morphology, spatial distribution and firing patterns in rat layer 1 neurons

The cortical layer 1 contains mainly small interneurons, which have traditionally been classified according to their axonal morphology. The dendritic morphology of these cells, however, has received little attention and remains ill defined. Very little is known about how the dendritic morphology and spatial distribution of these cells may relate to functional neuronal properties. We used biocytin labeling and whole cell patch clamp recordings, associated with digital reconstruction and quantitative morphological analysis, to assess correlations between dendritic morphology, spatial distribution and membrane properties of rat layer 1 neurons. A total of 106 cells were recorded, labeled and subjected to morphological analysis. Based on the quantitative patterns of their dendritic arbor, cells were divided into four major morphotypes: horizontal, radial, ascendant, and descendant cells. Descendant cells exhibited a highly distinct spatial distribution in relation to other morphotypes, suggesting that they may have a distinct function in these cortical circuits. A significant difference was also found in the distribution of firing patterns between each morphotype and between the neuronal populations of each sublayer. Passive membrane properties were, however, statistically homogeneous among all subgroups. We speculate that the differences observed in active membrane properties might be related to differences in the synaptic input of specific types of afferent fibers and to differences in the computational roles of each morphotype in layer 1 circuits. Our findings provide new insights into dendritic morphology and neuronal spatial distribution in layer 1 circuits, indicating that variations in these properties may be correlated with distinct physiological functions.

Escherichia coli STb toxin induces apoptosis in intestinal epithelial cell lines

La toxine stable à la chaleur de type b (STb) est une des toxines produites par les souches Enterotoxigenic Escherichia coli (ETEC) impliquée dans le développement de la diarrhée. Une étude antérieure par Goncalves et al. (2009) a démontré que les cellules ayant internalisé la toxine STb démontraient une morphologie qui rappelle l’apoptose. Le changement du potentiel membranaire observé par Goncalves et al. (2009) nous a incité à vérifier la capacité de la toxine STb à induire l’apoptose des cellules HRT-18 et IEC-18 par la voie intrinsèque. Les cellules HRT-18 et IEC-18 ont été traitées avec de la toxine purifiée pour une durée de 24 heures puis ells ont été récoltées et examinées pour des caratéristiques de l’apoptose. L’activation des caspases-9 et -3, mais pas de la caspase-8, a été observée dans les deux lignées cellulaires à l’aide des substrats fluorescents spécifiques pour chaque caspase. L’ADN extrait des cellules HRT-18 et IEC-18 a révélé une fragmentation lorsque migré sur gel d’agarose. La condensation et la fragmentation des noyaux ont été observées en microscopie à fluorescence suite à une coloration de l’ADN au Hoechst 33342. Les indices apoptotiques des cellules HRT-18 et IEC-18 traitées avec des quantités croissantes de STb montrent une dose-réponse pour les deux lignées. L’activation de la caspase-9 est une indication que la voie intrinsèque de l’apoptose est activée dans les cellules HRT-18 et IEC-18. L’absence de l’activation de la caspase-8 démontre que la voie extrinsèque n’est pas impliquée dans la mort cellulaire médiée par STb.

Vegetative and reproductive morphology of Kallymenia patens (Kallymeniaceae, Rhodophyta) in the Mediterranean Sea

Reproductive morphology of the Mediterranean red alga Kallymenia patens is described for the first time, confirming its position in the genus. K. patens is characterized by a non-procarpic female reproductive apparatus, carpogonial branch systems consisting of supporting cells bearing both three-celled carpogonial branches and subsidiary cells that lack a hypogynous cell and carpogonium; fusion cells develop numerous connecting filaments, and tetrasporangia are scattered over the thallus and are probably cruciately divided. Old fertile spathulate specimens of K. patens are morphologically similar to K. spathulata, but they can be distinguished by the length of spathulated proliferations (up to 0.6 cm and 6 cm, respectively), the length of inner cortical cells (up to 70 and 30 μm, respectively), and the gonimoblast location (in proliferations from the perennial part of the blade and over all the thallus surface, respectively)

Cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus replication

In eukaryotic cells, cell growth and division occur in a stepwise, orderly fashion described by a process known as the cell cycle. The relationship between positive-strand RNA viruses and the cell cycle and the concomitant effects on virus replication are not clearly understood. We have shown that infection of asynchronously replicating and synchronized replicating cells with the avian coronavirus infectious bronchitis virus (IBV), a positive-strand RNA virus, resulted in the accumulation of infected cells in the G(2)/M phase of the cell cycle. Analysis of various cell cycle-regulatory proteins and cellular morphology indicated that there was a down-regulation of cyclins D1 and D2 (G(2) regulatory cyclins) and that a proportion of virus-infected cells underwent aberrant cytokinesis, in which the cells underwent nuclear, but not cytoplasmic, division. We assessed the impact of the perturbations on the cell cycle for virus-infected cells and found that IBV-infected G(2)/M-phase-synchronized cells exhibited increased viral protein production when released from the block when compared to cells synchronized in the Go phase or asynchronously replicating cells. Our data suggested that IBV induces a G(2)/M phase arrest in infected cells to promote favorable conditions for viral replication.

Changes in nucleolar morphology and proteins during infection with the coronavirus infectious bronchitis virus

The nucleolus is a dynamic subnuclear structure involved in ribosome subunit biogenesis, cell cycle control and mediating responses to cell stress, among other functions. While many different viruses target proteins to the nucleolus and recruit nucleolar proteins to facilitate virus replication, the effect of infection on the nucleolus in terms of morphology and protein content is unknown. Previously we have shown that the coronavirus nucleocapsid protein will localize to the nucleolus. In this study, using the avian infectious bronchitis coronavirus, we have shown that virus infection results in a number of changes to the nucleolus both in terms of gross morphology and protein content. Using confocal microscopy coupled with fluorescent labelled nucleolar marker proteins we observed changes in the morphology of the nucleolus including an enlarged fibrillar centre. We found that the tumour suppressor protein, p53, which localizes normally to the nucleus and nucleolus, was redistributed predominately to the cytoplasm.

Modification of cell wall properties in lettuce improves shelf life

It is proposed that post-harvest longevity and appearance of salad crops is closely linked to pre-harvest leaf morphology (cell and leaf size) and biophysical structure (leaf strength). Transgenic lettuce plants (Lactuca sativa cv. Valeria) were produced in which the production of the cell wall-modifying enzyme xyloglucan endotransglucosylase/hydrolase (XTH) was down-regulated by antisense inhibition. Independently transformed lines were shown to have multiple members of the LsXTH gene family down-regulated in mature leaves of 6-week-old plants and during the course of shelf life. Consequently, xyloglucan endotransglucosylase (XET) enzyme activity and action were down-regulated in the cell walls of these leaves and it was established that leaf area and fresh weight were decreased while leaf strength was increased in the transgenic lines. Membrane permeability was reduced towards the end of shelf life in the transgenic lines relative to the controls and bacteria were evident inside the leaves of control plants only. Most importantly, an extended shelf-life of transgenic lines was observed relative to the non-transgenic control plants. These data illustrate the potential for engineering cell wall traits for improving quality and longevity of salad crops using either genetic modification directly, or by using markers associated with XTH genes to inform a commercial breeding programme.

Relation between neuronal morphology and electrophysiology in the Kainate lesion model of Alzheimer's Disease

This paper describes a computational and statistical study of the influence of morphological changes on the electrophysiological response of neurons from an animal model of Alzheimer's Disease (AD). We combined experimental morphological data from rat hippocampal CA1 pyramidal cells with a well-established model of active membrane properties. Dendritic morphology and the somatic response to simulated current clamp conditions were then compared for cells from the control and the AD group. The computational approach allowed us to single out the influences of neuromorphology on neuronal response by eliminating the effects of active channel variability. The results did not reveal a simple relationship between morphological changes associated with AD and changes in neural response. However, they did suggest the existence of more complex than anticipated relationships between dendritic morphology and single-cell electrophysiology.

Regulation of cell migration in cancer: investigation into the regulation of cancer cell blebbing in the extracellular matrix

Cell migration is a highly coordinated process and any aberration in the regulatory mechanisms could result in pathological conditions such as cancer. The ability of cancer cells to disseminate to distant sites within the body has made it difficult to treat. Cancer cells also exhibit plasticity that makes them able to interconvert from an elongated, mesenchymal morphology to an amoeboid blebbing form under different physiological conditions. Blebs are spherical membrane protrusions formed by actomyosin-mediated contractility of cortical actin resulting in increased hydrostatic pressure and subsequent detachment of the membrane from the cortex. Tumour cells use blebbing as an alternative mode of migration by squeezing through preexisting gaps in the ECM, and bleb formation is believed to be mediated by the Rho-ROCK signaling pathway. However, the involvement of transmembrane water and ion channels in cell blebbing has not been examined. In the present study, the role of the transmembrane water channels, aquaporins, transmembrane ion transporters and lipid signaling enzymes in the regulation of blebbing was investigated. Using 3D matrigel matrix as an in vitro model to mimic normal extracellular matrix, and a combination of confocal and time-lapse microscopy, it was found that AQP1 knockdown by siRNA ablated blebbing of HT1080 and ACHN cells, and overexpression of AQP1-GFP not only significantly increased bleb size with a corresponding decrease in bleb numbers, but also induced bleb formation in non-blebbing cell lines. Importantly, AQP1 overexpression reduces bleb lifespan due to faster bleb retraction. This novel finding of AQP1-facilitated bleb retraction requires the activity of the Na+/H+ pump as inhibition of the ion transporter, which was found localized to intracellular vesicles, blocked bleb retraction in both cell lines. This study also demonstrated that a differential regulation of cell blebbing by AQP isoforms exists as knockdown of AQP5 had no effect on bleb formation. Data from this study also demonstrates that the lipid signaling PLD2 signals through PA in the LPA-LPAR-Rho-ROCK axis to positively regulate bleb formation in both cell lines. Taken together, this work provides a novel role of AQP1 and Na+/H+ pump in regulation of cell blebbing, and this could be exploited in the development of new therapy to treat cancer.

Immunohistochemical Analysis of Proliferating Cell Nuclear Antigen (PCNA) in Dental Follicles of Impacted Third Molars

This study investigated the immunodetection of PCNA in epithelial components of dental follicles associated with impacted third molars without radiographical and morphological signs of pathosis. A total of 105 specimens of dental follicles associated with impacted third molars with incomplete rhizogenesis (between Nolla`s stage 6 and 9) were surgically removed from 56 patients. Epithelial cell proliferating was determined by using immunohistochemical labeling. Statistical analysis was performed using the Fisher exact test. Of the 105 dental follicles collected, 6 were PCNA-positive (approximate to 6%). The specimens with squamous metaplasia and epithelial hyperplasia had higher rates of positivity for PCNA, as well as those with proliferative remnants of odontogenic epithelium. In conclusion, this study shows that dental follicles at this stage of development have low proliferative potential, but suggests that squamous metaplasia, hyperplasia of the epithelial lining and presence of proliferative odontogenic epithelial rests in the connective tissue may be early signs of developing lesions of odontogenic origin.

Morphology and anatomy of inflorescence and inflorescence axis in Paepalanthus sect. Diphyomene Ruhland (Eriocaulaceae, Poales) and its taxonomic implications

Paepalanthus sect. Diphyomene has inflorescences arranged in umbels. The underlying bauplan seems however to be more complex and composed of several distinct subunits. Despite appearing superficially very similar, the morphology and anatomy of the inflorescences can supply useful information for the understanding of the phylogeny and taxonomy of the group. Inflorescences of Paepalanthus erectifolius, Paepalanthus flaccidus, Paepalanthus giganteus, and Paepalanthus polycladus were analyzed in regard to branching pattern and anatomy. In P. erectifolius, P. giganteus and P. polycladus the structure is a tribotryum, with terminal dibotryum, and with pherophylls bearing lateral dibotrya. In P. flaccidus, the inflorescence is a pleiobotryum, with terminal subunit, and without pherophylls. Secondary inflorescences may occur in all species without regular pattern. Especially when grown in sites without a pronounced seasonality, the distinction between enrichment zone (part of the same inflorescence) and new inflorescences may be obscured. The main anatomical features supplying diagnostic and phylogenetic information are as follows: (a) in the elongated axis, the thickness of the epidermal cell walls and the cortex size; (b) in the bracts, the quantity of parenchyma cells (c) in the scapes, the shape and the presence of a pith tissue. Therefore, P. sect. Diphyomene can be divided in two groups; group A is represented by P. erectifolius, P. giganteus and P. polycladus, and group B is represented by P. flaccidus. The differentiation is based in both, inflorescence structure and anatomy. Group A presents a life cycle and anatomical features similar to species of Actinocephalus. Molecular trees also point that these two groups are closely related. However, inflorescence morphology and blooming sequence are different. Species of group B present an inflorescence structure and anatomical features shared with many genera and species in Eriocaulaceae. The available molecular and morphology based phylogenies still do not allow a precise allocation of the group in the bulk of basal species of Paepalanthus collocated in P. sect. Variabiles. The characters described and used here supply however important information towards this goal. (C) 2009 Elsevier GmbH. All rights reserved.

Propagation of a Brazilian isolate of Anaplasma marginale with appendage in a tick cell line (BME26) derived from Rhipicephalus (Boophilus) microplus

Anaplasma marginale is a tick-borne pathogen of cattle responsible for the disease anaplasmosis. Data suggest that Rhipicephalus (Boophilus) microplus and R. annulatus may be the major tick vectors of A. marginale in tropical and subtropical regions of the world. In this work we demonstrated the first infection and propagation of a Brazilian isolate of A. marginale (UFMG1) in the BME26 cell line derived originally from embryos of R. (Boophilus) microplus. The establishment of A. marginale infection in a cell line derived from R. (Boophilus) microplus is relevant for studying the A. marginale/tick interface. (C) 2008 Elsevier B.V. All rights reserved.

Mycoplasma synoviae cell invasion: Elucidation of the Mycoplasma pathogenesis in chicken

Fluorochrome-labelled cells of two field isolates and Mycoplasma synoviae (Ms) were inoculated onto monolayer cultures of fluorochrome-labelled HEp-2 cells and monitored by confocal laser scanning microscopy (CLSM). Ms was detected initially adhered to and subsequently inside the host cells. Between 24 and 48 h of infection, Ms was detected in the perinuclear region, and after 72 h of infection was confirmed by gentamicin invasion assay. High and low passage Ms strains showed no differences in adherence or invasion. The morphology and the actin filaments of the infected HEp-2 cells were preserved throughout the study period. The observed invasion by Ms is consistent with the biology of Mollicutes, and could explain the difficulties in recovering field isolates of the mycoplasma and in controlling the infection in birds even after long-term antibiotic treatment. (C) 2009 Elsevier Ltd. All rights reserved.

The interaction of an antiparasitic peptide active against African Sleeping Sickness with cell membrane models

Zwitterionic peptides with trypanocidal activity are promising lead compounds for the treatment of African Sleeping Sickness, and have motivated research into the design of compounds capable of disrupting the protozoan membrane. In this study, we use the Langmuir monolayer technique to investigate the surface properties of an antiparasitic peptide, namely S-(2,4-dinitrophenyl)glutathione di-2-propyl ester, and its interaction with a model membrane comprising a phospholipid monolayer. The drug formed stable Langmuir monolayers. whose main feature was a phase transition accompanied by a negative surface elasticity. This was attributed to aggregation upon compression due to intermolecular bond associations of the molecules, inferred from surface pressure and surface potential isotherms. Brewster angle microscopy (BAM) images, infrared spectroscopy and dynamic elasticity measurements. When co-spread with dipalmitoyl phosphatidyl choline (DPPC). the drug affected both the surface pressure and the monolayer morphology, even at high surface pressures and with low amounts of the drug. The results were interpreted by assuming a repulsive, cooperative interaction between the drug and DPPC molecules. Such repulsive interaction and the large changes in fluidity arising from drug aggregation may be related to the disruption of the membrane, which is key for the parasite killing property. (C) 2009 Elsevier B.V. All rights reserved.