Loss Of Membrane Polarity Research Articles

The clinicopathological and microrna expression signature associated with lymphovascular invasion in squamous cell carcinoma: A basic descriptive study.

Lymphovascular invasion (LVI) is an indicator of lymph node metastasis and poor prognosis in various cancers including squamous cell carcinoma (SCC). Despite being easily resectable and having little potential for LVI; SCC displays aggressive behavior and often results in the death of the patient. With this in mind, it may be useful to investigate the clinical, pathological, and microRNA expression profile associated with LVI in SCC. We evaluated the histological hallmarks associated with LVI from 16 formalin fixed paraffin embedded (FFPE) tissue samples (10 LVI-, 6 LVI+). We also quantified the expression of 10 microRNAs (hsa-miR-21-5p, hsa-miR-21-3p, hsa-miR-155-5p, hsa-miR-196a-5p, hsa-miR-375, hsa-let-7d-5p, hsa-miR-146b-3p, hsa-miR-221-5p, hsa-miR-205-5p, hsa-miR-491-5p), which have been previously identified to play a role in SCC development, using real time-PCR with the Qiagen miRCURY LNA SYBR Green PCR Kit. We observed a significant upregulation of microRNA-155, microRNA-196a, microRNA-375, and microRNA-221 in cases with lymphovascular invasion. Morphologically, we identified poor differentiation, dysplasia, loss of membrane polarity, high nuclear to cytoplasmic ratio, and the presence of squamous nests as defining features of LVI. Additionally, we found a gender bias and observed a tendency toward lymphatic invasion in lesions presenting around the perineal and abdominal regions. We speculate that this profile may have prognostic significance and could guide the clinician in their treatment protocols for patients matching our genetic, demographic, and morphologic profile.

Inhibition of Murine Pulmonary Microvascular Endothelial Cell Apoptosis Promotes Recovery of Barrier Function under Septic Conditions.

Sepsis is characterized by injury of the pulmonary microvasculature and the pulmonary microvascular endothelial cells (PMVEC), leading to barrier dysfunction and acute respiratory distress syndrome (ARDS). Our recent work identified a strong correlation between PMVEC apoptosis and microvascular leak in septic mice in vivo, but the specific role of apoptosis in septic PMVEC barrier dysfunction remains unclear. Thus, we hypothesize that PMVEC apoptosis is likely required for PMVEC barrier dysfunction under septic conditions in vitro. Septic stimulation (mixture of tumour necrosis factor α, interleukin 1β, and interferon γ [cytomix]) of isolated murine PMVEC resulted in a significant loss of barrier function as early as 4 h after stimulation, which persisted until 24 h. PMVEC apoptosis, as reflected by caspase activation, DNA fragmentation, and loss of membrane polarity, was first apparent at 8 h after cytomix. Pretreatment of PMVEC with the pan-caspase inhibitor Q-VD significantly decreased septic PMVEC apoptosis and was associated with reestablishment of PMVEC barrier function at 16 and 24 h after stimulation but had no effect on septic PMVEC barrier dysfunction over the first 8 h. Collectively, our data suggest that early septic murine PMVEC barrier dysfunction driven by proinflammatory cytokines is not mediated through apoptosis, but PMVEC apoptosis contributes to late septic PMVEC barrier dysfunction.

In Response

In Response As is often the case, the genesis for our article1 was an observation of something occurring outside our normal expectation, during routine care of donation after cardiac death (DCD). The bispectral index (BIS) machine was initially used as an academic exercise to illustrate the difference between organ procurement during non\Nheart-beating donation and procurement from a brain-dead donor. To confirm these findings of increased BIS values during DCD, the second and third cases were recorded, after IRB approval. One recurring theme in the letters by Rady, Verheijde, Windokun, Thomas and Thomas2–4 is the need to further evaluate the true meaning of these observations of increased BIS values during the dying process, because these observations are not only reproducible with DCD patients, but in other end-of-life arenas as well. For clarity, all changes in BIS values occurred before declaration of death and all BIS values returned to zero before any organ procurement procedures. DCD is a valuable process to obtain organs for transplantation and we did not intend to provide observations that might question the use of DCD for organ procurement. If anything, understanding the dying process by monitoring electroencephalogram (EEG) may make families, health care professionals, and future donors more comfortable with end-of-life care. The secondary theme is the need to focus on informing our colleagues of best practice for end-of-life situations, and we find strong agreement for this theme. We neither intend nor anticipate that one would draw final conclusions based solely on these data. Rather, our findings, coupled with those of Chawla et al.,5 support a need for further research. We agree with Rady et al. and reiterate that dosing opioids and/or hypnotic agents may be reasonable if changes in processed EEG are observed during withdrawal of care. Perhaps these EEG changes reflect a physiologic process such as loss of membrane polarization or cellular death that may not be preventable with opioids or hypnotics. However, in end-of-life situations, we would much rather err in dosing these agents based on an EEG artifact than withholding these agents on the assumption of artifact when they might be truly indicated. David B. Auyong, MD Department of Anesthesiology Virginia Mason Medical Center Seattle, Washington [email protected] Stephen M. Klein, MD Tong J. Gan, MB, BS, FRCA Anthony M. Roche, MB, ChB DaiWai Olson, PhD, RN, CCRN Ashraf S. Habib, MB, ChB, MSc, FRCA Duke University Medical Center Durham, North Carolina

Interactions of Membrane-Active Peptides with Thick, Neutral, Nonzwitterionic Bilayers

Alamethicin is a well-studied channel-forming peptide that has a prototypical amphipathic helix structure. It permeabilizes both microbial and mammalian cell membranes, causing loss of membrane polarization and leakage of endogenous contents. Antimicrobial peptide-lipid systems have been studied quite extensively and have led to significant advancements in membrane biophysics. These studies have been performed on lipid bilayers that are generally charged or zwitterionic and restricted to a thickness range of 3-5 nm. Bilayers of amphiphilic diblock copolymers are a relatively new class of membranes that can have significantly different physicochemical properties compared with those of lipid membranes. In particular, they can be made uncharged, nonzwitterionic, and much thicker than their lipid counterparts. In an effort to extend studies of membrane-protein interactions to these synthetic membranes, we have characterized the interactions of alamethicin and several other membrane-active peptides with diblock copolymer bilayers. We find that although alamethicin is too small to span the bilayer, the peptide interacts with, and ruptures, thick polymer membranes.

Decreased actin solubility observed during ATP-depletion is mimicked by severing agents but not depolymerizing agents in isolated and cultured proximal tubular cells.

The microvilli of the apical membrane of proximal tubule (PT) cells are supported by the underlying actin cytoskeleton. Ischaemic or anoxic ATP-depletion leads to the disruption of the actin cytoskeleton, resulting in microvillar retraction and loss of membrane polarity. Using isolated PT cells, we have previously demonstrated that actin filaments (F-actin) are likely severed during ATP-depletion. A sequential extraction protocol revealed a decrease in actin solubility, resulting in the sequestration of a distinct F-actin pool with the insoluble cellular complex in ATP-depleted PT cells. We demonstrate here that decreased actin solubility is not only a reliable end-marker of ATP-depletion induced injury in freshly isolated PT cells, but also serves as a biochemical marker in the cultured proximal tubular cell line LLC-PK1. In the present studies, we also investigated specific actin-binding drugs to determine if they mimic the effects observed during energy depletion. Jasplakinolide (JP), a compound which binds F-actin and prevents depolymerization, did not effect actin solubility during ATP-depletion. Furthermore, swinholide A (SA), an F-actin severing agent, resulted in decreased actin solubility, mimicking the effects of ATP-depletion. Interestingly, latrunculin A (LA), an agent which depolymerizes F-actin, did not reduce actin solubility, but rather resulted in an increase in digitonin-soluble actin. Taken collectively, our results support previous work and suggest that disruption of the actin cytoskeleton during ATP-depletion is mediated by F-actin severing/fragmentation and not depolymerization. The differential effects of F-actin disrupting agents and the consistencies observed in both models of ischaemic injury will provide a basis for a more detailed understanding of the pathological events of PT-cell dysfunction.

The Drosophila genes crumbs and stardust are involved in the biogenesis of adherens junctions.

Morphogenetic movements of epithelia during development underlie the normal elaboration of the final body plan. The tissue integrity critical for these movements is conferred by anchorage of the cytoskeleton by adherens junctions, initially spot and later belt-like, zonular structures, which encircle the apical side of the cell. Loss-of-function mutations in the Drosophila genes crumbs and stardust lead to the loss of cell polarity in most ectodermally derived epithelia, followed in some, such as the epidermis, by extensive apoptosis. Here we show that both mutants fail to establish proper zonulae adherentes in the epidermis. Our results suggest that the two genes are involved in different aspects of this process. Further, they are compatible with the hypothesis that crumbs delimits the apical border, where the zonula adherens usually forms and where Crumbs protein is normally most abundant. In contrast, stardust seems to be required at an earlier stage for the assembly of the spot adherence junctions. In both mutants, the defect observed at the ultrastructural level are preceded by a misdistribution of Armadillo and DE-cadherin, the homologues of beta-catenin and E-cadherin, respectively, which are two constituents of the vertebrate adherens junctions. Strikingly, expansion of the apical membrane domain in epidermal cells by overexpression of crumbs also abolishes the formation of adherens junctions and results in the disruption of tissue integrity, but without loss of membrane polarity. This result supports the view that membrane polarity is independent of the formation of adherens junctions in epidermal cells.