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Cell Integrity (cell + integrity)
Selected AbstractsCellular effects of monohydrochloride of l -arginine, N, -lauroyl ethylester (LAE) on exposure to Salmonella typhimurium and Staphylococcus aureusJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2004E. Rodríguez Abstract Aims:, Here we study the effect of monohydrochloride of l -arginine, N, -lauroyl ethylester (LAE), a cationic preservative derived from lauric acid and arginine, on the cell envelopes of Salmonella typhimurium and Staphylococcus aureus at sub-lethal concentration such as their respective minimal inhibitory concentrations, 32 and 8 ,g ml,1, respectively. Methods and Results:, Bacterial populations were studied by using transmission electron and fluorescence microscopy (TEM and FM), flow cytometry (FC) and ion-flux across the cellular membrane. Cell integrity was altered mainly in the outer membrane of S. typhimurium, but there was no significant change in the cytoplasm. However, in Staph. aureus, clear zones, abnormal septation and mesosome-like structures were observed in the cytoplasm. Bacterial populations were double-stained with propidium iodide (PI) and SYTO-13 for FC analysis. In S. typhimurium the proportion of damaged cells after 24 h was 97% and in Staph. aureus 56·3%. LAE induced transmembrane ion flux, the increase of potassium leakage after 30 min of contact was 7·7 and 3·34 ,g ml,1 for Staph. aureus and S. typhimurium, respectively. Membrane disruption was detected by measuring the proton flow across the membrane. Conclusions:, Disturbance in membrane potential and structural changes was caused by LAE, although cells were not disrupted. Significance and Impact of the Study:, This is the first time the cellular effects of LAE on bacterial cells were studied. [source] Development of a small-scale bioreactor: Application to in vivo NMR measurementBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2005Dorra Gmati Abstract A perfused bioreactor allowing in vivo NMR measurement was developed and validated for Eschscholtzia californica cells. The bioreactor was made of a 10-mm NMR tube. NMR measurement of the signal-to-noise ratio was optimized using a sedimented compact bed of cells that were retained in the bioreactor by a supporting filter. Liquid medium flow through the cell bed was characterized from a mass balance on oxygen and a dispersive hydrodynamic model. Cell bed oxygen demand for 4 h perfusion required a minimal medium flow rate of 0.8 mL/min. Residence time distribution assays at 0.8,2.6 mL/min suggest that the cells are subjected to a uniform nutrient environment along the cell bed. Cell integrity was maintained for all culture conditions since the release of intracellular esterases was not significant even after 4 h of perfusion. In vivo NMR was performed for 31P NMR and the spectrum can be recorded after only 10 min of spectral accumulation (500 scans) with peaks identified as G-6P, F-6P, cytoplasmic Pi, vacuolar Pi, ATP, and ADP,, ATP, and ADP,, NADP and NDPG, NDPG and ATP,. Cell viability was shown to be maintained as 31P chemical shifts were constant with time for all the identified nuclei, thus suggesting constant intracellular pH. © 2004 Wiley Periodicals, Inc. [source] Novel insights into the osmotic stress response of yeastFEMS YEAST RESEARCH, Issue 3 2002Willem H Mager Abstract Response to hyperosmolarity in the baker's yeast Saccharomyces cerevisiae has attracted a great deal of attention of molecular and cellular biologists in recent years, from both the fundamental scientific and applied viewpoint. Indeed the underlying molecular mechanisms form a clear demonstration of the intricate interplay of (environmental) signalling events, regulation of gene expression and control of metabolism that is pivotal to any living cell. In this article we briefly review the cellular response to conditions of hyperosmolarity, with focus on the high-osmolarity glycerol mitogen-activated protein kinase pathway as the major signalling route governing cellular adaptations. Special attention will be paid to the recent finding that in the yeast cell also major structural changes occur in order to ensure maintenance of cell integrity. The intriguing role of glycerol in growth of yeast under (osmotic) stress conditions is highlighted. [source] Role of hepatocytes and bile duct cells in preservation-reperfusion injury of liver graftsLIVER TRANSPLANTATION, Issue 5 2001Marián Kukan In liver transplantation, it is currently hypothesized that nonparenchymal cell damage and/or activation is the major cause of preservation-related graft injury. Because parenchymal cells (hepatocytes) appear morphologically well preserved even after extended cold preservation, their injury after warm reperfusion is ascribed to the consequences of nonparenchymal cell damage and/or activation. However, accumulating evidence over the past decade indicated that the current hypothesis cannot fully explain preservation-related liver graft injury. We review data obtained in animal and human liver transplantation and isolated perfused animal livers, as well as isolated cell models to highlight growing evidence of the importance of hepatocyte disturbances in the pathogenesis of normal and fatty graft injury. Particular attention is given to preservation time-dependent decreases in high-energy adenine nucleotide levels in liver cells, a circumstance that (1) sensitizes hepatocytes to various stimuli and insults, (2) correlates well with graft function after liver transplantation, and (3) may also underlie the preservation time-dependent increase in endothelial cell damage. We also review damage to bile duct cells, which is increasingly being recognized as important in the long-lasting phase of reperfusion injury. The role of hydrophobic bile salts in that context is particularly assessed. Finally, a number of avenues aimed at preserving hepatocyte and bile duct cell integrity are discussed in the context of liver transplantation therapy as a complement to reducing nonparenchymal cell damage and/or activation. [source] Rga2 is a Rho2 GAP that regulates morphogenesis and cell integrity in S. pombeMOLECULAR MICROBIOLOGY, Issue 4 2008Ma Antonia Villar-Tajadura Summary Schizosaccharomyces pombe Rho2 GTPase regulates ,-D-glucan synthesis and acts upstream of Pck2 to activate the MAP kinase pathway for cell integrity. However, little is known about its regulation. Here we describe Rga2 as a Rho2 GTPase-activating protein (GAP) that regulates cell morphology. rga2+ gene is not essential for growth but its deletion causes longer and thinner cells whereas rga2+ overexpression causes shorter and broader cells. rga2+ overexpression also causes abnormal accumulation of Calcofluor-stained material and cell lysis, suggesting that it also participates in cell wall integrity. Rga2 localizes to growth tips and septum region. The N-terminal region of the protein is required for its correct localization whereas the PH domain is necessary exclusively for Rga2 localization to the division area. Also, Rga2 localization depends on polarity markers and on actin polymerization. Rga2 interacts with Rho2 and possesses in vitro and in vivo GAP activity for this GTPase. Accordingly, rga2, cells contain more ,-D-glucan and therefore partially suppress the thermosensitivity of mok1,664 cells, which have a defective ,-D-glucan synthase. Additionally, genetic interactions and biochemical analysis suggest that Rga2 regulates Rho2,Pck2 interaction and might participate in the regulation of the MAPK cell integrity pathway. [source] The Cryptococcus neoformans MAP kinase Mpk1 regulates cell integrity in response to antifungal drugs and loss of calcineurin functionMOLECULAR MICROBIOLOGY, Issue 5 2003Peter R. Kraus Summary Cell wall integrity is crucial for fungal growth, development and stress survival. In the model yeast Saccharomyces cerevisiae, the cell integrity Mpk1/Slt2 MAP kinase and calcineurin pathways monitor cell wall integrity and promote cell wall remodelling under stress conditions. We have identified the Cryptococcus neoformans homologue of the S. cerevisiae Mpk1/Slt2 MAP kinase and have characterized its role in the maintenance of cell integrity in response to elevated growth temperature and in the presence of cell wall synthesis inhibitors. C. neoformans Mpk1 is required for growth at 37°C in vitro, and this growth defect is suppressed by osmotic stabilization. C. neoformans mutants lacking Mpk1 are attenuated for virulence in the mouse model of cryptococcosis. Phosphorylation of Mpk1 is induced in response to perturbations of cell wall biosynthesis by the antifungal drugs nikkomycin Z (a chitin synthase inhibitor), caspofungin (a ,-1,3-glucan synthase inhibitor), or FK506 (a calcineurin inhibitor), and mutants lacking Mpk1 display enhanced sensitivity to nikkomycin Z and caspofungin. Lastly, we show that calcineurin and Mpk1 play complementing roles in regulating cell integrity in C. neoformans. Our studies demonstrate that pharmacological inhibition of the cell integrity pathway would enhance the activity of antifungal drugs that target the cell wall. [source] Rga5p is a specific Rho1p GTPase-activating protein that regulates cell integrity in Schizosaccharomyces pombeMOLECULAR MICROBIOLOGY, Issue 2 2003Teresa M. Calonge Summary Schizosaccharomyces pombe Rho1p regulates (1,3),- d -glucan synthesis and is required for cell integrity maintenance and actin cytoskeleton organization, but nothing is known about the regulation of this protein. At least nine different S. pombe genes code for proteins predicted to act as Rho GTPase-activating proteins (GAPs). The results shown in this paper demonstrate that the protein encoded by the gene named rga5+ is a GAP specific for Rho1p. rga5+ overexpression is lethal and causes morphological alterations similar to those reported for Rho1p inactivation. rga5+ deletion is not lethal and causes a mild general increase in cell wall biosynthesis and morphological alterations when cells are grown at 37°C. Upon mild overexpression, Rga5p localizes to growth areas and possesses both in vivo and in vitro GAP activity specific for Rho1p. Overexpression of rho1+ in rga5, cells is lethal, with a morphological phenotype resembling that of the overexpression of the constitutively active allele rho1G15V. In addition (1,3),- d -glucan synthase activity, regulated by Rho1p, is increased in rga5, cells and decreased in rga5 -overexpressing cells. Moreover, the increase in (1,3),- d -glucan synthase activity caused by rho1+ overexpression is considerably higher in rga5, than in wild-type cells. Genetic interactions suggest that Rga5p is also important for the regulation of the other known Rho1p effectors, Pck1p and Pck2p. [source] Role of Nitric Oxide in the Development of Distant Metastasis From Squamous Cell Carcinoma,THE LARYNGOSCOPE, Issue 2 2007Richard L. Scher MD Abstract Background: Metastasis, the dissemination of malignant cells to distant sites, remains one of the most significant factors responsible for death from cancer. Recent studies have shown some improvement in the rate of distant metastasis (DM) with the addition of chemotherapy to surgery and radiation for treatment of head and neck squamous cell carcinoma (HNSCC). However, diagnosis and treatment at an early stage ultimately leads to a better prognosis. The prediction of which patients will develop metastasis and the selection of treatment most effective at preventing and treating metastasis remains dependent on an incomplete understanding of prognostic factors and the biological and molecular basis for metastatic development. This study was undertaken using an in vivo model to investigate the possible role of nitric oxide (NO) in the development of metastasis from HNSCC. The findings will result in better understanding of the metastatic process for HNSCC, with the potential to develop and implement therapies that could prevent and treat metastasis in patients. Objectives/Hypothesis: 1) To analyze whether in vivo videomicroscopy (IVVM) is useful for the study of DM from squamous cell carcinoma of the head and neck; 2) with use of IVVM, investigate the effect of the biological mediators NO and interleukin (IL)-1 on the adhesion of circulating human HNSCC cells in the hepatic microcirculation. Study Design: Prospective study using an animal model. Methods: Phase 1: athymic nude rats and mice were used for IVVM experiments. The cremaster muscle and liver, used as arterial and venous flow models, were tested to determine whether IVVM was useful for the study of human HNSCC interactions with the microcirculation. A human squamous cell carcinoma cell line (FaDu) labeled with the intracytoplasmic fluorescent marker BCECF-am. was used for all experiments. Videomicroscopic images of FaDu cells in the microcirculation were analyzed for cell adhesion, morphology, deformation, circulation, location of adhesion within the microcirculation, and alteration of microvascular circulation. Phase 2: the effect of IL-1, NO, and NO inhibitors on HNSCC cell adhesion in the hepatic microcirculation of nude mice was analyzed by IVVM. This was followed by histologic determination of the ratio of FaDu cells present for liver area analyzed. Nude mice were treated with 1) IL-1; 2) L-arginine (an NO substrate); or 3) L-N-monomethyl-L-arginine (an NO synthase inhibitor) alone or in combination. These data were analyzed statistically to determine the effect on cell adhesion in the liver. Results: IVVM provided a method for the study of circulating HNSCC with the microcirculation in both the cremaster and liver models. FaDu cells were arrested at the inflow side of the circulation, with maintenance of cell integrity. L-arginine and IL-1 both increased FaDu cell arrest in the liver above baseline (P = .00008 and P = .03), and the combination of these agents potentiated the effect (P = .000009). Conclusions: IVVM allows direct assessment of circulating HNSCC with the microcirculation and is a powerful model for the study of DM. NO and IL-1 play a role in increasing the arrest of HNSCC in the liver and are important in the generation of DM in patients with HNSCC. [source] Flow cytometric detection of ,- D -glucuronidase gene in wild-type bacterial cells using in-situ PCRBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2003Ramaiah Sachidanandham Abstract An in situ PCR-based flow cytometry method useful for monitoring the presence or absence of the ,- D -glucuronidase gene in Escherichia coli has been developed. A single-step fixation and permeabilization procedure, which maintained cell integrity at the elevated temperatures used during thermal cycling in the presence of PCR reagents, was demonstrated. We have chosen a shorter DNA sequence of length 147 bp for the PCR. Cells subjected to in situ PCR using fluorescein-12-dUTP as a label, showed the presence of uid both in epifluorescence microscopic examination and flow cytometric analysis. Multi-parametric analysis of flow cytometric profiles revealed that the efficiency of labeling was found to be high. The potential of in situ PCR for the detection of uid in intact coliform cells was then successfully tested with a fecal coliform isolated from the coastal waters of Singapore. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 127,133, 2003. [source] Positive allosteric modulation of ,7 neuronal nicotinic acetylcholine receptors: lack of cytotoxicity in PC12 cells and rat primary cortical neuronsBRITISH JOURNAL OF PHARMACOLOGY, Issue 8 2009Min Hu Background and purpose:, ,7-Nicotinic acetylcholine receptors (,7 nAChRs) play an important role in cognitive function. Positive allosteric modulators (PAMs) amplify effects of ,7 nAChR agonist and could provide an approach for treatment of cognitive deficits in neuropsychiatric diseases. PAMs can either predominantly affect the apparent peak current response (type I) or increase both the apparent peak current response and duration of channel opening, due to prolonged desensitization (type II). The delay of receptor desensitization by type II PAMs raises the possibility of Ca2+ -induced toxicity through prolonged activation of ,7 nAChRs. The present study addresses whether type I and II PAMs exhibit different cytotoxicity profiles. Experimental approach:, The present studies evaluated cytotoxic effects of type I PAM [N-(4-chlorophenyl)]-,-[(4-chloro-phenyl)-aminomethylene]-3-methyl-5-isoxazoleacet-amide (CCMI) and type II PAM 1-[5-chloro-2,4-dimethoxy-phenyl]-3-[5-methyl-isoxazol-3-yl]-urea (PNU-120596), or 4-[5-(4chloro-phenyl)-2-methyl-3-propionyl-pyrrol-1-yl]-benzenesulphonamide (A-867744). The studies used cultures of PC12 cells and primary cultures of rat cortical neuronal cells. Key results:, Our results showed that neither type I nor type II PAMs had any detrimental effect on cell integrity or cell viability. In particular, type II PAMs did not affect neuron number and neurite outgrowth under conditions when ,7 nAChR activity was measured by Ca2+ influx and extracellular signal-regulated kinases 1 and 2 phosphorylation, following exposure to ,7 nAChR agonists. Conclusions and implications:, This study demonstrated that both type I and type II ,7 nAChR selective PAMs, although exhibiting differential electrophysiological profiles, did not exert cytotoxic effects in cells endogenously expressing ,7 nAChRs. [source] Cross-talk between L-type Ca2+ channels and mitochondriaCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 2 2010Helena M Viola Summary 1. Calcium is necessary for myocardial function, including contraction and maintenance of cardiac output. Calcium is also necessary for myocardial energetics and production of ATP by mitochondria, but the mechanisms for calcium regulation by mitochondria are still not fully resolved. 2. The cytoskeleton plays an important role in maintaining a cell's integrity. It is now recognized that cytoskeletal proteins can also assist in the transmission of signals from the plasma membrane to intracellular organelles. Cytoskeletal proteins can regulate the function of the L-type Ca2+ channel and alter intracellular calcium homeostasis. 3. Recent evidence suggests that calcium influx through the L-type Ca2+ channel is sufficient to alter a number of mitochondrial functional parameters, including superoxide production, NADH production and metabolic activity, assessed as the formation of formazan from tetrazolium salt. This occurs in a calcium-dependent manner. 4. Activation of the L-type Ca2+ channel also alters mitochondrial membrane potential in a calcium-independent manner and this is assisted by movement of the auxiliary ,2 -subunit through F-actin filaments. 5. Because the L-type Ca2+ channel is the initiator of contraction, a functional coupling between the channels and mitochondria may assist in meeting myocardial energy demand on a beat-to-beat basis. [source] | |||||||||||||