IRBIT a Master Regulator of Cell Physiology

(excerpt) Hormones and neurotransmitters regulate cell functions by binding to their receptors, which activate intracellular signaling and produce the physiological response [1]. There are several intracellular pathways, including but not limited to, leading to the activation of protein kinases, phosphatases and increase in intracellular calcium (Ca2+) [1]

Effect of Media and Estrogen on Morphological Change in Candida albicans

Introduction: Candida albicans (C. albicans), an opportunistic pathogen, lives symbiotically within the intestine of its human host. Temperature and chemical factors have been shown to induce a morphological change in C. albicans from yeast to filamentous form turning C. albicans pathogenic. In this study, we investigated the intestinal cues that might be responsible for the change. We found that different solid media impact the morphological phenotype so we focused on characterizing these before further testing. We tested Estradiol (E2) because of its known linkage to sepsis and higher levels during infections. Experiments were conducted to compare solid agar plates of YEPD, Minimal Media (MM), and Spider Media (SP) for C. albicans growth to choose the best one for further testing with E2 and other factors that could be prone to causing morphological changes. Methods: C. albicans was inoculated through streak method on different solid media (YEPD, MM, SP) and incubated at 30℃. The effect of 0.1nM E2 on C. albicans morphology was also tested. Morphological changes were assayed through bright-field microscopy. Results: Using the three different medias, we found three distinctive phenotypes: A, B, and C. Out of 6 experiments of 14 MM plates, the expressed phenotype was 86% A and 14% inconclusive of the time. 8 experiments of 17 SP plates showed 100% of phenotype B. 6 experiments of 14 YEPD plates presented phenotype C 92% of the time and 8% inconclusive. For E2 trials, 2 experiments, 6 MM plates showed 50% phenotype A and 50% inconclusive. 4 experiments, 10 SP plates had phenotype B 100%. YEPD 2 experiments, 2 plates had phenotype C at 100%. Conclusion: We have established experimental conditions of media controls for further testing whether E2 and other cues, such as inflammatory cytokines, have inhibitory or positive effects on the growth of C. albicans

IRBIT a Master Regulator of Cell Physiology

(excerpt) Hormones and neurotransmitters regulate cell functions by binding to their receptors, which activate intracellular signaling and produce the physiological response [1]. There are several intracellular pathways, including but not limited to, leading to the activation of protein kinases, phosphatases and increase in intracellular calcium (Ca2+) [1]

Candida albicans Infection Decreases The Expression Of The Na+-K+-2Cl– Cotransporter 1 In T84 and Madin Darby Canine Kidney Cells

The commensal human fungal pathogen, Candida albicans, prior to infect the human body, must penetrate the intestinal mucosal barrier. To do so, it needs to bypass the different protective mechanisms such as fluid secretion. The basolateral Na+-K+-2Cl– cotransporter 1 (NKCC1) is a key protein regulating fluid secretion in the intestine. We hypothesize that C. albicans decreases fluid secretion prior to invasion by inducing NKCC1 internalization. In our experiments, we used Madin Darby canine kidney (MDCK) cells expressing a GFP-NKCC1 fluorescent tag and T84 cells, a human colonic cell line. Cells were infected with 100,000 C. albicans for varying lengths of time, fixed, stained and mounted for fluorescence microscopy. The number of internalized vesicles was evaluated using FIJI. Our results show that in MDCK cells, C. albicans only increased NKCC1 internalization at the 30-minute time point (P\u3c0.05), all subsequent time points were not significant. Similarly, infecting T84 cells with C. albicans significantly induced NKCC1 internalization at the 30-minute (P\u3c0.05), 1 hour (P\u3c0.05), and 90-minute (P\u3c0.05) time points. Past 90 minutes, we observed a sharp decline in the number of internalized vesicles that continued to decrease through 6 hours of exposure to C. albicans. Finally, in C. albicans-T84 infected cells, using an immunoblot approach, we found that total NKCC1 protein expression was decreased by ~20% (P\u3c0.05) compared to uninfected cells. Our results suggest that C. albicans induces internalization of NKCC1, and subsequent degradation of NKCC1, which would decrease fluid secretion and allow adhesion, and invasion of the epithelium

Effect of Media and Estrogen on Morphological Change in Candida albicans

Introduction: Candida albicans (C. albicans), an opportunistic pathogen, lives symbiotically within the intestine of its human host. Temperature and chemical factors have been shown to induce a morphological change in C. albicans from yeast to filamentous form turning C. albicans pathogenic. In this study, we investigated the intestinal cues that might be responsible for the change. We found that different solid media impact the morphological phenotype so we focused on characterizing these before further testing. We tested Estradiol (E2) because of its known linkage to sepsis and higher levels during infections. Experiments were conducted to compare solid agar plates of YEPD, Minimal Media (MM), and Spider Media (SP) for C. albicans growth to choose the best one for further testing with E2 and other factors that could be prone to causing morphological changes. Methods: C. albicans was inoculated through streak method on different solid media (YEPD, MM, SP) and incubated at 30℃. The effect of 0.1nM E2 on C. albicans morphology was also tested. Morphological changes were assayed through bright-field microscopy. Results: Using the three different medias, we found three distinctive phenotypes: A, B, and C. Out of 6 experiments of 14 MM plates, the expressed phenotype was 86% A and 14% inconclusive of the time. 8 experiments of 17 SP plates showed 100% of phenotype B. 6 experiments of 14 YEPD plates presented phenotype C 92% of the time and 8% inconclusive. For E2 trials, 2 experiments, 6 MM plates showed 50% phenotype A and 50% inconclusive. 4 experiments, 10 SP plates had phenotype B 100%. YEPD 2 experiments, 2 plates had phenotype C at 100%. Conclusion: We have established experimental conditions of media controls for further testing whether E2 and other cues, such as inflammatory cytokines, have inhibitory or positive effects on the growth of C. albicans

Secreted Protein Acidic and Rich in Cysteine Is a Matrix Scavenger Chaperone

Secreted Protein Acidic and Rich in Cysteine (SPARC) is one of the major non-structural proteins of the extracellular matrix (ECM) in remodeling tissues. The functional significance of SPARC is emphasized by its origin in the first multicellular organisms and its high degree of evolutionary conservation. Although SPARC has been shown to act as a critical modulator of ECM remodeling with profound effects on tissue physiology and architecture, no plausible molecular mechanism of its action has been proposed. In the present study, we demonstrate that SPARC mediates the disassembly and degradation of ECM networks by functioning as a matricellular chaperone. While it has low affinity to its targets inside the cells where the Ca2+ concentrations are low, high extracellular concentrations of Ca2+ activate binding to multiple ECM proteins, including collagens. We demonstrated that in vitro, this leads to the inhibition of collagen I fibrillogenesis and disassembly of pre-formed collagen I fibrils by SPARC at high Ca2+ concentrations. In cell culture, exogenous SPARC was internalized by the fibroblast cells in a time- and concentration-dependent manner. Pulse-chase assay further revealed that internalized SPARC is quickly released outside the cell, demonstrating that SPARC shuttles between the cell and ECM. Fluorescently labeled collagen I, fibronectin, vitronectin, and laminin were co-internalized with SPARC by fibroblasts, and semi-quantitative Western blot showed that SPARC mediates internalization of collagen I. Using a novel 3-dimentional model of fluorescent ECM networks pre-deposited by live fibroblasts, we demonstrated that degradation of ECM depends on the chaperone activity of SPARC. These results indicate that SPARC may represent a new class of scavenger chaperones, which mediate ECM degradation, remodeling and repair by disassembling ECM networks and shuttling ECM proteins into the cell. Further understanding of this mechanism may provide insight into the pathogenesis of matrix-associated disorders and lead to the novel treatment strategies

Effect of estrogen on Candida albicans growth cultured on YEPD solid media

C. albicans is a commensal fungus which under certain environmental cues shifts morphology from spores to filamentous and becomes invasive within the human body. During sepsis, blood estrogen (E2) becomes elevated. E2 is also known to promote tissue growth, thus we hypothesize that E2 may influence C. albicans growth. In the present work, we investigated the effect of estrogen on C. albicans colony sizes grown on yeast extract peptone dextrose (YEPD). Using bright field microscopy, images of five colonies in each condition were captured on day one and pictures of the same colonies were captured on day two. The diameter of each colony was computed using ImageJ and the surface area was calculated using Excel. On day one, one-way ANOVA shows no difference (P=0.09) in colony size for the 4 conditions tested (control 0.19±0.09 mm2, 0.1 nM E2 0.20±0.08, fetal bovine serum (FBS) 0.23±0.11, FBS+ E2 0.24±0.12). On the second day, the size of each colony significantly increased compared to day one (P\u3c0.001, paired t-test) for each individual condition (i.e., control day 1 versus control day 2). Comparing colony size increase (surface area on day 2- surface area day1) a significant difference among the 4 conditions P\u3c0.001, one-way ANOVA. Dunnett post hoc test shows no significant difference between control 2.41±0.29 mm2 vs E2 2.46±0.35 (P=0.8) but a significant difference between control 2.41±0.29 versus FBS 2.69±0.27 (P\u3c0.001) and control 2.41±0.29 vs FBS+ E2 2.71±0.35 (P\u3c0.001). In conclusion, our results show that FBS significantly increased colony growth in YEPD, but E2 had no significant effect on colony growth. @font-face {font-family: Cambria Math ; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:3 0 0 0 1 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent: ; margin:0in; mso-pagination:widow-orphan; font-size:12.0pt; font-family: Times New Roman ,serif; mso-fareast-font-family: Times New Roman ;}.MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-family: Calibri ,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:Calibri; mso-fareast-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family: Times New Roman ; mso-bidi-theme-font:minor-bidi;}div.WordSection1 {page:WordSection1;

Mapping NKCC1 in the Endocytic Pathway During PKC Activation in Mardin Darby Canine Kidney Cells

Gut clearance represents an important defense mechanism of the gut by flushing out luminal bacteria and toxins. Transepithelial chloride fluid secretion is what drives gut clearance. The basolateral Na-K-2Cl cotransporter 1 (NKCC1) is the main mechanism for loading cells with chloride for its secretion by apical chloride channels (e.g., cystic fibrosis transmembrane regulator). We have previously shown that protein kinase C (PKC) activation causes internalization of NKCC1, thus blunting chloride secretion. However, the fate of NKCC1 remains unknown. To determine if NKCC1 is recycled or degraded, we used Mardin Darby Canine Kidney (MDCK) cells that stably expresses eGFP-NKCC1 to map the endocytic pathway. For immunocytochemistry, MDCK cells were cultured on coverslips in a six-well plate until confluence. Cells were exposed to either phorbol 12-myristate 13-acetate (PMA), an activator of PKC, or DMSO (vehicle). Cells were fixed with 1% paraformaldehyde, incubated with specific primary antibody against endosomal markers, and mounted for immunofluorescence. Images were acquired with an Olympus compound microscope equipped for fluorescence and processed using ImageJ. In these experiments, we did not find colocalization of NKCC1 with Rab5, a marker of the early endosome. We found occasional colocalization of NKCC1 and Rab11, a marker of vesicles recycling to the plasma membrane. Finally, we did not find colocalization of NKCC1 with either LAMP1 or P20S markers of the lysosome and the proteasome. Our results suggest that some NKCC1, after internalization recycles to the membrane in MDCK. Further investigation will be needed to determine the fate on NKCC1 in the endocytic pathway

Mapping NKCC1 in the Endocytic Pathway During PKC Activation in Mardin Darby Canine Kidney Cells

Gut clearance represents an important defense mechanism of the gut by flushing out luminal bacteria and toxins. Transepithelial chloride fluid secretion is what drives gut clearance. The basolateral Na-K-2Cl cotransporter 1 (NKCC1) is the main mechanism for loading cells with chloride for its secretion by apical chloride channels (e.g., cystic fibrosis transmembrane regulator). We have previously shown that protein kinase C (PKC) activation causes internalization of NKCC1, thus blunting chloride secretion. However, the fate of NKCC1 remains unknown. To determine if NKCC1 is recycled or degraded, we used Mardin Darby Canine Kidney (MDCK) cells that stably expresses eGFP-NKCC1 to map the endocytic pathway. For immunocytochemistry, MDCK cells were cultured on coverslips in a six-well plate until confluence. Cells were exposed to either phorbol 12-myristate 13-acetate (PMA), an activator of PKC, or DMSO (vehicle). Cells were fixed with 1% paraformaldehyde, incubated with specific primary antibody against endosomal markers, and mounted for immunofluorescence. Images were acquired with an Olympus compound microscope equipped for fluorescence and processed using ImageJ. In these experiments, we did not find colocalization of NKCC1 with Rab5, a marker of the early endosome. We found occasional colocalization of NKCC1 and Rab11, a marker of vesicles recycling to the plasma membrane. Finally, we did not find colocalization of NKCC1 with either LAMP1 or P20S markers of the lysosome and the proteasome. Our results suggest that some NKCC1, after internalization recycles to the membrane in MDCK. Further investigation will be needed to determine the fate on NKCC1 in the endocytic pathway

Secreted Protein Acidic and Rich in Cysteine Is a Matrix Scavenger Chaperone

Secreted Protein Acidic and Rich in Cysteine (SPARC) is one of the major non-structural proteins of the extracellular matrix (ECM) in remodeling tissues. The functional significance of SPARC is emphasized by its origin in the first multicellular organisms and its high degree of evolutionary conservation. Although SPARC has been shown to act as a critical modulator of ECM remodeling with profound effects on tissue physiology and architecture, no plausible molecular mechanism of its action has been proposed. In the present study, we demonstrate that SPARC mediates the disassembly and degradation of ECM networks by functioning as a matricellular chaperone. While it has low affinity to its targets inside the cells where the Ca2+ concentrations are low, high extracellular concentrations of Ca2+ activate binding to multiple ECM proteins, including collagens. We demonstrated that in vitro, this leads to the inhibition of collagen I fibrillogenesis and disassembly of pre-formed collagen I fibrils by SPARC at high Ca2+ concentrations. In cell culture, exogenous SPARC was internalized by the fibroblast cells in a time- and concentration-dependent manner. Pulse-chase assay further revealed that internalized SPARC is quickly released outside the cell, demonstrating that SPARC shuttles between the cell and ECM. Fluorescently labeled collagen I, fibronectin, vitronectin, and laminin were co-internalized with SPARC by fibroblasts, and semi-quantitative Western blot showed that SPARC mediates internalization of collagen I. Using a novel 3-dimentional model of fluorescent ECM networks pre-deposited by live fibroblasts, we demonstrated that degradation of ECM depends on the chaperone activity of SPARC. These results indicate that SPARC may represent a new class of scavenger chaperones, which mediate ECM degradation, remodeling and repair by disassembling ECM networks and shuttling ECM proteins into the cell. Further understanding of this mechanism may provide insight into the pathogenesis of matrix-associated disorders and lead to the novel treatment strategies.</p