3-Methylcholanthrene inhibits lymphocyte proliferation and increases intracellular calcium levels in common carp (Cyprinus carpio L)

International Nuclear Information System (INIS)

Reynaud, S.; Duchiron, C.; Deschaux, P.

2003-01-01

Polycyclic aromatic hydrocarbons (PAHs) are an important class of environmental pollutants that are known to be carcinogenic and immunotoxic. Many authors have focused on macrophage activities in fish exposed to PAHs. However, fewer studies have reported decrease in specific immunity in such fish. We investigated the intracellular mechanisms by which the 3-methylcholanthrene (3-MC) decreased lymphocyte proliferation in carp. T- and B-lymphocyte proliferation induced by Concanavalin A (Con A) and lipopolysaccharide (LPS) were inhibited by 3-MC (0.5-50 μM). 3-MC also produced a rapid and a sustained increase in intracellular calcium concentration ([Ca 2+ ] i ) (2 h minimum). However, the cytochrome P450 1A and Ah receptor inhibitor, α-naphtoflavone (a-NF), also inhibited lymphocyte proliferation and did not reverse the effects of 3-MC. Moreover, since a-NF and 3-MC increased [Ca 2+ ] i and inhibited lymphocyte proliferation it was possible that calcium release played a role in 3-MC-inhibited lymphocyte proliferation. The rise in [Ca 2+ ] i induced by 3-MC was potentiated by the inhibitor of the endoplasmic reticulum calcium ATPases, thapsigargin. Treating cells with 3-MC decreased calcium mobilization caused by thapsigargin. These results suggest that 3-MC acts on the endoplasmic reticulum, perhaps directly on calcium ATPases, to increase intracellular calcium levels in carp leucocytes

Increase of Intracellular Cyclic AMP by PDE4 Inhibitors Affects HepG2 Cell Cycle Progression and Survival.

Science.gov (United States)

Massimi, Mara; Cardarelli, Silvia; Galli, Francesca; Giardi, Maria Federica; Ragusa, Federica; Panera, Nadia; Cinque, Benedetta; Cifone, Maria Grazia; Biagioni, Stefano; Giorgi, Mauro

2017-06-01

Type 4 cyclic nucleotide phosphodiesterases (PDE4) are major members of a superfamily of enzymes (PDE) involved in modulation of intracellular signaling mediated by cAMP. Broadly expressed in most human tissues and present in large amounts in the liver, PDEs have in the last decade been key therapeutic targets for several inflammatory diseases. Recently, a significant body of work has underscored their involvement in different kinds of cancer, but with no attention paid to liver cancer. The present study investigated the effects of two PDE4 inhibitors, rolipram and DC-TA-46, on the growth of human hepatoma HepG2 cells. Treatment with these inhibitors caused a marked increase of intracellular cAMP level and a dose- and time-dependent effect on cell growth. The concentrations of inhibitors that halved cell proliferation to about 50% were used for cell cycle experiments. Rolipram (10 μM) and DC-TA-46 (0.5 μM) produced a decrease of cyclin expression, in particular of cyclin A, as well as an increase in p21, p27 and p53, as evaluated by Western blot analysis. Changes in the intracellular localization of cyclin D1 were also observed after treatments. In addition, both inhibitors caused apoptosis, as demonstrated by an Annexin-V cytofluorimetric assay and analysis of caspase-3/7 activity. Results demonstrated that treatment with PDE4 inhibitors affected HepG2 cell cycle and survival, suggesting that they might be useful as potential adjuvant, chemotherapeutic or chemopreventive agents in hepatocellular carcinoma. J. Cell. Biochem. 118: 1401-1411, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Insecticide resistance and intracellular proteases.

Science.gov (United States)

Wilkins, Richard M

2017-12-01

Pesticide resistance is an example of evolution in action with mechanisms of resistance arising from mutations or increased expression of intrinsic genes. Intracellular proteases have a key role in maintaining healthy cells and in responding to stressors such as pesticides. Insecticide-resistant insects have constitutively elevated intracellular protease activity compared to corresponding susceptible strains. This increase was shown for some cases originally through biochemical enzyme studies and subsequently putatively by transcriptomics and proteomics methods. Upregulation and expression of proteases have been characterised in resistant strains of some insect species, including mosquitoes. This increase in proteolysis results in more degradation products (amino acids) of intracellular proteins. These may be utilised in the resistant strain to better protect the cell from stress. There are changes in insect intracellular proteases shortly after insecticide exposure, suggesting a role in stress response. The use of protease and proteasome inhibitors or peptide mimetics as synergists with improved application techniques and through protease gene knockdown using RNA interference (possibly expressed in crop plants) may be potential pest management strategies, in situations where elevated intracellular proteases are relevant. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Intracellular calcium levels can regulate Importin-dependent nuclear import

International Nuclear Information System (INIS)

Kaur, Gurpreet; Ly-Huynh, Jennifer D.; Jans, David A.

2014-01-01

Highlights: • High intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import. • The effect of Ca 2+ on nuclear import does not relate to changes in the nuclear pore. • High intracellular calcium can result in mislocalisation of Impβ1, Ran and RCC1. - Abstract: We previously showed that increased intracellular calcium can modulate Importin (Imp)β1-dependent nuclear import of SRY-related chromatin remodeling proteins. Here we extend this work to show for the first time that high intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import generally. The basis of this relates to the mislocalisation of the transport factors Impβ1 and Ran, which show significantly higher nuclear localization in contrast to various other factors, and RCC1, which shows altered subnuclear localisation. The results here establish for the first time that intracellular calcium modulates conventional nuclear import through direct effects on the nuclear transport machinery

Intracellular calcium levels can regulate Importin-dependent nuclear import

Energy Technology Data Exchange (ETDEWEB)

Kaur, Gurpreet; Ly-Huynh, Jennifer D.; Jans, David A., E-mail: David.Jans@monash.edu

2014-07-18

Highlights: • High intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import. • The effect of Ca{sup 2+} on nuclear import does not relate to changes in the nuclear pore. • High intracellular calcium can result in mislocalisation of Impβ1, Ran and RCC1. - Abstract: We previously showed that increased intracellular calcium can modulate Importin (Imp)β1-dependent nuclear import of SRY-related chromatin remodeling proteins. Here we extend this work to show for the first time that high intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import generally. The basis of this relates to the mislocalisation of the transport factors Impβ1 and Ran, which show significantly higher nuclear localization in contrast to various other factors, and RCC1, which shows altered subnuclear localisation. The results here establish for the first time that intracellular calcium modulates conventional nuclear import through direct effects on the nuclear transport machinery.

Effect of bauhinia bauhinioides kallikrein inhibitor on endothelial proliferation and intracellular calcium concentration.

Science.gov (United States)

Bilgin, M; Burgazli, K M; Rafiq, A; Mericliler, M; Neuhof, C; Oliva, M L; Parahuleva, M; Soydan, N; Doerr, O; Abdallah, Y; Erdogan, A

2014-01-01

Proteinase inhibitors act as a defensive system against predators e.g. insects, in plants. Bauhinia bauhinioides kallikrein inhibitor (BbKI) is a serine proteinase inhibitor, isolated from seeds of Bauhinia bauhinioides and is structurally similar to plant Kunitz-type inhibitors but lacks disulfide bridges. In this study we evaluated the antiproliferative effect of BbKI on endothelial cells and its impact on changes in membrane potential and intracellular calcium. HUVEC proliferation was significantly reduced by incubation with BbKI 50 and 100 µM 12% and 13%. Furthermore, BbKI (100 µM) exposure caused a significant increase in intracellular Ca2+ concentration by 35% as compared to untreated control. The intracellular rise in calcium was not affected by the absence of extracellular calcium. BBKI also caused a significant change in the cell membrane potential but the antiproliferative effect was independent of changes in membrane potential. BBKI has an antiproliferative effect on HUVEC, which is independent of the changes in membrane potential, and it causes an increase in intracellular Ca2+.

Lansoprazole induces apoptosis of breast cancer cells through inhibition of intracellular proton extrusion

Energy Technology Data Exchange (ETDEWEB)

Zhang, Shangrong; Wang, Yifan; Li, Shu Jie, E-mail: shujieli@nankai.edu.cn

2014-06-13

Highlights: • Lansoprazole (LPZ) induces cell apoptosis in breast cancer cells. • LPZ markedly inhibits intracellular proton extrusion. • LPZ induces an increase in intracellular ATP level, lysosomal alkalinization and ROS accumulation. - Abstract: The increased glycolysis and proton secretion in tumors is proposed to contribute to the proliferation and invasion of cancer cells during the process of tumorigenesis and metastasis. Here, treatment of human breast cancer cells with proton pump inhibitor (PPI) lansoprazole (LPZ) induces cell apoptosis in a dose-dependent manner. In the implantation of the MDA-MB-231 xenografts in nude mice, administration of LPZ significantly inhibits tumorigenesis and induces large-scale apopotosis of tumor cells. LPZ markedly inhibits intracellular proton extrusion, induces an increase in intracellular ATP level, lysosomal alkalinization and accumulation of reactive oxygen species (ROS) in breast cancer cells. The ROS scavenger N-acetyl-L-cysteine (NAC) and diphenyleneiodonium (DPI), a specific pharmacological inhibitor of NADPH oxidases (NOX), significantly abolish LPZ-induced ROS accumulation in breast cancer cells. Our results suggested that LPZ may be used as a new therapeutic drug for breast tumor.

Lansoprazole induces apoptosis of breast cancer cells through inhibition of intracellular proton extrusion

International Nuclear Information System (INIS)

Zhang, Shangrong; Wang, Yifan; Li, Shu Jie

2014-01-01

Highlights: • Lansoprazole (LPZ) induces cell apoptosis in breast cancer cells. • LPZ markedly inhibits intracellular proton extrusion. • LPZ induces an increase in intracellular ATP level, lysosomal alkalinization and ROS accumulation. - Abstract: The increased glycolysis and proton secretion in tumors is proposed to contribute to the proliferation and invasion of cancer cells during the process of tumorigenesis and metastasis. Here, treatment of human breast cancer cells with proton pump inhibitor (PPI) lansoprazole (LPZ) induces cell apoptosis in a dose-dependent manner. In the implantation of the MDA-MB-231 xenografts in nude mice, administration of LPZ significantly inhibits tumorigenesis and induces large-scale apopotosis of tumor cells. LPZ markedly inhibits intracellular proton extrusion, induces an increase in intracellular ATP level, lysosomal alkalinization and accumulation of reactive oxygen species (ROS) in breast cancer cells. The ROS scavenger N-acetyl-L-cysteine (NAC) and diphenyleneiodonium (DPI), a specific pharmacological inhibitor of NADPH oxidases (NOX), significantly abolish LPZ-induced ROS accumulation in breast cancer cells. Our results suggested that LPZ may be used as a new therapeutic drug for breast tumor

The effect of high pressure on the intracellular trehalose synthase activity of Thermus aquaticus.

Science.gov (United States)

Dong, Yongsheng; Ma, Lei; Duan, Yuanliang

2016-01-01

To understand the effect of high pressure on the intracellular trehalose synthase activity, Thermus aquaticus (T. aquaticus) in the logarithmic growth phase was treated with high-pressure air, and its intracellular trehalose synthase (TSase) activity was determined. Our results indicated that pressure is a factor strongly affecting the cell growth. High pressure significantly attenuated the growth rate of T. aquaticus and shortened the duration of stationary phase. However, after 2 h of culture under 1.0 MPa pressure, the activity of intracellular TSase in T. aquaticus reached its maximum value, indicating that pressure can significantly increase the activity of intracellular TSase in T. aquaticus. Thus the present study provides an important guide for the enzymatic production of trehalose.

Increased intracellular Th1 cytokines in scid mice with inflammatory bowel disease

DEFF Research Database (Denmark)

Bregenholt, S; Claesson, Mogens Helweg

1998-01-01

Severe combined immunodeficient (scid) mice engrafted with small pieces of full thickness gut wall from immunocompetent syngenic donors develop a chronic and lethal colitis. Lymphocytes from the lamina propria of engrafted mice were analyzed for phorbol ester/ionomycin-induced cytokine production...... by intracellular staining. A 4-5-fold increase in the fraction of IFN-gamma-producing CD4+ lamina propria T cells was found in moderately and severely diseased mice when compared to healthy congenic C.B-17 control mice. The number of IL-2-producing T cells was increased by approximately 2-fold when comparing mice...... suffering from severe disease to healthy control mice. The fraction of TNF-alpha positive CD4+ T cells was increased by a factor of two in both moderately and severely diseased mice. When analyzing Th2 cytokines, it was found that the levels of IL-4-producing CD4+ T cells was not altered in diseased animals...

Induction of a stringent metabolic response in intracellular stages of Leishmania mexicana leads to increased dependence on mitochondrial metabolism.

Directory of Open Access Journals (Sweden)

Eleanor C Saunders

2014-01-01

Full Text Available Leishmania parasites alternate between extracellular promastigote stages in the insect vector and an obligate intracellular amastigote stage that proliferates within the phagolysosomal compartment of macrophages in the mammalian host. Most enzymes involved in Leishmania central carbon metabolism are constitutively expressed and stage-specific changes in energy metabolism remain poorly defined. Using (13C-stable isotope resolved metabolomics and (2H2O labelling, we show that amastigote differentiation is associated with reduction in growth rate and induction of a distinct stringent metabolic state. This state is characterized by a global decrease in the uptake and utilization of glucose and amino acids, a reduced secretion of organic acids and increased fatty acid β-oxidation. Isotopomer analysis showed that catabolism of hexose and fatty acids provide C4 dicarboxylic acids (succinate/malate and acetyl-CoA for the synthesis of glutamate via a compartmentalized mitochondrial tricarboxylic acid (TCA cycle. In vitro cultivated and intracellular amastigotes are acutely sensitive to inhibitors of mitochondrial aconitase and glutamine synthetase, indicating that these anabolic pathways are essential for intracellular growth and virulence. Lesion-derived amastigotes exhibit a similar metabolism to in vitro differentiated amastigotes, indicating that this stringent response is coupled to differentiation signals rather than exogenous nutrient levels. Induction of a stringent metabolic response may facilitate amastigote survival in a nutrient-poor intracellular niche and underlie the increased dependence of this stage on hexose and mitochondrial metabolism.

Gonadotropin stimulates oocyte translation by increasing magnesium activity through intracellular potassium-magnesium exchange

International Nuclear Information System (INIS)

Horowitz, S.B.; Tluczek, L.J.

1989-01-01

We previously showed that gonadotropin increases the K + activity in Xenopus oocytes and that this is a signal for increased translation. However, K + need not act to control synthesis directly but may act through an unidentified downstream effector. Using microinjection to vary the salt content of oocytes and concomitantly measuring [ 3 H]leucine incorporation, we found that small changes in Mg 2+ greatly affect translation rates. (Ca 2+ had little influence.) By measuring intracellular ion activities, we found that oocyte cations existed in a buffer-like (ion-exchange) equilibrium in which K + and Mg 2+ are the preponderant monovalent and divalent cations. Hence, increasing cellular K + activity might increase translation by causing Mg 2+ activity to rise. If so, the increased translation rates produced by hormone treatment or K + injection would be prevented by EDTA, a Mg 2+ chelating agent. This prediction was tested and confirmed. We conclude that, when gonadotropin increases K + activity, the cell's internal ion-exchange equilibrium is altered thereby increasing Mg 2+ activity and this up-regulates translation

Bauhinia bauhinioides cruzipain inhibitor reduces endothelial proliferation and induces an increase of the intracellular Ca2+ concentration.

Science.gov (United States)

Bilgin, Mehmet; Neuhof, Christiane; Doerr, Oliver; Benscheid, Utz; Andrade, Sheila S; Most, Astrid; Abdallah, Yaser; Parahuleva, Mariana; Guenduez, Dursun; Oliva, Maria L; Erdogan, Ali

2010-12-01

Proteinase inhibitors, isolated from different types of Bauhinia, have an effect on apoptosis, angiogenesis and inflammation. The Bauhinia bauhinioides cruzipain inhibitor (BbCI) is a Kunitz-type inhibitor and inactivates the cysteine proteinases cruzipain and cruzain from Trypanosoma cruzi. Cruzipain and tissue kallikrein have similar biochemical properties, e.g. the proteolytic cleavage of the kininogen precursor of lys-bradykinin. Tissue kallikrein stimulation in endothelial cells causes migration and capillary tube formation. The aim of this study was to examine whether the antiproliferative effect of BbCI is dependent on changes of the intracellular calcium concentration and membrane hyperpolarization. Endothelial cells were isolated from human umbilical cord veins (HUVEC). For proliferation experiments, HUVEC were incubated with BbCI (10-100 μmol/L) for 48 h. The proliferation was detected by cell counting with a Neubauer chamber. The effect of BbCI (10-100 μM) on the membrane potential was measured with the fluorescence dye DiBAC4(3) and the effect on [Ca+2]i with the fluorescence probe Fluo-3 AM. The change of the fluorescence intensity was determined with a GENios plate reader (Tecan). The experiments showed that BbCI (10-100 μmol/L) reduces the endothelial cell proliferation significantly in a concentration-dependent manner with a maximum effect at 100 μmol/L (35.1±1.8% as compared to control (p≤0.05; n=45)). As compared to the control, the addition of BbCI (100 μmol/L) caused a significant increase of systolic Ca2+ of 28.4±5.0% after 30 min incubation. HUVEC treatment with BbCI (100 μmol/L) showed a weak but significant decrease of the membrane potential of 9.5±0.9% as compared to control (p≤0.05; n=80). BbCI influenced significantly the endothelial proliferation, the intracellular Ca2+ concentration and the membrane potential.

Coinfection of Hepatic Cell Lines with Human Immunodeficiency Virus and Hepatitis B Virus Leads to an Increase in Intracellular Hepatitis B Surface Antigen▿

Science.gov (United States)

Iser, David M.; Warner, Nadia; Revill, Peter A.; Solomon, Ajantha; Wightman, Fiona; Saleh, Suha; Crane, Megan; Cameron, Paul U.; Bowden, Scott; Nguyen, Tin; Pereira, Cândida F.; Desmond, Paul V.; Locarnini, Stephen A.; Lewin, Sharon R.

2010-01-01

Liver-related mortality is increased in the setting of HIV-hepatitis B virus (HBV) coinfection. However, interactions between HIV and HBV to explain this observation have not been described. We hypothesized that HIV infection of hepatocytes directly affects the life cycle of HBV. We infected human hepatic cell lines expressing HBV (Hep3B and AD38 cells) or not expressing HBV (Huh7, HepG2, and AD43 cells) with laboratory strains of HIV (NL4-3 and AD8), as well as a vesicular stomatitis virus (VSV)-pseudotyped HIV expressing enhanced green fluorescent protein (EGFP). Following HIV infection with NL4-3 or AD8 in hepatic cell lines, we observed a significant increase in HIV reverse transcriptase activity which was infectious. Despite no detection of surface CD4, CCR5, and CXCR4 by flow cytometry, AD8 infection of AD38 cells was inhibited by maraviroc and NL4-3 was inhibited by AMD3100, demonstrating that HIV enters AD38 hepatic cell lines via CCR5 or CXCR4. High-level infection of AD38 cells (50%) was achieved using VSV-pseudotyped HIV. Coinfection of the AD38 cell line with HIV did not alter the HBV DNA amount or species as determined by Southern blotting or nucleic acid signal amplification. However, coinfection with HIV was associated with a significant increase in intracellular HBsAg when measured by Western blotting, quantitative HBsAg, and fluorescence microscopy. We conclude that HIV infection of HBV-infected hepatic cell lines significantly increased intracellular HBsAg but not HBV DNA synthesis and that increased intrahepatic HBsAg secondary to direct infection by HIV may contribute to accelerated liver disease in HIV-HBV-coinfected individuals. PMID:20357083

Coinfection of hepatic cell lines with human immunodeficiency virus and hepatitis B virus leads to an increase in intracellular hepatitis B surface antigen.

Science.gov (United States)

Iser, David M; Warner, Nadia; Revill, Peter A; Solomon, Ajantha; Wightman, Fiona; Saleh, Suha; Crane, Megan; Cameron, Paul U; Bowden, Scott; Nguyen, Tin; Pereira, Cândida F; Desmond, Paul V; Locarnini, Stephen A; Lewin, Sharon R

2010-06-01

Liver-related mortality is increased in the setting of HIV-hepatitis B virus (HBV) coinfection. However, interactions between HIV and HBV to explain this observation have not been described. We hypothesized that HIV infection of hepatocytes directly affects the life cycle of HBV. We infected human hepatic cell lines expressing HBV (Hep3B and AD38 cells) or not expressing HBV (Huh7, HepG2, and AD43 cells) with laboratory strains of HIV (NL4-3 and AD8), as well as a vesicular stomatitis virus (VSV)-pseudotyped HIV expressing enhanced green fluorescent protein (EGFP). Following HIV infection with NL4-3 or AD8 in hepatic cell lines, we observed a significant increase in HIV reverse transcriptase activity which was infectious. Despite no detection of surface CD4, CCR5, and CXCR4 by flow cytometry, AD8 infection of AD38 cells was inhibited by maraviroc and NL4-3 was inhibited by AMD3100, demonstrating that HIV enters AD38 hepatic cell lines via CCR5 or CXCR4. High-level infection of AD38 cells (50%) was achieved using VSV-pseudotyped HIV. Coinfection of the AD38 cell line with HIV did not alter the HBV DNA amount or species as determined by Southern blotting or nucleic acid signal amplification. However, coinfection with HIV was associated with a significant increase in intracellular HBsAg when measured by Western blotting, quantitative HBsAg, and fluorescence microscopy. We conclude that HIV infection of HBV-infected hepatic cell lines significantly increased intracellular HBsAg but not HBV DNA synthesis and that increased intrahepatic HBsAg secondary to direct infection by HIV may contribute to accelerated liver disease in HIV-HBV-coinfected individuals.

Intracellular alkalinization induces cytosolic Ca2+ increases by inhibiting sarco/endoplasmic reticulum Ca2+-ATPase (SERCA.

Directory of Open Access Journals (Sweden)

Sen Li

Full Text Available Intracellular pH (pHi and Ca(2+ regulate essentially all aspects of cellular activities. Their inter-relationship has not been mechanistically explored. In this study, we used bases and acetic acid to manipulate the pHi. We found that transient pHi rise induced by both organic and inorganic bases, but not acidification induced by acid, produced elevation of cytosolic Ca(2+. The sources of the Ca(2+ increase are from the endoplasmic reticulum (ER Ca(2+ pools as well as from Ca(2+ influx. The store-mobilization component of the Ca(2+ increase induced by the pHi rise was not sensitive to antagonists for either IP(3-receptors or ryanodine receptors, but was due to inhibition of the sarco/endoplasmic reticulum Ca(2+-ATPase (SERCA, leading to depletion of the ER Ca(2+ store. We further showed that the physiological consequence of depletion of the ER Ca(2+ store by pHi rise is the activation of store-operated channels (SOCs of Orai1 and Stim1, leading to increased Ca(2+ influx. Taken together, our results indicate that intracellular alkalinization inhibits SERCA activity, similar to thapsigargin, thereby resulting in Ca(2+ leak from ER pools followed by Ca(2+ influx via SOCs.

Estrogen-induced disruption of intracellular iron metabolism leads to oxidative stress, membrane damage, and cell cycle arrest in MCF-7 cells.

Science.gov (United States)

Bajbouj, Khuloud; Shafarin, Jasmin; Abdalla, Maher Y; Ahmad, Iman M; Hamad, Mawieh

2017-10-01

It is well established that several forms of cancer associate with significant iron overload. Recent studies have suggested that estrogen (E2) disrupts intracellular iron homeostasis by reducing hepcidin synthesis and maintaining ferroportin integrity. Here, the ability of E2 to alter intracellular iron status and cell growth potential was investigated in MCF-7 cells treated with increasing concentrations of E2. Treated cells were assessed for intracellular iron status, the expression of key proteins involved in iron metabolism, oxidative stress, cell survival, growth, and apoptosis. E2 treatment resulted in a significant reduction in hepcidin expression and a significant increase in hypoxia-inducible factor 1 alpha, ferroportin, transferrin receptor, and ferritin expression; a transient decrease in labile iron pool; and a significant increase in total intracellular iron content mainly at 20 nM/48 h E2 dose. Treated cells also showed increased total glutathione and oxidized glutathione levels, increased superoxide dismutase activity, and increased hemoxygenase 1 expression. Treatment with E2 at 20 nM for 48 h resulted in a significant reduction in cell growth (0.35/1 migration rate) and decreased cell survival (iron metabolism and precipitates adverse effects concerning cell viability, membrane integrity, and growth potential.

Intracellular calcium signal at the leading edge regulates mesodermal sheet migration during Xenopus gastrulation.

Science.gov (United States)

Hayashi, Kentaro; Yamamoto, Takamasa S; Ueno, Naoto

2018-02-05

During the gastrulation stage in animal embryogenesis, the cells leading the axial mesoderm migrate toward the anterior side of the embryo, vigorously extending cell protrusions such as lamellipodia. It is thought that the leading cells sense gradients of chemoattractants emanating from the ectodermal cells and translate them to initiate and maintain the cell movements necessary for gastrulation. However, it is unclear how the extracellular information is converted to the intracellular chemical reactions that lead to motion. Here we demonstrated that intracellular Ca 2+ levels in the protrusion-forming leading cells are markedly higher than those of the following cells and the axial mesoderm cells. We also showed that inhibiting the intracellular Ca 2+ significantly retarded the gastrulation cell movements, while increasing the intracellular Ca 2+ with an ionophore enhanced the migration. We further found that the ionophore treatment increased the active form of the small GTPase Rac1 in these cells. Our results suggest that transient intracellular Ca 2+ signals play an essential role in the active cell migration during gastrulation.

Nanoparticles for intracellular-targeted drug delivery

International Nuclear Information System (INIS)

Paulo, Cristiana S O; Pires das Neves, Ricardo; Ferreira, Lino S

2011-01-01

Nanoparticles (NPs) are very promising for the intracellular delivery of anticancer and immunomodulatory drugs, stem cell differentiation biomolecules and cell activity modulators. Although initial studies in the area of intracellular drug delivery have been performed in the delivery of DNA, there is an increasing interest in the use of other molecules to modulate cell activity. Herein, we review the latest advances in the intracellular-targeted delivery of short interference RNA, proteins and small molecules using NPs. In most cases, the drugs act at different cellular organelles and therefore the drug-containing NPs should be directed to precise locations within the cell. This will lead to the desired magnitude and duration of the drug effects. The spatial control in the intracellular delivery might open new avenues to modulate cell activity while avoiding side-effects.

Allopregnanolone-induced rise in intracellular calcium in embryonic hippocampal neurons parallels their proliferative potential

Directory of Open Access Journals (Sweden)

Brinton Roberta

2008-12-01

Full Text Available Abstract Background Factors that regulate intracellular calcium concentration are known to play a critical role in brain function and neural development, including neural plasticity and neurogenesis. We previously demonstrated that the neurosteroid allopregnanolone (APα; 5α-pregnan-3α-ol-20-one promotes neural progenitor proliferation in vitro in cultures of rodent hippocampal and human cortical neural progenitors, and in vivo in triple transgenic Alzheimer's disease mice dentate gyrus. We also found that APα-induced proliferation of neural progenitors is abolished by a calcium channel blocker, nifedipine, indicating a calcium dependent mechanism for the proliferation. Methods In the present study, we investigated the effect of APα on the regulation of intracellular calcium concentration in E18 rat hippocampal neurons using ratiometric Fura2-AM imaging. Results Results indicate that APα rapidly increased intracellular calcium concentration in a dose-dependent and developmentally regulated manner, with an EC50 of 110 ± 15 nM and a maximal response occurring at three days in vitro. The stereoisomers 3β-hydroxy-5α-hydroxy-pregnan-20-one, and 3β-hydroxy-5β-hydroxy-pregnan-20-one, as well as progesterone, were without significant effect. APα-induced intracellular calcium concentration increase was not observed in calcium depleted medium and was blocked in the presence of the broad spectrum calcium channel blocker La3+, or the L-type calcium channel blocker nifedipine. Furthermore, the GABAA receptor blockers bicuculline and picrotoxin abolished APα-induced intracellular calcium concentration rise. Conclusion Collectively, these data indicate that APα promotes a rapid, dose-dependent, stereo-specific, and developmentally regulated increase of intracellular calcium concentration in rat embryonic hippocampal neurons via a mechanism that requires both the GABAA receptor and L-type calcium channel. These data suggest that AP�

HYPERTHERMIA, INTRACELLULAR FREE CALCIUM AND CALCIUM IONOPHORES

NARCIS (Netherlands)

STEGE, GJJ; WIERENGA, PK; KAMPINGA, HH; KONINGS, AWT

1993-01-01

It is shown that heat-induced increase of intracellular calcium does not correlate with hyperthermic cell killing. Six different cell lines were investigated; in four (EAT, HeLa S3, L5178Y-R and L5178Y-S) heat treatments killing 90% of the cells did not affect the levels of intracellular free

N-Acetylcysteine-induced vasodilatation is modulated by KATP channels, Na+/K+-ATPase activity and intracellular calcium concentration: An in vitro study.

Science.gov (United States)

Vezir, Özden; Çömelekoğlu, Ülkü; Sucu, Nehir; Yalın, Ali Erdinç; Yılmaz, Şakir Necat; Yalın, Serap; Söğüt, Fatma; Yaman, Selma; Kibar, Kezban; Akkapulu, Merih; Koç, Meryem İlkay; Seçer, Didem

2017-08-01

In this study, we aimed to investigate the role of ATP-sensitive potassium (K ATP ) channel, Na + /K + -ATPase activity, and intracellular calcium levels on the vasodilatory effect of N-acetylcysteine (NAC) in thoracic aorta by using electrophysiological and molecular techniques. Rat thoracic aorta ring preparations and cultured thoracic aorta cells were divided into four groups as control, 2mM NAC, 5mM NAC, and 10mM NAC. Thoracic aorta rings were isolated from rats for measurements of relaxation responses and Na + /K + -ATPase activity. In the cultured thoracic aorta cells, we measured the currents of K ATP channel, the concentration of intracellular calcium and mRNA expression level of K ATP channel subunits (KCNJ8, KCNJ11, ABCC8 and ABCC9). The relaxation rate significantly increased in all NAC groups compared to control. Similarly, Na + /K + - ATPase activity also significantly decreased in NAC groups. Outward K ATP channel current significantly increased in all NAC groups compared to the control group. Intracellular calcium concentration decreased significantly in all groups with compared control. mRNA expression level of ABCC8 subunit significantly increased in all NAC groups compared to the control group. Pearson correlation analysis showed that relaxation rate was significantly associated with K ATP current, intracellular calcium concentration, Na + /K + -ATPase activity and mRNA expression level of ABCC8 subunit. Our findings suggest that NAC relaxes vascular smooth muscle cells through a direct effect on K ATP channels, by increasing outward K+ flux, partly by increasing mRNA expression of K ATP subunit ABCC8, by decreasing in intracellular calcium and by decreasing in Na + /K + -ATPase activity. Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Magnetic separation of algae genetically modified for increased intracellular iron uptake

Energy Technology Data Exchange (ETDEWEB)

Buck, Amy [Case Western Reserve University, Cleveland, OH (United States); Cleveland Clinic, Cleveland, OH (United States); Moore, Lee R. [Cleveland Clinic, Cleveland, OH (United States); Lane, Christopher D.; Kumar, Anil; Stroff, Clayton; White, Nicolas [Phycal Inc., Cleveland, OH (United States); Xue, Wei; Chalmers, Jeffrey J. [The Ohio State University, Columbus, OH (United States); Zborowski, Maciej, E-mail: zborowm@ccf.org [Cleveland Clinic, Cleveland, OH (United States)

2015-04-15

Algae were investigated in the past as a potential source of biofuel and other useful chemical derivatives. Magnetic separation of algae by iron oxide nanoparticle binding to cells has been proposed by others for dewatering of cellular mass prior to lipid extraction. We have investigated feasibility of magnetic separation based on the presence of natural iron stores in the cell, such as the ferritin in Auxenochlorella protothecoides (A. protothecoides) strains. The A. protothecoides cell constructs were tested for inserted genes and for increased intracellular iron concentration by inductively coupled plasma atomic absorption (ICP–AA). They were grown in Sueoka’s modified high salt media with added vitamin B1 and increasing concentration of soluble iron compound (FeCl{sub 3} EDTA, from 1× to 8× compared to baseline). The cell magnetic separation conditions were tested using a thin rectangular flow channel pressed against interpolar gaps of a permanent magnet forming a separation system of a well-defined fluid flow and magnetic fringing field geometry (up to 2.2 T and 1000 T/m) dubbed “magnetic deposition microscopy”, or MDM. The presence of magnetic cells in suspension was detected by formation of characteristic deposition bands at the edges of the magnet interpolar gaps, amenable to optical scanning and microscopic examination. The results demonstrated increasing cellular Fe uptake with increasing Fe concentration in the culture media in wild type strain and in selected genetically-modified constructs, leading to magnetic separation without magnetic particle binding. The throughput in this study is not sufficient for an economical scale harvest. - Highlights: • Auxenochlorella protothecoides algae were genetically modified for biofuel production. • Algal iron metabolism was sufficient for their label-less magnetic separation. • High magnetic field and low flow required make the separation scale-up uneconomical.

Magnetic separation of algae genetically modified for increased intracellular iron uptake

International Nuclear Information System (INIS)

Buck, Amy; Moore, Lee R.; Lane, Christopher D.; Kumar, Anil; Stroff, Clayton; White, Nicolas; Xue, Wei; Chalmers, Jeffrey J.; Zborowski, Maciej

2015-01-01

Algae were investigated in the past as a potential source of biofuel and other useful chemical derivatives. Magnetic separation of algae by iron oxide nanoparticle binding to cells has been proposed by others for dewatering of cellular mass prior to lipid extraction. We have investigated feasibility of magnetic separation based on the presence of natural iron stores in the cell, such as the ferritin in Auxenochlorella protothecoides (A. protothecoides) strains. The A. protothecoides cell constructs were tested for inserted genes and for increased intracellular iron concentration by inductively coupled plasma atomic absorption (ICP–AA). They were grown in Sueoka’s modified high salt media with added vitamin B1 and increasing concentration of soluble iron compound (FeCl 3 EDTA, from 1× to 8× compared to baseline). The cell magnetic separation conditions were tested using a thin rectangular flow channel pressed against interpolar gaps of a permanent magnet forming a separation system of a well-defined fluid flow and magnetic fringing field geometry (up to 2.2 T and 1000 T/m) dubbed “magnetic deposition microscopy”, or MDM. The presence of magnetic cells in suspension was detected by formation of characteristic deposition bands at the edges of the magnet interpolar gaps, amenable to optical scanning and microscopic examination. The results demonstrated increasing cellular Fe uptake with increasing Fe concentration in the culture media in wild type strain and in selected genetically-modified constructs, leading to magnetic separation without magnetic particle binding. The throughput in this study is not sufficient for an economical scale harvest. - Highlights: • Auxenochlorella protothecoides algae were genetically modified for biofuel production. • Algal iron metabolism was sufficient for their label-less magnetic separation. • High magnetic field and low flow required make the separation scale-up uneconomical

Role of UBIAD1 in Intracellular Cholesterol Metabolism and Vascular Cell Calcification.

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Sha Liu

Full Text Available Vascular calcification is an important risk factor associated with mortality among patients with chronic kidney disease. Intracellular cholesterol metabolism is involved in the process of vascular cell calcification. In this study, we investigated the role of UbiA prenyltransferase domain containing 1 (UBIAD1 in intracellular cholesterol metabolism and vascular cell calcification, and identified its subcellular location. Primary human umbilical vein smooth muscle cells (HUVSMCs were incubated with either growth medium (1.4 mmol/L Pi or calcification medium (CM (3.0 mmol/L Pi. Under treatment with CM, HUVSMCs were further incubated with exogenous cholesterol, or menaquinone-4, a product of UBIAD1. The plasmid and small interfering RNA were transfected in HUVSMCs to alter the expression of UBIAD1. Matrix calcium quantitation, alkaline phosphatase activity, intracellular cholesterol level and menaquinone-4 level were measured. The expression of several genes involved in cholesterol metabolism were analyzed. Using an anti-UBIAD1 antibody, an endoplasmic reticulum marker and a Golgi marker, the subcellular location of UBIAD1 in HUVSMCs was analyzed. CM increased matrix calcium, alkaline phosphatase activity and intracellular cholesterol level, and reduced UBIAD1 expression and menaquinone-4 level. Addition of cholesterol contributed to increased matrix calcification and alkaline phosphatase activity in a dose-dependent manner. Elevated expression of UBIAD1 or menaquinone-4 in HUVSMCs treated with CM significantly reduced intracellular cholesterol level, matrix calcification and alkaline phosphatase activity, but increased menaquinone-4 level. Elevated expression of UBIAD1 or menaquinone-4 reduced the gene expression of sterol regulatory element-binding protein-2, and increased gene expression of ATP binding cassette transporters A1, which are in charge of cholesterol synthesis and efflux. UBIAD1 co-localized with the endoplasmic reticulum marker and

Mycobacterium intracellulare Infection Mimicking Progression of Scleroderma

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Krabbe, Simon; Engelhart, Merete; Thybo, Sören

2017-01-01

This case report describes a patient with scleroderma who developed Mycobacterium intracellulare infection, which for more than a year mimicked worsening of her connective tissue disorder. The patient was diagnosed with scleroderma based on puffy fingers that developed into sclerodactyly, abnormal......, unfortunately with significant scarring. Immunodeficiency testing was unremarkable. In summary, an infection with Mycobacterium intracellulare was mistaken for an unusually severe progression of scleroderma....

Resveratrol inhibits the intracellular calcium increase and angiotensin/endothelin system activation induced by soluble uric acid in mesangial cells

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Albertoni, G.; Schor, N. [Divisão de Nefrologia, Departamento de Medicina, Universidade Federal de São Paulo, São Paulo, SP (Brazil)

2014-10-24

Resveratrol (Resv) is natural polyphenol found in grapes. This study evaluated the protective effect of Resv against the effects of uric acid (UA) in immortalized human mesangial cells (ihMCs). ihMCs were preincubated with Resv (12.5 µM) for 1 h and treated with UA (10 mg/dL) for 6 or 12 h. The intracellular calcium concentration [Ca{sup 2+}]i was quantified by fluorescence using flow cytometry. Angiotensinogen (AGT) and pre-pro endothelin-1 (ppET-1) mRNA were assayed by quantitative real-time RT-PCR. Angiotensin II (AII) and endothelin-1 (ET-1) were assayed by ELISA. UA significantly increased [Ca{sup 2+}]i. Pre-incubation with Resv significantly reduced the change in [Ca{sup 2+}]i induced by UA. Incubation with UA for 6 or 12 h also increased AGT mRNA expression and AII protein synthesis. Resv blunted these increases in AGT mRNA expression and AII protein. Incubation with UA in the ihMCs increased ppET-1 expression and ET-1 protein synthesis at 6 and 12 h. When ihMCs were pre-incubated with Resv, UA had a significantly diminished effect on ppET-1 mRNA expression and ET-1 protein synthesis at 6 and 12 h, respectively. Our results suggested that UA triggers reactions including AII and ET-1 production in mesangial cells. The renin-angiotensin system may contribute to the pathogenesis of renal function and chronic kidney disease. Resv can minimize the impact of UA on AII, ET-1 and the increase of [Ca{sup 2+}]i in mesangial cells, suggesting that, at least in part, Resv can prevent the effects of soluble UA in mesangial cells.

Development of an in vitro photosafety evaluation method utilizing intracellular ROS production in THP-1 cells.

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Toyoda, Akemi; Itagaki, Hiroshi

2018-01-01

Photoreactive compounds that may experience exposure to ultraviolet (UV) radiation can lead to the intracellular production of reactive oxygen species (ROS), which may cause phototoxic and photoallergenic responses. Here, we developed a novel in vitro photosafety assay and investigated whether it could be used to predict phototoxicity and photosensitivity by measuring changes in intracellular ROS production. THP-1 cells that had previously taken up 5-(and-6)-carboxy-2',7'-difluorodihydrofluorescein diacetate (carboxy-H 2 DFFDA), a ROS-sensitive fluorescent reagent, were exposed to photoreactive substances such as phototoxic and photoallergenic materials and then subjected to with UV-A irradiation (5 J/cm 2 ). The fluorescence intensity was subsequently measured using a flow cytometer, and the intracellular ROS production was calculated. A statistically significant increase in ROS following treatment with photoreactive substances was observed in cells irradiated with UV-A. In contrast, no significant increase was observed for non-photoreactive substances in comparison to the control solution. Next, to confirm the impact of intracellular ROS on the photosensitive response, changes in CD86 and CD54 expression were measured following quencher addition during the photo human cell line activation test (photo h-CLAT). The results confirmed the reduction of CD86 and CD54 expression in response to photoallergenic substances following quencher addition. Together, these findings suggest that intracellular ROS production is involved in photosensitizing reactions. Therefore, we suggest that the developed method utilizing intracellular ROS production as an index may be useful as a novel in vitro evaluation tool for photoreactive substances.

Dynamics of postirradiation intracellular cysteine and aspartic proteinases profiles in proliferating and nonproliferating mammalian cells

International Nuclear Information System (INIS)

Korbelik, M.; Osmak, M.; Suhar, A.; Turk, V.; Skrk, J.

1990-01-01

Dynamics of postirradiation intracellular cysteine and aspartic proteinases profiles were examined in proliferating and nonproliferating Chinese hamster fibroblasts (V 79). The results show that there are significant alterations in cysteine and aspartic intracellular proteinases activity already in the early postirradiation period, which are different in proliferating and nonproliferating cells. Irradiation of the cells examined to low doses and up to 15 Gy induced an increase in cysteine proteinases activity in the early postexposure period, while at higher irradiation doses applied, the activity of these proteinases was decreased. These observations suggest that intracellular proteinases are actively participating in process involving recovery from radiation injury or cell killing. (orig.) [de

Advances in genetic manipulation of obligate intracellular bacterial pathogens

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Paul eBeare

2011-05-01

Full Text Available Infections by obligate intracellular bacterial pathogens result in significant morbidity and mortality worldwide. These bacteria include Chlamydia spp., which causes millions of cases of sexually transmitted disease and blinding trachoma annually, and members of the α-proteobacterial genera Anaplasma, Ehrlichia, Orientia and Rickettsia, agents of serious human illnesses including epidemic typhus. Coxiella burnetii, the agent of human Q fever, has also been considered a prototypical obligate intracellular bacterium, but recent host cell-free (axenic growth has rescued it from obligatism. The historic genetic intractability of obligate intracellular bacteria has severely limited molecular dissection of their unique lifestyles and virulence factors involved in pathogenesis. Host cell restricted growth is a significant barrier to genetic transformation that can make simple procedures for free-living bacteria, such as cloning, exceedingly difficult. Low transformation efficiency requiring long term culture in host cells to expand small transformant populations is another obstacle. Despite numerous technical limitations, the last decade has witnessed significant gains in genetic manipulation of obligate intracellular bacteria including allelic exchange. Continued development of genetic tools should soon enable routine mutation and complementation strategies for virulence factor discovery and stimulate renewed interest in these refractory pathogens. In this review, we discuss the technical challenges associated with genetic transformation of obligate intracellular bacteria and highlight advances made with individual genera.

Epigenetic silencing of host cell defense genes enhances intracellular survival of the rickettsial pathogen Anaplasma phagocytophilum.

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Jose C Garcia-Garcia

2009-06-01

Full Text Available Intracellular bacteria have evolved mechanisms that promote survival within hostile host environments, often resulting in functional dysregulation and disease. Using the Anaplasma phagocytophilum-infected granulocyte model, we establish a link between host chromatin modifications, defense gene transcription and intracellular bacterial infection. Infection of THP-1 cells with A. phagocytophilum led to silencing of host defense gene expression. Histone deacetylase 1 (HDAC1 expression, activity and binding to the defense gene promoters significantly increased during infection, which resulted in decreased histone H3 acetylation in infected cells. HDAC1 overexpression enhanced infection, whereas pharmacologic and siRNA HDAC1 inhibition significantly decreased bacterial load. HDAC2 does not seem to be involved, since HDAC2 silencing by siRNA had no effect on A. phagocytophilum intracellular propagation. These data indicate that HDAC up-regulation and epigenetic silencing of host cell defense genes is required for A. phagocytophilum infection. Bacterial epigenetic regulation of host cell gene transcription could be a general mechanism that enhances intracellular pathogen survival while altering cell function and promoting disease.

Increased extracellular and intracellular Ca{sup 2+} lead to adipocyte accumulation in bone marrow stromal cells by different mechanisms

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Hashimoto, Ryota, E-mail: hryota@juntendo.ac.jp [Department of Physiology, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Katoh, Youichi, E-mail: katoyo@juntendo-urayasu.jp [Juntendo University Faculty of International Liberal Arts, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Department of Cardiology, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Miyamoto, Yuki [Juntendo University Faculty of Health Care and Nursing, Takasu 2-5-1, Urayasu-shi, Chiba 279-0023 (Japan); Itoh, Seigo; Daida, Hiroyuki [Department of Cardiology, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Nakazato, Yuji [Center for Environmental Research, Department of Cardiology, Juntendo University Faculty of Medicine Urayasu Hospital, Tomioka 2-1-1, Urayasu-shi, Chiba 279-0022 (Japan); Okada, Takao [Department of Physiology, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan)

2015-02-20

Mesenchymal stem cells found in bone marrow stromal cells (BMSCs) are the common progenitors for both adipocyte and osteoblast. An increase in marrow adipogenesis is associated with age-related osteopenia and anemia. Both extracellular and intracellular Ca{sup 2+} ([Ca{sup 2+}]{sub o} and [Ca{sup 2+}]{sub i}) are versatile signaling molecules that are involved in the regulation of cell functions, including proliferation and differentiation. We have recently reported that upon treatment of BMSCs with insulin and dexamethasone, both high [Ca{sup 2+}]{sub o} and high [Ca{sup 2+}]{sub i} enhanced adipocyte accumulation, which suggested that increases in [Ca{sup 2+}]{sub o} caused by bone resorption may accelerate adipocyte accumulation in aging and diabetic patients. In this study, we used primary mouse BMSCs to investigate the mechanisms by which high [Ca{sup 2+}]{sub o} and high [Ca{sup 2+}]{sub i} may enhance adipocyte accumulation. In the process of adipocyte accumulation, two important keys are adipocyte differentiation and the proliferation of BMSCs, which have the potential to differentiate into adipocytes. Use of MTT assay and real-time RT-PCR revealed that high [Ca{sup 2+}]{sub i} (ionomycin)-dependent adipocyte accumulation is caused by enhanced proliferation of BMSCs but not enhanced differentiation into adipocytes. Using fura-2 fluorescence-based approaches, we showed that high [Ca{sup 2+}]{sub o} (addition of CaCl{sub 2}) leads to increases in [Ca{sup 2+}]{sub i}. Flow cytometric methods revealed that high [Ca{sup 2+}]{sub o} suppressed the phosphorylation of ERK independently of intracellular Ca{sup 2+}. The inhibition of ERK by U0126 and PD0325901 enhanced the differentiation of BMSCs into adipocytes. These data suggest that increased extracellular Ca{sup 2+} provides the differentiation of BMSCs into adipocytes by the suppression of ERK activity independently of increased intracellular Ca{sup 2+}, which results in BMSC proliferation. - Highlights: �

[Alpha but not beta-adrenergic stimulation has a positive inotropic effect associated with alkalinization of intracellular pH].

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Gambassi, G; Lakatta, E G; Capogrossi, M C

1991-01-01

There is increasing evidence that alpha-adrenoceptors also exist in the myocardium and that an increase in force of contraction may be produced by stimulation of these sites. This positive inotropism seems to be dependent either on an increased amount of Ca++ released into the cytosol with each action potential or on increased myofilament responsiveness. In contrast, beta-adrenergic stimulation reduces the sensitivity of the contractile proteins and the positive inotropic effect is due to the activation of L-type calcium channels on the sarcolemma. We used single, isolated, enzymatically dissociated, adult rat ventricular myocytes. Cells were loaded either with the ester derivative of the Ca++ probe Indo-1 or with the intracellular pH probe Snarf-1 and at the same time we measured the contractile parameters and monitored the fluorescence as an index of intracellular calcium concentration or pH value. The single cells (bicarbonate buffer continuously gassed with O2 95%, CO2 5%, Ca++ 1.5 mM, field stimulation 0.5 Hz) were exposed to phenylephrine (50 microM) and nadolol (1 microM). Alpha-adrenergic stimulation increased twitch amplitude (delta ES = 1.93 +/- 0.77, n = 8; p less than 0.05) and showed only a slight increase in Ca++ transient. On the other end, the positive inotropic effect (delta ES = 2.84 +/- 0.86, n = 4; p less than 0.02) obtained with beta-adrenergic stimulation (isoproterenol 50 nM, bicarbonate buffer, Ca++ 0.5 mM, field stimulation 0.2 Hz) was always associated with a large increase in intracellular Ca++ concentration. Isoproterenol did not change intracellular pH (delta pH = 0.006 +/- 0.006, n = 4; NS) while phenylephrine increased it significantly (delta pH = 0.055 +/- 0.011, n = 8; p less than 0.002). Moreover, there was a statistically significant correlation between delta ES and delta pH (R2 = 0.532; p less than 0.05) when phenylephrine was present. This alkalinization as well as the increased contractility was antagonized by treatment with

Cross talk between increased intracellular zinc (Zn2+) and accumulation of reactive oxygen species in chemical ischemia.

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Slepchenko, Kira G; Lu, Qiping; Li, Yang V

2017-10-01

Both zinc (Zn 2+ ) and reactive oxygen species (ROS) have been shown to accumulate during hypoxic-ischemic stress and play important roles in pathological processes. To understand the cross talk between the two of them, here we studied Zn 2+ and ROS accumulation by employing fluorescent probes in HeLa cells to further the understanding of the cause and effect relationship of these two important cellular signaling systems during chemical-ischemia, stimulated by oxygen and glucose deprivation (OGD). We observed two Zn 2+ rises that were divided into four phases in the course of 30 min of OGD. The first Zn 2+ rise was a transient, which was followed by a latent phase during which Zn 2+ levels recovered; however, levels remained above a basal level in most cells. The final phase was the second Zn 2+ rise, which reached a sustained plateau called Zn 2+ overload. Zn 2+ rises were not observed when Zn 2+ was removed by TPEN (a Zn 2+ chelator) or thapsigargin (depleting Zn 2+ from intracellular stores) treatment, indicating that Zn 2+ was from intracellular storage. Damaging mitochondria with FCCP significantly reduced the second Zn 2+ rise, indicating that the mitochondrial Zn 2+ accumulation contributes to Zn 2+ overload. We also detected two OGD-induced ROS rises. Two Zn 2+ rises preceded two ROS rises. Removal of Zn 2+ reduced or delayed OGD- and FCCP-induced ROS generation, indicating that Zn 2+ contributes to mitochondrial ROS generation. There was a Zn 2+ -induced increase in the functional component of NADPH oxidase, p47 phox , thus suggesting that NADPH oxidase may mediate Zn 2+ -induced ROS accumulation. We suggest a new mechanism of cross talk between Zn 2+ and mitochondrial ROS through positive feedback processes that eventually causes excessive free Zn 2+ and ROS accumulations during the course of ischemic stress. Copyright © 2017 the American Physiological Society.

TNF-α as a potential mediator of cardiac dysfunction due to intracellular Ca2+-overload

International Nuclear Information System (INIS)

Zhang Ming; Xu Yanjun; Saini, Harjot K.; Turan, Belma; Liu, Peter P.; Dhalla, Naranjan S.

2005-01-01

TNF-α has been shown to be involved in cardiac dysfunction during ischemia/reperfusion injury; however, no information regarding the status of TNF-α production in myocardial injury due to intracellular Ca 2+ -overload is available in the literature. The intracellular Ca 2+ -overload was induced in the isolated rat hearts subjected to 5 min Ca 2+ -depletion and 30 min Ca 2+ -repletion (Ca 2+ -paradox). The Ca 2+ -paradox hearts exhibited a dramatic depression in left ventricular developed pressure, a marked elevation in left ventricular end diastolic pressure, and more than a 4-fold increase in TNF-α content. The ratio of cytosolic to homogenate nuclear factor-κB (NFκB) was decreased whereas the ratio of phospho-NFκB to total NFκB was increased in the Ca 2+ -paradox hearts. All these changes due to Ca 2+ -paradox were significantly attenuated upon treating the hearts with 100 μM pentoxifylline. These results suggest that activation of NFκB and increased production of TNF-α may play an important role in cardiac injury due to intracellular Ca 2+ -overload

Epithelial Cell Gene Expression Induced by Intracellular Staphylococcus aureus

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Xianglu Li

2009-01-01

Full Text Available HEp-2 cell monolayers were cocultured with intracellular Staphylococcus aureus, and changes in gene expression were profiled using DNA microarrays. Intracellular S. aureus affected genes involved in cellular stress responses, signal transduction, inflammation, apoptosis, fibrosis, and cholesterol biosynthesis. Transcription of stress response and signal transduction-related genes including atf3, sgk, map2k1, map2k3, arhb, and arhe was increased. In addition, elevated transcription of proinflammatory genes was observed for tnfa, il1b, il6, il8, cxcl1, ccl20, cox2, and pai1. Genes involved in proapoptosis and fibrosis were also affected at transcriptional level by intracellular S. aureus. Notably, intracellular S. aureus induced strong transcriptional down-regulation of several cholesterol biosynthesis genes. These results suggest that epithelial cells respond to intracellular S. aureus by inducing genes affecting immunity and in repairing damage caused by the organism, and are consistent with the possibility that the organism exploits an intracellular environment to subvert host immunity and promote colonization.

Effects of exposure to high glucose on primary cultured hippocampal neurons: involvement of intracellular ROS accumulation.

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Liu, Di; Zhang, Hong; Gu, Wenjuan; Zhang, Mengren

2014-06-01

Recent studies showed that hyperglycemia is the main trigger of diabetic cognitive impairment and can cause hippocampus abnormalities. The goal of this study is to explore the effects of different concentrations of high glucose for different exposure time on cell viability as well as intracellular reactive oxygen species (ROS) generation of primary cultured hippocampal neurons. Hippocampal neurons were exposed to different concentrations of high glucose (50, 75, 100, 125, and 150 mM) for 24, 48, 72 and 96 h. Cell viability and nuclear morphology were evaluated by MTT and Hoechst assays, respectively. Intracellular ROS were monitored using the fluorescent probe DCFH-DA. The results showed that, compared with control group, the cell viability of all high glucose-treated groups decreased significantly after 72 h and there also was a significant increase of apoptotic nuclei in high glucose-treated groups from 72 to 96 h. Furthermore, 50 mM glucose induced a peak rise in ROS generation at 24 h and the intracellular ROS levels of 50 mM glucose group were significantly higher than the corresponding control group from 6 to 72 h. These results suggest that hippocampal neurons could be injured by high glucose exposure and the neuronal injury induced by high glucose is potentially mediated through intracellular ROS accumulation.

Release of intracellular Calcium increase production of mitochondrial reactive oxygen species in renal distal epithelial cells

DEFF Research Database (Denmark)

Bjerregaard, Henning F.

peroxide (H2O2) has traditionally been regarded as toxic by-products of aerobic metabolism. However, recent findings indicate that H2O2 act as a signalling molecule. The aim of the present study was to monitor, in real time, the rates of ROS generation in order to directly determine their production......Release of intracellular Calcium increase production of mitochondrial reactive oxygen species in renal distal epithelial cells. Henning F. Bjerregaard, Roskilde University, Department of Science, Systems and Models , 4000 Roskilde, Denmark. HFB@ RUC.DK Reactive oxygen species (ROS) like, hydrogen...... to G-protein stimulation of phospholipase C and release of inositol -3 phosphate. Cd (0.4 mM) treatment of A6 cells enhanced the ROS production after one minutes incubation. The production rate was constant for at least 10 to 20 min. Experiments showed that the Cd induced increase in ROS production...

Intracellular trafficking of new anticancer therapeutics: antibody–drug conjugates

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Kalim, Muhammad; Chen, Jie; Wang, Shenghao; Lin, Caiyao; Ullah, Saif; Liang, Keying; Ding, Qian; Chen, Shuqing; Zhan, Jinbiao

2017-01-01

Antibody–drug conjugate (ADC) is a milestone in targeted cancer therapy that comprises of monoclonal antibodies chemically linked to cytotoxic drugs. Internalization of ADC takes place via clathrin-mediated endocytosis, caveolae-mediated endocytosis, and pinocytosis. Conjugation strategies, endocytosis and intracellular trafficking optimization, linkers, and drugs chemistry present a great challenge for researchers to eradicate tumor cells successfully. This inventiveness of endocytosis and intracellular trafficking has given considerable momentum recently to develop specific antibodies and ADCs to treat cancer cells. It is significantly advantageous to emphasize the endocytosis and intracellular trafficking pathways efficiently and to design potent engineered conjugates and biological entities to boost efficient therapies enormously for cancer treatment. Current studies illustrate endocytosis and intracellular trafficking of ADC, protein, and linker strategies in unloading and also concisely evaluate practically applicable ADCs. PMID:28814834

Intracellular trafficking of new anticancer therapeutics: antibody-drug conjugates.

Science.gov (United States)

Kalim, Muhammad; Chen, Jie; Wang, Shenghao; Lin, Caiyao; Ullah, Saif; Liang, Keying; Ding, Qian; Chen, Shuqing; Zhan, Jinbiao

2017-01-01

Antibody-drug conjugate (ADC) is a milestone in targeted cancer therapy that comprises of monoclonal antibodies chemically linked to cytotoxic drugs. Internalization of ADC takes place via clathrin-mediated endocytosis, caveolae-mediated endocytosis, and pinocytosis. Conjugation strategies, endocytosis and intracellular trafficking optimization, linkers, and drugs chemistry present a great challenge for researchers to eradicate tumor cells successfully. This inventiveness of endocytosis and intracellular trafficking has given considerable momentum recently to develop specific antibodies and ADCs to treat cancer cells. It is significantly advantageous to emphasize the endocytosis and intracellular trafficking pathways efficiently and to design potent engineered conjugates and biological entities to boost efficient therapies enormously for cancer treatment. Current studies illustrate endocytosis and intracellular trafficking of ADC, protein, and linker strategies in unloading and also concisely evaluate practically applicable ADCs.

Chronic ethanol exposure induces SK-N-SH cell apoptosis by increasing N-methyl-D-aspartic acid receptor expression and intracellular calcium.

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Wang, Hongbo; Wang, Xiaolong; Li, Yan; Yu, Hao; Wang, Changliang; Feng, Chunmei; Xu, Guohui; Chen, Jiajun; You, Jiabin; Wang, Pengfei; Wu, Xu; Zhao, Rui; Zhang, Guohua

2018-04-01

It has been identified that chronic ethanol exposure damages the nervous system, particularly neurons. There is scientific evidence suggesting that neuronal loss caused by chronic ethanol exposure has an association with neuron apoptosis and intracellular calcium oscillation is one of the primary inducers of apoptosis. Therefore, the present study aimed to investigate the inductive effects of intracellular calcium oscillation on apoptosis in SK-N-SH human neuroblastoma cells and the protective effects of the N-methyl-D-aspartic acid receptor (NMDAR) antagonist, memantine, on SK-N-SH cell apoptosis caused by chronic ethanol exposure. SK-N-SH cells were treated with 100 mM ethanol and memantine (4 µM) for 2 days. Protein expression of NR1 was downregulated by RNA interference (RNAi). Apoptosis was detected by Annexin V/propidium iodide (PI) double-staining and flow cytometry and cell viability was detected using an MTS kit. Fluorescence dual wavelength spectrophotometry was used to determine the intracellular calcium concentration and the levels of NR1 and caspase-3 were detected using western blotting. NR1 mRNA levels were also detected using qPCR. It was found that chronic ethanol exposure reduced neuronal cell viability and caused apoptosis of SK-N-SH cells, and the extent of damage in SK-N-SH cells was associated with ethanol exposure concentration and time. In addition, chronic ethanol exposure increased the concentration of intracellular calcium in SK-N-SH cells by inducing the expression of NMDAR, resulting in apoptosis, and memantine treatment reduced ethanol-induced cell apoptosis. The results of the present study indicate that the application of memantine may provide a novel strategy for the treatment of alcoholic dementia.

Effective modification of cell death-inducing intracellular peptides by means of a photo-cleavable peptide array-based screening system.

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Kozaki, Ikko; Shimizu, Kazunori; Honda, Hiroyuki

2017-08-01

Intracellular functional peptides that play a significant role inside cells have been receiving a lot of attention as regulators of cellular activity. Previously, we proposed a novel screening system for intracellular functional peptides; it combined a photo-cleavable peptide array system with cell-penetrating peptides (CPPs). Various peptides can be delivered into cells and intracellular functions of the peptides can be assayed by means of our system. The aim of the present study was to demonstrate that the proposed screening system can be used for assessing the intracellular activity of peptides. The cell death-inducing peptide (LNLISKLF) identified in a mitochondria-targeting domain (MTD) of the Noxa protein served as an original peptide sequence for screening of peptides with higher activity via modification of the peptide sequence. We obtained 4 peptides with higher activity, in which we substituted serine (S) at the fifth position with phenylalanine (F), valine (V), tryptophan (W), or tyrosine (Y). During analysis of the mechanism of action, the modified peptides induced an increase in intracellular calcium concentration, which was caused by the treatment with the original peptide. Higher capacity for cell death induction by the modified peptides may be caused by increased hydrophobicity or an increased number of aromatic residues. Thus, the present work suggests that the intracellular activity of peptides can be assessed using the proposed screening system. It could be used for identifying intracellular functional peptides with higher activity through comprehensive screening. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Intracellular insulin processing is altered in monocytes from patients with type II diabetes mellitus

International Nuclear Information System (INIS)

Trischitta, V.; Benzi, L.; Brunetti, A.; Cecchetti, P.; Marchetti, P.; Vigneri, R.; Navalesi, R.

1987-01-01

We studied total cell-associated A14-[ 125 I]insulin radioactivity (including surface-bound and internalized radioactivity), insulin internalization, and its intracellular degradation at 37 C in monocytes from nonobese type II untreated diabetic patients (n = 9) and normal subjects (n = 7). Total cell-associated radioactivity was decreased in diabetic patients [2.65 +/- 1.21% (+/- SD) vs. 4.47 +/- 1.04% of total radioactivity. Insulin internalization was also reduced in diabetic patients (34.0 +/- 6.8% vs. 59.0 +/- 11.3% of cell-associated radioactivity. Using high performance liquid chromatography six intracellular forms of radioactivity derived from A14-[ 125 I] insulin were identified; 10-20% of intracellular radioactivity had approximately 300,000 mol wt and was identified as radioactivity bound to the insulin receptor, and the remaining intracellular radioactivity included intact A14-[ 125 I]insulin, [ 125 I]iodide, or [ 125 I]tyrosine, and three intermediate compounds. A progressive reduction of intact insulin and a corresponding increase in iodine were found when the incubation time was prolonged. Intracellular insulin degradation was reduced in monocytes from diabetic patients; intracellular intact insulin was 65.6 +/- 18.1% vs. 37.4 +/- 18.0% of intracellular radioactivity after 2 min and 23.6 +/- 22.3% vs. 3.9 +/- 2.3% after 60 min in diabetic patients vs. normal subjects, respectively. In conclusion, 1) human monocytes internalize and degrade insulin in the intracellular compartment in a stepwise time-dependent manner; and 2) in monocytes from type II diabetic patients total cell-associated radioactivity, insulin internalization, and insulin degradation are significantly reduced. These defects may be related to the cellular insulin resistance present in these patients

Intracellular Trafficking Modulation by Ginsenoside Rg3 Inhibits Brucella abortus Uptake and Intracellular Survival within RAW 264.7 Cells.

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Huy, Tran Xuan Ngoc; Reyes, Alisha Wehdnesday Bernardo; Hop, Huynh Tan; Arayan, Lauren Togonon; Min, WonGi; Lee, Hu Jang; Rhee, Man Hee; Chang, Hong Hee; Kim, Suk

2017-03-28

Ginsenoside Rg3, a saponin extracted from ginseng, has various pharmacological and biological activities; however, its effects against Brucella infection are still unclear. Herein, the inhibitory effects of ginsenoside Rg3 against intracellular parasitic Brucella infection were evaluated through bacterial infection, adherence assays, and LAMP-1 colocalization, as well as immunoblotting and FACS for detecting MAPK signaling proteins and F-actin polymerization, respectively. The internalization, intracellular growth, and adherence of Brucella abortus in Rg3-treated RAW 264.7 cells were significantly decreased compared with the Rg3-untreated control. Furthermore, an apparent reduction of F-actin content and intensity of F-actin fluorescence in Rg3-treated cells was observed compared with B. abortus -infected cells without treatment by flow cytometry analysis and confocal microscopy, respectively. In addition, treating cells with Rg3 decreased the phosphorylation of MAPK signaling proteins such as ERK 1/2 and p38 compared with untreated cells. Moreover, the colocalization of B. abortus -containing phagosomes with LAMP-1 was markedly increased in Rg3-treated cells. These findings suggest that ginsenoside Rg3 inhibits B. abortus infection in mammalian cells and can be used as an alternative approach in the treatment of brucellosis.

Gas revenue increasingly significant

International Nuclear Information System (INIS)

Megill, R.E.

1991-01-01

This paper briefly describes the wellhead prices of natural gas compared to crude oil over the past 70 years. Although natural gas prices have never reached price parity with crude oil, the relative value of a gas BTU has been increasing. It is one of the reasons that the total amount of money coming from natural gas wells is becoming more significant. From 1920 to 1955 the revenue at the wellhead for natural gas was only about 10% of the money received by producers. Most of the money needed for exploration, development, and production came from crude oil. At present, however, over 40% of the money from the upstream portion of the petroleum industry is from natural gas. As a result, in a few short years natural gas may become 50% of the money revenues generated from wellhead production facilities

Silver ions increase plasma membrane permeability through modulation of intracellular calcium levels in tobacco BY-2 cells.

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Klíma, Petr; Laňková, Martina; Vandenbussche, Filip; Van Der Straeten, Dominique; Petrášek, Jan

2018-05-01

Silver ions increase plasma membrane permeability for water and small organic compounds through their stimulatory effect on plasma membrane calcium channels, with subsequent modulation of intracellular calcium levels and ion homeostasis. The action of silver ions at the plant plasma membrane is largely connected with the inhibition of ethylene signalling thanks to the ability of silver ion to replace the copper cofactor in the ethylene receptor. A link coupling the action of silver ions and cellular auxin efflux has been suggested earlier by their possible direct interaction with auxin efflux carriers or by influencing plasma membrane permeability. Using tobacco BY-2 cells, we demonstrate here that besides a dramatic increase of efflux of synthetic auxins 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-naphthalene acetic acid (NAA), treatment with AgNO 3 resulted in enhanced efflux of the cytokinin trans-zeatin (tZ) as well as the auxin structural analogues tryptophan (Trp) and benzoic acid (BA). The application of AgNO 3 was accompanied by gradual water loss and plasmolysis. The observed effects were dependent on the availability of extracellular calcium ions (Ca 2+ ) as shown by comparison of transport assays in Ca 2+ -rich and Ca 2+ -free buffers and upon treatment with inhibitors of plasma membrane Ca 2+ -permeable channels Al 3+ and ruthenium red, both abolishing the effect of AgNO 3 . Confocal microscopy of Ca 2+ -sensitive fluorescence indicator Fluo-4FF, acetoxymethyl (AM) ester suggested that the extracellular Ca 2+ availability is necessary to trigger the response to silver ions and that the intracellular Ca 2+ pool alone is not sufficient for this effect. Altogether, our data suggest that in plant cells the effects of silver ions originate from the primal modification of the internal calcium levels, possibly by their interaction with Ca 2+ -permeable channels at the plasma membrane.

Nuclear magnetic resonance studies of intracellular ions in perfused from heart

International Nuclear Information System (INIS)

Burnstein, D.; Fossel, E.T.

1987-01-01

Intracellular sodium, potassium, and lithium were observed in a perfused frog heart by nuclear magnetic resonance (NMR) spectroscopy. A perfusate buffer containing the shift reagent, dysprosium tripolyphosphate, was used in combination with mathematical filtering or presaturation of the extracellular resonance to separate the intra- and extracellular sodium NMR signals. Addition of 10 μM ouabain to the perfusate, perfusion with a zero potassium, low-calcium buffer, and replacement of 66% of the perfusate sodium with lithium resulted in changes in the intracellular sodium levels. An increase of 45% in the intracellular sodium was observed when changing the pacing rate from 0 to 60 beats/min (with proportional changes for intermediate pacing rates). The ratio of intracellular potassium to sodium concentration was determined to be 2.3 by NMR, indicating that a substantial amount of the intracellular potassium is undetectable with these NMR method. In addition, intracellular lithium was observed during perfusion with a lithium-containing perfusate

Intracellular Complement Activation Sustains T Cell Homeostasis and Mediates Effector Differentiation

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Liszewski, M. Kathryn; Kolev, Martin; Le Friec, Gaelle; Leung, Marilyn; Bertram, Paula G.; Fara, Antonella F.; Subias, Marta; Pickering, Matthew C.; Drouet, Christian; Meri, Seppo; Arstila, T. Petteri; Pekkarinen, Pirkka T.; Ma, Margaret; Cope, Andrew; Reinheckel, Thomas; Rodriguez de Cordoba, Santiago; Afzali, Behdad; Atkinson, John P.; Kemper, Claudia

2013-01-01

Summary Complement is viewed as a critical serum-operative component of innate immunity, with processing of its key component, C3, into activation fragments C3a and C3b confined to the extracellular space. We report here that C3 activation also occurred intracellularly. We found that the T cell-expressed protease cathepsin L (CTSL) processed C3 into biologically active C3a and C3b. Resting T cells contained stores of endosomal and lysosomal C3 and CTSL and substantial amounts of CTSL-generated C3a. While “tonic” intracellular C3a generation was required for homeostatic T cell survival, shuttling of this intracellular C3-activation-system to the cell surface upon T cell stimulation induced autocrine proinflammatory cytokine production. Furthermore, T cells from patients with autoimmune arthritis demonstrated hyperactive intracellular complement activation and interferon-γ production and CTSL inhibition corrected this deregulated phenotype. Importantly, intracellular C3a was observed in all examined cell populations, suggesting that intracellular complement activation might be of broad physiological significance. PMID:24315997

Effect of insulin resistance on intracellular signal transduction of vessels in diabetic

International Nuclear Information System (INIS)

Cen Rongguang; Wei Shaoying; Mo Xingju

2003-01-01

To investigate the relationship between the insulin resistance (IR) and the intracellular signal transduction of vessels, changes in fasting blood glucose (FBG), fasting insulin (FINS), triglyceride (TG), total cholesterol (TC), inositol triphosphate (IP 3 ), protein kinase C(PKC) and intracellular total calcium concentration in 31 diabetic patients were compared with those of 39 normal controls. The levels of FBG, FINS, TG and TC in diabetic patients were significantly higher than those of normal controls (P 3 and PKC in diabetic patients were significantly lower than those of normal controls (P<0.01). The results suggest that there is a causal relation between insulin resistance and abnormalities of cellular calcium metabolism and intracellular signal transduction of vessels

Extremely low frequency electromagnetic fields promote mesenchymal stem cell migration by increasing intracellular Ca2+ and activating the FAK/Rho GTPases signaling pathways in vitro.

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Zhang, Yingchi; Yan, Jiyuan; Xu, Haoran; Yang, Yong; Li, Wenkai; Wu, Hua; Liu, Chaoxu

2018-05-21

The ability of mesenchymal stem cells (MSCs) to migrate to the desired tissues or lesions is crucial for stem cell-based regenerative medicine and tissue engineering. Optimal therapeutics for promoting MSC migration are expected to become an effective means for tissue regeneration. Electromagnetic fields (EMF), as a noninvasive therapy, can cause a lot of biological changes in MSCs. However, whether EMF can promote MSC migration has not yet been reported. We evaluated the effects of EMF on cell migration in human bone marrow-derived MSCs. With the use of Helmholtz coils and an EMF stimulator, 7.5, 15, 30, 50, and 70 Hz/1 mT EMF was generated. Additionally, we employed the L-type calcium channel blocker verapamil and the focal adhesion kinase (FAK) inhibitor PF-573228 to investigate the role of intracellular calcium content, cell adhesion proteins, and the Rho GTPase protein family (RhoA, Rac1, and Cdc42) in EMF-mediated MSC migration. Cell adhesion proteins (FAK, talin, and vinculin) were detected by Western blot analysis. The Rho GTPase protein family activities were assessed by G-LISA, and F-actin levels, which reflect actin cytoskeletal organization, were detected using immunofluorescence. All the 7.5, 15, 30, 50, and 70 Hz/1 mT EMF promoted MSC migration. EMF increased MSC migration in an intracellular calcium-dependent manner. Notably, EMF-enhanced migration was mediated by FAK activation, which was critical for the formation of focal contacts, as evidenced by increased talin and vinculin expression. Moreover, RhoA, Rac1, and Cdc42 were activated by FAK to increase cytoskeletal organization, thus promoting cell contraction. EMF promoted MSC migration by increasing intracellular calcium and activating the FAK/Rho GTPase signaling pathways. This study provides insights into the mechanisms of MSC migration and will enable the rational design of targeted therapies to improve MSC engraftment.

Dextran sulfate sodium upregulates MAPK signaling for the uptake and subsequent intracellular survival of Brucella abortus in murine macrophages.

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Reyes, Alisha Wehdnesday Bernardo; Arayan, Lauren Togonon; Simborio, Hannah Leah Tadeja; Hop, Huynh Tan; Min, WonGi; Lee, Hu Jang; Kim, Dong Hee; Chang, Hong Hee; Kim, Suk

2016-02-01

Brucellosis is one of the major zoonoses worldwide that inflicts important health problems in animal and human. Here, we demonstrated that dextran sulfate sodium (DSS) significantly increased adhesion of Brucella (B.) abortus in murine macrophages compared to untreated cells. Even without infection, Brucella uptake into macrophages increased and F-actin reorganization was induced compared with untreated cells. Furthermore, DSS increased the phosphorylation of MAPKs (ERK1/2 and p38α) in Brucella-infected, DSS-treated cells compared with the control cells. Lastly, DSS markedly increased the intracellular survival of Brucella abortus in macrophages by up to 48 h. These results suggest that DSS enhanced the adhesion and phagocytosis of B. abortus into murine macrophages by stimulating the MAPK signaling proteins phospho-ERK1/2 and p38α and that DSS increased the intracellular survival of B. abortus by inhibiting colocalization of Brucella-containing vacuoles (BCVs) with the late endosome marker LAMP-1. This study emphasizes the enhancement of the phagocytic and intracellular modulatory effects of DSS, which may suppress the innate immune system and contribute to prolonged Brucella survival and chronic infection. Copyright © 2015 Elsevier Ltd. All rights reserved.

Melatonin-Mediated Intracellular Insulin during 2-Deoxy-d-glucose Treatment Is Reduced through Autophagy and EDC3 Protein in Insulinoma INS-1E Cells

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Han Sung Kim

2016-01-01

Full Text Available 2-DG triggers glucose deprivation without altering other nutrients or metabolic pathways and then activates autophagy via activation of AMPK and endoplasmic reticulum (ER stress. We investigated whether 2-DG reduced intracellular insulin increased by melatonin via autophagy/EDC3 in insulinoma INS-1E cells. p-AMPK and GRP78/BiP level were significantly increased by 2-DG in the presence/absence of melatonin, but IRE1α level was reduced in 2-DG treatment. Levels of p85α, p110, p-Akt (Ser473, Thr308, and p-mTOR (Ser2481 were also significantly reduced by 2-DG in the presence/absence of melatonin. Mn-SOD increased with 2-DG plus melatonin compared to groups treated with/without melatonin alone. Bcl-2 was decreased and Bax increased with 2-DG plus melatonin. LC3II level increased with 2-DG treatment in the presence/absence of melatonin. Intracellular insulin production increased in melatonin plus 2-DG but reduced in treatment with 2-DG with/without melatonin. EDC3 was increased by 2-DG in the presence/absence of melatonin. Rapamycin, an mTOR inhibitor, increased GRP78/BiP and EDC3 levels in a dose-dependent manner and subsequently resulted in a decrease in intracellular production of insulin. These results suggest that melatonin-mediated insulin synthesis during 2-DG treatment involves autophagy and EDC3 protein in rat insulinoma INS-1E cells and subsequently results in a decrease in intracellular production of insulin.

MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb.

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Wang, Jinli; Yang, Kun; Zhou, Lin; Minhaowu; Wu, Yongjian; Zhu, Min; Lai, Xiaomin; Chen, Tao; Feng, Lianqiang; Li, Meiyu; Huang, Chunyu; Zhong, Qiu; Huang, Xi

2013-01-01

Mycobacterium tuberculosis is a hard-to-eradicate intracellular pathogen that infects one-third of the global population. It can live within macrophages owning to its ability to arrest phagolysosome biogenesis. Autophagy has recently been identified as an effective way to control the intracellular mycobacteria by enhancing phagosome maturation. In the present study, we demonstrate a novel role of miR-155 in regulating the autophagy-mediated anti-mycobacterial response. Both in vivo and in vitro studies showed that miR-155 expression was significantly enhanced after mycobacterial infection. Forced expression of miR-155 accelerated the autophagic response in macrophages, thus promoting the maturation of mycobacterial phagosomes and decreasing the survival rate of intracellular mycobacteria, while transfection with miR-155 inhibitor increased mycobacterial survival. However, macrophage-mediated mycobacterial phagocytosis was not affected after miR-155 overexpression or inhibition. Furthermore, blocking autophagy with specific inhibitor 3-methyladenine or silencing of autophagy related gene 7 (Atg7) reduced the ability of miR-155 to promote autophagy and mycobacterial elimination. More importantly, our study demonstrated that miR-155 bound to the 3'-untranslated region of Ras homologue enriched in brain (Rheb), a negative regulator of autophagy, accelerated the process of autophagy and sequential killing of intracellular mycobacteria by suppressing Rheb expression. Our results reveal a novel role of miR-155 in regulating autophagy-mediated mycobacterial elimination by targeting Rheb, and provide potential targets for clinical treatment.

MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb.

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Jinli Wang

Full Text Available Mycobacterium tuberculosis is a hard-to-eradicate intracellular pathogen that infects one-third of the global population. It can live within macrophages owning to its ability to arrest phagolysosome biogenesis. Autophagy has recently been identified as an effective way to control the intracellular mycobacteria by enhancing phagosome maturation. In the present study, we demonstrate a novel role of miR-155 in regulating the autophagy-mediated anti-mycobacterial response. Both in vivo and in vitro studies showed that miR-155 expression was significantly enhanced after mycobacterial infection. Forced expression of miR-155 accelerated the autophagic response in macrophages, thus promoting the maturation of mycobacterial phagosomes and decreasing the survival rate of intracellular mycobacteria, while transfection with miR-155 inhibitor increased mycobacterial survival. However, macrophage-mediated mycobacterial phagocytosis was not affected after miR-155 overexpression or inhibition. Furthermore, blocking autophagy with specific inhibitor 3-methyladenine or silencing of autophagy related gene 7 (Atg7 reduced the ability of miR-155 to promote autophagy and mycobacterial elimination. More importantly, our study demonstrated that miR-155 bound to the 3'-untranslated region of Ras homologue enriched in brain (Rheb, a negative regulator of autophagy, accelerated the process of autophagy and sequential killing of intracellular mycobacteria by suppressing Rheb expression. Our results reveal a novel role of miR-155 in regulating autophagy-mediated mycobacterial elimination by targeting Rheb, and provide potential targets for clinical treatment.

CIRRHOSIS INDUCES APOPTOSIS IN RENAL TISSUE THROUGH INTRACELLULAR OXIDATIVE STRESS

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Keli Cristina Simões da SILVEIRA

2015-03-01

Full Text Available Background Renal failure is a frequent and serious complication in patients with decompensated cirrhosis. Objectives We aimed to evaluate the renal oxidative stress, cell damage and impaired cell function in animal model of cirrhosis. Methods Secondary biliary cirrhosis was induced in rats by ligation of the common bile duct. We measured TBARS, ROS and mitochondrial membrane potential in kidney as markers of oxidative stress, and activities of the antioxidant enzymes. Relative cell viability was determined by trypan blue dye-exclusion assay. Annexin V-PE was used with a vital dye, 7-AAD, to distinguish apoptotic from necrotic cells and comet assay was used for determined DNA integrity in single cells. Results In bile duct ligation animals there was significant increase in the kidney lipoperoxidation and an increase of the level of intracellular ROS. There was too an increase in the activity of all antioxidant enzymes evaluated in the kidney. The percentage viability was above 90% in the control group and in bile duct ligation was 64.66% and the dominant cell death type was apoptosis. DNA damage was observed in the bile duct ligation. There was a decreased in the mitochondrial membrane potential from 71.40% ± 6.35% to 34.48% ± 11.40% in bile duct ligation. Conclusions These results indicate that intracellular increase of ROS cause damage in the DNA and apoptosis getting worse the renal function in cirrhosis.

Concentration-dependent fluorescence live-cell imaging and tracking of intracellular nanoparticles

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Seo, Ji Hye; Joo, Sang-Woo [Department of Chemistry, Soongsil University, Seoul 156-743 (Korea, Republic of); Cho, Keunchang [Logos Biosystems, Incorporated, Anyang 431-070 (Korea, Republic of); Lee, So Yeong, E-mail: leeso@snu.ac.kr, E-mail: sjoo@ssu.ac.kr [Laboratory of Pharmacology, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 151-742 (Korea, Republic of)

2011-06-10

Using live-cell imaging techniques we investigated concentration-dependent intracellular movements of fluorescence nanoparticles (NPs) in real-time after their entry into HeLa cells via incubation. Intracellular particle traces appeared to be a mixture of both random and fairly unidirectional movements of the particles. At rather low concentrations of NPs, a majority of the non-random intracellular particle trajectories are assumed to mostly go along microtubule networks after endocytosis, as evidenced from the inhibition test with nocodazole. On the other hand, as the concentrations of NPs increased, random motions were more frequently observed inside the cells.

Concentration-dependent fluorescence live-cell imaging and tracking of intracellular nanoparticles

International Nuclear Information System (INIS)

Seo, Ji Hye; Joo, Sang-Woo; Cho, Keunchang; Lee, So Yeong

2011-01-01

Using live-cell imaging techniques we investigated concentration-dependent intracellular movements of fluorescence nanoparticles (NPs) in real-time after their entry into HeLa cells via incubation. Intracellular particle traces appeared to be a mixture of both random and fairly unidirectional movements of the particles. At rather low concentrations of NPs, a majority of the non-random intracellular particle trajectories are assumed to mostly go along microtubule networks after endocytosis, as evidenced from the inhibition test with nocodazole. On the other hand, as the concentrations of NPs increased, random motions were more frequently observed inside the cells.

Pathway analysis for intracellular Porphyromonas gingivalis using a strain ATCC 33277 specific database

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Wang Tiansong

2009-09-01

Full Text Available Abstract Background Porphyromonas gingivalis is a Gram-negative intracellular pathogen associated with periodontal disease. We have previously reported on whole-cell quantitative proteomic analyses to investigate the differential expression of virulence factors as the organism transitions from an extracellular to intracellular lifestyle. The original results with the invasive strain P. gingivalis ATCC 33277 were obtained using the genome sequence available at the time, strain W83 [GenBank: AE015924]. We present here a re-processed dataset using the recently published genome annotation specific for strain ATCC 33277 [GenBank: AP009380] and an analysis of differential abundance based on metabolic pathways rather than individual proteins. Results Qualitative detection was observed for 1266 proteins using the strain ATCC 33277 annotation for 18 hour internalized P. gingivalis within human gingival epithelial cells and controls exposed to gingival cell culture medium, an improvement of 7% over the W83 annotation. Internalized cells showed increased abundance of proteins in the energy pathway from asparagine/aspartate amino acids to ATP. The pathway producing one short chain fatty acid, propionate, showed increased abundance, while that of another, butyrate, trended towards decreased abundance. The translational machinery, including ribosomal proteins and tRNA synthetases, showed a significant increase in protein relative abundance, as did proteins responsible for transcription. Conclusion Use of the ATCC 33277 specific genome annotation resulted in improved proteome coverage with respect to the number of proteins observed both qualitatively in terms of protein identifications and quantitatively in terms of the number of calculated abundance ratios. Pathway analysis showed a significant increase in overall protein synthetic and transcriptional machinery in the absence of significant growth. These results suggest that the interior of host cells

Intracellular transport of fat-soluble vitamins A and E.

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Kono, Nozomu; Arai, Hiroyuki

2015-01-01

Vitamins are compounds that are essential for the normal growth, reproduction and functioning of the human body. Of the 13 known vitamins, vitamins A, D, E and K are lipophilic compounds and are therefore called fat-soluble vitamins. Because of their lipophilicity, fat-soluble vitamins are solubilized and transported by intracellular carrier proteins to exert their actions and to be metabolized properly. Vitamin A and its derivatives, collectively called retinoids, are solubilized by intracellular retinoid-binding proteins such as cellular retinol-binding protein (CRBP), cellular retinoic acid-binding protein (CRABP) and cellular retinal-binding protein (CRALBP). These proteins act as chaperones that regulate the metabolism, signaling and transport of retinoids. CRALBP-mediated intracellular retinoid transport is essential for vision in human. α-Tocopherol, the main form of vitamin E found in the body, is transported by α-tocopherol transfer protein (α-TTP) in hepatic cells. Defects of α-TTP cause vitamin E deficiency and neurological disorders in humans. Recently, it has been shown that the interaction of α-TTP with phosphoinositides plays a critical role in the intracellular transport of α-tocopherol and is associated with familial vitamin E deficiency. In this review, we summarize the mechanisms and biological significance of the intracellular transport of vitamins A and E. © 2014 The Authors. Traffic published by John Wiley & Sons Ltd.

The Slow Dynamics of Intracellular Sodium Concentration Increase the Time Window of Neuronal Integration: A Simulation Study

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Asaph Zylbertal

2017-09-01

Full Text Available Changes in intracellular Na+ concentration ([Na+]i are rarely taken into account when neuronal activity is examined. As opposed to Ca2+, [Na+]i dynamics are strongly affected by longitudinal diffusion, and therefore they are governed by the morphological structure of the neurons, in addition to the localization of influx and efflux mechanisms. Here, we examined [Na+]i dynamics and their effects on neuronal computation in three multi-compartmental neuronal models, representing three distinct cell types: accessory olfactory bulb (AOB mitral cells, cortical layer V pyramidal cells, and cerebellar Purkinje cells. We added [Na+]i as a state variable to these models, and allowed it to modulate the Na+ Nernst potential, the Na+-K+ pump current, and the Na+-Ca2+ exchanger rate. Our results indicate that in most cases [Na+]i dynamics are significantly slower than [Ca2+]i dynamics, and thus may exert a prolonged influence on neuronal computation in a neuronal type specific manner. We show that [Na+]i dynamics affect neuronal activity via three main processes: reduction of EPSP amplitude in repeatedly active synapses due to reduction of the Na+ Nernst potential; activity-dependent hyperpolarization due to increased activity of the Na+-K+ pump; specific tagging of active synapses by extended Ca2+ elevation, intensified by concurrent back-propagating action potentials or complex spikes. Thus, we conclude that [Na+]i dynamics should be considered whenever synaptic plasticity, extensive synaptic input, or bursting activity are examined.

Evaluation of intracellular anion superoxide level, heat shock protein A2 and protamine positive spermatozoa percentages in teratoasthenozoospermia

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Parvin Sabeti

2017-09-01

Full Text Available Background: Teratoasthenozoospermia (TA is a severe form of male infertility with no clear etiology. Objective: To compare the level of intracellular anion superoxide (O2–, heat shock protein A2 (HSPA2 and protamine deficiencies in ejaculated spermatozoa between teratoasthenozoospermic and normozoospermic men. Materials and Methods: In this case- control study, semen samples of 20 infertile men, with TA (with normal morphology lower than 4%_ and total motility lower than 40% as the case group and 20 normozoospermic fertile men as the control group were evaluated for intracellular O2 – and HSPA2 by flow cytometry and protamine deficiency by Chromomycin A3 (CMA3 test. Results: The rate of CMA3+ spermatozoa in the case group was higher than controls (p=0.001. The percentages of HSPA2+ spermatozoa in the cases were significantly lower than controls (p=0.001. Also, intracellular O2 – levels in the case group were significantly higher than controls (p=0.001 and had positive correlations with sperm apoptosis (r=0.79, p=0.01 and CMA3 positive sperm (r=0.76, p=0.01, but negative correlations with normal morphology (r=-0.81, p=0.01 and motility (r=-0.81, p=0.01. There was no significant correlation between intracellular O2 – and HSPA2 in the case group (r=0.041, p=0.79. Conclusion: We suggest that the increase in intracellular O2 –, decrease in spermatozoa HSPA2+, and high percentages of spermatozoa with immature chromatin might be considered as etiologies of infertility in TA patients

Semiconductor quantum dots as Förster resonance energy transfer donors for intracellularly-based biosensors

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Field, Lauren D.; Walper, Scott A.; Susumu, Kimihiro; Oh, Eunkeu; Medintz, Igor L.; Delehanty, James B.

2017-02-01

Förster resonance energy transfer (FRET)-based assemblies currently comprise a significant portion of intracellularly based sensors. Although extremely useful, the fluorescent protein pairs typically utilized in such sensors are still plagued by many photophysical issues including significant direct acceptor excitation, small changes in FRET efficiency, and limited photostability. Luminescent semiconductor nanocrystals or quantum dots (QDs) are characterized by many unique optical properties including size-tunable photoluminescence, broad excitation profiles coupled to narrow emission profiles, and resistance to photobleaching, which can cumulatively overcome many of the issues associated with use of fluorescent protein FRET donors. Utilizing QDs for intracellular FRET-based sensing still requires significant development in many areas including materials optimization, bioconjugation, cellular delivery and assay design and implementation. We are currently developing several QD-based FRET sensors for various intracellular applications. These include sensors targeting intracellular proteolytic activity along with those based on theranostic nanodevices for monitoring drug release. The protease sensor is based on a unique design where an intracellularly expressed fluorescent acceptor protein substrate assembles onto a QD donor following microinjection, forming an active complex that can be monitored in live cells over time. In the theranostic configuration, the QD is conjugated to a carrier protein-drug analogue complex to visualize real-time intracellular release of the drug from its carrier in response to an external stimulus. The focus of this talk will be on the design, properties, photophysical characterization and cellular application of these sensor constructs.

Polycaprolactone/maltodextrin nanocarrier for intracellular drug delivery: formulation, uptake mechanism, internalization kinetics, and subcellular localization.

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Korang-Yeboah, Maxwell; Gorantla, Yamini; Paulos, Simon A; Sharma, Pankaj; Chaudhary, Jaideep; Palaniappan, Ravi

2015-01-01

Prostate cancer (PCa) disease progression is associated with significant changes in intracellular and extracellular proteins, intracellular signaling mechanism, and cancer cell phenotype. These changes may have direct impact on the cellular interactions with nanocarriers; hence, there is the need for a much-detailed understanding, as nanocarrier cellular internalization and intracellular sorting mechanism correlate directly with bioavailability and clinical efficacy. In this study, we report the differences in the rate and mechanism of cellular internalization of a biocompatible polycaprolactone (PCL)/maltodextrin (MD) nanocarrier system for intracellular drug delivery in LNCaP, PC3, and DU145 PCa cell lines. PCL/MD nanocarriers were designed and characterized. PCL/MD nanocarriers significantly increased the intracellular concentration of coumarin-6 and fluorescein isothiocyanate-labeled bovine serum albumin, a model hydrophobic and large molecule, respectively. Fluorescence microscopy and flow cytometry analysis revealed rapid internalization of the nanocarrier. The extent of nanocarrier cellular internalization correlated directly with cell line aggressiveness. PCL/MD internalization was highest in PC3 followed by DU145 and LNCaP, respectively. Uptake in all PCa cell lines was metabolically dependent. Extraction of endogenous cholesterol by methyl-β-cyclodextrin reduced uptake by 75%±4.53% in PC3, 64%±6.01% in LNCaP, and 50%±4.50% in DU145, indicating the involvement of endogenous cholesterol in cellular internalization. Internalization of the nanocarrier in LNCaP was mediated mainly by macropinocytosis and clathrin-independent pathways, while internalization in PC3 and DU145 involved clathrin-mediated endocytosis, clathrin-independent pathways, and macropinocytosis. Fluorescence microscopy showed a very diffused and non-compartmentalized subcellular localization of the PCL/MD nanocarriers with possible intranuclear localization and minor colocalization in

Pathogenic mechanisms of intracellular bacteria.

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Niller, Hans Helmut; Masa, Roland; Venkei, Annamária; Mészáros, Sándor; Minarovits, Janos

2017-06-01

We wished to overview recent data on a subset of epigenetic changes elicited by intracellular bacteria in human cells. Reprogramming the gene expression pattern of various host cells may facilitate bacterial growth, survival, and spread. DNA-(cytosine C5)-methyltransferases of Mycoplasma hyorhinis targeting cytosine-phosphate-guanine (CpG) dinucleotides and a Mycobacterium tuberculosis methyltransferase targeting non-CpG sites methylated the host cell DNA and altered the pattern of gene expression. Gene silencing by CpG methylation and histone deacetylation, mediated by cellular enzymes, also occurred in M. tuberculosis-infected macrophages. M. tuberculosis elicited cell type-specific epigenetic changes: it caused increased DNA methylation in macrophages, but induced demethylation, deposition of euchromatic histone marks and activation of immune-related genes in dendritic cells. A secreted transposase of Acinetobacter baumannii silenced a cellular gene, whereas Mycobacterium leprae altered the epigenotype, phenotype, and fate of infected Schwann cells. The 'keystone pathogen' oral bacterium Porphyromonas gingivalis induced local DNA methylation and increased the level of histone acetylation in host cells. These epigenetic changes at the biofilm-gingiva interface may contribute to the development of periodontitis. Epigenetic regulators produced by intracellular bacteria alter the epigenotype and gene expression pattern of host cells and play an important role in pathogenesis.

Involvement of intracellular Zn2+ signaling in LTP at perforant pathway-CA1 pyramidal cell synapse.

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Tamano, Haruna; Nishio, Ryusuke; Takeda, Atsushi

2017-07-01

Physiological significance of synaptic Zn 2+ signaling was examined at perforant pathway-CA1 pyramidal cell synapses. In vivo long-term potentiation (LTP) at perforant pathway-CA1 pyramidal cell synapses was induced using a recording electrode attached to a microdialysis probe and the recording region was locally perfused with artificial cerebrospinal fluid (ACSF) via the microdialysis probe. Perforant pathway LTP was not attenuated under perfusion with CaEDTA (10 mM), an extracellular Zn 2+ chelator, but attenuated under perfusion with ZnAF-2DA (50 μM), an intracellular Zn 2+ chelator, suggesting that intracellular Zn 2+ signaling is required for perforant pathway LTP. Even in rat brain slices bathed in CaEDTA in ACSF, intracellular Zn 2+ level, which was measured with intracellular ZnAF-2, was increased in the stratum lacunosum-moleculare where perforant pathway-CA1 pyramidal cell synapses were contained after tetanic stimulation. These results suggest that intracellular Zn 2+ signaling, which originates in internal stores/proteins, is involved in LTP at perforant pathway-CA1 pyramidal cell synapses. Because the influx of extracellular Zn 2+ , which originates in presynaptic Zn 2+ release, is involved in LTP at Schaffer collateral-CA1 pyramidal cell synapses, synapse-dependent Zn 2+ dynamics may be involved in plasticity of postsynaptic CA1 pyramidal cells. © 2017 Wiley Periodicals, Inc.

Effects of hirsutine, an antihypertensive indole alkaloid from Uncaria rhynchophylla, on intracellular calcium in rat thoracic aorta.

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Horie, S; Yano, S; Aimi, N; Sakai, S; Watanabe, K

1992-01-01

The effects of hirsutine, an indole alkaloid from Uncaria rhynchophylla (MIQ.) Jackson, on cytosolic Ca2+ level ([Ca2+]cyt) were studied by using fura-2-Ca2+ fluorescence in smooth muscle of the isolated rat aorta. Noradrenaline and high K+ solution produced a sustained increase in [Ca2+]cyt. Application of hirsutine after the increases in [Ca2+]cyt induced by noradrenaline and high K+ notably decreased [Ca2+]cyt, suggesting that hirsutine inhibits Ca2+ influx mainly through a voltage-dependent Ca2+ channel. Furthermore, the effect of hirsutine on intracellular Ca2+ store was studied by using contractile responses to caffeine under the Ca(2+)-free nutrient condition in the rat aorta. When hirsutine was added at 30 microM before caffeine treatment, the agent slightly but significantly reduced the caffeine-induced contraction. When added during Ca2+ loading, hirsutine definitely augmented the contractile response to caffeine. These results suggest that hirsutine inhibits Ca2+ release from the Ca2+ store and increases Ca2+ uptake into the Ca2+ store, leading to a reduction of intracellular Ca2+ level. It is concluded that hirsutine reduces intracellular Ca2+ level through its effect on the Ca2+ store as well as through its effect on the voltage-dependent Ca2+ channel.

Intracellular, genetic or congenital immunisation--transgenic approaches to increase disease resistance of farm animals.

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Müller, M; Brem, G

1996-01-26

Novel approaches to modify disease resistance or susceptibility in livestock are justified not only by economical reasons and with respect to animal welfare but also by recent advancements in molecular genetics. The control or elimination of infectious pathogens in farm animals is historically achieved by the use of vaccines and drugs and by quarantine safeguards and eradication. Currently, research on the improvement of disease resistance based on nucleic acid technology focuses on two main issues: additive gene transfer and the development of nucleic acid vaccines. The strategies aim at the stable or transient expression of components known to influence non-specific or specific host defence mechanisms against infectious pathogens. Thus, candidates for gene transfer experiments include all genes inducing or conferring innate and acquired immunity as well as specific disease resistance genes. Referring to the site and mode of action and the source of the effective agent the strategies are termed 'intracellular', 'genetic' and 'congenital' immunisation. The targeted disruption (deletive gene transfer) of disease susceptibility genes awaits the establishment of totipotential embryonic cell lineages in farm animals. The cytokine network regulates cellular viability, growth and differentiation in physiological and pathophysiological states. The identification of the JAK-STAT pathway used by many cytokines for their intracellular signal propagation has provided not only new target molecules for modulating the immune response but will also permit the further elucidation of host-pathogen interactions and resistance mechanisms.

Type II iodothyronine deiodinase provides intracellular 3,5,3′-triiodothyronine to normal and regenerating mouse skeletal muscle

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Marsili, Alessandro; Tang, Dan; Harney, John W.; Singh, Prabhat; Zavacki, Ann Marie; Dentice, Monica; Salvatore, Domenico

2011-01-01

The FoxO3-dependent increase in type II deiodinase (D2), which converts the prohormone thyroxine (T4) to 3,5,3′-triiodothyronine (T3), is required for normal mouse skeletal muscle differentiation and regeneration. This implies a requirement for an increase in D2-generated intracellular T3 under these conditions, which has not been directly demonstrated despite the presence of D2 activity in skeletal muscle. We directly show that D2-mediated T4-to-T3 conversion increases during differentiation in C2C12 myoblast and primary cultures of mouse neonatal skeletal muscle precursor cells, and that blockade of D2 eliminates this. In adult mice given 125I-T4 and 131I-T3, the intracellular 125I-T3/131I-T3 ratio is significantly higher than in serum in both the D2-expressing cerebral cortex and the skeletal muscle of wild-type, but not D2KO, mice. In D1-expressing liver and kidney, the 125I-T3/131I-T3 ratio does not differ from that in serum. Hypothyroidism increases D2 activity, and in agreement with this, the difference in 125I-T3/131I-T3 ratio is increased further in hypothyroid wild-type mice but not altered in the D2KO. Notably, in wild-type but not in D2KO mice, the muscle production of 125I-T3 is doubled after skeletal muscle injury. Thus, D2-mediated T4-to-T3 conversion generates significant intracellular T3 in normal mouse skeletal muscle, with the increased T3 required for muscle regeneration being provided by increased D2 synthesis, not by T3 from the circulation. PMID:21771965

Electron Microscopy of Intracellular Protozoa

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1988-12-20

Classification) " ELECTRON MICROSCOPY OF INTRACELLULAR PROTOZOA 12. PERSONAL AUTHOR(S) Aikawa, Masamichi 13a. TYPE OF REPORT I13b. TIME COVERED 114...authors suggest that anti-CS protein antibody is important in reducing the prevalence of malaria with increasing age among persons in such areas and... Hygine 33, 220-226. 0Giudice, G.D., Engers, H.D., Tougne, C., Biro, S.S., Weiss, N., Verdini, A.S., Pessi, A., Degremont, A.A., Freyvogel, T.A., Lambert

Novel pathways to erythropoiesis induced by dimerization of intracellular C-Mpl in human hematopoietic progenitors.

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Parekh, Chintan; Sahaghian, Arineh; Kim, William; Scholes, Jessica; Ge, Shundi; Zhu, Yuhua; Asgharzadeh, Shahab; Hollis, Roger; Kohn, Donald; Ji, Lingyun; Malvar, Jemily; Wang, Xiaoyan; Crooks, Gay

2012-04-01

The cytokine thrombopoietin (Tpo) plays a critical role in hematopoiesis by binding to the extracellular domain and inducing homodimerization of the intracellular signaling domain of its receptor, c-Mpl. Mpl homodimerization can also be accomplished by binding of a synthetic ligand to a constitutively expressed fusion protein F36VMpl consisting of a ligand binding domain (F36V) and the intracellular signaling domain of Mpl. Unexpectedly, in contrast to Tpo stimulation, robust erythropoiesis is induced after dimerization of F36VMpl in human CD34+ progenitor cells. The goal of this study was to define the hematopoietic progenitor stages at which dimerization of intracellular Mpl induces erythropoiesis and the downstream molecular events that mediate this unanticipated effect. Dimerization (in the absence of erythropoietin and other cytokines) in human common myeloid progenitors and megakaryocytic erythroid progenitors caused a significant increase in CD34+ cells (p Mpl in human myeloerythroid progenitors induces progenitor expansion and erythropoiesis through molecular mechanisms that are not shared by Tpo stimulation of endogenous Mpl. Copyright © 2012 AlphaMed Press.

Fragrance chemicals lyral and lilial decrease viability of HaCat cells' by increasing free radical production and lowering intracellular ATP level: protection by antioxidants.

Science.gov (United States)

Usta, Julnar; Hachem, Yassmine; El-Rifai, Omar; Bou-Moughlabey, Yolla; Echtay, Karim; Griffiths, David; Nakkash-Chmaisse, Hania; Makki, Rajaa Fakhoury

2013-02-01

We investigate in this study the biochemical effects on cells in culture of two commonly used fragrance chemicals: lyral and lilial. Whereas both chemicals exerted a significant effect on primary keratinocyte(s), HaCat cells, no effect was obtained with any of HepG2, Hek293, Caco2, NIH3T3, and MCF7 cells. Lyral and lilial: (a) decreased the viability of HaCat cells with a 50% cell death at 100 and 60 nM respectively; (b) decreased significantly in a dose dependant manner the intracellular ATP level following 12-h of treatment; (c) inhibited complexes I and II of electron transport chain in liver sub-mitochondrial particles; and (d) increased reactive oxygen species generation that was reversed by N-acetyl cysteine and trolox and the natural antioxidant lipoic acid, without influencing the level of free and/or oxidized glutathione. Lipoic acid protected HaCat cells against the decrease in viability induced by either compound. Dehydrogenation of lyral and lilial produce α,β-unsaturated aldehydes, that reacts with lipoic acid requiring proteins resulting in their inhibition. We propose lyral and lilial as toxic to mitochondria that have a direct effect on electron transport chain, increase ROS production, derange mitochondrial membrane potential, and decrease cellular ATP level, leading thus to cell death. Copyright © 2012 Elsevier Ltd. All rights reserved.

Synthesis of interleukin 6 (interferon-β2/B cell stimulatory factor 2) in human fibroblasts is triggered by an increase in intracellular cyclic AMP

International Nuclear Information System (INIS)

Zhange, Y.; Lin, J.X.; Vilcek, J.

1988-01-01

Interleukin 6 (IL-6; also referred to as interferon-β 2 , 26-kDa protein, and B cell stimulatory factor 2) is a cytokine whose actions include a stimulation of immunoglobulin synthesis, enhancement of B cell growth, and modulation of acute phase protein synthesis by hepatocytes. Synthesis of IL-6 is stimulated by interleukin 1 (IL-1), tumor necrosis factor (TNF), or platelet-derived growth factor. The authors examined the role of the cyclic AMP (cAMP)-dependent signal transduction pathway in IL-6 gene expression. Several activators of adenylate cyclase, including prostaglandin E1, forskolin, and cholera toxin, as well as the phosphodiesterase inhibitor isobutylmethylxanthine and the cAMP analog dibutyryl cAMP, shared the ability to cause a dramatic and sustained increase in IL-6 mRNA levels in human FS-4 fibroblasts. Actinomycin D treatment abolished this enhancement. Treatments that increased intracellular cAMP also stimulated the secretion of the IL-6 protein in a biologically active form. Increased intracellular cAMP appears to enhance IL-6 gene expression by a protein kinase C-independent mechanism because down-regulation of protein kinase C by a chronic exposure of cells to a high dose of 12-O-tetradecanoylphorbol 13-acetate did not abolish the enhancement of IL-6 expression by treatments that increase cAMP. IL-1 and TNF too increased IL-6 mRNA levels by a protein kinase C-independent mechanism. The results suggest a role for the cAMP-dependent pathway(s) in IL-6 gene activation by TNF and IL-1

Imaging and controlling intracellular reactions: Lysosome transport as a function of diameter and the intracellular synthesis of conducting polymers

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Payne, Christine

2014-03-01

Eukaryotic cells are the ultimate complex environment with intracellular chemical reactions regulated by the local cellular environment. For example, reactants are sequestered into specific organelles to control local concentration and pH, motor proteins transport reactants within the cell, and intracellular vesicles undergo fusion to bring reactants together. Current research in the Payne Lab in the School of Chemistry and Biochemistry at Georgia Tech is aimed at understanding and utilizing this complex environment to control intracellular chemical reactions. This will be illustrated using two examples, intracellular transport as a function of organelle diameter and the intracellular synthesis of conducting polymers. Using single particle tracking fluorescence microscopy, we measured the intracellular transport of lysosomes, membrane-bound organelles, as a function of diameter as they underwent transport in living cells. Both ATP-dependent active transport and diffusion were examined. As expected, diffusion scales with the diameter of the lysosome. However, active transport is unaffected suggesting that motor proteins are insensitive to cytosolic drag. In a second example, we utilize intracellular complexity, specifically the distinct micro-environments of different organelles, to carry out chemical reactions. We show that catalase, found in the peroxisomes of cells, can be used to catalyze the polymerization of the conducting polymer PEDOT:PSS. More importantly, we have found that a range of iron-containing biomolecules are suitable catalysts with different iron-containing biomolecules leading to different polymer properties. These experiments illustrate the advantage of intracellular complexity for the synthesis of novel materials.

The wheat NHX antiporter gene TaNHX2 confers salt tolerance in transgenic alfalfa by increasing the retention capacity of intracellular potassium.

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Zhang, Yan-Min; Zhang, Hong-Mei; Liu, Zi-Hui; Li, Hui-Cong; Guo, Xiu-Lin; Li, Guo-Liang

2015-02-01

Previous studies have shown that TaNHX2 transgenic alfalfa (Medicago sativa L.) accumulated more K(+) and less Na(+) in leaves than did the wild-type plants. To investigate whether the increased K(+) accumulation in transgenic plants is attributed to TaNHX2 gene expression and whether the compartmentalization of Na(+) into vacuoles or the intracellular compartmentalization of potassium is the critical mechanism for TaNHX2-dependent salt tolerance in transgenic alfalfa, aerated hydroponic culture was performed under three different stress conditions: control condition (0.1 mM Na(+) and 6 mM K(+) inside culture solution), K(+)-sufficient salt stress (100 mM NaCl and 6 mM K(+)) and K(+)-insufficient salt stress (100 mM NaCl and 0.1 mM K(+)). The transgenic alfalfa plants had lower K(+) efflux through specific K(+) channels and higher K(+) absorption through high-affinity K(+) transporters than did the wild-type plants. Therefore, the transgenic plants had greater K(+) contents and [K(+)]/[Na(+)] ratios in leaf tissue and cell sap. The intracellular compartmentalization of potassium is critical for TaNHX2-induced salt tolerance in transgenic alfalfa.

UV light induces premature senescence in Akt1-null mouse embryonic fibroblasts by increasing intracellular levels of ROS

International Nuclear Information System (INIS)

Jee, Hye Jin; Kim, Hyun-Ju; Kim, Ae Jeong; Bae, Yoe-Sik; Bae, Sun Sik; Yun, Jeanho

2009-01-01

Akt/PKB plays a pivotal role in cell survival and proliferation. Previously, we reported that UV-irradiation induces extensive cell death in Akt2 -/- mouse embryonic fibroblasts (MEFs) while Akt1 -/- MEFs show cell cycle arrest. Here, we find that Akt1 -/- MEFs exhibit phenotypic changes characteristics of senescence upon UV-irradiation. An enlarged and flattened morphology, a reduced cell proliferation and an increased senescence-associated β-galactosidase (SA β-gal) staining indicate that Akt1 -/- MEFs undergo premature senescence after UV-irradiation. Restoring Akt1 expression in Akt1 -/- MEFs suppressed SA β-gal activity, indicating that UV-induced senescence is due to the absence of Akt1 function. Notably, levels of ROS were rapidly increased upon UV-irradiation and the ROS scavenger NAC inhibits UV-induced senescence of Akt1 -/- MEFs, suggesting that UV light induces premature senescence in Akt1 -/- MEFs by modulating intracellular levels of ROS. In conjunction with our previous work, this indicates that different isoforms of Akt have distinct function in response to UV-irradiation.

Potential role of sodium-proton exchangers in the low concentration arsenic trioxide-increased intracellular pH and cell proliferation.

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Carmen Aravena

Full Text Available Arsenic main inorganic compound is arsenic trioxide (ATO presented in solution mainly as arsenite. ATO increases intracellular pH (pHi, cell proliferation and tumor growth. Sodium-proton exchangers (NHEs modulate the pHi, with NHE1 playing significant roles. Whether ATO-increased cell proliferation results from altered NHEs expression and activity is unknown. We hypothesize that ATO increases cell proliferation by altering pHi due to increased NHEs-like transport activity. Madin-Darby canine kidney (MDCK cells grown in 5 mmol/L D-glucose-containing DMEM were exposed to ATO (0.05, 0.5 or 5 µmol/L, 0-48 hours in the absence or presence of 5-N,N-hexamethylene amiloride (HMA, 5-100 µmol/L, NHEs inhibitor, PD-98059 (30 µmol/L, MAPK1/2 inhibitor, Gö6976 (10 µmol/L, PKCα, βI and μ inhibitor, or Schering 28080 (10 µmol/L, H(+/K(+ATPase inhibitor plus concanamycin (0.1 µmol/L, V type ATPases inhibitor. Incorporation of [(3H]thymidine was used to estimate cell proliferation, and counting cells with a hemocytometer to determine the cell number. The pHi was measured by fluorometry in 2,7-bicarboxyethyl-5,6-carboxyfluorescein loaded cells. The Na(+-dependent HMA-sensitive NHEs-like mediated proton transport kinetics, NHE1 protein abundance in the total, cytoplasm and plasma membrane protein fractions, and phosphorylated and total p42/44 mitogen-activated protein kinases (p42/44(mapk were also determined. Lowest ATO (0.05 µmol/L, ~0.01 ppm used in this study increased cell proliferation, pHi, NHEs-like transport and plasma membrane NHE1 protein abundance, effects blocked by HMA, PD-98059 or Gö6976. Cell-buffering capacity did not change by ATO. The results show that a low ATO concentration increases MDCK cells proliferation by NHEs (probably NHE1-like transport dependent-increased pHi requiring p42/44(mapk and PKCα, βI and/or μ activity. This finding could be crucial in diseases where uncontrolled cell growth occurs, such as tumor growth, and

Modulation of Connexin-36 Gap Junction Channels by Intracellular pH and Magnesium Ions.

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Rimkute, Lina; Kraujalis, Tadas; Snipas, Mindaugas; Palacios-Prado, Nicolas; Jotautis, Vaidas; Skeberdis, Vytenis A; Bukauskas, Feliksas F

2018-01-01

Connexin-36 (Cx36) protein forms gap junction (GJ) channels in pancreatic beta cells and is also the main Cx isoform forming electrical synapses in the adult mammalian brain. Cx36 GJs can be regulated by intracellular pH (pH i ) and cytosolic magnesium ion concentration ([Mg 2+ ] i ), which can vary significantly under various physiological and pathological conditions. However, the combined effect and relationship of these two factors over Cx36-dependent coupling have not been previously studied in detail. Our experimental results in HeLa cells expressing Cx36 show that changes in both pH i and [Mg 2+ ] i affect junctional conductance (g j ) in an interdependent manner; in other words, intracellular acidification cause increase or decay in g j depending on whether [Mg 2+ ] i is high or low, respectively, and intracellular alkalization cause reduction in g j independently of [Mg 2+ ] i . Our experimental and modelling data support the hypothesis that Cx36 GJ channels contain two separate gating mechanisms, and both are differentially sensitive to changes in pH i and [Mg 2+ ] i . Using recombinant Cx36 we found that two glutamate residues in the N-terminus could be partly responsible for the observed interrelated effect of pH i and [Mg 2+ ] i . Mutation of glutamate at position 8 attenuated the stimulatory effect of intracellular acidification at high [Mg 2+ ] i , while mutation at position 12 and double mutation at both positions reversed stimulatory effect to inhibition. Moreover, Cx36 * E8Q lost the initial increase of g j at low [Mg 2+ ] i and double mutation lost the sensitivity to high [Mg 2+ ] i . These results suggest that E8 and E12 are involved in regulation of Cx36 GJ channels by Mg 2+ and H + ions.

INTRACELLULAR ION CONCENTRATIONS IN BRANCHIAL EPITHELIAL CELLS OF BROWN TROUT (SALMO TRUTTA L.) DETERMINED BY X-RAY MICROANALYSIS

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Morgan; Potts; Oates

1994-09-01

The intracellular concentrations of sodium, chloride, phosphorus and potassium under normal conditions in pavement epithelial (PE) cells of brown trout (Salmo trutta) gill were 66, 51, 87 and 88 mmol l-1 respectively. The concentrations of these elements under identical conditions in mitochondria-rich (MR) cells were not significantly different, except for that of chlorine, which was lower in MR cells (40 mmol l-1). The concentration of sodium in the PE cells decreased slightly after exposure of the fish to low external [Na+] (25 µmol l-1) for 7 days but increased greatly within 5 min of subsequent exposure to 1 mmol l-1 external Na+. These changes in external [Na+] had no significant effect on MR cells. Exposure of fish to low [Cl-] (25 µmol l-1) had no effect on PE or MR cells, but on exposure to 1 mmol l-1 Cl- the concentrations of chlorine, phosphorus and potassium in both types of cells increased, whilst the intracellular sodium concentration decreased only in MR cells. The PE cells were little affected by exposure of the fish to the carbonic anhydrase inhibitor acetazolamide. In contrast, 0.5 mmol l-1 external acetazolamide caused a significant decrease in intracellular phosphorus, chlorine and potassium concentrations in MR cells. This suggests that the PE cells are the sites of sodium uptake in the gills of the brown trout and that chloride uptake occurs via the MR cells. These results are discussed with respect to the sites and possible mechanisms of ionic exchange in freshwater vertebrates.

Efficient intracellular delivery and improved biocompatibility of colloidal silver nanoparticles towards intracellular SERS immuno-sensing.

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Bhardwaj, Vinay; Srinivasan, Supriya; McGoron, Anthony J

2015-06-21

High throughput intracellular delivery strategies, electroporation, passive and TATHA2 facilitated diffusion of colloidal silver nanoparticles (AgNPs) are investigated for cellular toxicity and uptake using state-of-art analytical techniques. The TATHA2 facilitated approach efficiently delivered high payload with no toxicity, pre-requisites for intracellular applications of plasmonic metal nanoparticles (PMNPs) in sensing and therapeutics.

The Effect of Size and Species on Lens Intracellular Hydrostatic Pressure

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Gao, Junyuan; Sun, Xiurong; Moore, Leon C.; Brink, Peter R.; White, Thomas W.; Mathias, Richard T.

2013-01-01

Purpose. Previous experiments showed that mouse lenses have an intracellular hydrostatic pressure that varied from 335 mm Hg in central fibers to 0 mm Hg in surface cells. Model calculations predicted that in larger lenses, all else equal, pressure should increase as the lens radius squared. To test this prediction, lenses of different radii from different species were studied. Methods. All studies were done in intact lenses. Intracellular hydrostatic pressures were measured with a microelectrode-manometer–based system. Membrane conductances were measured by frequency domain impedance analysis. Intracellular Na+ concentrations were measured by injecting the Na+-sensitive dye sodium-binding benzofuran isophthalate. Results. Intracellular hydrostatic pressures were measured in lenses from mice, rats, rabbits, and dogs with radii (cm) 0.11, 0.22, 0.49, and 0.57, respectively. In each species, pressure varied from 335 ± 6 mm Hg in central fiber cells to 0 mm Hg in surface cells. Further characterization of transport in lenses from mice and rats showed that the density of fiber cell gap junction channels was approximately the same, intracellular Na+ concentrations varied from 17 mM in central fiber cells to 7 mM in surface cells, and intracellular voltages varied from −45 mV in central fiber cells to −60 mV in surface cells. Fiber cell membrane conductance was a factor of 2.7 times larger in mouse than in rat lenses. Conclusions. Intracellular hydrostatic pressure is an important physiological parameter that is regulated in lenses from these different species. The most likely mechanism of regulation is to reduce the density of open Na+-leak channels in fiber cells of larger lenses. PMID:23211824

Intracellular survival of Staphylococcus aureus during persistent infection in the insect Tenebrio molitor.

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McGonigle, John E; Purves, Joanne; Rolff, Jens

2016-06-01

Survival of bacteria within host cells and tissues presents a challenge to the immune systems of higher organisms. Escape from phagocytic immune cells compounds this issue, as immune cells become potential vehicles for pathogen dissemination. However, the duration of persistence within phagocytes and its contribution to pathogen load has yet to be determined. We investigate the immunological significance of intracellular persistence within the insect model Tenebrio molitor, assessing the extent, duration and location of bacterial recovery during a persistent infection. Relative abundance of Staphylococcus aureus in both intracellular and extracellular fractions was determined over 21 days, and live S. aureus were successfully recovered from both the hemolymph and within phagocytic immune cells across the entire time course. The proportion of bacteria recovered from within phagocytes also increased over time. Our results show that to accurately estimate pathogen load it is vital to account for bacteria persisting within immune cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

MR imaging of intracellular and extracellular deoxyhemoglobin

International Nuclear Information System (INIS)

Janick, P.A.; Grossman, R.I.; Asakura, T.

1989-01-01

MR imaging was performed on varying concentrations of intracellular and extracellular deoxyhemoglobin as well as varying proportions of deoxyhemoglobin and oxyhemoglobin in vitro at 1.5T with use of standard spin-echo and gradient-refocused spin sequences. This study indicates that susceptibility-induced T2 shortening occurs over a broad range of intracellular deoxyhemoglobin concentrations (maximal at hematocrits between 20% and 45%), reflecting diffusional effects at the cellular level. T2* gradient-echo imaging enhances the observed hypointensity in images of intracellular deoxyhemoglobin. The characteristic MR appearance of acute hemotomas can be modeled by the behavior of intracellular and extracellular deoxyhemoglobin and oxyhemoglobin

The tripeptide feG regulates the production of intracellular reactive oxygen species by neutrophils

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Davison Joseph S

2006-06-01

Full Text Available Abstract Background The D-isomeric form of the tripeptide FEG (feG is a potent anti-inflammatory agent that suppresses type I hypersensitivity (IgE-mediated allergic reactions in several animal species. One of feG's primary actions is to inhibit leukocyte activation resulting in loss of their adhesive and migratory properties. Since activation of neutrophils is often associated with an increase in respiratory burst with the generation of reactive oxygen species (ROS, we examined the effect of feG on the respiratory burst in neutrophils of antigen-sensitized rats. A role for protein kinase C (PKC in the actions of feG was evaluated by using selective isoform inhibitors for PKC. Results At 18h after antigen (ovalbumin challenge of sensitized Sprague-Dawley rats a pronounced neutrophilia occurred; a response that was reduced in animals treated with feG (100 μg/kg. With antigen-challenged animals the protein kinase C (PKC activator, PMA, significantly increased intracellular ROS of circulating neutrophils, as determined by flow cytometry using the fluorescent probe dihydrorhodamine-123. This increase was prevented by treatment with feG at the time of antigen challenge. The inhibitor of PKCδ, rottlerin, which effectively prevented intracellular ROS production by circulating neutrophils of animals receiving a naïve antigen, failed to inhibit PMA-stimulated ROS production if the animals were challenged with antigen. feG treatment, however, re-established the inhibitory effects of the PKCδ inhibitor on intracellular ROS production. The extracellular release of superoxide anion, evaluated by measuring the oxidative reduction of cytochrome C, was neither modified by antigen challenge nor feG treatment. However, hispidin, an inhibitor of PKCβ, inhibited the release of superoxide anion from circulating leukocytes in all groups of animals. feG prevented the increased expression of the β1-integrin CD49d on the circulating neutrophils elicited by antigen

Optimizing Nanoelectrode Arrays for Scalable Intracellular Electrophysiology.

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Abbott, Jeffrey; Ye, Tianyang; Ham, Donhee; Park, Hongkun

2018-03-20

Electrode technology for electrophysiology has a long history of innovation, with some decisive steps including the development of the voltage-clamp measurement technique by Hodgkin and Huxley in the 1940s and the invention of the patch clamp electrode by Neher and Sakmann in the 1970s. The high-precision intracellular recording enabled by the patch clamp electrode has since been a gold standard in studying the fundamental cellular processes underlying the electrical activities of neurons and other excitable cells. One logical next step would then be to parallelize these intracellular electrodes, since simultaneous intracellular recording from a large number of cells will benefit the study of complex neuronal networks and will increase the throughput of electrophysiological screening from basic neurobiology laboratories to the pharmaceutical industry. Patch clamp electrodes, however, are not built for parallelization; as for now, only ∼10 patch measurements in parallel are possible. It has long been envisioned that nanoscale electrodes may help meet this challenge. First, nanoscale electrodes were shown to enable intracellular access. Second, because their size scale is within the normal reach of the standard top-down fabrication, the nanoelectrodes can be scaled into a large array for parallelization. Third, such a nanoelectrode array can be monolithically integrated with complementary metal-oxide semiconductor (CMOS) electronics to facilitate the large array operation and the recording of the signals from a massive number of cells. These are some of the central ideas that have motivated the research activity into nanoelectrode electrophysiology, and these past years have seen fruitful developments. This Account aims to synthesize these findings so as to provide a useful reference. Summing up from the recent studies, we will first elucidate the morphology and associated electrical properties of the interface between a nanoelectrode and a cellular membrane

Cationic Antimicrobial Peptide LL-37 Is Effective against both Extra- and Intracellular Staphylococcus aureus

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Noore, Jabeen; Noore, Adly

2013-01-01

The increasing resistance of bacteria to conventional antibiotics and the challenges posed by intracellular bacteria, which may be responsible for chronic and recurrent infections, have driven the need for advanced antimicrobial drugs for effective elimination of both extra- and intracellular pathogens. The purpose of this study was to determine the killing efficacy of cationic antimicrobial peptide LL-37 compared to conventional antibiotics against extra- and intracellular Staphylococcus aureus. Bacterial killing assays and an infection model of osteoblasts and S. aureus were studied to determine the bacterial killing efficacy of LL-37 and conventional antibiotics against extra- and intracellular S. aureus. We found that LL-37 was effective in killing extracellular S. aureus at nanomolar concentrations, while lactoferricin B was effective at micromolar concentrations and doxycycline and cefazolin at millimolar concentrations. LL-37 was surprisingly more effective in killing the clinical strain than in killing an ATCC strain of S. aureus. Moreover, LL-37 was superior to conventional antibiotics in eliminating intracellular S. aureus. The kinetic studies further revealed that LL-37 was fast in eliminating both extra- and intracellular S. aureus. Therefore, LL-37 was shown to be very potent and prompt in eliminating both extra- and intracellular S. aureus and was more effective in killing extra- and intracellular S. aureus than commonly used conventional antibiotics. LL-37 could potentially be used to treat chronic and recurrent infections due to its effectiveness in eliminating not only extracellular but also intracellular pathogens. PMID:23274662

Intracellular Bacterial Infections: A Challenge for Developing Cellular Mediated Immunity Vaccines for Farmed Fish

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Hetron Mweemba Munang’andu

2018-04-01

Full Text Available Aquaculture is one of the most rapidly expanding farming systems in the world. Its rapid expansion has brought with it several pathogens infecting different fish species. As a result, there has been a corresponding expansion in vaccine development to cope with the increasing number of infectious diseases in aquaculture. The success of vaccine development for bacterial diseases in aquaculture is largely attributed to empirical vaccine designs based on inactivation of whole cell (WCI bacteria vaccines. However, an upcoming challenge in vaccine design is the increase of intracellular bacterial pathogens that are not responsive to WCI vaccines. Intracellular bacterial vaccines evoke cellular mediated immune (CMI responses that “kill” and eliminate infected cells, unlike WCI vaccines that induce humoral immune responses whose protective mechanism is neutralization of extracellular replicating pathogens by antibodies. In this synopsis, I provide an overview of the intracellular bacterial pathogens infecting different fish species in aquaculture, outlining their mechanisms of invasion, replication, and survival intracellularly based on existing data. I also bring into perspective the current state of CMI understanding in fish together with its potential application in vaccine development. Further, I highlight the immunological pitfalls that have derailed our ability to produce protective vaccines against intracellular pathogens for finfish. Overall, the synopsis put forth herein advocates for a shift in vaccine design to include CMI-based vaccines against intracellular pathogens currently adversely affecting the aquaculture industry.

Light irradiation helps magnetotactic bacteria eliminate intracellular reactive oxygen species.

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Li, Kefeng; Wang, Pingping; Chen, Chuanfang; Chen, Changyou; Li, Lulu; Song, Tao

2017-09-01

Magnetotactic bacteria (MTB) demonstrate photoresponse. However, little is known about the biological significance of this behaviour. Magnetosomes exhibit peroxidase-like activity and can scavenge reactive oxygen species (ROS). Magnetosomes extracted from the Magnetospirillum magneticum strain AMB-1 show enhanced peroxidase-like activity under illumination. The present study investigated the effects of light irradiation on nonmagnetic (without magnetosomes) and magnetic (with magnetosomes) AMB-1 cells. Results showed that light irradiation did not affect the growth of nonmagnetic and magnetic cells but significantly increased magnetosome synthesis and reduced intracellular ROS level in magnetic cells. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to analyse the expression level of magnetosome formation-associated genes (mamA, mms6, mms13 and mmsF) and stress-related genes (recA, oxyR, SOD, amb0664 and amb2684). Results showed that light irradiation upregulated the expression of mms6, mms13 and mmsF. Furthermore, light irradiation upregulated the expression of stress-related genes in nonmagnetic cells but downregulated them in magnetic cells. Additionally, magnetic cells exhibited stronger phototactic behaviour than nonmagnetic ones. These results suggested that light irradiation could heighten the ability of MTB to eliminate intracellular ROS and help them adapt to lighted environments. This phenomenon may be related to the enhanced peroxidase-like activity of magnetosomes under light irradiation. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Simple Recovery of Intracellular Gold Nanoparticles from Peanut Seedling Roots.

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Raju, D; Mehta, Urmil J; Ahmad, Absar

2015-02-01

Fabrication of inorganic nanomaterials via a biological route witnesses the formation either extracellularly, intracellulary or both. Whereas extracellular formation of these nanomaterials is cherished owing to their easy and economical extraction and purification processes; the intracellular formation of nanomaterials, due to the lack of a proper recovery protocol has always been dreaded, as the extraction processes used so far were tedious, costly, time consuming and often resulting in very low recovery. The aim of the present study was to overcome the problems related with the extraction and recovery of intracellularly synthesized inorganic nanoparticles, and to devise a method to increasing the output, the shape, size, composition and dispersal of nanoparticles is not altered. Water proved to be much better system as it provided well dispersed, stable gold nanoparticles and higher recovery. This is the first report, where intracellular nanoparticles have been recovered using a very cost-effective and eco-friendly approach.

Rising intracellular zinc by membrane depolarization and glucose in insulin-secreting clonal HIT-T15 beta cells.

Science.gov (United States)

Slepchenko, Kira G; Li, Yang V

2012-01-01

Zinc (Zn(2+)) appears to be intimately involved in insulin metabolism since insulin secretion is correlated with zinc secretion in response to glucose stimulation, but little is known about the regulation of zinc homeostasis in pancreatic beta-cells. This study set out to identify the intracellular zinc transient by imaging free cytosolic zinc in HIT-T15 beta-cells with fluorescent zinc indicators. We observed that membrane depolarization by KCl (30-60 mM) was able to induce a rapid increase in cytosolic concentration of zinc. Multiple zinc transients of similar magnitude were elicited during repeated stimulations. The amplitude of zinc responses was not affected by the removal of extracellular calcium or zinc. However, the half-time of the rising slope was significantly slower after removing extracellular zinc with zinc chelator CaEDTA, suggesting that extracellular zinc affect the initial rising phase of zinc response. Glucose (10 mM) induced substantial and progressive increases in intracellular zinc concentration in a similar way as KCl, with variation in the onset and the duration of zinc mobilization. It is known that the depolarization of beta-cell membrane is coupled with the secretion of insulin. Rising intracellular zinc concentration may act as a critical signaling factor in insulin metabolism of pancreatic beta-cells.

Monitoring intracellular oxidative events using dynamic spectral unmixing microscopy

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There is increasing interest in using live-cell imaging to monitor not just individual intracellular endpoints, but to investigate the interplay between multiple molecular events as they unfold in real time within the cell. A major impediment to simultaneous acquisition of multip...

NAD+-Glycohydrolase Promotes Intracellular Survival of Group A Streptococcus.

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Onkar Sharma

2016-03-01

Full Text Available A global increase in invasive infections due to group A Streptococcus (S. pyogenes or GAS has been observed since the 1980s, associated with emergence of a clonal group of strains of the M1T1 serotype. Among other virulence attributes, the M1T1 clone secretes NAD+-glycohydrolase (NADase. When GAS binds to epithelial cells in vitro, NADase is translocated into the cytosol in a process mediated by streptolysin O (SLO, and expression of these two toxins is associated with enhanced GAS intracellular survival. Because SLO is required for NADase translocation, it has been difficult to distinguish pathogenic effects of NADase from those of SLO. To resolve the effects of the two proteins, we made use of anthrax toxin as an alternative means to deliver NADase to host cells, independently of SLO. We developed a novel method for purification of enzymatically active NADase fused to an amino-terminal fragment of anthrax toxin lethal factor (LFn-NADase that exploits the avid, reversible binding of NADase to its endogenous inhibitor. LFn-NADase was translocated across a synthetic lipid bilayer in vitro in the presence of anthrax toxin protective antigen in a pH-dependent manner. Exposure of human oropharyngeal keratinocytes to LFn-NADase in the presence of protective antigen resulted in cytosolic delivery of NADase activity, inhibition of protein synthesis, and cell death, whereas a similar construct of an enzymatically inactive point mutant had no effect. Anthrax toxin-mediated delivery of NADase in an amount comparable to that observed during in vitro infection with live GAS rescued the defective intracellular survival of NADase-deficient GAS and increased the survival of SLO-deficient GAS. Confocal microscopy demonstrated that delivery of LFn-NADase prevented intracellular trafficking of NADase-deficient GAS to lysosomes. We conclude that NADase mediates cytotoxicity and promotes intracellular survival of GAS in host cells.

Single-cell intracellular nano-pH probes†

OpenAIRE

Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

2015-01-01

Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular p...

ß-Adrenergic stimulation increases RyR2 activity via intracellular Ca2+ and Mg2+ regulation.

Directory of Open Access Journals (Sweden)

Jiao Li

Full Text Available Here we investigate how ß-adrenergic stimulation of the heart alters regulation of ryanodine receptors (RyRs by intracellular Ca(2+ and Mg(2+ and the role of these changes in SR Ca(2+ release. RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 min and subject to 1 min perfusion with 1 µM isoproterenol or without (control and snap frozen in liquid N2 to capture their phosphorylation state. Western Blots show that RyR2 phosphorylation was increased by isoproterenol, confirming that RyR2 were subject to normal ß-adrenergic signaling. Under basal conditions, S2808 and S2814 had phosphorylation levels of 69% and 15%, respectively. These levels were increased to 83% and 60%, respectively, after 60 s of ß-adrenergic stimulation consistent with other reports that ß-adrenergic stimulation of the heart can phosphorylate RyRs at specific residues including S2808 and S2814 causing an increase in RyR activity. At cytoplasmic [Ca(2+] 1 µM, ß-adrenergic stimulation only decreased cytoplasmic Mg(2+ and Ca(2+ inhibition of RyRs. The Ka and maximum levels of cytoplasmic Ca(2+ activation site were not affected by ß-adrenergic stimulation. Our RyR2 gating model was fitted to the single channel data. It predicted that in diastole, ß-adrenergic stimulation is mediated by 1 increasing the activating potency of Ca(2+ binding to the luminal Ca(2+ site and decreasing its affinity for luminal Mg(2+ and 2 decreasing affinity of the low-affinity Ca(2+/Mg(2+ cytoplasmic inhibition site. However in systole, ß-adrenergic stimulation is mediated mainly by the latter.

The small molecule triclabendazole decreases the intracellular level of cyclic AMP and increases resistance to stress in Saccharomyces cerevisiae.

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Yong Joo Lee

Full Text Available The Ras-adenylyl cyclase-protein kinase A nutrient-sensing pathway controls metabolism, proliferation and resistance to stress in Saccharomyces cerevisiae. The genetic disruption of this pathway increases resistance to a variety of stresses. We show here that the pharmacological inhibition of this pathway by the drug triclabendazole increases resistance to oxidants, heat stress and extends the chronological life. Evidence is presented that triclabendazole decreases the intracellular level of cyclic AMP by inhibiting adenylyl cyclase and triggers the parallel rapid translocation of the stress-resistance transcription factor Msn2 from the cytosol into the nucleus, as deduced from experiments employing a strain in which MSN2 is replaced with MSN2-GFP (GFP, green fluorescent protein. Msn2 and Msn4 are responsible for activating the transcription of numerous genes that encode proteins that protect cells from stress. The results are consistent with triclabendazole either inhibiting the association of Ras with adenylyl cyclase or directly inhibiting adenylyl cyclase, which in turn triggers Msn2/4 to enter the nucleus and activate stress-responsible element gene expression.

FLIPR assays of intracellular calcium in GPCR drug discovery

DEFF Research Database (Denmark)

Hansen, Kasper Bø; Bräuner-Osborne, Hans

2009-01-01

Fluorescent dyes sensitive to changes in intracellular calcium have become increasingly popular in G protein-coupled receptor (GPCR) drug discovery for several reasons. First of all, the assays using the dyes are easy to perform and are of low cost compared to other assays. Second, most non...

Intracellular renin disrupts chemical communication between heart cells. Pathophysiological implications

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Walmor eDe Mello

2015-01-01

Full Text Available The influence of intracellular renin on the process of chemical communication between cardiac cells was investigated in cell pairs isolated from the left ventricle of adult Wistar Kyoto rats. The enzyme together with Lucifer yellow CH was dialyzed into one cell of the pair using the whole cell clamp technique. The diffusion of the dye in the dialyzed and in non-dialyzed cell was followed by measuring the intensity of fluorescence in both cells as a function of time. The results indicated that; 1 under normal conditions, Lucifer Yellow flows from cell-to-cell through gap junctions; 2 the intracellular dialysis of renin (100nM disrupts chemical communication-an effect enhanced by simultaneous administration of angiotensinogen (100nM; 3 enalaprilat (10-9M administered to the cytosol together with renin reduced drastically the uncoupling action of the enzyme; 4 aliskiren (10-8M inhibited the effect of renin on chemical communication;5 the possible role of intracellular renin independently of angiotensin II (Ang II was evaluated including the increase of the inward calcium current elicited by the enzyme and the possible role of oxidative stress on the disruption of cell communication; 6 the possible harmful versus the beneficial effect of intracellular renin during myocardial infarction was discussed;7 the present results indicate that intracellular renin due to internalization or in situ synthesis, causes a severe impairment of chemical communication in the heart resulting in derangement of metabolic cooperation with serious consequences for heart function.

β adrenergic receptor/cAMP/PKA signaling contributes to the intracellular Ca2+ release by tentacle extract from the jellyfish Cyanea capillata.

Science.gov (United States)

Wang, Qianqian; Zhang, Hui; Wang, Bo; Wang, Chao; Xiao, Liang; Zhang, Liming

2017-07-25

Intracellular Ca 2+ overload induced by extracellular Ca 2+ entry has previously been confirmed to be an important mechanism for the cardiotoxicity as well as the acute heart dysfunction induced by jellyfish venom, while the underlying mechanism remains to be elucidated. Under extracellular Ca 2+ -free or Ca 2+ -containing conditions, the Ca 2+ fluorescence in isolated adult mouse cardiomyocytes pre-incubated with tentacle extract (TE) from the jellyfish Cyanea capillata and β blockers was scanned by laser scanning confocal microscope. Then, the cyclic adenosine monophosphate (cAMP) concentration and protein kinase A (PKA) activity in primary neonatal rat ventricular cardiomyocytes were determined by ELISA assay. Furthermore, the effect of propranolol against the cardiotoxicity of TE was evaluated in Langendorff-perfused rat hearts and intact rats. The increase of intracellular Ca 2+ fluorescence signal by TE was significantly attenuated and delayed when the extracellular Ca 2+ was removed. The β adrenergic blockers, including propranolol, atenolol and esmolol, partially inhibited the increase of intracellular Ca 2+ in the presence of 1.8 mM extracellular Ca 2+ and completely abolished the Ca 2+ increase under an extracellular Ca 2+ -free condition. Both cAMP concentration and PKA activity were stimulated by TE, and were inhibited by the β adrenergic blockers. Cardiomyocyte toxicity of TE was antagonized by β adrenergic blockers and the PKA inhibitor H89. Finally, the acute heart dysfuction by TE was antagonized by propranolol in Langendorff-perfused rat hearts and intact rats. Our findings indicate that β adrenergic receptor/cAMP/PKA signaling contributes to the intracellular Ca 2+ overload through intracellular Ca 2+ release by TE from the jellyfish C. capillata.

Metformin induces an intracellular reductive state that protects oesophageal squamous cell carcinoma cells against cisplatin but not copper-bis(thiosemicarbazones)

International Nuclear Information System (INIS)

Damelin, Leonard Howard; Jivan, Rupal; Veale, Robin Bruce; Rousseau, Amanda Louise; Mavri-Damelin, Demetra

2014-01-01

Oesophageal squamous cell carcinoma (OSCC) is a highly aggressive carcinoma with a poor survival rate. One of the most commonly used chemotherapeutic drugs, cisplatin, displays varied and often poor efficacy in vivo. Therefore, alternative, cost-effective and more efficacious treatments are required. Metformin has been previously shown to reduce proliferative rates in various carcinoma cell lines. We report for the first time, the effect of metformin on OSCC cell proliferation and show that it antagonises cisplatin-induced but not copper-bis(thiosemicarbazone)-induced cytotoxicity in OSCC cells. Cell proliferation and stage of the cell cycle were quantified by trypan blue counts and flow cytometry, respectively. All cytotoxicity measurements were made using the tetrazolium based MTT assay. Metabolic alterations to cells were determined as follows: glycolysis via a lactate dehydrogenase assay, reducing equivalents by MTT reduction and reduced intracellular thiols by monobromobimane-thiol fluorescence, and glutathione depletion using buthionine sulfoximine. Inductively coupled plasma mass spectrometry was used to quantify cisplatin-DNA adduct formation. Metformin was found to reduce cell proliferation significantly in all OSCC cell lines, with an accumulation of cells in G0/G1 phase of the cell cycle. However, metformin significantly protected OSCC cells against cisplatin toxicity. Our results indicate that a major mechanism of metformin-induced cisplatin resistance results from a significant increase in glycolysis, intracellular NAD(P)H levels with a concomitant increase in reduced intracellular thiols, leading to decreased cisplatin-DNA adduct formation. The glutathione synthesis inhibitor buthionine sulfoximine significantly ablated the protective effect of metformin. We subsequently show that the copper-bis(thiosemicarbazones), Cu-ATSM and Cu-GTSM, which are trapped in cells under reducing conditions, cause significant OSCC cytotoxicity, both alone and in

ß-Adrenergic Stimulation Increases RyR2 Activity via Intracellular Ca2+ and Mg2+ Regulation

Science.gov (United States)

Li, Jiao; Imtiaz, Mohammad S.; Beard, Nicole A.; Dulhunty, Angela F.; Thorne, Rick; vanHelden, Dirk F.; Laver, Derek R.

2013-01-01

Here we investigate how ß-adrenergic stimulation of the heart alters regulation of ryanodine receptors (RyRs) by intracellular Ca2+ and Mg2+ and the role of these changes in SR Ca2+ release. RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 min and subject to 1 min perfusion with 1 µM isoproterenol or without (control) and snap frozen in liquid N2 to capture their phosphorylation state. Western Blots show that RyR2 phosphorylation was increased by isoproterenol, confirming that RyR2 were subject to normal ß-adrenergic signaling. Under basal conditions, S2808 and S2814 had phosphorylation levels of 69% and 15%, respectively. These levels were increased to 83% and 60%, respectively, after 60 s of ß-adrenergic stimulation consistent with other reports that ß-adrenergic stimulation of the heart can phosphorylate RyRs at specific residues including S2808 and S2814 causing an increase in RyR activity. At cytoplasmic [Ca2+] adrenergic stimulation increased luminal Ca2+ activation of single RyR channels, decreased luminal Mg2+ inhibition and decreased inhibition of RyRs by mM cytoplasmic Mg2+. At cytoplasmic [Ca2+] >1 µM, ß-adrenergic stimulation only decreased cytoplasmic Mg2+ and Ca2+ inhibition of RyRs. The Ka and maximum levels of cytoplasmic Ca2+ activation site were not affected by ß-adrenergic stimulation. Our RyR2 gating model was fitted to the single channel data. It predicted that in diastole, ß-adrenergic stimulation is mediated by 1) increasing the activating potency of Ca2+ binding to the luminal Ca2+ site and decreasing its affinity for luminal Mg2+ and 2) decreasing affinity of the low-affinity Ca2+/Mg2+ cytoplasmic inhibition site. However in systole, ß-adrenergic stimulation is mediated mainly by the latter. PMID:23533585

Factors influencing intracellular uptake and radiosensitization by 2-nitroimidazoles in vitro

International Nuclear Information System (INIS)

Brown, D.M.; Gonzalez-Mendez, R.; Brown, J.M.

1983-01-01

In this study it is shown that the radiosensitization of hypoxic Chinese hamster ovary (HA-1) cells in vitro by misonidazole (MIS) and other 1-substituted 2-nitroimidazoles depends on the rate and extent of intracellular uptake of these radiosensitizers, which in turn is governed by their lipophilicity [expressed as the octanol:water partition coefficient (P)]. As the lipophilicity of the compounds decreased, the rate of drug entry into the cells was slower, and below P values of approximately 0.05, peak intracellular drug concentrations were found to be lower than that of MIS (P=0.43). In addition, the number of hydroxyl groups on the side chain of the nitroimidazole molecule influenced the uptake of drug into the cells. For compounds of similar P, but differing in the number of side-chain hydroxyl groups, the addition of a single hydroxyl group to the molecule decreased the amount of drug entering the cell by a factor of approximately 2. These compounds enter the cell by nonmediated passive diffusion since altering the energy (ATP) capacity of the cell by 2-deoxyglucose did not affect uptake. It is also shown that increases in temperature or decreases in pH can increase the intracellular uptake of MIS. For example, equal intracellular and extracellular concentrations (100% uptake) of MIS were obtained if cells were heated to 44-45 0 C for 15 min compared to 20-40% uptake at 37 0 C. Increases in MIS uptake by factors of 2 to 3 could be demonstrated within 30 min when cells were incubated in Hanks' balanced salt solution at pH between 6.0 and 6.3 without loss of cell viability. In addition, MIS uptake in aerobic cultured cells varied from 15 to 60% depending on the cell line and culure conditions used

Hypertonic saline enhances host response to bacterial challenge by augmenting receptor-independent neutrophil intracellular superoxide formation.

LENUS (Irish Health Repository)

Shields, Conor J

2012-02-03

OBJECTIVE: This study sought to determine whether hypertonic saline (HTS) infusion modulates the host response to bacterial challenge. METHODS: Sepsis was induced in 30 Balb-C mice by intraperitoneal injection of Escherichia coli (5 x 107 organisms per animal). In 10 mice, resuscitation was performed at 0 and 24 hours with a 4 mL\\/kg bolus of HTS (7.5% NaCl), 10 animals received 4 mL\\/kg of normal saline (0.9% NaCl), and the remaining animals received 30 mL\\/kg of normal saline. Samples of blood, spleen, and lung were cultured at 8 and 36 hours. Polymorphonucleocytes were incubated in isotonic or hypertonic medium before culture with E. coli. Phagocytosis was assessed by flow cytometry, whereas intracellular bacterial killing was measured after inhibition of phagocytosis with cytochalasin B. Intracellular formation of free radicals was assessed by the molecular probe CM-H(2)DCFDA. Mitogen-activated protein (MAP) kinase p38 and ERK-1 phosphorylation, and nuclear factor kappa B (NFkappaB) activation were determined. Data are represented as means (SEM), and an analysis of variance test was performed to gauge statistical significance. RESULTS: Significantly reduced bacterial culture was observed in the animals resuscitated with HTS when compared with their NS counterparts, in blood (51.8 +\\/- 4.3 vs. 82.0 +\\/- 3.3 and 78.4 +\\/- 4.8, P = 0.005), lung (40.0 +\\/- 4.1 vs. 93.2 +\\/- 2.1 and 80.9 +\\/- 4.7, P = 0.002), and spleen (56.4 +\\/- 3.8 vs. 85.4 +\\/- 4.2 and 90.1 +\\/- 5.9, P = 0.05). Intracellular killing of bacteria increased markedly (P = 0.026) and superoxide generation was enhanced upon exposure to HTS (775.78 +\\/- 23.6 vs. 696.57 +\\/- 42.2, P = 0.017) despite inhibition of MAP kinase and NFkappaB activation. CONCLUSIONS: HTS significantly enhances intracellular killing of bacteria while attenuating receptor-mediated activation of proinflammatory cascades.

CONTRIBUTIONS OF INTRACELLULAR IONS TO Kv CHANNEL VOLTAGE SENSOR DYNAMICS.

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Samuel eGoodchild

2012-06-01

Full Text Available Voltage sensing domains of Kv channels control ionic conductance through coupling of the movement of charged residues in the S4 segment to conformational changes at the cytoplasmic region of the pore domain, that allow K+ ions to flow. Conformational transitions within the voltage sensing domain caused by changes in the applied voltage across the membrane field are coupled to the conducting pore region and the gating of ionic conductance. However, several other factors not directly linked to the voltage dependent movement of charged residues within the voltage sensor impact the dynamics of the voltage sensor, such as inactivation, ionic conductance, intracellular ion identity and block of the channel by intracellular ligands. The effect of intracellular ions on voltage sensor dynamics is of importance in the interpretation of gating current measurements and the physiology of pore/voltage sensor coupling. There is a significant amount of variability in the reported kinetics of voltage sensor deactivation kinetics of Kv channels attributed to different mechanisms such as open state stabilization, immobilization and relaxation processes of the voltage sensor. Here we separate these factors and focus on the causal role that intracellular ions can play in allosterically modulating the dynamics of Kv voltage sensor deactivation kinetics. These considerations are of critical importance in understanding the molecular determinants of the complete channel gating cycle from activation to deactivation.

Galectin-3 guides intracellular trafficking of some human serotransferrin glycoforms

DEFF Research Database (Denmark)

Carlsson, Carl Michael; Bengtson, Per; Cucak, Helena

2013-01-01

these transferrin glycoforms differently after preloading with exogenously added galectin-3. In all, this study provides the first evidence of a functional role for transferrin glycans, in intracellular trafficking after uptake. Moreover, the galectin-3 bound glycoform increased in cancer, suggesting...

Rising Intracellular Zinc by Membrane Depolarization and Glucose in Insulin-Secreting Clonal HIT-T15 Beta Cells

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Kira G. Slepchenko

2012-01-01

Full Text Available Zinc (Zn2+ appears to be intimately involved in insulin metabolism since insulin secretion is correlated with zinc secretion in response to glucose stimulation, but little is known about the regulation of zinc homeostasis in pancreatic beta-cells. This study set out to identify the intracellular zinc transient by imaging free cytosolic zinc in HIT-T15 beta-cells with fluorescent zinc indicators. We observed that membrane depolarization by KCl (30–60 mM was able to induce a rapid increase in cytosolic concentration of zinc. Multiple zinc transients of similar magnitude were elicited during repeated stimulations. The amplitude of zinc responses was not affected by the removal of extracellular calcium or zinc. However, the half-time of the rising slope was significantly slower after removing extracellular zinc with zinc chelator CaEDTA, suggesting that extracellular zinc affect the initial rising phase of zinc response. Glucose (10 mM induced substantial and progressive increases in intracellular zinc concentration in a similar way as KCl, with variation in the onset and the duration of zinc mobilization. It is known that the depolarization of beta-cell membrane is coupled with the secretion of insulin. Rising intracellular zinc concentration may act as a critical signaling factor in insulin metabolism of pancreatic beta-cells.

Intracellular Cholesterol Trafficking and Impact in Neurodegeneration

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Fabian Arenas

2017-11-01

Full Text Available Cholesterol is a critical component of membrane bilayers where it plays key structural and functional roles by regulating the activity of diverse signaling platforms and pathways. Particularly enriched in brain, cholesterol homeostasis in this organ is singular with respect to other tissues and exhibits a heterogeneous regulation in distinct brain cell populations. Due to the key role of cholesterol in brain physiology and function, alterations in cholesterol homeostasis and levels have been linked to brain diseases and neurodegeneration. In the case of Alzheimer disease (AD, however, this association remains unclear with evidence indicating that either increased or decreased total brain cholesterol levels contribute to this major neurodegenerative disease. Here, rather than analyzing the role of total cholesterol levels in neurodegeneration, we focus on the contribution of intracellular cholesterol pools, particularly in endolysosomes and mitochondria through its trafficking via specialized membrane domains delineated by the contacts between endoplasmic reticulum and mitochondria, in the onset of prevalent neurodegenerative diseases such as AD, Parkinson disease, and Huntington disease as well as in lysosomal disorders like Niemann-Pick type C disease. We dissect molecular events associated with intracellular cholesterol accumulation, especially in mitochondria, an event that results in impaired mitochondrial antioxidant defense and function. A better understanding of the mechanisms involved in the distribution of cholesterol in intracellular compartments may shed light on the role of cholesterol homeostasis disruption in neurodegeneration and may pave the way for specific intervention opportunities.

Measurement of Intracellular Ionized Calcium in a Free-living Soil Nematode, Caenorhabditis elegans.

Science.gov (United States)

Kawaii, S; Yoshizawa, Y; Mizutani, J

1993-01-01

A calcium chelating fluorescence indicator, fura-2, was used to measure intracellular ionized calcium in Caenorhabditis elegans. The indicator loading process was harmless to the nematode, and completed within 2-3 h. Fura-2 was loaded mainly at its intestinal tract. The effects of DOPA on locomotion and the level of intracellular calcium were investigated and measured by using a microfluorometer. The addition of DOPA temporarily increased [Ca(2+)]i for several minutes.

Weakened Intracellular Zn2+-Buffering in the Aged Dentate Gyrus and Its Involvement in Erasure of Maintained LTP.

Science.gov (United States)

Takeda, Atsushi; Tamano, Haruna; Murakami, Taku; Nakada, Hiroyuki; Minamino, Tatsuya; Koike, Yuta

2018-05-01

Memory is lost by the increased influx of extracellular Zn 2+ into neurons. It is possible that intracellular Zn 2+ dynamics is modified even at non-zincergic medial perforant pathway-dentate granule cell synapses along with aging and that vulnerability to the modification is linked to age-related cognitive decline. To examine these possibilities, vulnerability of long-term potentiation (LTP) maintenance, which underlies memory retention, to modification of synaptic Zn 2+ dynamics was compared between young and aged rats. The influx of extracellular Zn 2+ into dentate granule cells was increased in aged rats after injection of high K + into the dentate gyrus, but not in young rats. This increase impaired maintained LTP in aged rats. However, the impairment was rescued by co-injection of CaEDTA, an extracellular Zn 2+ chelator, or CNQX, an AMPA receptor antagonist, which suppressed the Zn 2+ influx. Maintained LTP was also impaired in aged rats after injection of ZnAF-2DA into the dentate gyrus that chelates intracellular Zn 2+ , but not in young rats. Interestingly, the capacity of chelating intracellular Zn 2+ with intracellular ZnAF-2 was almost lost in the aged dentate gyrus 2 h after injection of ZnAF-2DA into the dentate gyrus, suggesting that intracellular Zn 2+ -buffering is weakened in the aged dentate gyrus, compared to the young dentate gyrus. In the dentate gyrus of aged rats, maintained LTP is more vulnerable to modification of intracellular Zn 2+ dynamics than in young rats, probably due to weakened intracellular Zn 2+ -buffering.

Disruption of Intracellular ATP Generation and Tight Junction Protein Expression during the Course of Brain Edema Induced by Subacute Poisoning of 1,2-Dichloroethane

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Gaoyang Wang

2018-01-01

Full Text Available The aim of this study was to explore changes in intracellular ATP generation and tight junction protein expression during the course of brain edema induced by subacute poisoning of 1,2-dichloroethane (1,2-DCE. Mice were exposed to 1.2 g/m3 1,2-DCE for 3.5 h per day for 1, 2, or 3 days, namely group A, B, and C. Na+-K+-ATPase and Ca2+-ATPase activity, ATP and lactic acid content, intracellular free Ca2+ concentration and ZO-1 and occludin expression in the brain were measured. Results of present study disclosed that Ca2+-ATPase activities in group B and C, and Na+/K+-ATPase activity in group C decreased, whereas intracellular free Ca2+ concentrations in group B and C increased significantly compared with control. Moreover, ATP content decreased, whereas lactic acid content increased significantly in group C compared with control. On the other hand, expressions of ZO-1 and occludin at both the protein and gene levels in group B and C decreased significantly compared with control. In conclusion, findings from this study suggest that calcium overload and depressed expression of tight junction associated proteins, such as ZO-1 and occludin might play an important role in the early phase of brain edema formation induced by subacute poisoning of 1,2-DCE.

Intracellular generation of single-strand template increases the knock-in efficiency by combining CRISPR/Cas9 with AAV.

Science.gov (United States)

Xiao, Qing; Min, Taishan; Ma, Shuangping; Hu, Lingna; Chen, Hongyan; Lu, Daru

2018-04-18

Targeted integration of transgenes facilitates functional genomic research and holds prospect for gene therapy. The established microhomology-mediated end-joining (MMEJ)-based strategy leads to the precise gene knock-in with easily constructed donor, yet the limited efficiency remains to be further improved. Here, we show that single-strand DNA (ssDNA) donor contributes to efficient increase of knock-in efficiency and establishes a method to achieve the intracellular linearization of long ssDNA donor. We identified that the CRISPR/Cas9 system is responsible for breaking double-strand DNA (dsDNA) of palindromic structure in inverted terminal repeats (ITRs) region of recombinant adeno-associated virus (AAV), leading to the inhibition of viral second-strand DNA synthesis. Combing Cas9 plasmids targeting genome and ITR with AAV donor delivery, the precise knock-in of gene cassette was achieved, with 13-14% of the donor insertion events being mediated by MMEJ in HEK 293T cells. This study describes a novel method to integrate large single-strand transgene cassettes into the genomes, increasing knock-in efficiency by 13.6-19.5-fold relative to conventional AAV-mediated method. It also provides a comprehensive solution to the challenges of complicated production and difficult delivery with large exogenous fragments.

Intracellular Protein Delivery and Gene Transfection by Electroporation Using a Microneedle Electrode Array

Science.gov (United States)

Choi, Seong-O; Kim, Yeu-Chun; Lee, Jeong Woo; Park, Jung-Hwan

2012-01-01

The impact of many biopharmaceuticals, including protein- and gene-based therapies, has been limited by the need for better methods of delivery into cells within tissues. Here, we present intracellular delivery of molecules and transfection with plasmid DNA by electroporation using a novel microneedle electrode array designed for targeted treatment of skin and other tissue surfaces. The microneedle array is molded out of polylactic acid. Electrodes and circuitry required for electroporation are applied to the microneedle array surface by a new metal-transfer micromolding method. The microneedle array maintains mechanical integrity after insertion into pig cadaver skin and is able to electroporate human prostate cancer cells in vitro. Quantitative measurements show that increasing electroporation pulse voltage increases uptake efficiency of calcein and bovine serum albumin, whereas increasing pulse length has lesser effects over the range studied. Uptake of molecules by up to 50 % of cells and transfection of 12 % of cells with a gene for green fluorescent protein is demonstrated at high cell viability. We conclude that the microneedle electrode array is able to electroporate cells, resulting in intracellular uptake of molecules, and has potential applications to improve intracellular delivery of proteins, DNA and other biopharmaceuticals. PMID:22328093

The In Vitro Antioxidant Activity and Inhibition of Intracellular Reactive Oxygen Species of Sweet Potato Leaf Polyphenols

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Hongnan Sun

2018-01-01

Full Text Available The in vitro antioxidant activity and inhibition of intracellular reactive oxygen species (ROS of the total and individual phenolic compounds from Yuzi No. 7 sweet potato leaves were investigated in this study. Sweet potato leaf polyphenols possessed significantly higher antioxidant activity than ascorbic acid, tea polyphenols, and grape seed polyphenols. Among the individual phenolic compounds, caffeic acid showed the highest antioxidant activity, followed by monocaffeoylquinic acids and dicaffeoylquinic acids, while 3,4,5-tri-O-caffeoylquinic acid showed the lowest value. Sweet potato leaf polyphenols could significantly decrease the level of intracellular ROS in a dose-dependent manner. The order of the inhibiting effect of individual phenolic compounds on the intracellular ROS level was not in accordance with that of antioxidant activity, suggesting that there was no direct relationship between antioxidant activity and intracellular ROS-inhibiting effect. Sweet potato leaves could be a good source of biologically active polyphenols with multiple applications in the development of foods, health products, pharmaceuticals, and cosmetics.

The In Vitro Antioxidant Activity and Inhibition of Intracellular Reactive Oxygen Species of Sweet Potato Leaf Polyphenols

Science.gov (United States)

Sun, Hongnan; Mu, Bona; Song, Zhen; Ma, Zhimin

2018-01-01

The in vitro antioxidant activity and inhibition of intracellular reactive oxygen species (ROS) of the total and individual phenolic compounds from Yuzi No. 7 sweet potato leaves were investigated in this study. Sweet potato leaf polyphenols possessed significantly higher antioxidant activity than ascorbic acid, tea polyphenols, and grape seed polyphenols. Among the individual phenolic compounds, caffeic acid showed the highest antioxidant activity, followed by monocaffeoylquinic acids and dicaffeoylquinic acids, while 3,4,5-tri-O-caffeoylquinic acid showed the lowest value. Sweet potato leaf polyphenols could significantly decrease the level of intracellular ROS in a dose-dependent manner. The order of the inhibiting effect of individual phenolic compounds on the intracellular ROS level was not in accordance with that of antioxidant activity, suggesting that there was no direct relationship between antioxidant activity and intracellular ROS-inhibiting effect. Sweet potato leaves could be a good source of biologically active polyphenols with multiple applications in the development of foods, health products, pharmaceuticals, and cosmetics. PMID:29643978

Self-organization of intracellular gradients during mitosis

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Fuller Brian G

2010-01-01

Full Text Available Abstract Gradients are used in a number of biological systems to transmit spatial information over a range of distances. The best studied are morphogen gradients where information is transmitted over many cell lengths. Smaller mitotic gradients reflect the need to organize several distinct events along the length of the mitotic spindle. The intracellular gradients that characterize mitosis are emerging as important regulatory paradigms. Intracellular gradients utilize intrinsic auto-regulatory feedback loops and diffusion to establish stable regions of activity within the mitotic cytosol. We review three recently described intracellular mitotic gradients. The Ran GTP gradient with its elaborate cascade of nuclear transport receptors and cargoes is the best characterized, yet the dynamics underlying the robust gradient of Ran-GTP have received little attention. Gradients of phosphorylation have been observed on Aurora B kinase substrates both before and after anaphase onset. In both instances the phosphorylation gradient appears to result from a soluble gradient of Aurora B kinase activity. Regulatory properties that support gradient formation are highlighted. Intracellular activity gradients that regulate localized mitotic events bare several hallmarks of self-organizing biologic systems that designate spatial information during pattern formation. Intracellular pattern formation represents a new paradigm in mitotic regulation.

Antimycobacterial Efficacy of Andrographis paniculata Leaf Extracts Under Intracellular and Hypoxic Conditions.

Science.gov (United States)

Bhatter, Purva; Gupta, Pooja; Daswani, Poonam; Tetali, Pundarikakshudu; Birdi, Tannaz

2015-01-01

The inhibition of the growth of Mycobacterium tuberculosis by the extracts of Andrographis paniculata has been studied using intracellular and axenic hypoxic conditions. The inhibition (confirmed using the gold standard colony forming unit assay) was found to increase with "double stimuli" or higher concentration of the extract. Organic solvent extracts were found to inhibit bacterial growth more than the aqueous extracts under microaerophilic conditions mimicked through axenic and intracellular assays. This could be further explored to evaluate the potential of the plant to be used against nonreplicating/dormant bacilli. © The Author(s) 2014.

Carbon black nanoparticles promote endothelial activation and lipid accumulation in macrophages independently of intracellular ROS production

DEFF Research Database (Denmark)

Cao, Yi; Roursgaard, Martin; Danielsen, Pernille Høgh

2014-01-01

, the concentrations of CB to induce lipid accumulation were lower than the concentrations to promote intracellular ROS production in THP-1a cells. In conclusion, exposure to nano-sized CB induced endothelial dysfunction and foam cell formation, which was not dependent on intracellular ROS production....... and WST-1 assays, especially in THP-1 and THP-1a cells. The CB exposure decreased the glutathione (GSH) content in THP-1 and THP-1a cells, whereas GSH was increased in HUVECs. The reactive oxygen species (ROS) production was increased in all cell types after CB exposure. A reduction of the intracellular...... GSH concentration by buthionine sulfoximine (BSO) pre-treatment further increased the CB-induced ROS production in THP-1 cells and HUVECs. The expression of adhesion molecules ICAM-1 and VCAM-1, but not adhesion of THP-1 to HUVECs or culture dishes, was elevated by CB exposure, whereas these effects...

Hemochromatosis enhances tumor progression via upregulation of intracellular iron in head and neck cancer.

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Michelle Lenarduzzi

Full Text Available Despite improvements in treatment strategies for head and neck squamous cell carcinoma (HNSCC, outcomes have not significantly improved; highlighting the importance of identifying novel therapeutic approaches to target this disease. To address this challenge, we proceeded to evaluate the role of iron in HNSCC.Expression levels of iron-related genes were evaluated in HNSCC cell lines using quantitative RT-PCR. Cellular phenotypic effects were assessed using viability (MTS, clonogenic survival, BrdU, and tumor formation assays. The prognostic significance of iron-related proteins was determined using immunohistochemistry.In a panel of HNSCC cell lines, hemochromatosis (HFE was one of the most overexpressed genes involved in iron regulation. In vitro knockdown of HFE in HNSCC cell lines significantly decreased hepcidin (HAMP expression and intracellular iron level. This in turn, resulted in a significant decrease in HNSCC cell viability, clonogenicity, DNA synthesis, and Wnt signalling. These cellular changes were reversed by re-introducing iron back into HNSCC cells after HFE knockdown, indicating that iron was mediating this phenotype. Concordantly, treating HNSCC cells with an iron chelator, ciclopirox olamine (CPX, significantly reduced viability and clonogenic survival. Finally, patients with high HFE expression experienced a reduced survival compared to patients with low HFE expression.Our data identify HFE as potentially novel prognostic marker in HNSCC that promotes tumour progression via HAMP and elevated intracellular iron levels, leading to increased cellular proliferation and tumour formation. Hence, these findings suggest that iron chelators might have a therapeutic role in HNSCC management.

Hemochromatosis Enhances Tumor Progression via Upregulation of Intracellular Iron in Head and Neck Cancer

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Lenarduzzi, Michelle; Hui, Angela B. Y.; Yue, Shijun; Ito, Emma; Shi, Wei; Williams, Justin; Bruce, Jeff; Sakemura-Nakatsugawa, Noriko; Xu, Wei; Schimmer, Aaron; Liu, Fei-Fei

2013-01-01

Introduction Despite improvements in treatment strategies for head and neck squamous cell carcinoma (HNSCC), outcomes have not significantly improved; highlighting the importance of identifying novel therapeutic approaches to target this disease. To address this challenge, we proceeded to evaluate the role of iron in HNSCC. Experimental Design Expression levels of iron-related genes were evaluated in HNSCC cell lines using quantitative RT-PCR. Cellular phenotypic effects were assessed using viability (MTS), clonogenic survival, BrdU, and tumor formation assays. The prognostic significance of iron-related proteins was determined using immunohistochemistry. Results In a panel of HNSCC cell lines, hemochromatosis (HFE) was one of the most overexpressed genes involved in iron regulation. In vitro knockdown of HFE in HNSCC cell lines significantly decreased hepcidin (HAMP) expression and intracellular iron level. This in turn, resulted in a significant decrease in HNSCC cell viability, clonogenicity, DNA synthesis, and Wnt signalling. These cellular changes were reversed by re-introducing iron back into HNSCC cells after HFE knockdown, indicating that iron was mediating this phenotype. Concordantly, treating HNSCC cells with an iron chelator, ciclopirox olamine (CPX), significantly reduced viability and clonogenic survival. Finally, patients with high HFE expression experienced a reduced survival compared to patients with low HFE expression. Conclusions Our data identify HFE as potentially novel prognostic marker in HNSCC that promotes tumour progression via HAMP and elevated intracellular iron levels, leading to increased cellular proliferation and tumour formation. Hence, these findings suggest that iron chelators might have a therapeutic role in HNSCC management. PMID:23991213

LDL Receptors as Gateways for Intracellular Porphyrin Uptake

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Novick, S.; Laster, B.; Quastel, M.

2004-01-01

Boronated compounds are currently being studied for possible use in Boron Neutron Capture Therapy (BNCT). We found that one of these agents, BOPP (tetrakis-carborane-carboxylate, esters of 2,4-bis (a,b- dihydroxyethyl) deuteroporphyrin IX), could also be labeled with indium (In-BOPP) and, therefore, could also be used potentially to transport high Z atoms into tumor cell DNA for AET (Auger Electron Therapy). In order to assess the uptake of these agents into cells, the role of the LDL receptor in the intracellular accumulation of BOPP and In-BOPP was investigated. Pre-incubation of V-79 Chinese hamster cells in medium containing delipidized fetal bovine serum (FBS) markedly increased the subsequent uptake of intracellular boron transported by both BOPP and In-BOPP when compared with cells that had been pre-incubated with medium containing 10% normal FBS (lipidized). The increased uptake was characterized by elevated levels of receptor, and greater affinity was shown for both BOPP and In-BOPP, although less marked with the latter. Positive cooperativity was demonstrated by sigmoid saturation curves, Scatchard analysis and Hill plots. Increasing the amount of LDL in the incubation medium had a relatively small effect on the total accumulation of either indium or boron atoms inside the cell. Furthermore, chemical acetylation of LDL did not decrease the intracellular uptake of either boron or indium transported by BOPP or In-BOPP. It is thus concluded that BOPP and In-BOPP preferentially enter the cells directly by way of the LDL receptor and that only a small fraction of these molecules are transported into the cells indirectly using serum LDLs as their carriers. These data suggest a novel way of bringing greater amounts of boron and indium (and perhaps other agents) into tissues. Porphyrins can be used to transport different agents into tumor cells because they are tumor affinic molecules. Tumors express a higher number of LDL receptors than do most normal tissues

An effective intracellular delivery system of monoclonal antibody for treatment of tumors: erythrocyte membrane-coated self-associated antibody nanoparticles

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Gao, Lipeng; Han, Lin; Ding, Xiaoling; Xu, Jiaojiao; Wang, Jing; Zhu, Jianzhong; Lu, Weiyue; Sun, Jihong; Yu, Lei; Yan, Zhiqiang; Wang, Yiting

2017-08-01

Antibody-based drugs have attracted much attention for their targeting ability, high efficacy and low toxicity. But it is difficult for those intrabodies, a kind of antibody whose targets are intracellular biomarkers, to become effective drugs due to the lack of intracellular delivery strategy and their short circulation time in blood. Human telomerase reverse transcriptase (hTERT), an important biomarker for tumors, is expressed only in cytoplasm instead of on cell membrane. In this study, the anti-hTERT blocking monoclonal antibody (mAb), as the model intrabody, was used to prepare nanoparticles (NPs), followed by the encapsulation of erythrocyte membrane (EM), to obtain the EM-coated anti-hTERT mAb NPs delivery system. The final NPs showed a z-average hydrodynamic diameter of about 197.3 nm. The in vitro cellular uptake by HeLa cells confirmed that compared with free anti-hTERT mAb, the EM-coated anti-hTERT mAb NPs exhibited a significantly increased uptake by tumor cells. Besides, the pharmacokinetic study confirmed that the EM encapsulation can remarkably prolong the circulation time and increase the area under curve (AUC) of NPs in blood. The EM-coated anti-hTERT mAb NPs exhibited a remarkably decreased uptake by macrophages than uncoated NPs, which may be responsible for the prolonged circulation time and increased AUC. Furthermore, the frozen section of tumor tissue was performed and proved that the EM-coated anti-hTERT mAb NPs can be more effectively accumulated in tumor tissues than the free mAb and uncoated NPs. In summary, this study indicated that EM-coated anti-hTERT mAb NPs are an effective delivery system for the long circulation and intracellular delivery of an intrabody, and make it possible for the intracellular biomarkers to become the potential targets of drugs.

Intracellular delivery of poly(I:C) induces apoptosis of fibroblast-like synoviocytes via an unknown dsRNA sensor

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Karpus, Olga N.; Hsiao, Cheng-Chih; Kort, Hanneke de; Tak, Paul P.; Hamann, Jörg, E-mail: j.hamann@amc.uva.nl

2016-08-26

Fibroblast-like synoviocytes (FLS) express functional membranous and cytoplasmic sensors for double-stranded (ds)RNA. Notably, FLS undergo apoptosis upon transfection with the synthetic dsRNA analog poly(I:C). We here studied the mechanism of intracellular poly(I:C) recognition and subsequent cell death in FLS. FLS responded similarly to poly(I:C) or 3pRNA transfection; however, only intracellular delivery of poly(I:C) induced significant cell death, accompanied by upregulation of pro-apoptotic proteins Puma and Noxa, caspase 3 cleavage, and nuclear segregation. Knockdown of the DExD/H-box helicase MDA5 did not affect the response to intracellular poly(I:C); in contrast, knockdown of RIG-I abrogated the response to 3pRNA. Knockdown of the downstream adaptor proteins IPS, STING, and TRIF or inhibition of TBK1 did not affect the response to intracellular poly(I:C), while knockdown of IFNAR blocked intracellular poly(I:C)-mediated signaling and cell death. We conclude that a so far unknown intracellular sensor recognizes linear dsRNA and induces apoptosis in FLS. - Highlights: • Intracellular poly(I:C) and 3pRNA evoke immune responses in FLS. • Only intracellular delivery of poly(I:C) induces FLS apoptosis. • FLS do not require MDA5 for their response to intracellular poly(I:C). • FLS respond to intracellular poly(I:C) independent of IPS and STING. • An unknown intracellular sensor recognizes linear dsRNA in FLS.

Intracellular zinc flux causes reactive oxygen species mediated mitochondrial dysfunction leading to cell death in Leishmania donovani.

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Anjali Kumari

Full Text Available Leishmaniasis caused by Leishmania parasite is a global threat to public health and one of the most neglected tropical diseases. Therefore, the discovery of novel drug targets and effective drug is a major challenge and an important goal. Leishmania is an obligate intracellular parasite that alternates between sand fly and human host. To survive and establish infections, Leishmania parasites scavenge and internalize nutrients from the host. Nevertheless, host cells presents mechanism like nutrient restriction to inhibit microbial growth and control infection. Zinc is crucial for cellular growth and disruption in its homeostasis hinders growth and survival in many cells. However, little is known about the role of zinc in Leishmania growth and survival. In this study, the effect of zinc on the growth and survival of L.donovani was analyzed by both Zinc-depletion and Zinc-supplementation using Zinc-specific chelator N, N, N', N'-tetrakis (2-pyridylmethyl ethylenediamine (TPEN and Zinc Sulfate (ZnSO4. Treatment of parasites with TPEN rather than ZnSO4 had significantly affected the growth in a dose- and time-dependent manner. The pre-treatment of promastigotes with TPEN resulted into reduced host-parasite interaction as indicated by decreased association index. Zn depletion resulted into flux in intracellular labile Zn pool and increased in ROS generation correlated with decreased intracellular total thiol and retention of plasma membrane integrity without phosphatidylserine exposure in TPEN treated promastigotes. We also observed that TPEN-induced Zn depletion resulted into collapse of mitochondrial membrane potential which is associated with increase in cytosolic calcium and cytochrome-c. DNA fragmentation analysis showed increased DNA fragments in Zn-depleted cells. In summary, intracellular Zn depletion in the L. donovani promastigotes led to ROS-mediated caspase-independent mitochondrial dysfunction resulting into apoptosis-like cell death

Effects of prolonged acclimation to cold on the extra--and intracellular acid-base status in the land snail Helix lucorum (L.).

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Staikou, A; Stiakakis, M; Michaelidis, B

2001-01-01

The aim of this study was to examine the effect of prolonged acclimation to cold on the acid-base status of extra- and intracellular fluids in the land snail Helix lucorum. For this purpose, acid-base parameters in the hemolymph and tissues were determined. In addition, the buffer values of hemolymph and tissues were determined in order to examine whether they change in the snails during acclimation to cold. According to the results presented, there is an inverse pH-temperature relationship in the hemolymph within the first day of acclimation, which is consistent with alphastat regulation. The Pco2 decreased, and pH in the hemolymph (pH(e)) increased by 0.32 U within the first day of acclimation to cold, which corresponds to a change of 0.013 U degrees C(-1). After the first day of acclimation, Pco2 increased in the hemolymph, resulting in a significant drop in pH(e) by 90 d of acclimation to cold. Acclimation of snails to low temperatures did not change the buffer value of the hemolymph. Also, intracellular pH (pH(i)) and intracellular buffer values remained stable during acclimation to cold for prolonged periods. The latter results in conjunction with those obtained by the in vitro determination of the passive component of intracellular fluids indicate an active regulation of pH(i) in H. lucorum during acclimation to cold.

Dual-layered nanogel-coated hollow lipid/polypeptide conjugate assemblies for potential pH-triggered intracellular drug release.

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Wen-Hsuan Chiang

Full Text Available To achieve effective intracellular anticancer drug delivery, the polymeric vesicles supplemented with the pH-responsive outlayered gels as a delivery system of doxorubicin (DOX were developed from self-assembly of the lipid/polypeptide adduct, distearin grafted poly(γ-glutamic acid (poly(γ-GA, followed by sequential deposition of chitosan and poly(γ-GA-co-γ-glutamyl oxysuccinimide-g-monomethoxy poly(ethylene glycol in combination with in situ covalent cross-linking on assembly surfaces. The resultant gel-caged polymeric vesicles (GCPVs showed superior performance in regulating drug release in response to the external pH change. Under typical physiological conditions (pH 7.4 and 37 °C at which the γ-GA/DOX ionic pairings remained mostly undisturbed, the dense outlayered gels of GCPVs significantly reduced the premature leakage of the uncomplexed payload. With the environmental pH being reduced from pH 7.4 to 4.7, the drug liberation was appreciably promoted by the massive disruption of the ionic γ-GA/DOX complexes along with the significant swelling of nanogel layers upon the increased protonation of chitosan chain segments. After being internalized by HeLa cells via endocytosis, GCPVs exhibited cytotoxic effect comparable to free DOX achieved by rapidly releasing the payload in intracellular acidic endosomes and lysosomes. This strongly implies the great promise of such unique GCPVs as an intracellular drug delivery carrier for potential anticancer treatment.

Loperamide Restricts Intracellular Growth of Mycobacterium tuberculosis in Lung Macrophages.

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Juárez, Esmeralda; Carranza, Claudia; Sánchez, Guadalupe; González, Mitzi; Chávez, Jaime; Sarabia, Carmen; Torres, Martha; Sada, Eduardo

2016-12-01

New approaches for improving tuberculosis (TB) control using adjunct host-directed cellular and repurposed drug therapies are needed. Autophagy plays a crucial role in the response to TB, and a variety of autophagy-inducing drugs that are currently available for various medical conditions may serve as an adjunct treatment in pulmonary TB. Here, we evaluated the potential of loperamide, carbamazepine, valproic acid, verapamil, and rapamycin to enhance the antimicrobial immune response to Mycobacterium tuberculosis (Mtb). Human monocyte-derived macrophages (MDMs) and murine alveolar cells (MACs) were infected with Mtb and treated with loperamide, carbamazepine, valproic acid, verapamil, and rapamycin in vitro. Balb/c mice were intraperitoneally administered loperamide, valproic acid, and verapamil, and MACs were infected in vitro with Mtb. The induction of autophagy, the containment of Mtb within autophagosomes and the intracellular Mtb burden were determined. Autophagy was induced by all of the drugs in human and mouse macrophages, and loperamide significantly increased the colocalization of microtubule-associated protein 1 light chain 3 with Mtb in MDMs. Carbamazepine, loperamide, and valproic acid induced microtubule-associated protein 1 light chain 3 and autophagy related 16- like protein 1 gene expression in MDMs and in MACs. Loperamide also induced a reduction in TNF-α production. Loperamide and verapamil induced autophagy, which was associated with a significant reduction in the intracellular growth of Mtb in MACs and alveolar macrophages. The intraperitoneal administration of loperamide and valproic acid induced autophagy in freshly isolated MACs. The antimycobacterial activity in MACs was higher after loperamide treatment and was associated with the degradation of p62. In conclusion, loperamide shows potential as an adjunctive therapy for the treatment of TB.

Intracellularly Induced Cyclophilins Play an Important Role in Stress Adaptation and Virulence of Brucella abortus

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García Fernández, Lucía; DelVecchio, Vito G.; Briones, Gabriel

2013-01-01

Brucella is an intracellular bacterial pathogen that causes the worldwide zoonotic disease brucellosis. Brucella virulence relies on its ability to transition to an intracellular lifestyle within host cells. Thus, this pathogen must sense its intracellular localization and then reprogram gene expression for survival within the host cell. A comparative proteomic investigation was performed to identify differentially expressed proteins potentially relevant for Brucella intracellular adaptation. Two proteins identified as cyclophilins (CypA and CypB) were overexpressed in the intracellular environment of the host cell in comparison to laboratory-grown Brucella. To define the potential role of cyclophilins in Brucella virulence, a double-deletion mutant was constructed and its resulting phenotype was characterized. The Brucella abortus ΔcypAB mutant displayed increased sensitivity to environmental stressors, such as oxidative stress, pH, and detergents. In addition, the B. abortus ΔcypAB mutant strain had a reduced growth rate at lower temperature, a phenotype associated with defective expression of cyclophilins in other microorganisms. The B. abortus ΔcypAB mutant also displays reduced virulence in BALB/c mice and defective intracellular survival in HeLa cells. These findings suggest that cyclophilins are important for Brucella virulence and survival in the host cells. PMID:23230297

Model Linking Plasma and Intracellular Tenofovir/Emtricitabine with Deoxynucleoside Triphosphates.

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Xinhui Chen

Full Text Available The coformulation of the nucleos(tide analogs (NA tenofovir (TFV disoproxil fumarate (TDF and emtricitabine (FTC is approved for HIV-infection treatment and prevention. Plasma TFV and FTC undergo complicated hybrid processes to form, accumulate, and retain as their active intracellular anabolites: TFV-diphosphate (TFV-DP and FTC-triphosphate (FTC-TP. Such complexities manifest in nonlinear intracellular pharmacokinetics (PK. In target cells, TFV-DP/FTC-TP compete with endogenous deoxynucleoside triphosphates (dNTP at the active site of HIV reverse transcriptase, underscoring the importance of analog:dNTP ratios for antiviral efficacy. However, NA such as TFV and FTC have the potential to disturb the dNTP pool, which could augment or reduce their efficacies. We conducted a pharmacokinetics-pharmacodynamics (PKPD study among forty subjects receiving daily TDF/FTC (300 mg/200 mg from the first-dose to pharmacological intracellular steady-state (30 days. TFV/FTC in plasma, TFV-DP/FTC-TP and dNTPs in peripheral blood mononuclear cells (PBMC were quantified using validated LC/MS/MS methodologies. Concentration-time data were analyzed using nonlinear mixed effects modeling (NONMEM. Formations and the accumulation of intracellular TFV-DP/FTC-TP was driven by plasma TFV/FTC, which was described by a hybrid of first-order formation and saturation. An indirect response link model described the interplay between TFV-DP/FTC-TP and the dNTP pool change. The EC50 (interindividual variability, (%CV of TFV-DP and FTC-TP on the inhibition of deoxyadenosine triphosphate (dATP and deoxycytidine triphosphate (dCTP production were 1020 fmol/106 cells (130% and 44.4 pmol/106 cells (82.5%, resulting in (90% prediction interval 11% (0.45%, 53% and 14% (2.6%, 35% reductions. Model simulations of analog:dNTP molar ratios using IPERGAY dosing suggested that FTC significantly contributes to the protective effect of preexposure prophylaxis (PrEP. Simulation

Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk.

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El-Awady, Ahmed R; Miles, Brodie; Scisci, Elizabeth; Kurago, Zoya B; Palani, Chithra D; Arce, Roger M; Waller, Jennifer L; Genco, Caroline A; Slocum, Connie; Manning, Matthew; Schoenlein, Patricia V; Cutler, Christopher W

2015-02-01

Signaling via pattern recognition receptors (PRRs) expressed on professional antigen presenting cells, such as dendritic cells (DCs), is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs) and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of Porphyromonas gingivalis in human monocyte-derived DCs (MoDCs). We employed a panel of P. gingivalis isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+P. gingivalis strains by MoDCs resulted in lower intracellular killing and higher intracellular content of P. gingivalis. Moreover, Mfa1+P. gingivalis was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, P. gingivalis uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.

Reexamination of the Physiological Role of PykA in Escherichia coli Revealed that It Negatively Regulates the Intracellular ATP Levels under Anaerobic Conditions.

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Zhao, Chunhua; Lin, Zhao; Dong, Hongjun; Zhang, Yanping; Li, Yin

2017-06-01

Pyruvate kinase is one of the three rate-limiting glycolytic enzymes that catalyze the last step of glycolysis, conversion of phosphoenolpyruvate (PEP) into pyruvate, which is associated with ATP generation. Two isozymes of pyruvate kinase, PykF and PykA, are identified in Escherichia coli PykF is considered important, whereas PykA has a less-defined role. Prior studies inactivated the pykA gene to increase the level of its substrate, PEP, and thereby increased the yield of end products derived from PEP. We were surprised when we found a pykA ::Tn 5 mutant in a screen for increased yield of an end product derived from pyruvate ( n -butanol), suggesting that the role of PykA needs to be reexamined. We show that the pykA mutant exhibited elevated intracellular ATP levels, biomass concentrations, glucose consumption, and n -butanol production. We also discovered that the pykA mutant expresses higher levels of a presumed pyruvate transporter, YhjX, permitting the mutant to recapture and metabolize excreted pyruvate. Furthermore, we demonstrated that the nucleotide diphosphate kinase activity of PykA leads to negative regulation of the intracellular ATP levels. Taking the data together, we propose that inactivation of pykA can be considered a general strategy to enhance the production of pyruvate-derived metabolites under anaerobic conditions. IMPORTANCE This study showed that knocking out pykA significantly increased the intracellular ATP level and thus significantly increased the levels of glucose consumption, biomass formation, and pyruvate-derived product formation under anaerobic conditions. pykA was considered to be encoding a dispensable pyruvate kinase; here we show that pykA negatively regulates the anaerobic glycolysis rate through regulating the energy distribution. Thus, knocking out pykA can be used as a general strategy to increase the level of pyruvate-derived fermentative products. Copyright © 2017 American Society for Microbiology.

The alteration of intracellular enzymes. III. The effect of temperature on the kinetics of altered and unaltered yeast catalase.

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FRASER, M J; KAPLAN, J G

1955-03-20

1. The very large increase in catalase activity (Euler effect) which follows treatment of yeast cells with CHCl(3), UV and n-propanol is accompanied by highly significant changes in kinetic properties. With respect to the enzymatic decomposition of H(2)O(2), the thermodynamic constants of the activation process micro, DeltaHdouble dagger, DeltaSdouble dagger, DeltaFdouble dagger, decrease, following treatment of the intracellular enzyme, by 4.5 kcal., 4.5 kcal., 10.1 e.u. and 1.7 kcal., respectively, all these differences being significant at the 1 per cent level. 2. Similar differences exist between the untreated, intracellular enzyme on the one hand, and the extracted yeast and crystalline beef liver catalases on the other. Significant differences in these thermodynamic constants do not exist among the treated intracellular, extracted yeast, and crystalline liver catalases. 3. These data provide unequivocal confirmation of the phenomenon of enzyme alteration reported previously, and confirm previous evidence that the extracted and crystalline enzymes have also undergone enzyme alteration and have properties which are identical with, or very similar to, those of the catalase altered in situ. 4. With respect to the process of heat destruction of catalase, the greatly diminished stability to heat of the altered enzymes, previously reported, has been confirmed. The thermodynamic constants of activation of this process have likewise changed following alteration, in the case of micro, DeltaHdouble dagger, and DeltaSdouble dagger an increase of 20.6 kcal., 20.6 kcal., and 70 e.u., respectively, and of DeltaFdouble dagger a decrease of 2.8 kcal. 5. All these data have been shown to be consistent with, and in some cases predictable from, the interfacial hypothesis, which states that the unaltered catalase exists within the cell adsorbed to some interface, in a partially, but reversibly, unfolded configuration of relatively low specificity; enzyme alteration consists, in

Human gastric signet ring carcinoma (KATO-III) cell apoptosis induced by Vitex agnus-castus fruit extract through intracellular oxidative stress.

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Ohyama, Kunio; Akaike, Takenori; Imai, Masahiko; Toyoda, Hiroo; Hirobe, Chieko; Bessho, Toshio

2005-07-01

We have previously reported that an ethanol extract of the dried ripe fruit of Vitex agnus-castus (Vitex) displays cytotoxic activity against certain kinds of human cancer cell line resulting in the induction of apoptosis. In this paper, we investigate the molecular mechanism of apoptosis induced by Vitex using a human gastric signet ring carcinoma cell line, KATO-III. DNA fragmentation was observed in Vitex-treated KATO-III cells in a time- and dose-dependent manner. DNA fragmentation was accompanied by the following phenomena: elevation in the level of hemeoxygenase-1 protein and thioredoxin reductase mRNA; repression of Mn-superoxide dismutase and catalase mRNAs; release of cytochrome c from mitochondria into the cytosol; activation of caspases-8, -9 and -3; decrease in the level of Bcl-2, Bcl-XL and Bid protein; increase in the level of Bad protein. The intracellular oxidized state, measured using 2',7'-dichlorofluorescin diacetate, increased after Vitex treatment. While the amount of intracellular GSH decreased significantly after treatment with Vitex, the level of GSSG was unaffected. Furthermore, no significant perturbation in the amount of proteins/mRNAs related to glutathione metabolism could be detected. These apoptotic alterations induced by exposure to Vitex were blocked by the presence of an anti-oxidative reagent, N-acetyl-l-cysteine, or the addition of exogenous GSH. Our results demonstrate that intracellular oxidative stress and mitochondrial membrane damage is responsible for Vitex-induced apoptosis, which may be mediated by a diminution of reduced type glutathione within the cell.

Tissue- and Cell-Specific Co-localization of Intracellular Gelatinolytic Activity and Matrix Metalloproteinase 2

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Solli, Ann Iren; Fadnes, Bodil; Winberg, Jan-Olof; Uhlin-Hansen, Lars

2013-01-01

Matrix metalloproteinase 2 (MMP-2) is a proteolytic enzyme that degrades extracellular matrix proteins. Recent studies indicate that MMP-2 also has a role in intracellular proteolysis during various pathological conditions, such as ischemic injuries in heart and brain and in tumor growth. The present study was performed to map the distribution of intracellular MMP-2 activity in various mouse tissues and cells under physiological conditions. Samples from normal brain, heart, lung, liver, spleen, pancreas, kidney, adrenal gland, thyroid gland, gonads, oral mucosa, salivary glands, esophagus, intestines, and skin were subjected to high-resolution in situ gelatin zymography and immunohistochemical staining. In hepatocytes, cardiac myocytes, kidney tubuli cells, epithelial cells in the oral mucosa as well as in excretory ducts of salivary glands, and adrenal cortical cells, we found strong intracellular gelatinolytic activity that was significantly reduced by the metalloprotease inhibitor EDTA but not by the cysteine protease inhibitor E-64. Furthermore, the gelatinolytic activity was co-localized with MMP-2. Western blotting and electron microscopy combined with immunogold labeling revealed the presence of MMP-2 in different intracellular compartments of isolated hepatocytes. Our results indicate that MMP-2 takes part in intracellular proteolysis in specific tissues and cells during physiological conditions. PMID:23482328

A general mechanism for intracellular toxicity of metal-containing nanoparticles

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Sabella, Stefania; Carney, Randy P.; Brunetti, Virgilio; Malvindi, Maria Ada; Al-Juffali, Noura; Vecchio, Giuseppe; Janes, Sam M.; Bakr, Osman M.; Cingolani, Roberto; Stellacci, Francesco; Pompa, Pier Paolo

2014-05-01

The assessment of the risks exerted by nanoparticles is a key challenge for academic, industrial, and regulatory communities worldwide. Experimental evidence points towards significant toxicity for a range of nanoparticles both in vitro and in vivo. Worldwide efforts aim at uncovering the underlying mechanisms for this toxicity. Here, we show that the intracellular ion release elicited by the acidic conditions of the lysosomal cellular compartment - where particles are abundantly internalized - is responsible for the cascading events associated with nanoparticles-induced intracellular toxicity. We call this mechanism a ``lysosome-enhanced Trojan horse effect'' since, in the case of nanoparticles, the protective cellular machinery designed to degrade foreign objects is actually responsible for their toxicity. To test our hypothesis, we compare the toxicity of similar gold particles whose main difference is in the internalization pathways. We show that particles known to pass directly through cell membranes become more toxic when modified so as to be mostly internalized by endocytosis. Furthermore, using experiments with chelating and lysosomotropic agents, we found that the toxicity mechanism for different metal containing NPs (such as metallic, metal oxide, and semiconductor NPs) is mainly associated with the release of the corresponding toxic ions. Finally, we show that particles unable to release toxic ions (such as stably coated NPs, or diamond and silica NPs) are not harmful to intracellular environments.The assessment of the risks exerted by nanoparticles is a key challenge for academic, industrial, and regulatory communities worldwide. Experimental evidence points towards significant toxicity for a range of nanoparticles both in vitro and in vivo. Worldwide efforts aim at uncovering the underlying mechanisms for this toxicity. Here, we show that the intracellular ion release elicited by the acidic conditions of the lysosomal cellular compartment - where

Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors.

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Hannah Karlsson

Full Text Available CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs.

Intracellular Signalling by C-Peptide

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Claire E. Hills

2008-01-01

Full Text Available C-peptide, a cleavage product of the proinsulin molecule, has long been regarded as biologically inert, serving merely as a surrogate marker for insulin release. Recent findings demonstrate both a physiological and protective role of C-peptide when administered to individuals with type I diabetes. Data indicate that C-peptide appears to bind in nanomolar concentrations to a cell surface receptor which is most likely to be G-protein coupled. Binding of C-peptide initiates multiple cellular effects, evoking a rise in intracellular calcium, increased PI-3-kinase activity, stimulation of the Na+/K+ ATPase, increased eNOS transcription, and activation of the MAPK signalling pathway. These cell signalling effects have been studied in multiple cell types from multiple tissues. Overall these observations raise the possibility that C-peptide may serve as a potential therapeutic agent for the treatment or prevention of long-term complications associated with diabetes.

Characterization of Leptin Intracellular Trafficking

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E Walum

2009-12-01

Full Text Available Leptin is produced by adipose tissue, and its concentration in plasma is related to the amount of fat in the body. The leptin receptor (OBR is a member of the class I cytokine receptor family and several different isoforms, produced by alternative mRNA splicing are found in many tissues, including the hypothalamus. The two predominant isoforms includes a long form (OBRl with an intracellular domain of 303 amino acids and a shorter form (OBRs with an intracellular domain of 34 amino acids. Since OBRl is mainly expressed in the hypotalamus, it has been suggested to be the main signalling form. The peripheral production of leptin by adipocyte tissue and its effects as a signal of satiety in the central nervous system imply that leptin gains access to regions of the brain regulating in energy balance by crossing the blood-brain barrier. In an attempt to characterize the intracellular transport of leptin, we have followed binding internalization and degradation of leptin in HEK293 cells. We have also monitored the intracellular transport pathway of fluorescent conjugated leptin in HEK293 cells. Phenylarsine oxide, a general inhibitor of endocytosis, as well as incubation at mild hypertonic conditions, prevented the uptake of leptin, confirming a receptor-mediated internalization process. When internalized, 125I-leptin was rapidly accumulated inside the cells and reached a maximum after 10 min. After 70 minutes about 40-50% of total counts in each time point were found in the medium as TCA-soluble material. Leptin sorting, at the level of early endosomes, did not seem to involve recycling endosomes, since FITC-leptin was sorted from Cy3- transferrin containing compartments at 37°C. At 45 minutes of continuos internalization, FITC-leptin appeared mainly accumulated in late endocytic structures colocalizing with internalized rhodamine coupled epidermial growth factor (EGF and the lysosomal marker protein lamp-1. The transport of leptin was also shown

Mechanisms of cellular invasion by intracellular parasites.

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Walker, Dawn M; Oghumu, Steve; Gupta, Gaurav; McGwire, Bradford S; Drew, Mark E; Satoskar, Abhay R

2014-04-01

Numerous disease-causing parasites must invade host cells in order to prosper. Collectively, such pathogens are responsible for a staggering amount of human sickness and death throughout the world. Leishmaniasis, Chagas disease, toxoplasmosis, and malaria are neglected diseases and therefore are linked to socio-economical and geographical factors, affecting well-over half the world's population. Such obligate intracellular parasites have co-evolved with humans to establish a complexity of specific molecular parasite-host cell interactions, forming the basis of the parasite's cellular tropism. They make use of such interactions to invade host cells as a means to migrate through various tissues, to evade the host immune system, and to undergo intracellular replication. These cellular migration and invasion events are absolutely essential for the completion of the lifecycles of these parasites and lead to their for disease pathogenesis. This review is an overview of the molecular mechanisms of protozoan parasite invasion of host cells and discussion of therapeutic strategies, which could be developed by targeting these invasion pathways. Specifically, we focus on four species of protozoan parasites Leishmania, Trypanosoma cruzi, Plasmodium, and Toxoplasma, which are responsible for significant morbidity and mortality.

Stochastic models of intracellular transport

KAUST Repository

Bressloff, Paul C.

2013-01-09

The interior of a living cell is a crowded, heterogenuous, fluctuating environment. Hence, a major challenge in modeling intracellular transport is to analyze stochastic processes within complex environments. Broadly speaking, there are two basic mechanisms for intracellular transport: passive diffusion and motor-driven active transport. Diffusive transport can be formulated in terms of the motion of an overdamped Brownian particle. On the other hand, active transport requires chemical energy, usually in the form of adenosine triphosphate hydrolysis, and can be direction specific, allowing biomolecules to be transported long distances; this is particularly important in neurons due to their complex geometry. In this review a wide range of analytical methods and models of intracellular transport is presented. In the case of diffusive transport, narrow escape problems, diffusion to a small target, confined and single-file diffusion, homogenization theory, and fractional diffusion are considered. In the case of active transport, Brownian ratchets, random walk models, exclusion processes, random intermittent search processes, quasi-steady-state reduction methods, and mean-field approximations are considered. Applications include receptor trafficking, axonal transport, membrane diffusion, nuclear transport, protein-DNA interactions, virus trafficking, and the self-organization of subcellular structures. © 2013 American Physical Society.

Exploring Anti-Bacterial Compounds against Intracellular Legionella

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Harrison, Christopher F.; Kicka, Sébastien; Trofimov, Valentin; Berschl, Kathrin; Ouertatani-Sakouhi, Hajer; Ackermann, Nikolaus; Hedberg, Christian; Cosson, Pierre; Soldati, Thierry; Hilbi, Hubert

2013-01-01

Legionella pneumophila is a ubiquitous fresh-water bacterium which reproduces within its erstwhile predators, environmental amoeba, by subverting the normal pathway of phagocytosis and degradation. The molecular mechanisms which confer resistance to amoeba are apparently conserved and also allow replication within macrophages. Thus, L. pneumophila can act as an ‘accidental’ human pathogen and cause a severe pneumonia known as Legionnaires’ disease. The intracellular localisation of L. pneumophila protects it from some antibiotics, and this fact must be taken into account to develop new anti-bacterial compounds. In addition, the intracellular lifestyle of L. pneumophila may render the bacteria susceptible to compounds diminishing bacterial virulence and decreasing intracellular survival and replication of this pathogen. The development of a single infection cycle intracellular replication assay using GFP-producing L. pneumophila and Acanthamoeba castellanii amoeba is reported here. This fluorescence-based assay allows for continuous monitoring of intracellular replication rates, revealing the effect of bacterial gene deletions or drug treatment. To examine how perturbations of the host cell affect L. pneumophila replication, several known host-targeting compounds were tested, including modulators of cytoskeletal dynamics, vesicle scission and Ras GTPase localisation. Our results reveal a hitherto unrealized potential antibiotic property of the β-lactone-based Ras depalmitoylation inhibitor palmostatin M, but not the closely related inhibitor palmostatin B. Further characterisation indicated that this compound caused specific growth inhibition of Legionella and Mycobacterium species, suggesting that it may act on a common bacterial target. PMID:24058631

Exploring anti-bacterial compounds against intracellular Legionella.

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Christopher F Harrison

Full Text Available Legionella pneumophila is a ubiquitous fresh-water bacterium which reproduces within its erstwhile predators, environmental amoeba, by subverting the normal pathway of phagocytosis and degradation. The molecular mechanisms which confer resistance to amoeba are apparently conserved and also allow replication within macrophages. Thus, L. pneumophila can act as an 'accidental' human pathogen and cause a severe pneumonia known as Legionnaires' disease. The intracellular localisation of L. pneumophila protects it from some antibiotics, and this fact must be taken into account to develop new anti-bacterial compounds. In addition, the intracellular lifestyle of L. pneumophila may render the bacteria susceptible to compounds diminishing bacterial virulence and decreasing intracellular survival and replication of this pathogen. The development of a single infection cycle intracellular replication assay using GFP-producing L. pneumophila and Acanthamoebacastellanii amoeba is reported here. This fluorescence-based assay allows for continuous monitoring of intracellular replication rates, revealing the effect of bacterial gene deletions or drug treatment. To examine how perturbations of the host cell affect L. pneumophila replication, several known host-targeting compounds were tested, including modulators of cytoskeletal dynamics, vesicle scission and Ras GTPase localisation. Our results reveal a hitherto unrealized potential antibiotic property of the β-lactone-based Ras depalmitoylation inhibitor palmostatin M, but not the closely related inhibitor palmostatin B. Further characterisation indicated that this compound caused specific growth inhibition of Legionella and Mycobacterium species, suggesting that it may act on a common bacterial target.

Intracellular pH-sensing using core/shell silica nanoparticles.

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Korzeniowska, B; Woolley, R; DeCourcey, J; Wencel, D; Loscher, C E; McDonagh, C

2014-07-01

An in-depth understanding of biochemical processes occurring within biological systems is key for early diagnosis of disease and identification of appropriate treatments. Nanobiophotonics offers huge potential benefits for intracellular diagnostics and therapeutics. Intracellular sensing using fluorescent nanoparticles is a potentially useful tool for real-time, in vivo monitoring of important cellular analytes. This work is focused on synthesis of optical chemical nanosensors for the quantitative analysis of pH inside living cells. The structure of the nanosensor comprises a biofriendly silica matrix with co-encapsulated Texas Red, acting as a reference dye, and pH-sensitive fluorescein isothiocyanate enabling ratiometric quantitative environmental detection. In order to obtain silica-based nanoparticles -70 nm in size, a modified sol-gel-based Stöber method was employed. The potential of these nanosensors for intracellular pH monitoring is demonstrated inside a live human embryonic kidney cell line whereby a significant change in fluorescence is observed when the cell pH is switched from acidic to basic. High loading efficiencies of nanoparticles into the cells is seen, with little effect on cell morphology even following extended nanoparticle exposure (up to 72 h). Nanoparticle incubation time and the fast response of the nanosensor (-2 s) make it a very powerful tool in monitoring the processes occurring within the cytosol.

Temperature dependence of intracellular Ca2+ homeostasis in rat meiotic and postmeiotic spermatogenic cells.

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Herrera, E; Salas, K; Lagos, N; Benos, D J; Reyes, J G

2001-10-01

The hypothesis that intracellular [Ca2+] is a cell parameter responsive to extreme temperatures in rat meiotic and postmeiotic spermatogenic cells was tested using intracellular fluorescent probes for Ca2+ and pH. In agreement with this hypothesis, extreme temperatures induced a rapid increase of cytosolic [Ca2+] in rat pachytene spermatocytes and round spermatids. Oscillatory changes in temperature can induce oscillations in cytosolic [Ca2+] in these cells. Intracellular [Ca2+] homeostasis in round spermatids was more sensitive to high temperatures compared with pachytene spermatocytes. The calculated activation energies for SERCA ATPase-mediated fluxes in pachytene spermatocytes and round spermatids were 62 and 75 kJ mol(-1), respectively. The activation energies for leak fluxes from intracellular Ca2+ stores were 55 and 68 kJ mol(-1) for pachytene spermatocytes and round spermatids, respectively. Together with changes in cytosolic [Ca2+], round spermatids undergo a decrease in pH(i) at high temperatures. This temperature-induced decrease in pH(i) appears to be partially responsible for the increase in cytosolic [Ca2+] of round spermatids induced by high temperatures. This characteristic of rat meiotic and postmeiotic spermatogenic cells to undergo an increment in cytosolic Ca2+ at temperatures > 33 degrees C can be related to the induction of programmed cell death by high temperatures in these cells.

Promotion and Rescue of Intracellular Brucella neotomae Replication during Coinfection with Legionella pneumophila.

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Kang, Yoon-Suk; Kirby, James E

2017-05-01

We established a new Brucella neotomae in vitro model system for study of type IV secretion system-dependent (T4SS) pathogenesis in the Brucella genus. Importantly, B. neotomae is a rodent pathogen, and unlike B. abortus , B. melitensis , and B. suis , B. neotomae has not been observed to infect humans. It therefore can be handled more facilely using biosafety level 2 practices. More particularly, using a series of novel fluorescent protein and lux operon reporter systems to differentially label pathogens and track intracellular replication, we confirmed T4SS-dependent intracellular growth of B. neotomae in macrophage cell lines. Furthermore, B. neotomae exhibited early endosomal (LAMP-1) and late endoplasmic reticulum (calreticulin)-associated phagosome maturation. These findings recapitulate prior observations for human-pathogenic Brucella spp. In addition, during coinfection experiments with Legionella pneumophila , we found that defective intracellular replication of a B. neotomae T4SS virB4 mutant was rescued and baseline levels of intracellular replication of wild-type B. neotomae were significantly stimulated by coinfection with wild-type but not T4SS mutant L. pneumophila Using confocal microscopy, it was determined that intracellular colocalization of B. neotomae and L. pneumophila was required for rescue and that colocalization came at a cost to L. pneumophila fitness. These findings were not completely expected based on known temporal and qualitative differences in the intracellular life cycles of these two pathogens. Taken together, we have developed a new system for studying in vitro Brucella pathogenesis and found a remarkable T4SS-dependent interplay between Brucella and Legionella during macrophage coinfection. Copyright © 2017 American Society for Microbiology.

Alterations of the intracellular peptidome in response to the proteasome inhibitor bortezomib.

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Julia S Gelman

Full Text Available Bortezomib is an antitumor drug that competitively inhibits proteasome beta-1 and beta-5 subunits. While the impact of bortezomib on protein stability is known, the effect of this drug on intracellular peptides has not been previously explored. A quantitative peptidomics technique was used to examine the effect of treating human embryonic kidney 293T (HEK293T cells with 5-500 nM bortezomib for various lengths of time (30 minutes to 16 hours, and human neuroblastoma SH-SY5Y cells with 500 nM bortezomib for 1 hour. Although bortezomib treatment decreased the levels of some intracellular peptides, the majority of peptides were increased by 50-500 nM bortezomib. Peptides requiring cleavage at acidic and hydrophobic sites, which involve beta-1 and -5 proteasome subunits, were among those elevated by bortezomib. In contrast, the proteasome inhibitor epoxomicin caused a decrease in the levels of many of these peptides. Although bortezomib can induce autophagy under certain conditions, the rapid bortezomib-mediated increase in peptide levels did not correlate with the induction of autophagy. Taken together, the present data indicate that bortezomib alters the balance of intracellular peptides, which may contribute to the biological effects of this drug.

Bovine lactoferrin and lactoferricin interfere with intracellular trafficking of Herpes simplex virus-1.

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Marr, A K; Jenssen, H; Moniri, M Roshan; Hancock, R E W; Panté, N

2009-01-01

Although both lactoferrin (Lf), a component of the innate immune system of living organisms, and its N-terminal pepsin cleavage product lactoferricin (Lfcin) have anti-herpes activity, the precise mechanisms by which Lf and Lfcin bring about inhibition of herpes infections are not fully understood. In the present study, experiments were carried out to characterize the activity of bovine Lf and Lfcin (BLf and BLfcin) against the Herpes simplex virus-1 (HSV-1). HSV-1 cellular uptake and intracellular trafficking were studied by immunofluorescence microscopy. In comparison to the untreated infected control cells, both the BLf- and BLfcin-treated cells showed a significant reduction in HSV-1 cellular uptake. The few virus particles that were internalized appeared to have a delayed intracellular trafficking. Thus, in addition to their interference with the uptake of the virus into host cells, Lf and Lfcin also exert their antiviral effect intracellularly.

Intracellular compartmentalization of skeletal muscle glycogen metabolism and insulin signalling

DEFF Research Database (Denmark)

Prats Gavalda, Clara; Gomez-Cabello, Alba; Vigelsø Hansen, Andreas

2011-01-01

The interest in skeletal muscle metabolism and insulin signalling has increased exponentially in recent years as a consequence of their role in the development of type 2 diabetes mellitus. Despite this, the exact mechanisms involved in the regulation of skeletal muscle glycogen metabolism...... and insulin signalling transduction remain elusive. We believe that one of the reasons is that the role of intracellular compartmentalization as a regulator of metabolic pathways and signalling transduction has been rather ignored. This paper briefly reviews the literature to discuss the role of intracellular...... compartmentalization in the regulation of skeletal muscle glycogen metabolism and insulin signalling. As a result, a hypothetical regulatory mechanism is proposed by which cells could direct glycogen resynthesis towards different pools of glycogen particles depending on the metabolic needs. Furthermore, we discuss...

Acrolein produces nitric oxide through the elevation of intracellular calcium levels to induce apoptosis in human umbilical vein endothelial cells: implications for smoke angiopathy.

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Misonou, Yoshiko; Asahi, Michio; Yokoe, Shunichi; Miyoshi, Eiji; Taniguchi, Naoyuki

2006-03-01

Acrolein is a highly electrophilic alpha, beta-unsaturated aldehyde, the levels of which are increased in the blood of smokers. To determine if acrolein is involved in the pathology of smoke angiopathy, the effect of acrolein on human umbilical vein endothelial cells (HUVEC) was examined. Intracellular nitric oxide (NO) levels, determined using diaminofluorescein-2 diacetate (DAF-2 DA), an NO sensitive fluorescent dye, were found to be increased after treatment in HUVEC with 10 microM acrolein. The measurement of nitrite with 2,3-diaminonaphthalene and a Western blot analysis revealed that nitrite and S-nitroso-cysteine levels were increased in a dose-dependent manner, confirming that NO production is increased by acrolein. The increase was not reduced by treatment with 10mM N-acetyl-l-cysteine (NAC), an anti-oxidant, but was reduced with 10 microM of the intracellular calcium chelator, 1,2-bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester. Acrolein-stimulated NO production was significantly reduced by pretreatment with 1mM N(G)-nitro-l-arginine-methyl ester (L-NAME), an NO synthase inhibitor. The cytotoxicity of acrolein was reduced by pretreatment with 10 microM 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO), an intracellular NO scavenger, or 1mM L-NAME, whereas it was not reduced by 10mM NAC, 20 microM Curcumin, another peroxide scavenger, or 100 microM Mn(III)TMPyP, a superoxide dismutase mimic. Nuclear staining and a Western blot analysis using an anti-cleaved caspase 3 antibody revealed that the reduced viability of HUVEC by acrolein was due to apoptosis, which was reversed after pretreatment with 0.1mM carboxy-PTIO or 1mM L-NAME. Thus, acrolein increases intracellular calcium production to induce intracellular NO production by a calcium-dependent NO synthase, possibly eNOS, and the excess and rapid increase in NO might lead to the apoptosis of HUVEC. These data suggest that acrolein might be

Intracellular thiol levels and radioresistance: Studies with glutathione and glutathione mono ethyl ester

International Nuclear Information System (INIS)

Astor, M.B.; Meister, A.; Anderson, M.E.

1987-01-01

Intracellular thiols such as glutathione (GSH) protect cells against free radicals formed during oxidative metabolism or from exposure to drugs or ionizing radiation. The role of intracellular GSH in the repair of radiation induced free radical damage was studied using GSH or its analog glutathione mono ethyl ester (GEE), which readily penetrates into the cell. Chinese hamster V79 cells with normal GSH levels were afforded equal protection under aerated and hypoxic conditions (DMF = 1.2 OER = 3.7) by both 10 mM GSH and GEE although GEE had raised interacellular GSH levels three-fold. Growth of V79 cells in cysteine free media resulted in undetectable levels of GSH and OER of 2.2 with no change in aerated survival. Restoration of intracellular GSH by 10 mM GEE resulted in an increase of the OER from 2.2. to 3.8 (DMF = 1.7). Only 14% of the intracellular GSH needs to be repleted to give an OER of 3.0. These experiments provide evidence that thiols do play a role in the oxygen effect and are present at levels in excess of what is necessary for maximal radioprotection

Modulating intracellular acidification by regulating the incubation time of proton caged compounds.

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Carbone, Marilena; Sabbatella, Gianfranco; Antonaroli, Simonetta; Orlando, Viviana; Biagioni, Stefano; Nucara, Alessandro

2016-09-01

A proton caged compound, the 1-(2-nitrophenyl)- ethylhexadecyl sulfonate (HDNS), was dosed into HEK-293 at different incubation times. Samples were irradiated with filtered UV light for inducing photolysis of the HDNS and then probed by infrared spectroscopy. The intracellular acidification reaction can be followed by monitoring the consequent CO2 peak intensity variation. The total CO2 produced is similar for all the samples, hence it is only a function of the initial HDNS concentration. The way it is achieved, though, is different for the different incubation times and follows kinetics, which results in a combination of a linear CO2 increase and a steep CO2 increase followed by a decay. This is interpreted in terms of confinement of the HDNS into intracellular vesicles of variable average size and sensitive to UV light when they reach critical dimensions.

Modeling HIV-1 intracellular replication: two simulation approaches

NARCIS (Netherlands)

Zarrabi, N.; Mancini, E.; Tay, J.; Shahand, S.; Sloot, P.M.A.

2010-01-01

Many mathematical and computational models have been developed to investigate the complexity of HIV dynamics, immune response and drug therapy. However, there are not many models which consider the dynamics of virus intracellular replication at a single level. We propose a model of HIV intracellular

Intermittent Hypoxia Inhibits Na+-H+ Exchange-Mediated Acid Extrusion Via Intracellular Na+ Accumulation in Cardiomyocytes

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Huai-Ren Chang

2018-04-01

Full Text Available Background/Aims: Intermittent hypoxia (IH has been shown to exert preconditioning-like cardioprotective effects. It also has been reported that IH preserves intracellular pH (pHi during ischemia and protects cardiomyocytes against ischemic reperfusion injury. However, the exact mechanism is still unclear. Methods: In this study, we used proton indicator BCECF-AM to analyze the rate of pHi recovery from acidosis in the IH model of rat neonatal cardiomyocytes. Neonatal cardiomyocytes were first treated with repetitive hypoxia-normoxia cycles for 1-4 days. Cells were then acid loaded with NH4Cl, and the rate of pHi recovery from acidosis was measured. Results: We found that the pHi recovery rate from acidosis was much slower in the IH group than in the room air (RA group. When we treated cardiomyocytes with Na+-H+ exchange (NHE inhibitors (Amiloride and HOE642 or Na+-free Tyrode solution during the recovery, there was no difference between RA and IH groups. We also found intracellular Na+ concentration ([Na+]i significantly increased after IH exposure for 4 days. However, the phenomenon could be abolished by pretreatment with ROS inhibitors (SOD and phenanathroline, intracellular calcium chelator or Na+-Ca2+ exchange (NCX inhibitor. Furthermore, the pHi recovery rate from acidosis became faster in the IH group than in the RA group when inhibition of NCX activity. Conclusions: These results suggest that IH would induce the elevation of ROS production. ROS then activates Ca2+-efflux mode of NCX and results in intracellular Na+ accumulation. The rise of [Na+]i further inhibits the activity of NHE-mediated acid extrusion and retards the rate of pHi recovery from acidosis during IH.

Mobilization of Intracellular Copper by Gossypol and Apogossypolone Leads to Reactive Oxygen Species-Mediated Cell Death: Putative Anticancer Mechanism

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Haseeb Zubair

2016-06-01

Full Text Available There is compelling evidence that serum, tissue and intracellular levels of copper are elevated in all types of cancer. Copper has been suggested as an important co-factor for angiogenesis. It is also a major metal ion present inside the nucleus, bound to DNA bases, particularly guanine. We have earlier proposed that the interaction of phenolic-antioxidants with intracellular copper leads to the generation of reactive oxygen species (ROS that ultimately serve as DNA cleaving agents. To further validate our hypothesis we show here that the antioxidant gossypol and its semi-synthetic derivative apogossypolone induce copper-mediated apoptosis in breast MDA-MB-231, prostate PC3 and pancreatic BxPC-3 cancer cells, through the generation of ROS. MCF10A breast epithelial cells refractory to the cytotoxic property of these compounds become sensitized to treatment against gossypol, as well as apogossypolone, when pre-incubated with copper. Our present results confirm our earlier findings and strengthen our hypothesis that plant-derived antioxidants mobilize intracellular copper instigating ROS-mediated cellular DNA breakage. As cancer cells exist under significant oxidative stress, this increase in ROS-stress to cytotoxic levels could be a successful anticancer approach.

Intracellular diffusion restrictions in isolated cardiomyocytes from rainbow trout

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Birkedal Rikke

2009-12-01

Full Text Available Abstract Background Restriction of intracellular diffusion of adenine nucleotides has been studied intensively on adult rat cardiomyocytes. However, their cause and role in vivo is still uncertain. Intracellular membrane structures have been suggested to play a role. We therefore chose to study cardiomyocytes from rainbow trout (Oncorhynchus mykiss, which are thinner and have fewer intracellular membrane structures than adult rat cardiomyocytes. Previous studies suggest that trout permeabilized cardiac fibers also have diffusion restrictions. However, results from fibers may be affected by incomplete separation of the cells. This is avoided when studying permeabilized, isolated cardiomyocytes. The aim of this study was to verify the existence of diffusion restrictions in trout cardiomyocytes by comparing ADP-kinetics of mitochondrial respiration in permeabilized fibers, permeabilized cardiomyocytes and isolated mitochondria from rainbow trout heart. Experiments were performed at 10, 15 and 20°C in the absence and presence of creatine. Results Trout cardiomyocytes hypercontracted in the solutions used for mammalian cardiomyocytes. We developed a new solution in which they retained their shape and showed stable steady state respiration rates throughout an experiment. The apparent ADP-affinity of permeabilized cardiomyocytes was different from that of fibers. It was higher, independent of temperature and not increased by creatine. However, it was still about ten times lower than in isolated mitochondria. Conclusions The differences between fibers and cardiomyocytes suggest that results from trout heart fibers were affected by incomplete separation of the cells. However, the lower ADP-affinity of cardiomyocytes compared to isolated mitochondria indicate that intracellular diffusion restrictions are still present in trout cardiomyocytes despite their lower density of intracellular membrane structures. The lack of a creatine effect indicates that

Single-cell intracellular nano-pH probes†

Science.gov (United States)

Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

2016-01-01

Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution. PMID:27708772

Single-cell intracellular nano-pH probes.

Science.gov (United States)

Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

2015-01-01

Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution.

Controlled intracellular generation of reactive oxygen species in human mesenchymal stem cells using porphyrin conjugated nanoparticles.

Science.gov (United States)

Lavado, Andrea S; Chauhan, Veeren M; Zen, Amer Alhaj; Giuntini, Francesca; Jones, D Rhodri E; Boyle, Ross W; Beeby, Andrew; Chan, Weng C; Aylott, Jonathan W

2015-09-14

Nanoparticles capable of generating controlled amounts of intracellular reactive oxygen species (ROS), that advance the study of oxidative stress and cellular communication, were synthesized by functionalizing polyacrylamide nanoparticles with zinc(II) porphyrin photosensitisers. Controlled ROS production was demonstrated in human mesenchymal stem cells (hMSCs) through (1) production of nanoparticles functionalized with varying percentages of Zn(II) porphyrin and (2) modulating the number of doses of excitation light to internalized nanoparticles. hMSCs challenged with nanoparticles functionalized with increasing percentages of Zn(II) porphyrin and high numbers of irradiations of excitation light were found to generate greater amounts of ROS. A novel dye, which is transformed into fluorescent 7-hydroxy-4-trifluoromethyl-coumarin in the presence of hydrogen peroxide, provided an indirect indicator for cumulative ROS production. The mitochondrial membrane potential was monitored to investigate the destructive effect of increased intracellular ROS production. Flow cytometric analysis of nanoparticle treated hMSCs suggested irradiation with excitation light signalled controlled apoptotic cell death, rather than uncontrolled necrotic cell death. Increased intracellular ROS production did not induce phenotypic changes in hMSC subcultures.

Age-associated intracellular superoxide dismutase deficiency potentiates dermal fibroblast dysfunction during wound healing.

Science.gov (United States)

Fujiwara, Toshihiro; Dohi, Teruyuki; Maan, Zeshaan N; Rustad, Kristine C; Kwon, Sun Hyung; Padmanabhan, Jagannath; Whittam, Alexander J; Suga, Hirotaka; Duscher, Dominik; Rodrigues, Melanie; Gurtner, Geoffrey C

2017-07-04

Reactive oxygen species (ROS) impair wound healing through destructive oxidation of intracellular proteins, lipids and nucleic acids. Intracellular superoxide dismutase (SOD1) regulates ROS levels and plays a critical role in tissue homoeostasis. Recent evidence suggests that age-associated wound healing impairments may partially result from decreased SOD1 expression. We investigated the mechanistic basis by which increased oxidative stress links to age-associated impaired wound healing. Fibroblasts were isolated from unwounded skin of young and aged mice, and myofibroblast differentiation was assessed by measuring α-smooth muscle actin and collagen gel contraction. Excisional wounds were created on young and aged mice to study the healing rate, ROS levels and SOD1 expression. A mechanistic link between oxidative stress and fibroblast function was explored by assessing the TGF-β1 signalling pathway components in young and aged mice. Age-related wounds displayed reduced myofibroblast differentiation and delayed wound healing, consistent with a decrease in the in vitro capacity for fibroblast-myofibroblast transition following oxidative stress. Young fibroblasts with normal SOD1 expression exhibited increased phosphorylation of ERK in response to elevated ROS. In contrast, aged fibroblasts with reduced SOD1 expression displayed a reduced capacity to modulate intracellular ROS. Collectively, age-associated wound healing impairments are associated with fibroblast dysfunction that is likely the result of decreased SOD1 expression and subsequent dysregulation of intracellular ROS. Strategies targeting these mechanisms may suggest a new therapeutic approach in the treatment of chronic non-healing wounds in the aged population. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Uptake and intracellular activity of AM-1155 in phagocytic cells.

Science.gov (United States)

Yamamoto, T; Kusajima, H; Hosaka, M; Fukuda, H; Oomori, Y; Shinoda, H

1996-01-01

The uptake and intracellular activity of AM-1155 in murine J774.1 macrophages and human polymorphonuclear leukocytes were investigated. AM-1155 penetrated phagocytic cells rapidly and reversibly, although the penetration process was not affected by metabolic inhibitors such as sodium fluoride, cyanide m-chlorophenylhydrazone, or ouabain or by nucleoside transport system inhibitors such as adenosine. The intracellular concentration-to-extracellular concentration ratio of AM-1155 in both cell types of phagocytes ranged from 5 to 7. These ratios were almost equal to those for sparfloxacin. The intracellular activity of AM-1155 in J774.1 macrophages, examined with Staphylococcus aureus 209P as a test bacterium, was dependent on the extracellular concentration. AM-1155 at a concentration of 1 microgram/ml reduced the number of viable cells of S. aureus ingested by more than 90%. The intracellular activity of AM-1155 was more potent than those of sparfloxacin, ofloxacin, ciprofloxacin, flomoxef, and erythromycin. These results suggest that the potent intracellular activity of AM-1155 might mainly be due to the high intracellular concentration and its potent in vitro activity. PMID:9124835

Sodium transport and intracellular sodium activity in cultured human nasal epithelium

DEFF Research Database (Denmark)

Willumsen, Niels J.; Boucher, Richard C.

1991-01-01

 human nasal epithelium (HNE). Under control conditions, intracellular Na+ activity (acNa) was 23 +/- 1 mM (n = 44 preparations, 393 impalements).Amiloride (10(-4) M) hyperpolarized the apical membrane and increased the fractional apical membrane resistance but did not affect acNa. Exposure to...

Extracellular and Intracellular Mechanisms Mediating Metastatic Activity of Exogenous Osteopontin

Science.gov (United States)

Mandelin, Jami; Lin, Emme C. K.; Hu, Dana D.; Knowles, Susan K.; Do, Kim-Anh; Wang, Xuemei; Sage, E. Helene; Smith, Jeffrey W.; Arap, Wadih; Pasqualini, Renata

2009-01-01

BACKGROUND Osteopontin affects several steps of the metastatic cascade. Despite direct correlation with metastasis in experimental systems and in patient studies, the extracellular and intracellular basis for these observations remains unsolved. We used human melanoma and sarcoma cell lines to evaluate the effects of soluble osteopontin on metastasis. METHODS Exogenous osteopontin or negative controls, including a site-directed mutant osteopontin, were used in functional assays in vitro, ex vivo, and in vivo designed to test extracellular and intracellular mechanisms involved in experimental metastasis. RESULTS In the extracellular environment, we confirm that soluble osteopontin is required for its pro-metastatic effects; this phenomenon is specific, RGD-dependent, and evident in experimental models of metastasis. In the intracellular environment, osteopontin initially induces rapid Tyr-418 dephosphorylation of c-Src, with decreases in actin stress fibers and increased binding to the vascular endothelium. This heretofore undescribed Tyr dephosphorylation is followed by a tandem c-Src phosphorylation after tumor cell attachment to the metastatic site. CONCLUSION Our results reveal a complex molecular interaction as well as a dual role for osteopontin in metastasis that is dependent on whether tumor cells are in circulation or attached. Such context-dependent functional insights may contribute to anti-metastasis strategies. PMID:19224553

Relevance of intracellular polarity to accuracy of eukaryotic chemotaxis

International Nuclear Information System (INIS)

Hiraiwa, Tetsuya; Nishikawa, Masatoshi; Shibata, Tatsuo; Nagamatsu, Akihiro; Akuzawa, Naohiro

2014-01-01

Eukaryotic chemotaxis is usually mediated by intracellular signals that tend to localize at the front or back of the cell. Such intracellular polarities frequently require no extracellular guidance cues, indicating that spontaneous polarization occurs in the signal network. Spontaneous polarization activity is considered relevant to the persistent motions in random cell migrations and chemotaxis. In this study, we propose a theoretical model that connects spontaneous intracellular polarity and motile ability in a chemoattractant solution. We demonstrate that the intracellular polarity can enhance the accuracy of chemotaxis. Chemotactic accuracy should also depend on chemoattractant concentration through the concentration-dependent correlation time in the polarity direction. Both the polarity correlation time and the chemotactic accuracy depend on the degree of responsiveness to the chemical gradient. We show that optimally accurate chemotaxis occurs at an intermediate responsiveness of intracellular polarity. Experimentally, we find that the persistence time of randomly migrating Dictyostelium cells depends on the chemoattractant concentration, as predicted by our theory. At the optimum responsiveness, this ameboid cell can enhance its chemotactic accuracy tenfold. (paper)

Demand for Zn2+ in acid-secreting gastric mucosa and its requirement for intracellular Ca2+.

Directory of Open Access Journals (Sweden)

JingJing Liu

Full Text Available Recent work has suggested that Zn(2+ plays a critical role in regulating acidity within the secretory compartments of isolated gastric glands. Here, we investigate the content, distribution and demand for Zn(2+ in gastric mucosa under baseline conditions and its regulation during secretory stimulation.Content and distribution of zinc were evaluated in sections of whole gastric mucosa using X-ray fluorescence microscopy. Significant stores of Zn(2+ were identified in neural elements of the muscularis, glandular areas enriched in parietal cells, and apical regions of the surface epithelium. In in vivo studies, extraction of the low abundance isotope, (70Zn(2+, from the circulation was demonstrated in samples of mucosal tissue 24 hours or 72 hours after infusion (250 µg/kg. In in vitro studies, uptake of (70Zn(2+ from media was demonstrated in isolated rabbit gastric glands following exposure to concentrations as low as 10 nM. In additional studies, demand of individual gastric parietal cells for Zn(2+ was monitored using the fluorescent zinc reporter, fluozin-3, by measuring increases in free intracellular concentrations of Zn(2+ {[Zn(2+](i} during exposure to standard extracellular concentrations of Zn(2+ (10 µM for standard intervals of time. Under resting conditions, demand for extracellular Zn(2+ increased with exposure to secretagogues (forskolin, carbachol/histamine and under conditions associated with increased intracellular Ca(2+ {[Ca(2+](i}. Uptake of Zn(2+ was abolished following removal of extracellular Ca(2+ or depletion of intracellular Ca(2+ stores, suggesting that demand for extracellular Zn(2+ increases and depends on influx of extracellular Ca(2+.This study is the first to characterize the content and distribution of Zn(2+ in an organ of the gastrointestinal tract. Our findings offer the novel interpretation, that Ca(2+ integrates basolateral demand for Zn(2+ with stimulation of secretion of HCl into the lumen of the gastric

Long-term lithium treatment increases intracellular and extracellular brain-derived neurotrophic factor (BDNF) in cortical and hippocampal neurons at subtherapeutic concentrations.

Science.gov (United States)

De-Paula, Vanessa J; Gattaz, Wagner F; Forlenza, Orestes V

2016-12-01

The putative neuroprotective effects of lithium treatment rely on the fact that it modulates several homeostatic mechanisms involved in the neurotrophic response, autophagy, oxidative stress, inflammation, and mitochondrial function. Lithium is a well-established therapeutic option for the acute and long-term management of bipolar disorder and major depression. The aim of this study was to evaluate the effects of subtherapeutic and therapeutic concentrations of chronic lithium treatment on brain-derived neurotrophic factor (BDNF) synthesis and secretion. Primary cultures of cortical and hippocampal neurons were treated with different subtherapeutic (0.02 and 0.2 mM) and therapeutic (2 mM) concentrations of chronic lithium treatment in cortical and hippocampal cell culture. Lithium treatment increased the intracellular protein expression of cortical neurons (10% at 0.02 mM) and hippocampal neurons (28% and 14% at 0.02 mM and 0.2 mM, respectively). Extracellular BDNF of cortical neurons increased 30% and 428% at 0.02 and 0.2 mM, respectively and in hippocampal neurons increased 44% at 0.02 mM. The present study indicates that chronic, low-dose lithium treatment up-regulates BDNF production in primary neuronal cell culture. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Protective Effects of Extracellular and Intracellular Polysaccharides on Hepatotoxicity by Hericium erinaceus SG-02.

Science.gov (United States)

Cui, Fangyuan; Gao, Xia; Zhang, Jianjun; Liu, Min; Zhang, Chen; Xu, Nuo; Zhao, Huajie; Lin, Lin; Zhou, Meng; Jia, Le

2016-09-01

The protective effects of extracellular and intracellular polysaccharides from Hericium erinaceus SG-02 on the CCl4-induced hepatic injury of mice were investigated in this work. By the analysis of GC, the extracellular polysaccharides (EPS) were composed of arabinose, mannose, galactose, and glucose with a ratio of 1:7:14:52, and the composition of intracellular polysaccharides (IPS) was rhamnose, xylose, mannose, galactose, and glucose with a ratio of 3:4:7:14:137. The model of hepatic injury of mice was induced by CCl4 and three tested levels (200, 400, and 800 mg/kg) of EPS and IPS were set as the experimental group. Results showed that the aspartate aminotransferase and glutamic pyruvic transaminase activities in serum were reduced by the supplement of EPS and IPS, while the blood lipid levels including cholesterol, triglyceride, and albumin were improved. In liver tissue, the lipid peroxidation and malondialdehyde were largely decreased, and the superoxide dismutase and catalase activities were significantly increased. The evidence demonstrated that the EPS and IPS of H. erinaceus SG-02 were protective for liver injury. The histopathological observations of mice liver slices indicated that EPS and IPS had obvious effects on liver protection.

Analysis of Intracellular Metabolites from Microorganisms: Quenching and Extraction Protocols.

Science.gov (United States)

Pinu, Farhana R; Villas-Boas, Silas G; Aggio, Raphael

2017-10-23

Sample preparation is one of the most important steps in metabolome analysis. The challenges of determining microbial metabolome have been well discussed within the research community and many improvements have already been achieved in last decade. The analysis of intracellular metabolites is particularly challenging. Environmental perturbations may considerably affect microbial metabolism, which results in intracellular metabolites being rapidly degraded or metabolized by enzymatic reactions. Therefore, quenching or the complete stop of cell metabolism is a pre-requisite for accurate intracellular metabolite analysis. After quenching, metabolites need to be extracted from the intracellular compartment. The choice of the most suitable metabolite extraction method/s is another crucial step. The literature indicates that specific classes of metabolites are better extracted by different extraction protocols. In this review, we discuss the technical aspects and advancements of quenching and extraction of intracellular metabolite analysis from microbial cells.

Influence of intracellular acidosis on contractile function in the working rat heart

International Nuclear Information System (INIS)

Jeffrey, F.M.H.; Malloy, C.R.; Radda, G.K.

1987-01-01

The decrease in myocardial contractility during ischemia, hypoxia, and extracellular acidosis has been attributed to intracellular acidosis. Previous studies of the relationship between pH and contractile state have utilized respiratory or metabolic acidosis to alter intracellular pH. The authors developed a model in the working perfused rat heart to study the effects of intracellular acidosis with normal external pH and optimal O 2 delivery. Intracellular pH and high-energy phosphates were monitored by 31 P nuclear magnetic resonance spectroscopy. Hearts were perfused to a steady state with a medium containing 10 mM NH 4 Cl. Acidosis induced a substantial decrease in aortic flow and stroke volume which was associated with little change in peak systolic pressure. It was concluded that (1) for the same intracellular acidosis the influence on tension development was more pronounced with a combined extra- and intracellular acidosis than with an isolated intracellular acidosis, and (2) stroke volume at constant preload was impaired by intracellular acidosis even though changes in developed pressure were minimal. These observations suggest that isolated intracellular acidosis has adverse effects on diastolic compliance and/or relaxation

Spatial Cytoskeleton Organization Supports Targeted Intracellular Transport

Science.gov (United States)

Hafner, Anne E.; Rieger, Heiko

2018-03-01

The efficiency of intracellular cargo transport from specific source to target locations is strongly dependent upon molecular motor-assisted motion along the cytoskeleton. Radial transport along microtubules and lateral transport along the filaments of the actin cortex underneath the cell membrane are characteristic for cells with a centrosome. The interplay between the specific cytoskeleton organization and the motor performance realizes a spatially inhomogeneous intermittent search strategy. In order to analyze the efficiency of such intracellular search strategies we formulate a random velocity model with intermittent arrest states. We evaluate efficiency in terms of mean first passage times for three different, frequently encountered intracellular transport tasks: i) the narrow escape problem, which emerges during cargo transport to a synapse or other specific region of the cell membrane, ii) the reaction problem, which considers the binding time of two particles within the cell, and iii) the reaction-escape problem, which arises when cargo must be released at a synapse only after pairing with another particle. Our results indicate that cells are able to realize efficient search strategies for various intracellular transport tasks economically through a spatial cytoskeleton organization that involves only a narrow actin cortex rather than a cell body filled with randomly oriented actin filaments.

Vectorization efforts to increase Gram-negative intracellular drug concentration: a case study on HldE-K inhibitors.

Science.gov (United States)

Atamanyuk, Dmytro; Faivre, Fabien; Oxoby, Mayalen; Ledoussal, Benoit; Drocourt, Elodie; Moreau, François; Gerusz, Vincent

2013-03-14

In this paper, we present different strategies to vectorize HldE kinase inhibitors with the goal to improve their gram-negative intracellular concentration. Syntheses and biological effects of siderophoric, aminoglycosidic, amphoteric, and polycationic vectors are discussed. While siderophoric and amphoteric vectorization efforts proved to be disappointing in this series, aminoglycosidic and polycationic vectors were able for the first time to achieve synergistic effects of our inhibitors with erythromycin. Although these effects proved to be nonspecific, this study provides information about the required stereoelectronic arrangement of the polycationic amines and their basicity requirements to fulfill outer membrane destabilization resulting in better erythromycin synergies.

Thioredoxin 80-Activated-Monocytes (TAMs) Inhibit the Replication of Intracellular Pathogens

DEFF Research Database (Denmark)

Cortes-Bratti, Ximena; Brasseres, Eugenie; Herrera-Rodriquez, Fabiola

2011-01-01

Background: Thioredoxin 80 (Trx80) is an 80 amino acid natural cleavage product of Trx, produced primarily by monocytes. Trx80 induces differentiation of human monocytes into a novel cell type, named Trx80-activated-monocytes (TAMs). Principal Findings: In this investigation we present evidence...... for a role of TAMs in the control of intracellular bacterial infections. As model pathogens we have chosen Listeria monocytogenes and Brucella abortus which replicate in the cytosol and the endoplasmic reticulum respectively. Our data indicate that TAMs efficiently inhibit intracellular growth of both L...... in TAMs compared to that observed in control cells 24 h post-infection, indicating that TAMs kill bacteria by preventing their escape from the endosomal compartments, which progress into a highly degradative phagolysosome. Significance: Our results show that Trx80 potentiates the bactericidal activities...

Expression of intracellular interferon-alpha confers antiviral properties in transfected bovine fetal fibroblasts and does not affect the full development of SCNT embryos.

Directory of Open Access Journals (Sweden)

Dawei Yu

Full Text Available Foot-and-mouth disease, one of the most significant diseases of dairy herds, has substantial effects on farm economics, and currently, disease control measures are limited. In this study, we constructed a vector with a human interferon-α (hIFN-α (without secretory signal sequence gene cassette containing the immediate early promoter of human cytomegalovirus. Stably transfected bovine fetal fibroblasts were obtained by G418 selection, and hIFN-α transgenic embryos were produced by somatic cell nuclear transfer (SCNT. Forty-six transgenic embryos were transplanted into surrogate cows, and five cows (10.9% became pregnant. Two male cloned calves were born. Expression of hIFN-α was detected in transfected bovine fetal fibroblasts, transgenic SCNT embryos, and different tissues from a transgenic SCNT calf at two days old. In transfected bovine fetal fibroblasts, expression of intracellular IFN-α induced resistance to vesicular stomatitis virus infection, increased apoptosis, and induced the expression of double-stranded RNA-activated protein kinase gene (PKR and the 2'-5'-oligoadenylate synthetase gene (2'-5' OAS, which are IFN-inducible genes with antiviral activity. Analysis by qRT-PCR showed that the mRNA expression levels of PKR, 2'-5' OAS, and P53 were significantly increased in wild-type bovine fetal fibroblasts stimulated with extracellular recombinant human IFN-α-2b, showing that intracellular IFN-α induces biological functions similar to extracellular IFN-α. In conclusion, expression of intracellular hIFN-α conferred antiviral properties in transfected bovine fetal fibroblasts and did not significantly affect the full development of SCNT embryos. Thus, IFN-α transgenic technology may provide a revolutionary way to achieve elite breeding of livestock.

Polymeric nanoparticles affect the intracellular delivery, antiretroviral activity and cytotoxicity of the microbicide drug candidate dapivirine.

Science.gov (United States)

das Neves, José; Michiels, Johan; Ariën, Kevin K; Vanham, Guido; Amiji, Mansoor; Bahia, Maria Fernanda; Sarmento, Bruno

2012-06-01

To assess the intracellular delivery, antiretroviral activity and cytotoxicity of poly(ε-caprolactone) (PCL) nanoparticles containing the antiretroviral drug dapivirine. Dapivirine-loaded nanoparticles with different surface properties were produced using three surface modifiers: poloxamer 338 NF (PEO), sodium lauryl sulfate (SLS) and cetyl trimethylammonium bromide (CTAB). The ability of nanoparticles to promote intracellular drug delivery was assessed in different cell types relevant for vaginal HIV transmission/microbicide development. Also, antiretroviral activity of nanoparticles was determined in different cell models, as well as their cytotoxicity. Dapivirine-loaded nanoparticles were readily taken up by different cells, with particular kinetics depending on the cell type and nanoparticles, resulting in enhanced intracellular drug delivery in phagocytic cells. Different nanoparticles showed similar or improved antiviral activity compared to free drug. There was a correlation between increased antiviral activity and increased intracellular drug delivery, particularly when cell models were submitted to a single initial short-course treatment. PEO-PCL and SLS-PCL nanoparticles consistently showed higher selectivity index values than free drug, contrasting with high cytotoxicity of CTAB-PCL. These results provide evidence on the potential of PCL nanoparticles to affect in vitro toxicity and activity of dapivirine, depending on surface engineering. Thus, this formulation approach may be a promising strategy for the development of next generation microbicides.

Drosophila VAMP7 regulates Wingless intracellular trafficking.

Science.gov (United States)

Gao, Han; He, Fang; Lin, Xinhua; Wu, Yihui

2017-01-01

Drosophila Wingless (Wg) is a morphogen that determines cell fate during development. Previous studies have shown that endocytic pathways regulate Wg trafficking and signaling. Here, we showed that loss of vamp7, a gene required for vesicle fusion, dramatically increased Wg levels and decreased Wg signaling. Interestingly, we found that levels of Dally-like (Dlp), a glypican that can interact with Wg to suppress Wg signaling at the dorsoventral boundary of the Drosophila wing, were also increased in vamp7 mutant cells. Moreover, Wg puncta in Rab4-dependent recycling endosomes were Dlp positive. We hypothesize that VAMP7 is required for Wg intracellular trafficking and the accumulation of Wg in Rab4-dependent recycling endosomes might affect Wg signaling.

Tumor-promoting function and prognostic significance of the RNA-binding protein T-cell intracellular antigen-1 in esophageal squamous cell carcinoma.

Science.gov (United States)

Hamada, Junichi; Shoda, Katsutoshi; Masuda, Kiyoshi; Fujita, Yuji; Naruto, Takuya; Kohmoto, Tomohiro; Miyakami, Yuko; Watanabe, Miki; Kudo, Yasusei; Fujiwara, Hitoshi; Ichikawa, Daisuke; Otsuji, Eigo; Imoto, Issei

2016-03-29

T-cell intracellular antigen-1 (TIA1) is an RNA-binding protein involved in many regulatory aspects of mRNA metabolism. Here, we report previously unknown tumor-promoting activity of TIA1, which seems to be associated with its isoform-specific molecular distribution and regulation of a set of cancer-related transcripts, in esophageal squamous cell carcinoma (ESCC). Immunohistochemical overexpression of TIA1 ectopically localized in the cytoplasm of tumor cells was an independent prognosticator for worse overall survival in a cohort of 143 ESCC patients. Knockdown of TIA1 inhibited proliferation of ESCC cells. By exogenously introducing each of two major isoforms, TIA1a and TIA1b, only TIA1a, which was localized to both the nucleus and cytoplasm, promoted anchorage-dependent and anchorage-independent ESCC cell proliferation. Ribonucleoprotein immunoprecipitation, followed by microarray analysis or massive-parallel sequencing, identified a set of TIA1-binding mRNAs, including SKP2 and CCNA2. TIA1 increased SKP2 and CCNA2 protein levels through the suppression of mRNA decay and translational induction, respectively. Our findings uncover a novel oncogenic function of TIA1 in esophageal tumorigenesis, and implicate its use as a marker for prognostic evaluation and as a therapeutic target in ESCC.

Nucleation temperature-controlled synthesis and in vitro toxicity evaluation of L-cysteine-capped Mn:ZnS quantum dots for intracellular imaging.

Science.gov (United States)

Pandey, Vivek; Pandey, Gajanan; Tripathi, Vinay Kumar; Yadav, Sapna; Mudiam, Mohana Krishna Reddy

2016-03-01

Quantum dots (QDs), one of the fastest developing and most exciting fluorescent materials, have attracted increasing interest in bioimaging and biomedical applications. The long-term stability and emission in the visible region of QDs have proved their applicability as a significant fluorophore in cell labelling. In this study, an attempt has been made to explore the efficacy of L-cysteine as a capping agent for Mn-doped ZnS QD for intracellular imaging. A room temperature nucleation strategy was adopted to prepare non-toxic, water-dispersible and biocompatible Mn:ZnS QDs. Aqueous and room temperature QDs with L-cysteine as a capping agent were found to be non-toxic even at a concentration of 1500 µg/mL and have wide applications in intracellular imaging. Copyright © 2015 John Wiley & Sons, Ltd.

Analysis of Intracellular Metabolites from Microorganisms: Quenching and Extraction Protocols

Directory of Open Access Journals (Sweden)

Farhana R. Pinu

2017-10-01

Full Text Available Sample preparation is one of the most important steps in metabolome analysis. The challenges of determining microbial metabolome have been well discussed within the research community and many improvements have already been achieved in last decade. The analysis of intracellular metabolites is particularly challenging. Environmental perturbations may considerably affect microbial metabolism, which results in intracellular metabolites being rapidly degraded or metabolized by enzymatic reactions. Therefore, quenching or the complete stop of cell metabolism is a pre-requisite for accurate intracellular metabolite analysis. After quenching, metabolites need to be extracted from the intracellular compartment. The choice of the most suitable metabolite extraction method/s is another crucial step. The literature indicates that specific classes of metabolites are better extracted by different extraction protocols. In this review, we discuss the technical aspects and advancements of quenching and extraction of intracellular metabolite analysis from microbial cells.

Manipulation of Host Cholesterol by Obligate Intracellular Bacteria

Directory of Open Access Journals (Sweden)

Dhritiman Samanta

2017-05-01

Full Text Available Cholesterol is a multifunctional lipid that plays important metabolic and structural roles in the eukaryotic cell. Despite having diverse lifestyles, the obligate intracellular bacterial pathogens Chlamydia, Coxiella, Anaplasma, Ehrlichia, and Rickettsia all target cholesterol during host cell colonization as a potential source of membrane, as well as a means to manipulate host cell signaling and trafficking. To promote host cell entry, these pathogens utilize cholesterol-rich microdomains known as lipid rafts, which serve as organizational and functional platforms for host signaling pathways involved in phagocytosis. Once a pathogen gains entrance to the intracellular space, it can manipulate host cholesterol trafficking pathways to access nutrient-rich vesicles or acquire membrane components for the bacteria or bacteria-containing vacuole. To acquire cholesterol, these pathogens specifically target host cholesterol metabolism, uptake, efflux, and storage. In this review, we examine the strategies obligate intracellular bacterial pathogens employ to manipulate cholesterol during host cell colonization. Understanding how obligate intracellular pathogens target and use host cholesterol provides critical insight into the host-pathogen relationship.

A Thapsigargin-Resistant Intracellular Calcium Sequestering Compartment in Rat Brain

Science.gov (United States)

2000-03-31

have a major impact on neuronal intracellular signaling. Most of the ER in neurons and glia appears to accumulate calcium by energy driven ion pumps...secretion of exocrine, endocrine, and neurocrine products, regulation of glycogenolysis and gluconeogenesis , intracellular transport, secretion of fluids...the RyRs [140]. Furthermore, the intracellular expression of these receptor-channels in neuronal ER is also reciprocal with RyRs located primarily in

Glucocorticoids can affect Pseudomonas aeruginosa (ATCC 27853) internalization and intracellular calcium concentration in cystic fibrosis bronchial epithelial cells.

Science.gov (United States)

Hussain, Rashida; Shahror, Rami; Karpati, Ferenc; Roomans, Godfried M

2015-01-01

Glucocorticoids (GCs) are anti-inflammatory agents, but their use in cystic fibrosis (CF) is controversial. In CF, the early colonization with Pseudomonas aeruginosa is mainly due to nonmucoid strains that can internalize, and induce apoptosis in the epithelial cells. Uptake of P. aeruginosa by the epithelial cells and subsequent apoptosis may prevent colonization of P. aeruginosa in CF airways. In the airway epithelia, several other biological effects, including an anti-secretory role by decreasing intracellular Ca(2+) concentration have been described for this anti-inflammatory drug. However, the effects of GCs on the nonmucoid P. aeruginosa internalization and intracellular Ca(2+) in CF bronchial epithelial cells have not been evaluated. We used cultured human CF bronchial airway epithelial cell (CFBE) monolayers to determine P. aeruginosa internalization, apoptosis, and intracellular Ca(2+)concentration in CF bronchial epithelial cells. Cells were treated with IL-6, IL-8, dexamethasone, betamethasone, or budesonide. GCs in co-treatments with IL-6 reversed the effect of IL-6 by decreasing the internalization of P. aeruginosa in the CFBE cells. GCs decreased the extent of apoptosis in CFBE cells infected with internalized P. aeruginosa, and increased the intracellular Ca(2+) concentration. These findings suggest that if internalization of P. aeruginosa reduces infection, GC therapy would increase the risk of pulmonary infection by decreasing the internalization of P. aeruginosa in CF cells, but GCs may improve airway hydration by increasing the intracellular Ca(2+) concentration. Whether the benefits of GC treatment outweigh the negative effects is questionable, and further clinical studies need to be carried out.

A general mechanism for intracellular toxicity of metal-containing nanoparticles

KAUST Repository

Sabella, Stefania

2014-04-09

The assessment of the risks exerted by nanoparticles is a key challenge for academic, industrial, and regulatory communities worldwide. Experimental evidence points towards significant toxicity for a range of nanoparticles both in vitro and in vivo. Worldwide efforts aim at uncovering the underlying mechanisms for this toxicity. Here, we show that the intracellular ion release elicited by the acidic conditions of the lysosomal cellular compartment-where particles are abundantly internalized-is responsible for the cascading events associated with nanoparticles-induced intracellular toxicity. We call this mechanism a "lysosome-enhanced Trojan horse effect" since, in the case of nanoparticles, the protective cellular machinery designed to degrade foreign objects is actually responsible for their toxicity. To test our hypothesis, we compare the toxicity of similar gold particles whose main difference is in the internalization pathways. We show that particles known to pass directly through cell membranes become more toxic when modified so as to be mostly internalized by endocytosis. Furthermore, using experiments with chelating and lysosomotropic agents, we found that the toxicity mechanism for different metal containing NPs (such as metallic, metal oxide, and semiconductor NPs) is mainly associated with the release of the corresponding toxic ions. Finally, we show that particles unable to release toxic ions (such as stably coated NPs, or diamond and silica NPs) are not harmful to intracellular environments. The Royal Society of Chemistry 2014.

A general mechanism for intracellular toxicity of metal-containing nanoparticles

KAUST Repository

Sabella, Stefania; Carney, Randy P.; Brunetti, Virgilio; Malvindi, Maria Ada; Al-Juffali, Noura; Vecchio, Giuseppe; Janes, Sam M.; Bakr, Osman; Cingolani, Roberto; Stellacci, Francesco; Pompa, Pier Paolo

2014-01-01

The assessment of the risks exerted by nanoparticles is a key challenge for academic, industrial, and regulatory communities worldwide. Experimental evidence points towards significant toxicity for a range of nanoparticles both in vitro and in vivo. Worldwide efforts aim at uncovering the underlying mechanisms for this toxicity. Here, we show that the intracellular ion release elicited by the acidic conditions of the lysosomal cellular compartment-where particles are abundantly internalized-is responsible for the cascading events associated with nanoparticles-induced intracellular toxicity. We call this mechanism a "lysosome-enhanced Trojan horse effect" since, in the case of nanoparticles, the protective cellular machinery designed to degrade foreign objects is actually responsible for their toxicity. To test our hypothesis, we compare the toxicity of similar gold particles whose main difference is in the internalization pathways. We show that particles known to pass directly through cell membranes become more toxic when modified so as to be mostly internalized by endocytosis. Furthermore, using experiments with chelating and lysosomotropic agents, we found that the toxicity mechanism for different metal containing NPs (such as metallic, metal oxide, and semiconductor NPs) is mainly associated with the release of the corresponding toxic ions. Finally, we show that particles unable to release toxic ions (such as stably coated NPs, or diamond and silica NPs) are not harmful to intracellular environments. The Royal Society of Chemistry 2014.

Resolution of intracellular calcium metabolism in intact segments of rabbit aorta

International Nuclear Information System (INIS)

Phair, R.D.; Hai, C.M.

1986-01-01

A new method, based on computer-assisted kinetic analysis of 45 Ca efflux data, was used to measure calcium contents and fluxes for extracellular and intracellular compartments in intact segments of rabbit aorta. After a 1-hour loading period, efflux data were collected for 8 hours using a flow-through tissue chamber. These long-term effluxes were necessary because information on intracellular calcium metabolism was concentrated in the slow components of the efflux curves while earlier components appeared to be dominated by washout of extracellular calcium. Intracellular compartments were identified as those whose calcium contents were altered by 10 microM phenylephrine. This method complements previous approaches by providing simultaneous estimates of compartmental calcium contents and fluxes without requiring the assumption of isotopic equilibrium and without recourse to standard wash techniques for removal of extracellular calcium. In normal, calcium-containing, bicarbonate-buffered physiological salt solution these compartments contained a total of approximately 300 nmol Ca/g wet aorta. Of this total, 55 nmol/g were associated with the slowest resolvable compartment whose turnover time was 170 minutes and whose exchange flux was 0.32 nmol min-1g-1. Two other intracellular compartments had turnover times of 30 minutes. One of these was phenylephrine releasable and contained 145 nmol/g; it exchanged calcium at 4.9 nmol min-1g-1. In normal physiological salt solution the plasma membrane was, surprisingly, not rate limiting for Ca efflux; and in 10 microM phenylephrine the membrane Ca flux was even greater, increasing 3.5-fold compared to control

Intracellular pH homeostasis in Leishmania donovani amastigotes and promastigotes

International Nuclear Information System (INIS)

Glaser, T.A.; Baatz, J.E.; Kreishman, G.P.; Mukkada, A.J.

1988-01-01

Intracellular pH and pH gradients of Leishmania donovani amastigotes and promastigotes were determined over a broad range of extracellular pH values. Intracellular pH was determined by 31 P NMR and by equilibrium distribution studies with 5,5-dimethyloxazolidine-2,4-dione or methylamine. Promastigotes maintain intracellular pH values close to neutral between extracellular pH values of 5.0 and 7.4. Amastigote intracellular pH is maintained close to neutral at external pH values as low as 4.0. Both life stages maintain a positive pH gradient to an extracellular pH of 7.4, which is important for active transport of substrates. Treatment with ionophores, such as nigericin and carbonyl cyanide m-chlorophenylhydrazone and the ATPase inhibitor dicyclohexylcarbodiimide, reduced pH gradients in both stages. Maintenance of intracellular pH in the physiologic range is especially relevant for the survival of the amastigote in its acidic in vivo environment

Directed antigen delivery as a vaccine strategy for an intracellular bacterial pathogen

Science.gov (United States)

Bouwer, H. G. Archie; Alberti-Segui, Christine; Montfort, Megan J.; Berkowitz, Nathan D.; Higgins, Darren E.

2006-03-01

We have developed a vaccine strategy for generating an attenuated strain of an intracellular bacterial pathogen that, after uptake by professional antigen-presenting cells, does not replicate intracellularly and is readily killed. However, after degradation of the vaccine strain within the phagolysosome, target antigens are released into the cytosol for endogenous processing and presentation for stimulation of CD8+ effector T cells. Applying this strategy to the model intracellular pathogen Listeria monocytogenes, we show that an intracellular replication-deficient vaccine strain is cleared rapidly in normal and immunocompromised animals, yet antigen-specific CD8+ effector T cells are stimulated after immunization. Furthermore, animals immunized with the intracellular replication-deficient vaccine strain are resistant to lethal challenge with a virulent WT strain of L. monocytogenes. These studies suggest a general strategy for developing safe and effective, attenuated intracellular replication-deficient vaccine strains for stimulation of protective immune responses against intracellular bacterial pathogens. CD8+ T cell | replication-deficient | Listeria monocytogenes

Clinical significance of Phosphatidyl Inositol Synthase overexpression in oral cancer

International Nuclear Information System (INIS)

Kaur, Jatinder; Sawhney, Meenakshi; DattaGupta, Siddartha; Shukla, Nootan K; Srivastava, Anurag; Ralhan, Ranju

2010-01-01

We reported increased levels of Phosphatidyl Inositol synthase (PI synthase), (enzyme that catalyses phosphatidyl inositol (PI) synthesis-implicated in intracellular signaling and regulation of cell growth) in smokeless tobacco (ST) exposed oral cell cultures by differential display. This study determined the clinical significance of PI synthase overexpression in oral squamous cell carcinoma (OSCC) and premalignant lesions (leukoplakia), and identified the downstream signaling proteins in PI synthase pathway that are perturbed by smokeless tobacco (ST) exposure. Tissue microarray (TMA) Immunohistochemistry, Western blotting, Confocal laser scan microscopy, RT-PCR were performed to define the expression of PI synthase in clinical samples and in oral cell culture systems. Significant increase in PI synthase immunoreactivity was observed in premalignant lesions and OSCCs as compared to oral normal tissues (p = 0.000). Further, PI synthase expression was significantly associated with de-differentiation of OSCCs, (p = 0.005) and tobacco consumption (p = 0.03, OR = 9.0). Exposure of oral cell systems to smokeless tobacco (ST) in vitro confirmed increase in PI synthase, Phosphatidylinositol 3-kinase (PI3K) and cyclin D1 levels. Collectively, increased PI synthase expression was found to be an early event in oral cancer and a target for smokeless tobacco

Intracellular Enzymes Contribution to the Biocatalytic Removal of Pharmaceuticals by Trametes hirsuta.

Science.gov (United States)

Haroune, Lounès; Saibi, Sabrina; Cabana, Hubert; Bellenger, Jean-Philippe

2017-01-17

The use of white rot fungi (WRF) for bioremediation of recalcitrant trace organic contaminants (TrOCs) is becoming greatly popular. Biosorption and lignin modifying enzymes (LMEs) are the most often reported mechanisms of action. Intracellular enzymes, such as cytochrome P450 (CYP450), have also been suggested to contribute. However, direct evidence of TrOCs uptake and intracellular transformation is lacking. The aim of this study was to evaluate the relative contribution of biosorption, extracellular LMEs activity, TrOCs uptake, and intracellular CYP450 on the removal of six nonsteroidal anti-inflammatories (NSAIs) by Trametes hirsuta. Results show that for most tested NSAIs, LMEs activity and biosorption failed to explain the observed removal. Most tested TrOCs are quickly taken up and intracellularly transformed. Fine characterization of intracellular transformation using ketoprofen showed that CYP450 is not the sole intracellular enzyme responsible for intracellular transformation. The contribution of CYP450 in further transformation of ketoprofen byproducts is also reported. These results illustrate that TrOCs transformation by WRF is a more complex process than previously reported. Rapid uptake of TrOCs and intracellular transformation through diverse enzymatic systems appears to be important components of WRF efficiency toward TrOCs.

Protection of Dentate Hilar Cells from Prolonged Stimulation by Intracellular Calcium Chelation

Science.gov (United States)

Scharfman, Helen E.; Schwartzkroin, Philip A.

1989-10-01

Prolonged afferent stimulation of the rat dentate gyrus in vivo leads to degeneration only of those cells that lack immunoreactivity for the calcium binding proteins parvalbumin and calbindin. In order to test the hypothesis that calcium binding proteins protect against the effects of prolonged stimulation, intracellular recordings were made in hippocampal slices from cells that lack immunoreactivity for calcium binding proteins. Calcium binding protein--negative cells showed electrophysiological signs of deterioration during prolonged stimulation; cells containing calcium binding protein did not. When neurons without calcium binding proteins were impaled with microelectrodes containing the calcium chelator BAPTA, and BAPTA was allowed to diffuse into the cells, these cells showed no deterioration. These results indicate that, in a complex tissue of the central nervous system, an activity-induced increase in intracellular calcium can trigger processes leading to cell deterioration, and that increasing the calcium binding capacity of a cell decreases its vulnerability to damage.

Mitofusin2 decreases intracellular cholesterol of oxidized LDL-induced foam cells from rat vascular smooth muscle cells.

Science.gov (United States)

He, Chao; Chen, Ying; Liu, Chun; Cao, Ming; Fan, Yu-jin; Guo, Xiao-mei

2013-04-01

Mitofusin2 (Mfn2) plays a pivotal role in the proliferation and apoptosis of vascular smooth muscle cells (VSMCs). The purpose of this study was to investigate the effects of Mfn2 on the trafficking of intracellular cholesterol in the foam cells derived from rat VSMCs (rVSMCs) and also to investigate the effects of Mfn2 on the expression of adenosine triphosphate-binding cassette subfamily A member 1 (ABCA1), adenosine triphosphate-binding cassette subfamily G member 1 (ABCG1) and peroxisome proliferator-activated receptor gamma (PPARγ). The rVSMCs were co-cultured with oxidized low density lipoprotein (LDL, 80 μg/mL) to produce foam cells and cholesterol accumulation in cells. Before oxidized LDL treatment, different titers (20, 40 and 60 pfu/cell) of recombinant adenovirus containing Mfn2 gene (Adv-Mfn2) were added into the culture medium for 24 h to transfect the Mfn2 gene into the rVSMCs. Then the cells were harvested for analyses. The protein expression of Mfn2 was significantly higher in Adv-Mfn2-transfected group than in untransfected group (PLDL treatment, rVSMCs became irregular and their nuclei became larger, and their plasma abounded with red lipid droplets. However, the number of red lipid droplets was significantly decreased in Adv-Mfn2-transfected group as compared with untransfected group. At 48 h after oxidized LDL treatment, the intracellular cholesterol in rVSMCs was significantly increased (P0.05), the phosporylation levels of PPARγ were significantly decreased in Adv-Mfn2-transfected group as compared with untransfected group (Pcholesterol in oxidized LDL-induced rVSMCs possibly by decreasing PPARγ phosporylation and then increasing protein expression levels of ABCA1 and ABCG1, which may be helpful to suppress the formation of foam cells.

Ortholog-based screening and identification of genes related to intracellular survival.

Science.gov (United States)

Yang, Xiaowen; Wang, Jiawei; Bing, Guoxia; Bie, Pengfei; De, Yanyan; Lyu, Yanli; Wu, Qingmin

2018-04-20

Bioinformatics and comparative genomics analysis methods were used to predict unknown pathogen genes based on homology with identified or functionally clustered genes. In this study, the genes of common pathogens were analyzed to screen and identify genes associated with intracellular survival through sequence similarity, phylogenetic tree analysis and the λ-Red recombination system test method. The total 38,952 protein-coding genes of common pathogens were divided into 19,775 clusters. As demonstrated through a COG analysis, information storage and processing genes might play an important role intracellular survival. Only 19 clusters were present in facultative intracellular pathogens, and not all were present in extracellular pathogens. Construction of a phylogenetic tree selected 18 of these 19 clusters. Comparisons with the DEG database and previous research revealed that seven other clusters are considered essential gene clusters and that seven other clusters are associated with intracellular survival. Moreover, this study confirmed that clusters screened by orthologs with similar function could be replaced with an approved uvrY gene and its orthologs, and the results revealed that the usg gene is associated with intracellular survival. The study improves the current understanding of intracellular pathogens characteristics and allows further exploration of the intracellular survival-related gene modules in these pathogens. Copyright © 2018. Published by Elsevier B.V.

DMPD: Intracellular DNA sensors in immunity. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 18573338 Intracellular DNA sensors in immunity. Takeshita F, Ishii KJ. Curr Opin Im...munol. 2008 Aug;20(4):383-8. Epub 2008 Jun 23. (.png) (.svg) (.html) (.csml) Show Intracellular DNA sensors ...in immunity. PubmedID 18573338 Title Intracellular DNA sensors in immunity. Authors Takeshita F, Ishii KJ. P

Stochastic models of intracellular transport

KAUST Repository

Bressloff, Paul C.; Newby, Jay M.

2013-01-01

mechanisms for intracellular transport: passive diffusion and motor-driven active transport. Diffusive transport can be formulated in terms of the motion of an overdamped Brownian particle. On the other hand, active transport requires chemical energy, usually

Activation of a Neospora caninum EGFR-Like Kinase Facilitates Intracellular Parasite Proliferation

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Xiaoxia Jin

2017-10-01

Full Text Available The Apicomplexan parasite Neospora caninum, an obligate intracellular protozoan, causes serious diseases in a number of mammalian species, especially in cattle. Infection with N. caninum is associated with abortions in both dairy and beef cattle worldwide which have a major economic impact on the cattle industry. However, the mechanism by which N. caninum proliferates within host cells is poorly understood. Epidermal growth factor receptor (EGFR is a protein kinase ubiquitously expressed, present on cell surfaces in numerous species, which has been confirmed to be essential in signal transduction involved in cell growth, proliferation, survival, and many other intracellular processes. However, the presence of EGFR in N. caninum and its role in N. caninum proliferation remain unclear. In the present study, we identified a putative EGFR-like kinase in N. caninum, which could be activated in tachyzoites by infection or treatment with rNcMIC3 [containing four epidermal growth factor (EGF domains] or human EGF. Blockade of EGFR-like in tachyzoites by AG1478 significantly reduced parasite proliferation in host cells. Our data suggested that the activation of tachyzoite EGFR-like might facilitate the intracellular proliferation of N. caninum.

Intracellular chromium localization and cell physiological response in the unicellular alga Micrasterias

Science.gov (United States)

Volland, Stefanie; Lütz, Cornelius; Michalke, Bernhard; Lütz-Meindl, Ursula

2012-01-01

Various contaminants like metals and heavy metals are constantly released into the environment by anthropogenic activities. The heavy metal chromium has a wide industrial use and exists in two stable oxidation states: trivalent and hexavalent. Chromium can cause harm to cell metabolism and development, when it is taken up by plants instead of necessary micronutrients such as for example iron. The uptake of Cr VI into plant cells has been reported to be an active process via carriers of essential anions, while the cation Cr III seems to be taken up inactively. Micrasterias denticulata, an unicellular green alga of the family Desmidiaceae is a well-studied cell biological model organism. Cr III and VI had inhibiting effects on its cell development, while cell division rates were only impaired by Cr VI. Transmission electron microscopy (TEM) revealed ultrastructural changes such as increased vacuolization, condensed cytoplasm and dark precipitations in the cell wall after 3 weeks of Cr VI treatment. Electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) were applied to measure intracellular chromium distribution. Chromium was only detected after 3 weeks of 10 μM Cr VI treatment in electron dense precipitations found in bag-like structures along the inner side of the cell walls together with iron and elevated levels of oxygen, pointing toward an accumulation respectively extrusion of chromium in form of an iron–oxygen compound. Atomic emission spectroscopy (EMS) revealed that Micrasterias cells are able to accumulate considerable amounts of chromium and iron. During chromium treatment the Cr:Fe ratio shifted in favor of chromium, which implied that chromium may be taken up instead of iron. Significant and rapid increase of ROS production within the first 5 min of treatment confirms an active Cr VI uptake. SOD and CAT activity after Cr VI treatment did not show a response, while the glutathione pool determined by immuno-TEM decreased

Intracellular chromium localization and cell physiological response in the unicellular alga Micrasterias

Energy Technology Data Exchange (ETDEWEB)

Volland, Stefanie, E-mail: Stefanie.Volland@stud.sbg.ac.at [Plant Physiology Division, Cell Biology Department, University of Salzburg, Hellbrunnerstr 34, 5020 Salzburg (Austria); Luetz, Cornelius, E-mail: cornelius.luetz@uibk.ac.at [Institute of Botany, University of Innsbruck, Sternwartestrasse 15, 6020 Innsbruck (Austria); Michalke, Bernhard, E-mail: bernhard.michalke@helmholtz-muenchen.de [Helmholtz Zentrum Muenchen, German Research Centre for Environmental Health, Institute of Ecological Chemistry, Ingolstaedter Landstrasse 1, 85764 Neuherberg (Germany); Luetz-Meindl, Ursula, E-mail: ursula.luetz-meindl@sbg.ac.at [Plant Physiology Division, Cell Biology Department, University of Salzburg, Hellbrunnerstr 34, 5020 Salzburg (Austria)

2012-03-15

Various contaminants like metals and heavy metals are constantly released into the environment by anthropogenic activities. The heavy metal chromium has a wide industrial use and exists in two stable oxidation states: trivalent and hexavalent. Chromium can cause harm to cell metabolism and development, when it is taken up by plants instead of necessary micronutrients such as for example iron. The uptake of Cr VI into plant cells has been reported to be an active process via carriers of essential anions, while the cation Cr III seems to be taken up inactively. Micrasterias denticulata, an unicellular green alga of the family Desmidiaceae is a well-studied cell biological model organism. Cr III and VI had inhibiting effects on its cell development, while cell division rates were only impaired by Cr VI. Transmission electron microscopy (TEM) revealed ultrastructural changes such as increased vacuolization, condensed cytoplasm and dark precipitations in the cell wall after 3 weeks of Cr VI treatment. Electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) were applied to measure intracellular chromium distribution. Chromium was only detected after 3 weeks of 10 {mu}M Cr VI treatment in electron dense precipitations found in bag-like structures along the inner side of the cell walls together with iron and elevated levels of oxygen, pointing toward an accumulation respectively extrusion of chromium in form of an iron-oxygen compound. Atomic emission spectroscopy (EMS) revealed that Micrasterias cells are able to accumulate considerable amounts of chromium and iron. During chromium treatment the Cr:Fe ratio shifted in favor of chromium, which implied that chromium may be taken up instead of iron. Significant and rapid increase of ROS production within the first 5 min of treatment confirms an active Cr VI uptake. SOD and CAT activity after Cr VI treatment did not show a response, while the glutathione pool determined by immuno-TEM decreased

Intracellular chromium localization and cell physiological response in the unicellular alga Micrasterias

International Nuclear Information System (INIS)

Volland, Stefanie; Lütz, Cornelius; Michalke, Bernhard; Lütz-Meindl, Ursula

2012-01-01

Various contaminants like metals and heavy metals are constantly released into the environment by anthropogenic activities. The heavy metal chromium has a wide industrial use and exists in two stable oxidation states: trivalent and hexavalent. Chromium can cause harm to cell metabolism and development, when it is taken up by plants instead of necessary micronutrients such as for example iron. The uptake of Cr VI into plant cells has been reported to be an active process via carriers of essential anions, while the cation Cr III seems to be taken up inactively. Micrasterias denticulata, an unicellular green alga of the family Desmidiaceae is a well-studied cell biological model organism. Cr III and VI had inhibiting effects on its cell development, while cell division rates were only impaired by Cr VI. Transmission electron microscopy (TEM) revealed ultrastructural changes such as increased vacuolization, condensed cytoplasm and dark precipitations in the cell wall after 3 weeks of Cr VI treatment. Electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) were applied to measure intracellular chromium distribution. Chromium was only detected after 3 weeks of 10 μM Cr VI treatment in electron dense precipitations found in bag-like structures along the inner side of the cell walls together with iron and elevated levels of oxygen, pointing toward an accumulation respectively extrusion of chromium in form of an iron–oxygen compound. Atomic emission spectroscopy (EMS) revealed that Micrasterias cells are able to accumulate considerable amounts of chromium and iron. During chromium treatment the Cr:Fe ratio shifted in favor of chromium, which implied that chromium may be taken up instead of iron. Significant and rapid increase of ROS production within the first 5 min of treatment confirms an active Cr VI uptake. SOD and CAT activity after Cr VI treatment did not show a response, while the glutathione pool determined by immuno-TEM decreased

Downregulation of transferrin receptor surface expression by intracellular antibody

International Nuclear Information System (INIS)

Peng Jilin; Wu Sha; Zhao Xiaoping; Wang Min; Li Wenhan; Shen Xin; Liu Jing; Lei Ping; Zhu Huifen; Shen Guanxin

2007-01-01

To deplete cellular iron uptake, and consequently inhibit the proliferation of tumor cells, we attempt to block surface expression of transferrin receptor (TfR) by intracellular antibody technology. We constructed two expression plasmids (scFv-HAK and scFv-HA) coding for intracellular single-chain antibody against TfR with or without endoplasmic reticulum (ER) retention signal, respectively. Then they were transfected tumor cells MCF-7 by liposome. Applying RT-PCR, Western blotting, immunofluorescence microscopy and immunoelectron microscope experiments, we insure that scFv-HAK intrabody was successfully expressed and retained in ER contrasted to the secreted expression of scFv-HA. Flow cytometric analysis confirmed that the TfR surface expression was markedly decreased approximately 83.4 ± 2.5% in scFv-HAK transfected cells, while there was not significantly decrease in scFv-HA transfected cells. Further cell growth and apoptosis characteristics were evaluated by cell cycle analysis, nuclei staining and MTT assay. Results indicated that expression of scFv-HAK can dramatically induce cell cycle G1 phase arrest and apoptosis of tumor cells, and consequently significantly suppress proliferation of tumor cells compared with other control groups. For First time this study demonstrates the potential usage of anti-TfR scFv-intrabody as a growth inhibitor of TfR overexpressing tumors

Investigating Internalization and Intracellular Trafficking of GPCRs

DEFF Research Database (Denmark)

Foster, Simon R; Bräuner-Osborne, Hans

2017-01-01

for signal transduction. One of the major mechanisms for GPCR regulation involves their endocytic trafficking, which serves to internalize the receptors from the plasma membrane and thereby attenuate G protein-dependent signaling. However, there is accumulating evidence to suggest that GPCRs can signal...... independently of G proteins, as well as from intracellular compartments including endosomes. It is in this context that receptor internalization and intracellular trafficking have attracted renewed interest within the GPCR field. In this chapter, we will review the current understanding and methodologies...

Gelsolin-Cu/ZnSOD interaction alters intracellular reactive oxygen species levels to promote cancer cell invasion

KAUST Repository

Tochhawng, Lalchhandami

2016-07-07

The actin-binding protein, gelsolin, is a well known regulator of cancer cell invasion. However, the mechanisms by which gelsolin promotes invasion are not well established. As reactive oxygen species (ROS) have been shown to promote cancer cell invasion, we investigated on the hypothesis that gelsolin-induced changes in ROS levels may mediate the invasive capacity of colon cancer cells. Herein, we show that increased gelsolin enhances the invasive capacity of colon cancer cells, and this is mediated via gelsolin\\'s effects in elevating intracellular superoxide (O2 .-) levels. We also provide evidence for a novel physical interaction between gelsolin and Cu/ZnSOD, that inhibits the enzymatic activity of Cu/ZnSOD, thereby resulting in a sustained elevation of intracellular O2 .-. Using microarray data of human colorectal cancer tissues from Gene Omnibus, we found that gelsolin gene expression positively correlates with urokinase plasminogen activator (uPA), an important matrix-degrading protease invovled in cancer invasion. Consistent with the in vivo evidence, we show that increased levels of O2 .- induced by gelsolin overexpression triggers the secretion of uPA. We further observed reduction in invasion and intracellular O2 .- levels in colon cancer cells, as a consequence of gelsolin knockdown using two different siRNAs. In these cells, concurrent repression of Cu/ ZnSOD restored intracellular O2 .- levels and rescued invasive capacity. Our study therefore identified gelsolin as a novel regulator of intracellular O2 .- in cancer cells via interacting with Cu/ZnSOD and inhibiting its enzymatic activity. Taken together, these findings provide insight into a novel function of gelsolin in promoting tumor invasion by directly impacting the cellular redox milieu.

The role of intracellular redox imbalance in nanomaterial induced cellular damage and genotoxicity

DEFF Research Database (Denmark)

Kermanizadeh, Ali; Chauché, Caroline; Brown, David M

2015-01-01

The terms oxidative stress, free radical generation, and intracellular antioxidant protection have become part of everyday nanotoxicology terminology. In recent years, an ever increasing number of in vitro and in vivo studies have implicated disruptions to the redox balance and oxidative stress...

Optochemokine Tandem for Light-Control of Intracellular Ca2.

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Katrin Feldbauer

Full Text Available An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-gated Ca2+-permeable cation channel Channelrhodopsin-2(L132C, CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signaling pathway. The kinetics of internalization could be followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca2+ by tandem endosomes into the cytosol via CatCh was visualized using the Ca2+-sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca2+ in response to light.

In Vitro Evaluation of the Impact of the Probiotic E. coli Nissle 1917 on Campylobacter jejuni's Invasion and Intracellular Survival in Human Colonic Cells.

Science.gov (United States)

Helmy, Yosra A; Kassem, Issmat I; Kumar, Anand; Rajashekara, Gireesh

2017-01-01

Campylobacter jejuni is a leading cause of bacterial food poisoning in humans. Due to the rise in antibiotic-resistant Campylobacter , there exists a need to develop antibiotic-independent interventions to control infections in humans. Here, we evaluated the impact of Escherichia coli Nissle 1917 (EcN), a probiotic strain, on C. jejuni's invasion and intracellular survival in polarized human colonic cells (HT-29). To further understand how EcN mediates its impact, the expression of 84 genes associated with tight junctions and cell adhesion was profiled in HT-29 cells after treatment with EcN and challenge with C. jejuni . The pre-treatment of polarized HT-29 cells with EcN for 4 h showed a significant effect on C. jejuni 's invasion (∼2 log reduction) of the colonic cells. Furthermore, no intracellular C. jejuni were recovered from EcN pre-treated HT-29 cells at 24 h post-infection. Other probiotic strains tested had no significant impact on C. jejuni invasion and intracellular survival. C. jejuni decreased the expression of genes associated with epithelial cells permeability and barrier function in untreated HT-29 cells. However, EcN positively affected the expression of genes that are involved in enhanced intestinal barrier function, decreased cell permeability, and increased tight junction integrity. The results suggest that EcN impedes C. jejuni invasion and subsequent intracellular survival by affecting HT-29 cells barrier function and tight junction integrity. We conclude that EcN might be a viable alternative for controlling C. jejuni infections.

Intracellular localisation of proteins to specific cellular areas by nanocapsule mediated delivery.

Science.gov (United States)

Wang, Huabin; Chen, Ligang; Sun, Xianchao; Fu, Ailing

2017-09-01

Nanocapsules are promising carriers with great potential for intracellular protein transport. Although many studies have intended to improve cell uptake efficacy, there is an increasing interest in understanding of subcellular distribution of cargoes inside cells, which is essential for purposeful delivery of biomolecules into specific sites within cells. Herein, we interrogate the intracellular localisation of exogenous proteins, including fluorescein isothiocyanate (FITC)-labelled bovine serum albumin (BSA) and green fluorescent protein (GFP), mediated by specially designed nanocapsules. The results show that the designed nanocapsules can deliver the two types of fluorescent proteins into different cellular destinations (cytosol, nucleus or the whole cell), depending on the composition of nanocapsules. Meanwhile, several impact factors that influence the distribution of proteins in cells have also been investigated, and the results suggest that the localisation of capsule-mediated proteins in cells is strongly affected by the surface properties of nanocapsules, the types of stabilisers and proteins, and environmental temperatures. The rational control of intracellular localised delivery of exogenous proteins as we demonstrated in this study might open new avenues to obtain desired magnitude of drug effects for modulating cell activity.

Curcumin Mitigates the Intracellular Lipid Deposit Induced by Antipsychotics In Vitro.

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Alberto Canfrán-Duque

Full Text Available First- and second-generation antipsychotics (FGAs and SGAs, respectively, both inhibit cholesterol biosynthesis and impair the intracellular cholesterol trafficking, leading to lipid accumulation in the late endosome/lysosome compartment. In this study we examined if curcumin, a plant polyphenol that stimulates exosome release, can alleviate antipsychotic-induced intracellular lipid accumulation.HepG2 hepatocarcinoma cells were treated with antipsychotics or placebo and DiI-labelled LDL for 18 h and then exposed to curcumin for the last 2 h. Cells and media were collected separately and used for biochemical analyses, electron microscopy and immunocytochemistry. Exosomes were isolated from the incubation medium by ultracentrifugation.Curcumin treatment reduced the number of heterolysosomes and shifted their subcellular localization to the periphery, as revealed by electron microscopy, and stimulated the release of lysosomal β-hexosaminidase and exosome markers flotillin-2 and CD63 into the media. The presence of DiI in exosomes released by cells preloaded with DiI-LDL demonstrated the endolysosomal origin of the microvesicles. Furthermore, curcumin increased the secretion of cholesterol as well as LDL-derived DiI and [3H]-cholesterol, in association with a decrease of intracellular lipids. Thus, the disruption of lipid trafficking induced by FGAs or SGAs can be relieved by curcumin treatment. This polyphenol, however, did not mitigate the reduction of cholesterol esterification induced by antipsychotics.Curcumin stimulates exosome release to remove cholesterol (and presumably other lipids accumulated within the endolysosomal compartment, thereby normalizing intracellular lipid homeostasis. This action may help minimize the adverse metabolic effects of antipsychotic treatment, which should now be evaluated in clinical trials.

The epithelial cell cytoskeleton and intracellular trafficking. I. Shiga toxin B-subunit system: retrograde transport, intracellular vectorization, and more.

Science.gov (United States)

Johannes, Ludger

2002-07-01

Many intracellular transport routes are still little explored. This is particularly true for retrograde transport between the plasma membrane and the endoplasmic reticulum. Shiga toxin B subunit has become a powerful tool to study this pathway, and recent advances on the molecular mechanisms of transport in the retrograde route and on its physiological function(s) are summarized. Furthermore, it is discussed how the study of retrograde transport of Shiga toxin B subunit allows one to design new methods for the intracellular delivery of therapeutic compounds.

Allyl Isothiocyanate Inhibits Actin-Dependent Intracellular Transport in Arabidopsis thaliana

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Bjørnar Sporsheim

2015-12-01

Full Text Available Volatile allyl isothiocyanate (AITC derives from the biodegradation of the glucosinolate sinigrin and has been associated with growth inhibition in several plants, including the model plant Arabidopsis thaliana. However, the underlying cellular mechanisms of this feature remain scarcely investigated in plants. In this study, we present evidence of an AITC-induced inhibition of actin-dependent intracellular transport in A. thaliana. A transgenic line of A. thaliana expressing yellow fluorescent protein (YFP-tagged actin filaments was used to show attenuation of actin filament movement by AITC. This appeared gradually in a time- and dose-dependent manner and resulted in actin filaments appearing close to static. Further, we employed four transgenic lines with YFP-fusion proteins labeling the Golgi apparatus, endoplasmic reticulum (ER, vacuoles and peroxisomes to demonstrate an AITC-induced inhibition of actin-dependent intracellular transport of or, in these structures, consistent with the decline in actin filament movement. Furthermore, the morphologies of actin filaments, ER and vacuoles appeared aberrant following AITC-exposure. However, AITC-treated seedlings of all transgenic lines tested displayed morphologies and intracellular movements similar to that of the corresponding untreated and control-treated plants, following overnight incubation in an AITC-absent environment, indicating that AITC-induced decline in actin-related movements is a reversible process. These findings provide novel insights into the cellular events in plant cells following exposure to AITC, which may further expose clues to the physiological significance of the glucosinolate-myrosinase system.

Pulsed electromagnetic fields promote the proliferation and differentiation of osteoblasts by reinforcing intracellular calcium transients.

Science.gov (United States)

Tong, Jie; Sun, Lijun; Zhu, Bin; Fan, Yun; Ma, Xingfeng; Yu, Liyin; Zhang, Jianbao

2017-10-01

Pulsed electromagnetic fields (PEMF) can be used to treat bone-related diseases, but the underlying mechanism remains unclear, especially the process by which PEMFs initiate biological effects. In this study, we demonstrated the effects of PEMF on proliferation and differentiation of osteoblasts using the model of calcium transients induced by high extracellular calcium. Our results showed that PEMF can increase both the percentage of responding cells and amplitude of intracellular calcium transients induced by high extracellular calcium stimulation. Compared with corresponding extracellular calcium levels, PEMF stimulation increased proliferation and differentiation of osteoblasts and related gene expressions, such as insulin-like growth factor 1 (IGF-1), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteocalcin (OCN), which can be completely abolished by BAPTA-AM. Moreover, PEMF did not affect proliferation and differentiation of osteoblasts if no intracellular calcium transient was present in osteoblasts during PEMF exposure. Our results revealed that PEMF affects osteoblast proliferation and differentiation through enhanced intracellular calcium transients, which provided a cue to treat bone-related diseases with PEMF. Bioelectromagnetics. 38:541-549, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Dual Targeting of Intracellular Pathogenic Bacteria with a Cleavable Conjugate of Kanamycin and an Antibacterial Cell-Penetrating Peptide.

Science.gov (United States)

Brezden, Anna; Mohamed, Mohamed F; Nepal, Manish; Harwood, John S; Kuriakose, Jerrin; Seleem, Mohamed N; Chmielewski, Jean

2016-08-31

Bacterial infection caused by intracellular pathogens, such as Mycobacterium, Salmonella, and Brucella, is a burgeoning global health epidemic that necessitates urgent action. However, the therapeutic value of a number of antibiotics, including aminoglycosides, against intracellular pathogenic bacteria is compromised due to their inability to traverse eukaryotic membranes. For this significant problem to be addressed, a cleavable conjugate of the antibiotic kanamycin and a nonmembrane lytic, broad-spectrum antimicrobial peptide with efficient mammalian cell penetration, P14LRR, was prepared. This approach allows kanamycin to enter mammalian cells as a conjugate linked via a tether that breaks down in the reducing environment within cells. Potent antimicrobial activity of the P14KanS conjugate was demonstrated in vitro, and this reducible conjugate effectively cleared intracellular pathogenic bacteria within macrophages more potently than that of a conjugate lacking the disulfide moiety. Notably, successful clearance of Mycobacterium tuberculosis within macrophages was observed with the dual antibiotic conjugate, and Salmonella levels were significantly reduced in an in vivo Caenorhabditis elegans model.

Honey bee dopamine and octopamine receptors linked to intracellular calcium signaling have a close phylogenetic and pharmacological relationship.

Directory of Open Access Journals (Sweden)

Kyle T Beggs

Full Text Available BACKGROUND: Three dopamine receptor genes have been identified that are highly conserved among arthropod species. One of these genes, referred to in honey bees as Amdop2, shows a close phylogenetic relationship to the a-adrenergic-like octopamine receptor family. In this study we examined in parallel the functional and pharmacological properties of AmDOP2 and the honey bee octopamine receptor, AmOA1. For comparison, pharmacological properties of the honey bee dopamine receptors AmDOP1 and AmDOP3, and the tyramine receptor AmTYR1, were also examined. METHODOLOGY/PRINCIPAL FINDINGS: Using HEK293 cells heterologously expressing honey bee biogenic amine receptors, we found that activation of AmDOP2 receptors, like AmOA1 receptors, initiates a rapid increase in intracellular calcium levels. We found no evidence of calcium signaling via AmDOP1, AmDOP3 or AmTYR1 receptors. AmDOP2- and AmOA1-mediated increases in intracellular calcium were inhibited by 10 µM edelfosine indicating a requirement for phospholipase C-β activity in this signaling pathway. Edelfosine treatment had no effect on AmDOP2- or AmOA1-mediated increases in intracellular cAMP. The synthetic compounds mianserin and epinastine, like cis-(Z-flupentixol and spiperone, were found to have significant antagonist activity on AmDOP2 receptors. All 4 compounds were effective antagonists also on AmOA1 receptors. Analysis of putative ligand binding sites offers a possible explanation for why epinastine acts as an antagonist at AmDOP2 receptors, but fails to block responses mediated via AmDOP1. CONCLUSIONS/SIGNIFICANCE: Our results indicate that AmDOP2, like AmOA1, is coupled not only to cAMP, but also to calcium-signalling and moreover, that the two signalling pathways are independent upstream of phospholipase C-β activity. The striking similarity between the pharmacological properties of these 2 receptors suggests an underlying conservation of structural properties related to receptor

New perspective in the assessment of total intracellular magnesium

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Azzurra Sargenti

2014-01-01

Full Text Available Magnesium (Mg is essential for biological processes, but its cellular homeostasis has not been thoroughly elucidated, mainly because of the inadequacy of the available techniques to map intracellular Mg distribution. Recently, particular interest has been raised by a new family of fluorescent probes, diaza-18-crown-hydroxyquinoline (DCHQ, that shows remarkably high affinity and specificity for Mg, thus permitting the detection of the total intracellular Mg. The data obtained by fluori- metric and cytofluorimetric assays performed with DCHQ5 are in good agreement with atomic absorption spectroscopy, confirming that DCHQ5 probe allows both qualitative and quantitative determination of total intracellular Mg.

Glycine facilitates gamma-glutamylcysteinylethyl ester-mediated increase in liver glutathione level.

Science.gov (United States)

Nishida, K; Ohta, Y; Ishiguro, I

1997-08-27

gamma-Glutamylcysteinylethyl ester (gamma-GCE) increases reduced glutathione (GSH) levels in GSH-depleted rat hepatocytes. Because glycine, a constituent of GSH, exists at 0.3 to 0.4 mM in rat plasma, we examined the influence of glycine added to the medium on the action of gamma-GCE to increase GSH levels in the rat hepatocytes. Glycine (0.2-0.8 mM) dose-dependently enhanced gamma-GCE-mediated increase in intracellular GSH levels with an increase in intracellular gamma-GCE levels. These results indicate that exogenous glycine facilitates gamma-GCE-mediated increase in intracellular GSH levels in rat hepatocytes possibly by enhancing the uptake of gamma-GCE into the cells.

Intracellular Na+ regulation of Na+ pump sites in cultured vascular smooth muscle cells

International Nuclear Information System (INIS)

Allen, J.C.; Navran, S.S.; Seidel, C.L.; Dennison, D.K.; Amann, J.M.; Jemelka, S.K.

1989-01-01

Enzymatically dispersed cells from canine saphenous vein and femoral artery were grown in fetal calf serum and studied at day 0 (freshly dispersed) through confluence in primary culture. Intracellular Na levels (Nai), but not intracellular K (Ki), were increased after 24 h in culture and then decreased to a steady state by 4 days. Na+ pump site number [( 3 H] ouabain binding) increased through day 3 and remained elevated. Nai was still elevated at 2 days when the Na+ pump site number began to increase. Total pump turnover (maximum ouabain-inhibited 86 Rb uptake) reflected the increase in Na+ pump site number. These key events precede the observed increases in both protein production and cellular proliferation. If the same cells are maintained in defined medium, without fetal calf serum, Nai, Ki, and the number of [ 3 H]ouabain binding sites do not change with time. These data are consistent with the suggestion that the initial mitogenic response of vascular smooth muscle cells to fetal calf serum involves an increased Na+ influx, and a Nai accumulation, caused by low Na+ pump density. The synthesis of new pump sites effects a decrease in the accumulated Nai, which may be related to cell proliferation

Dimethylfumarate induces cell cycle arrest and apoptosis via regulating intracellular redox systems in HeLa cells.

Science.gov (United States)

Han, Guocan; Zhou, Qiang

2016-12-01

Dimethylfumarate (DMF) is cytotoxic to several kinds of cells and serves as an anti-tumor drug. This study was designed to investigate the effects and underlying mechanism of DMF on cervical cancer cells. HeLa cells were cultured and treated with 0, 50, 100, 150, and 200 μM DMF, respectively. After 24 h, cell growth was evaluated using Cell Counting Kit-8 (CCK-8) assay and the cell cycle was examined using flow cytometry. In addition, cell apoptosis was detected by Annexin V/propidium iodide (PI) staining and the expressions of caspase-3 and poly-ADP-ribose polymerase (PARP) were detected using western blotting. The redox-related factors were then assessed. Furthermore, all of the indicators were detected in HeLa cells after combined treatment of DMF and N-acetyl-L-cysteine (NAC, an oxygen-free radical scavenger). The cell number and cell growth of HeLa were obviously inhibited by DMF in a dose-dependent manner, as the cell cycle was arrested at G0/G1 phase (P HeLa cells were markedly increased, and the expression levels of caspase-3 and PARP were significantly increased in a DMF concentration-dependent way (P cell proliferation and apoptosis of HeLa cells was mainly related to the intracellular redox systems by depletion of intracellular GSH.

Ciprofloxacin nano-niosomes for targeting intracellular infections: an in vitro evaluation

International Nuclear Information System (INIS)

Akbari, Vajihe; Abedi, Daryoush; Pardakhty, Abbas; Sadeghi-Aliabadi, Hojjat

2013-01-01

In order to propose non-ionic surfactant vesicles (niosomes) for the treatment of intracellular infections, a remote loading method (active drug encapsulation) followed by sonication was used to prepare nano-niosome formulations containing ciprofloxacin (CPFX). Size analysis, size distribution and zeta potentials of niosomes were evaluated and then their antimicrobial activity, cellular uptake, cytotoxicity, intracellular distribution, and antibacterial activity against intracellular Staphylococcus aureus infection of murine macrophage-like, J774, cells were investigated in comparison to free drug. Our findings reveal that size and composition of the niosome formula can influence their in vitro biological properties. Vesicles in the 300–600 nm size range were phagocytosed to a greater degree by macrophages in comparison to other size vesicles. The minimum inhibitory concentrations (MICs) of CPFX-loaded niosomes were two to eightfold lower than MICs of free CPFX. In addition, niosome encapsulation of CPFX provided high intracellular antimicrobial activities while free CPFX is ineffective for eradicating intracellular forms of S. aureus. Encapsulation of CPFX in niosomes generally decreased its in vitro cytotoxicity. Our results show that niosomes are suitable drug delivery systems with good efficacy and safety properties to be proposed for drug targeting against intracellular infections.

Ciprofloxacin nano-niosomes for targeting intracellular infections: an in vitro evaluation

Energy Technology Data Exchange (ETDEWEB)

Akbari, Vajihe; Abedi, Daryoush [Isfahan University of Medical Sciences, Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Research Center, Faculty of Pharmacy (Iran, Islamic Republic of); Pardakhty, Abbas [Kerman University of Medical Sciences, Pharmaceutics Research Center (Iran, Islamic Republic of); Sadeghi-Aliabadi, Hojjat, E-mail: sadeghi@pharm.mui.ac.ir [Isfahan University of Medical Sciences, Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Research Center, Faculty of Pharmacy (Iran, Islamic Republic of)

2013-04-15

In order to propose non-ionic surfactant vesicles (niosomes) for the treatment of intracellular infections, a remote loading method (active drug encapsulation) followed by sonication was used to prepare nano-niosome formulations containing ciprofloxacin (CPFX). Size analysis, size distribution and zeta potentials of niosomes were evaluated and then their antimicrobial activity, cellular uptake, cytotoxicity, intracellular distribution, and antibacterial activity against intracellular Staphylococcus aureus infection of murine macrophage-like, J774, cells were investigated in comparison to free drug. Our findings reveal that size and composition of the niosome formula can influence their in vitro biological properties. Vesicles in the 300-600 nm size range were phagocytosed to a greater degree by macrophages in comparison to other size vesicles. The minimum inhibitory concentrations (MICs) of CPFX-loaded niosomes were two to eightfold lower than MICs of free CPFX. In addition, niosome encapsulation of CPFX provided high intracellular antimicrobial activities while free CPFX is ineffective for eradicating intracellular forms of S. aureus. Encapsulation of CPFX in niosomes generally decreased its in vitro cytotoxicity. Our results show that niosomes are suitable drug delivery systems with good efficacy and safety properties to be proposed for drug targeting against intracellular infections.

Azithromycin effectiveness against intracellular infections of Francisella

Directory of Open Access Journals (Sweden)

Mann Barbara J

2010-04-01

Full Text Available Abstract Background Macrolide antibiotics are commonly administered for bacterial respiratory illnesses. Azithromycin (Az is especially noted for extremely high intracellular concentrations achieved within macrophages which is far greater than the serum concentration. Clinical strains of Type B Francisella (F. tularensis have been reported to be resistant to Az, however our laboratory Francisella strains were found to be sensitive. We hypothesized that different strains/species of Francisella (including Type A may have different susceptibilities to Az, a widely used and well-tolerated antibiotic. Results In vitro susceptibility testing of Az confirmed that F. tularensis subsp. holarctica Live Vaccine Strain (LVS (Type B was not sensitive while F. philomiragia, F. novicida, and Type A F. tularensis (NIH B38 and Schu S4 strain were susceptible. In J774A.1 mouse macrophage cells infected with F. philomiragia, F. novicida, and F. tularensis LVS, 5 μg/ml Az applied extracellularly eliminated intracellular Francisella infections. A concentration of 25 μg/ml Az was required for Francisella-infected A549 human lung epithelial cells, suggesting that macrophages are more effective at concentrating Az than epithelial cells. Mutants of RND efflux components (tolC and ftlC in F. novicida demonstrated less sensitivity to Az by MIC than the parental strain, but the tolC disc-inhibition assay demonstrated increased sensitivity, indicating a complex role for the outer-membrane transporter. Mutants of acrA and acrB mutants were less sensitive to Az than the parental strain, suggesting that AcrAB is not critical for the efflux of Az in F. novicida. In contrast, F. tularensis Schu S4 mutants ΔacrB and ΔacrA were more sensitive than the parental strain, indicating that the AcrAB may be important for Az efflux in F. tularensis Schu S4. F. novicida LPS O-antigen mutants (wbtN, wbtE, wbtQ and wbtA were found to be less sensitive in vitro to Az compared to the wild

Induction of Intracellular Ca2+ and pH Changes in Sf9 Insect Cells by Rhodojaponin-III, A Natural Botanic Insecticide Isolated from Rhododendron molle

Directory of Open Access Journals (Sweden)

Yan-Bo Zhang

2011-04-01

Full Text Available Many studies on intracellular calcium ([Ca2+]i and intracellular pH (pHi have been carried out due to their importance in regulation of different cellular functions. However, most of the previous studies are focused on human or mammalian cells. The purpose of the present study was to characterize the effect of Rhodojaponin-III (R-III on [Ca2+]i and pHi and the proliferation of Sf9 cells. R-III strongly inhibited Sf9 cells proliferation with a time- and dose-dependent manner. Flow cytometry established that R-III interfered with Sf9 cells division and arrested them in G2/M. By using confocal scanning technique, effects of R-III on intracellular free calcium ([Ca2+]i and intracellular pH (pHi in Sf9 cells were determined. R-III induced a significant dose-dependent (1, 10, 100, 200 μg/mL increase in [Ca2+]i and pHi of Sf9 cells in presence of Ca2+-containing solution (Hanks and an irreversible decrease in the absence of extra cellular Ca2+. We also found that both extra cellular Ca2+ and intracellular Ca2+ stores contributed to the increase of [Ca2+]i, because completely treating Sf9 cells with CdCl2 (5 mM, a Ca2+ channels blocker, R-III (100 μg/mL induced a transient elevation of [Ca2+]i in case of cells either in presence of Ca2+ containing or Ca2+ free solution. In these conditions, pHi showed similar changes with that of [Ca2+]i on the whole. Accordingly, we supposed that there was a certain linkage for change of [Ca2+]i, cell cycle arrest, proliferation inhibition in Sf9 cells induced by R-III.

A novel intracellular pool of LFA-1 is critical for asymmetric CD8+ T cell activation and differentiation.

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Capece, Tara; Walling, Brandon L; Lim, Kihong; Kim, Kyun-Do; Bae, Seyeon; Chung, Hung-Li; Topham, David J; Kim, Minsoo

2017-11-06

The integrin lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18) is a key T cell adhesion receptor that mediates stable interactions with antigen-presenting cell (APC), as well as chemokine-mediated migration. Using our newly generated CD11a-mYFP knock-in mice, we discovered that naive CD8 + T cells reserve a significant intracellular pool of LFA-1 in the uropod during migration. Intracellular LFA-1 quickly translocated to the cell surface with antigenic stimulus. Importantly, the redistribution of intracellular LFA-1 at the contact with APC was maintained during cell division and led to an unequal inheritance of LFA-1 in divided T cells. The daughter CD8 + T cells with disparate LFA-1 expression showed different patterns of migration on ICAM-1, APC interactions, and tissue retention, as well as altered effector functions. In addition, we identified Rab27 as an important regulator of the intracellular LFA-1 translocation. Collectively, our data demonstrate that an intracellular pool of LFA-1 in naive CD8 + T cells plays a key role in T cell activation and differentiation. © 2017 Capece et al.

Sixteen-Day Bedrest Significantly Increases Plasma Colloid Osmotic Pressure

Science.gov (United States)

Hargens, Alan R.; Hsieh, S. T.; Murthy, G.; Ballard, R. E.; Convertino, V. A.; Wade, Charles E. (Technical Monitor)

1994-01-01

Upon exposure to microgravity, astronauts lose up to 10% of their total plasma volume, which may contribute to orthostatic intolerance after space flight. Because plasma colloid osmotic pressure (COP) is a primary factor maintaining plasma volume, our objective was to measure time course changes in COP during microgravity simulated by 6 deg. head-down tilt (HDT). Seven healthy male subjects (30-55 years of age) were placed in HDT for 16 days. For the purpose of another study, three of the seven subjects were chosen to exercise on a cycle ergometer on day 16. Blood samples were drawn immediately before bedrest on day 14 of bedrest, 18-24 hours following exercise while all subjects were still in HDT and 1 hour following bedrest termination. Plasma COP was measured in all 20 microliter EDTA-treated samples using an osmometer fitted with a PM 30 membrane. Data were analyzed with paired and unpaired t-tests. Plasma COP on day 14 of bedrest (29.9 +/- 0.69 mmHg) was significantly higher (p less than 0.005) than the control, pre-bedrest value (23.1 +/- 0.76 mmHg). At one hour of upright recovery after HDT, plasma COP remained significantly elevated (exercise: 26.9 +/- 0.87 mmHg; no exercise: 26.3 +/- 0.85 mmHg). Additionally, exercise had no significant effect on plasma COP 18-24 hours following exercise (exercise: 27.8 +/- 1.09 mmHg; no exercise: 27.1 +/- 0.78 mmHg). Our results demonstrate that plasma COP increases significantly with microgravity simulated by HDT. However, preliminary results indicate exercise during HDT does not significantly affect plasma COP.

A bacteriophage endolysin that eliminates intracellular streptococci

Science.gov (United States)

Shen, Yang; Barros, Marilia; Vennemann, Tarek; Gallagher, D Travis; Yin, Yizhou; Linden, Sara B; Heselpoth, Ryan D; Spencer, Dennis J; Donovan, David M; Moult, John; Fischetti, Vincent A; Heinrich, Frank; Lösche, Mathias; Nelson, Daniel C

2016-01-01

PlyC, a bacteriophage-encoded endolysin, lyses Streptococcus pyogenes (Spy) on contact. Here, we demonstrate that PlyC is a potent agent for controlling intracellular Spy that often underlies refractory infections. We show that the PlyC holoenzyme, mediated by its PlyCB subunit, crosses epithelial cell membranes and clears intracellular Spy in a dose-dependent manner. Quantitative studies using model membranes establish that PlyCB interacts strongly with phosphatidylserine (PS), whereas its interaction with other lipids is weak, suggesting specificity for PS as its cellular receptor. Neutron reflection further substantiates that PlyC penetrates bilayers above a PS threshold concentration. Crystallography and docking studies identify key residues that mediate PlyCB–PS interactions, which are validated by site-directed mutagenesis. This is the first report that a native endolysin can traverse epithelial membranes, thus substantiating the potential of PlyC as an antimicrobial for Spy in the extracellular and intracellular milieu and as a scaffold for engineering other functionalities. DOI: http://dx.doi.org/10.7554/eLife.13152.001 PMID:26978792

Dependence of the Ce(iii)/Ce(iv) ratio on intracellular localization in ceria nanoparticles internalized by human cells

KAUST Repository

Ferraro, Daniela

2017-01-09

CeO2 nanoparticles (CNPs) have been investigated as promising antioxidant agents with significant activity in the therapy of diseases involving free radicals or oxidative stress. However, the exact mechanism responsible for CNP activity has not been completely elucidated. In particular, in situ evidence of modification of the oxidative state of CNPs in human cells and their evolution during cell internalization and subsequent intracellular distribution has never been presented. In this study we investigated modification of the Ce(iii)/Ce(iv) ratio following internalization in human cells by X-ray absorption near edge spectroscopy (XANES). From this analysis on cell pellets, we observed that CNPs incubated for 24 h showed a significant increase in Ce(iii). By coupling on individual cells synchrotron micro-X-ray fluorescence (μXRF) with micro-XANES (μXANES) we demonstrated that the Ce(iii)/Ce(iv) ratio is also dependent on CNP intracellular localization. The regions with the highest CNP concentrations, suggested to be endolysosomes by transmission electron microscopy, were characterized by Ce atoms in the Ce(iv) oxidation state, while a higher Ce(iii) content was observed in regions surrounding these areas. These observations suggest that the interaction of CNPs with cells involves a complex mechanism in which different cellular areas play different roles.

Dependence of the Ce(iii)/Ce(iv) ratio on intracellular localization in ceria nanoparticles internalized by human cells

KAUST Repository

Ferraro, Daniela; Tredici, Ilenia G.; Ghigna, Paolo; Castillio-Michel, Hiram; Falqui, Andrea; Di Benedetto, Cristiano; Alberti, Giancarla; Ricci, Vittorio; Anselmi-Tamburini, Umberto; Sommi, Patrizia

2017-01-01

CeO2 nanoparticles (CNPs) have been investigated as promising antioxidant agents with significant activity in the therapy of diseases involving free radicals or oxidative stress. However, the exact mechanism responsible for CNP activity has not been completely elucidated. In particular, in situ evidence of modification of the oxidative state of CNPs in human cells and their evolution during cell internalization and subsequent intracellular distribution has never been presented. In this study we investigated modification of the Ce(iii)/Ce(iv) ratio following internalization in human cells by X-ray absorption near edge spectroscopy (XANES). From this analysis on cell pellets, we observed that CNPs incubated for 24 h showed a significant increase in Ce(iii). By coupling on individual cells synchrotron micro-X-ray fluorescence (μXRF) with micro-XANES (μXANES) we demonstrated that the Ce(iii)/Ce(iv) ratio is also dependent on CNP intracellular localization. The regions with the highest CNP concentrations, suggested to be endolysosomes by transmission electron microscopy, were characterized by Ce atoms in the Ce(iv) oxidation state, while a higher Ce(iii) content was observed in regions surrounding these areas. These observations suggest that the interaction of CNPs with cells involves a complex mechanism in which different cellular areas play different roles.

Intracellular trafficking of a pH-responsive drug metal complex.

Science.gov (United States)

Kheirolomoom, Azadeh; Ingham, Elizabeth S; Commisso, Joel; Abushaban, Neveen; Ferrara, Katherine W

2016-12-10

We previously developed a pH-responsive copper-doxorubicin (CuDox) cargo in lysolipid-based temperature-sensitive liposomes (LTSLs). The CuDox complex is released from the particle by elevated temperature; however, full release of doxorubicin from CuDox requires a reduced pH, such as that expected in lysosomes. The primary goal of this study is to evaluate the cellular uptake and intracellular trafficking of the drug-metal complex in comparison with intact liposomes and free drug. We found that the CuDox complex was efficiently internalized by mammary carcinoma cells after release from LTSLs. Intracellular doxorubicin and copper were 6-fold and 5-fold greater, respectively, after a 0.5h incubation with the released CuDox complex, as compared to incubation with intact liposomes containing the complex. Total cellular doxorubicin fluorescence was similar following CuDox and free doxorubicin incubation. Imaging and mass spectrometry assays indicated that the CuDox complex was initially internalized intact but breaks down over time within cells, with intracellular copper decreasing more rapidly than intracellular doxorubicin. Doxorubicin fluorescence was reduced when complexed with copper, and nuclear fluorescence was reduced when cells were incubated with the CuDox complex as compared with free doxorubicin. Therapeutic efficacy, which typically results from intercalation of doxorubicin with DNA, was equivalent for the CuDox complex and free doxorubicin and was superior to that of liposomal doxorubicin formulations. Taken together, the results suggest that quenched CuDox reaches the nucleus and remains efficacious. In order to design protocols for the use of these temperature-sensitive particles in cancer treatment, the timing of hyperthermia relative to drug administration must be examined. When cells were heated to 42°C prior to the addition of free doxorubicin, nuclear drug accumulation increased by 1.8-fold in cancer cells after 5h, and cytotoxicity increased 1

Investigation on the Metabolic Regulation of pgi gene knockout Escherichia coli by Enzyme Activities and Intracellular Metabolite Concentrations

Directory of Open Access Journals (Sweden)

Nor ‘Aini, A. R.

2006-01-01

Full Text Available An integrated analysis of the cell growth characteristics, enzyme activities, intracellular metabolite concentrations was made to investigate the metabolic regulation of pgi gene knockout Escherichia coli based on batch culture and continuous culture which was performed at the dilution rate of 0.2h-1. The enzymatic study identified that pathways of pentose phosphate, ED pathway and glyoxylate shunt were all active in pgi mutant. The glycolysis enzymes i.e glyceraldehyde-3-phosphate dehydrogenase, fructose diphosphatase, pyruvate kinase, triose phosphate isomerase were down regulated implying that the inactivation of pgi gene reduced the carbon flux through glycolytic pathway. Meanwhile, the pentose phosphate pathway was active as a major route for intermediary carbohydrate metabolism instead of glycolysis. The pentose phosphate pathway generates most of the major reducing co-factor NADPH as shown by the increased of NADPH/NADP+ ratio in the mutant when compared with the parent strain. The fermentative enzymes such as acetate kinase and lactate dehydrogenase were down regulated in the mutant. Knockout of pgi gene results in the significant increase in the intracellular concentration of glucose-6-phosphate and decrease in the concentration of oxaloacetate. The slow growth rate of the mutant was assumed to be affected by the accumulation of glucose-6-phosphate and imbalance of NADPH reoxidation.

The transition between monostable and bistable states induced by time delay in intracellular calcium oscillation

International Nuclear Information System (INIS)

Duan, Wei-Long

2013-01-01

The revised role of the time delay of active processes with colored noises of transmission of intracellular Ca 2+ in intracellular calcium oscillation (ICO) is investigated by means of a first-order algorithm based on stochastic simulation. The simulation results indicate that time delay induces a double critical phenomenon and a transition between the monostable and bistable states of the ICO system. In addition, as the time delay increases, for a cytosolic Ca 2+ concentration with weak colored noises there appears a calcium burst, and the Ca 2+ concentration of the calcium store shows nonmonotonic variation. (paper)

Variety in intracellular diffusion during the cell cycle

DEFF Research Database (Denmark)

Selhuber-Unkel, C.; Yde, P.; Berg-Sørensen, Kirstine

2009-01-01

During the cell cycle, the organization of the cytoskeletal network undergoes dramatic changes. In order to reveal possible changes of the viscoelastic properties in the intracellular space during the cell cycle we investigated the diffusion of endogenous lipid granules within the fission yeast...... Schizosaccharomyces Pombe using optical tweezers. The cell cycle was divided into interphase and mitotic cell division, and the mitotic cell division was further subdivided in its stages. During all stages of the cell cycle, the granules predominantly underwent subdiffusive motion, characterized by an exponent...... a that is also linked to the viscoelastic moduli of the cytoplasm. The exponent a was significantly smaller during interphase than during any stage of the mitotic cell division, signifying that the cytoplasm was more elastic during interphase than during division. We found no significant differences...

A new nano-enhanced technology proposed to quantify intracellular detection of radiation-induced metabolic processes.

Science.gov (United States)

Malak, Henryk; Richmond, Robert; Dicello, J F

2011-02-01

A new approach to intracellular detection and imaging of metabolic processes and pathways is presented that uses surface plasmon resonance to enhance interactions between photon-absorbing metabolites and metal nanoparticles in contact with cells in vitro or in vivo. Photon absorption in the nanoparticles creates plasmon fields, enhancing intrinsic metabolite fluorescence, thereby increasing absorption and emission rates, creating new spectral emission bands, shortening fluorescence lifetimes, becoming more photo-stable and increasing fluorescent resonance energy transfer efficiency. Because the cells remain viable, it is proposed that the method may be used to interrogate cells prior to and after irradiation, with the potential for automated analyses of intracellular interactive pathways associated with radiation exposures at lower doses than existing technologies. The design and concepts of the instrument are presented along with data for unexposed cells.

Profiling of Intracellular Metabolites: An Approach to Understanding the Characteristic Physiology of Mycobacterium leprae.

Science.gov (United States)

Miyamoto, Yuji; Mukai, Tetsu; Matsuoka, Masanori; Kai, Masanori; Maeda, Yumi; Makino, Masahiko

2016-08-01

Mycobacterium leprae is the causative agent of leprosy and also known to possess unique features such as inability to proliferate in vitro. Among the cellular components of M. leprae, various glycolipids present on the cell envelope are well characterized and some of them are identified to be pathogenic factors responsible for intracellular survival in host cells, while other intracellular metabolites, assumed to be associated with basic physiological feature, remain largely unknown. In the present study, to elucidate the comprehensive profile of intracellular metabolites, we performed the capillary electrophoresis-mass spectrometry (CE-MS) analysis on M. leprae and compared to that of M. bovis BCG. Interestingly, comparison of these two profiles showed that, in M. leprae, amino acids and their derivatives are significantly accumulated, but most of intermediates related to central carbon metabolism markedly decreased, implying that M. leprae possess unique metabolic features. The present study is the first report demonstrating the unique profiles of M. leprae metabolites and these insights might contribute to understanding undefined metabolism of M. leprae as well as pathogenic characteristics related to the manifestation of the disease.

Monitoring changes in the intracellular calcium concentration and synaptic efficacy in the mollusc Aplysia.

Science.gov (United States)

Ludwar, Bjoern Ch; Evans, Colin G; Cropper, Elizabeth C

2012-07-15

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation). These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 °C) that are natural for Aplysia, preparations are stable for long periods of time. To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3). Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record

Controlled intracellular generation of reactive oxygen species in human mesenchymal stem cells using porphyrin conjugated nanoparticles

Science.gov (United States)

Lavado, Andrea S.; Chauhan, Veeren M.; Alhaj Zen, Amer; Giuntini, Francesca; Jones, D. Rhodri E.; Boyle, Ross W.; Beeby, Andrew; Chan, Weng C.; Aylott, Jonathan W.

2015-08-01

Nanoparticles capable of generating controlled amounts of intracellular reactive oxygen species (ROS), that advance the study of oxidative stress and cellular communication, were synthesized by functionalizing polyacrylamide nanoparticles with zinc(ii) porphyrin photosensitisers. Controlled ROS production was demonstrated in human mesenchymal stem cells (hMSCs) through (1) production of nanoparticles functionalized with varying percentages of Zn(ii) porphyrin and (2) modulating the number of doses of excitation light to internalized nanoparticles. hMSCs challenged with nanoparticles functionalized with increasing percentages of Zn(ii) porphyrin and high numbers of irradiations of excitation light were found to generate greater amounts of ROS. A novel dye, which is transformed into fluorescent 7-hydroxy-4-trifluoromethyl-coumarin in the presence of hydrogen peroxide, provided an indirect indicator for cumulative ROS production. The mitochondrial membrane potential was monitored to investigate the destructive effect of increased intracellular ROS production. Flow cytometric analysis of nanoparticle treated hMSCs suggested irradiation with excitation light signalled controlled apoptotic cell death, rather than uncontrolled necrotic cell death. Increased intracellular ROS production did not induce phenotypic changes in hMSC subcultures.Nanoparticles capable of generating controlled amounts of intracellular reactive oxygen species (ROS), that advance the study of oxidative stress and cellular communication, were synthesized by functionalizing polyacrylamide nanoparticles with zinc(ii) porphyrin photosensitisers. Controlled ROS production was demonstrated in human mesenchymal stem cells (hMSCs) through (1) production of nanoparticles functionalized with varying percentages of Zn(ii) porphyrin and (2) modulating the number of doses of excitation light to internalized nanoparticles. hMSCs challenged with nanoparticles functionalized with increasing percentages of Zn

MRP8/14 induces autophagy to eliminate intracellular Mycobacterium bovis BCG.

Science.gov (United States)

Wang, Jinli; Huang, Chunyu; Wu, Minhao; Zhong, Qiu; Yang, Kun; Li, Miao; Zhan, Xiaoxia; Wen, Jinsheng; Zhou, Lin; Huang, Xi

2015-04-01

To explore the role of myeloid-related protein 8/14 in mycobacterial infection. The mRNA and protein expression levels of MRP8 or MRP14 were measured by real-time PCR and flow cytometry, respectively. Role of MRP8/14 was tested by overexpression or RNA interference assays. Flow cytometry and colony forming unit were used to test the phagocytosis and the survival of intracellular Mycobacterium bovis BCG (BCG), respectively. Autophagy mediated by MRP8/14 was detected by Western blot and immunofluorescence. The colocalization of BCG phagosomes with autophagosomes or lysosomes was by detected by confocal microscopy. ROS production was detected by flow cytometry. MRP8/14 expressions were up-regulated in human monocytic THP1 cells and primary macrophages after mycobacterial challenge. Silencing of MRP8/14 suppressed bacterial killing, but had no influence on the phagocytosis of BCG. Importantly, silencing MRP8/14 decreased autophagy and BCG phagosome maturation in THP1-derived macrophages, thereby increasing the BCG survival. Additionally, we demonstrated that MRP8/14 promoted autophagy in a ROS-dependent manner. The present study revealed a novel role of MRP8/14 in the autophagy-mediated elimination of intracellular BCG by promoting ROS generation, which may provide a promising therapeutic target for tuberculosis and other intracellular bacterial infectious diseases. Copyright © 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Atazanavir intracellular concentrations remain stable during pregnancy in HIV-infected patients.

Science.gov (United States)

Focà, Emanuele; Calcagno, Andrea; Bonito, Andrea; Simiele, Marco; Domenighini, Elisabetta; D'Avolio, Antonio; Quiros Roldan, Eugenia; Trentini, Laura; Casari, Salvatore; Di Perri, Giovanni; Castelli, Francesco; Bonora, Stefano

2017-11-01

Atazanavir (300 mg) boosted by ritonavir (100 mg) is the preferred third drug in pregnancy. However, there is still discordance on atazanavir dose increase during the third trimester. To evaluate plasma and intracellular atazanavir and ritonavir concentrations in HIV-infected women during pregnancy and after delivery. This was an observational study. HIV-infected pregnant patients treated with atazanavir/ritonavir plus either tenofovir/emtricitabine or abacavir/lamivudine had been prospectively enrolled after having signed a written informed consent form. Plasma and intracellular atazanavir and ritonavir Ctrough (24 ± 3 h after drug intake) were measured at each visit during the first, second and third trimesters and post-partum using validated HPLC-MS and HPLC-photodiode array methods (with direct evaluation of cellular volume). Data are described as median (IQR) and compared through non-parametric tests. Twenty-five patients were enrolled; at baseline, the median age was 32 years (27-35). All patients had plasma HIV RNA  0.05). This is the first demonstration that intracellular atazanavir exposure remains unchanged during pregnancy supporting the standard 300/100 mg atazanavir/ritonavir dosing throughout pregnancy. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Differential intracellular calcium influx, nitric oxide production, ICAM-1 and IL8 expression in primary bovine endothelial cells exposed to nonesterified fatty acids.

Science.gov (United States)

Loaiza, Anitsi; Carretta, María D; Taubert, Anja; Hermosilla, Carlos; Hidalgo, María A; Burgos, Rafael A

2016-02-25

Nonesterified fatty acids (NEFAs) are involved in proinflammatory processes in cattle, including in the increased expression of adhesion molecules in endothelial cells. However, the mechanisms underlying these effects are still unknown. The aim of this study was to assess the effects of NEFAs on the intracellular calcium (Ca(2+) i) influx, nitric oxide production, and ICAM-1 and IL-8 expression in primary bovine umbilical vein endothelial cells (BUVECs). Myristic (MA), palmitic (PA), stearic (SA), oleic (OA) and linoleic acid (LA) rapidly increased Ca(2+) i. The calcium response to all tested NEFAs showed an extracellular calcium dependence and only the LA response was significantly inhibited until the intracellular calcium was chelated. The EC50 values for MA and LA were 125 μM and 37 μM, respectively, and the MA and LA effects were dependent on calcium release from the endoplasmic reticulum stores and on the L-type calcium channels. Only the calcium response to MA was significantly reduced by GW1100, a selective G-protein-coupled free fatty acid receptor (GPR40) antagonist. We also detected a functional FFAR1/GPR40 protein in BUVECs by using western blotting and the FFAR1/GPR40 agonist TAK-875. Only LA increased the cellular nitric oxide levels in a calcium-dependent manner. LA stimulation but not MA stimulation increased ICAM-1 and IL-8-expression in BUVECs. This effect was inhibited by GW1100, an antagonist of FFAR1/GPR40, but not by U-73122, a phospholipase C inhibitor. These findings strongly suggest that each individual NEFA stimulates endothelial cells in a different way, with clearly different effects on intracellular calcium mobilization, NO production, and IL-8 and ICAM-1 expression in primary BUVECs. These findings not only extend our understanding of NEFA-mediated diseases in ruminants, but also provide new insight into the different molecular mechanisms involved during endothelial cell activation by NEFAs.

Determination of Intracellular Vitrification Temperatures for Unicellular Micro Organisms under Conditions Relevant for Cryopreservation.

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Fonseca, Fernanda; Meneghel, Julie; Cenard, Stéphanie; Passot, Stéphanie; Morris, G John

2016-01-01

During cryopreservation ice nucleation and crystal growth may occur within cells or the intracellular compartment may vitrify. Whilst previous literature describes intracellular vitrification in a qualitative manner, here we measure the intracellular vitrification temperature of bacteria and yeasts under conditions relevant to cryopreservation, including the addition of high levels of permeating and nonpermeating additives and the application of rapid rates of cooling. The effects of growth conditions that are known to modify cellular freezing resistance on the intracellular vitrification temperature are also examined. For bacteria a plot of the activity on thawing against intracellular glass transition of the maximally freeze-concentrated matrix (Tg') shows that cells with the lowest value of intracellular Tg' survive the freezing process better than cells with a higher intracellular Tg'. This paper demonstrates the role of the physical state of the intracellular environment in determining the response of microbial cells to preservation and could be a powerful tool to be manipulated to allow the optimization of methods for the preservation of microorganisms.

Transient fluctuations of intracellular zinc ions in cell proliferation

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Li, Yuan [Division of Human Nutrition, Department of Preventive Medicine and Community Health, The University of Texas Medical Branch, Galveston, TX 77555 (United States); Maret, Wolfgang, E-mail: womaret@utmb.edu [Division of Human Nutrition, Department of Preventive Medicine and Community Health, The University of Texas Medical Branch, Galveston, TX 77555 (United States); Department of Anesthesiology, The University of Texas Medical Branch, Galveston, TX 77555 (United States)

2009-08-15

Zinc is essential for cell proliferation, differentiation, and viability. When zinc becomes limited for cultured cells, DNA synthesis ceases and the cell cycle is arrested. The molecular mechanisms of actions of zinc are believed to involve changes in the availability of zinc(II) ions (Zn{sup 2+}). By employing a fluorescent Zn{sup 2+} probe, FluoZin-3 acetoxymethyl ester, intracellular Zn{sup 2+} concentrations were measured in undifferentiated and in nerve growth factor (NGF)-differentiated rat pheochromocytoma (PC12) cells. Intracellular Zn{sup 2+} concentrations are pico- to nanomolar in PC12 cells and are higher in the differentiated than in the undifferentiated cells. When following cellular Zn{sup 2+} concentrations for 48 h after the removal of serum, a condition that is known to cause cell cycle arrest, Zn{sup 2+} concentrations decrease after 30 min but, remarkably, increase after 1 h, and then decrease again to about one half of the initial concentration. Cell proliferation, measured by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, decreases after both serum starvation and zinc chelation. Two peaks of Zn{sup 2+} concentrations occur within one cell cycle: one early in the G1 phase and the other in the late G1/S phase. Thus, fluctuations of intracellular Zn{sup 2+} concentrations and established modulation of phosphorylation signaling, via an inhibition of protein tyrosine phosphatases at commensurately low Zn{sup 2+} concentrations, suggest a role for Zn{sup 2+} in the control of the cell cycle. Interventions targeted at these picomolar Zn{sup 2+} fluctuations may be a way of controlling cell growth in hyperplasia, neoplasia, and diseases associated with aberrant differentiation.

Intracellular Hyper-Acidification Potentiated by Hydrogen Sulfide Mediates Invasive and Therapy Resistant Cancer Cell Death

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Zheng-Wei Lee

2017-10-01

Full Text Available Slow and continuous release of H2S by GYY4137 has previously been demonstrated to kill cancer cells by increasing glycolysis and impairing anion exchanger and sodium/proton exchanger activity. This action is specific for cancer cells. The resulting lactate overproduction and defective pH homeostasis bring about intracellular acidification-induced cancer cell death. The present study investigated the potency of H2S released by GYY4137 against invasive and radio- as well as chemo-resistant cancers, known to be glycolytically active. We characterized and utilized cancer cell line pairs of various organ origins, based on their aggressive behaviors, and assessed their response to GYY4137. We compared glycolytic activity, via lactate production, and intracellular pH of each cancer cell line pair after exposure to H2S. Invasive and therapy resistant cancers, collectively termed aggressive cancers, are receptive to H2S-mediated cytotoxicity, albeit at a higher concentration of GYY4137 donor. While lactate production was enhanced, intracellular pH of aggressive cancers was only modestly decreased. Inherently, the magnitude of intracellular pH decrease is a key determinant for cancer cell sensitivity to H2S. We demonstrated the utility of coupling GYY4137 with either simvastatin, known to inhibit monocarboxylate transporter 4 (MCT4, or metformin, to further boost glycolysis, in bringing about cell death for aggressive cancers. Simvastatin inhibiting lactate extrusion thence contained excess lactate induced by GYY4137 within intracellular compartment. In contrast, the combined exposure to both GYY4137 and metformin overwhelms cancer cells with lactate over-production exceeding its expulsion rate. Together, GYY4137 and simvastatin or metformin synergize to induce intracellular hyper-acidification-mediated cancer cell death.

Neurotrophin-3 Regulates Synapse Development by Modulating TrkC-PTPσ Synaptic Adhesion and Intracellular Signaling Pathways.

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Han, Kyung Ah; Woo, Doyeon; Kim, Seungjoon; Choii, Gayoung; Jeon, Sangmin; Won, Seoung Youn; Kim, Ho Min; Heo, Won Do; Um, Ji Won; Ko, Jaewon

2016-04-27

Neurotrophin-3 (NT-3) is a secreted neurotrophic factor that binds neurotrophin receptor tyrosine kinase C (TrkC), which in turn binds to presynaptic protein tyrosine phosphatase σ (PTPσ) to govern excitatory synapse development. However, whether and how NT-3 cooperates with the TrkC-PTPσ synaptic adhesion pathway and TrkC-mediated intracellular signaling pathways in rat cultured neurons has remained unclear. Here, we report that NT-3 enhances TrkC binding affinity for PTPσ. Strikingly, NT-3 treatment bidirectionally regulates the synaptogenic activity of TrkC: at concentrations of 10-25 ng/ml, NT-3 further enhanced the increase in synapse density induced by TrkC overexpression, whereas at higher concentrations, NT-3 abrogated TrkC-induced increases in synapse density. Semiquantitative immunoblotting and optogenetics-based imaging showed that 25 ng/ml NT-3 or light stimulation at a power that produced a comparable level of NT-3 (6.25 μW) activated only extracellular signal-regulated kinase (ERK) and Akt, whereas 100 ng/ml NT-3 (light intensity, 25 μW) further triggered the activation of phospholipase C-γ1 and CREB independently of PTPσ. Notably, disruption of TrkC intracellular signaling pathways, extracellular ligand binding, or kinase activity by point mutations compromised TrkC-induced increases in synapse density. Furthermore, only sparse, but not global, TrkC knock-down in cultured rat neurons significantly decreased synapse density, suggesting that intercellular differences in TrkC expression level are critical for its synapse-promoting action. Together, our data demonstrate that NT-3 is a key factor in excitatory synapse development that may direct higher-order assembly of the TrkC/PTPσ complex and activate distinct intracellular signaling cascades in a concentration-dependent manner to promote competition-based synapse development processes. In this study, we present several lines of experimental evidences to support the conclusion that

Thymosin β4 promotes the migration of endothelial cells without intracellular Ca2+ elevation

International Nuclear Information System (INIS)

Selmi, Anna; Malinowski, Mariusz; Brutkowski, Wojciech; Bednarek, Radoslaw; Cierniewski, Czeslaw S.

2012-01-01

Numerous studies have demonstrated the effects of Tβ4 on cell migration, proliferation, apoptosis and inflammation after exogenous treatment, but the mechanism by which Tβ4 functions is still unclear. Previously, we demonstrated that incubation of endothelial cells with Tβ4 induced synthesis and secretion of various proteins, including plasminogen activator inhibitor type 1 and matrix metaloproteinases. We also showed that Tβ4 interacts with Ku80, which may operate as a novel receptor for Tβ4 and mediates its intracellular activity. In this paper, we provide evidence that Tβ4 induces cellular processes without changes in the intracellular Ca 2+ concentration. External treatment of HUVECs with Tβ4 and its mutants deprived of the N-terminal tetrapeptide AcSDKP (Tβ4 AcSDKPT/4A ) or the actin-binding sequence KLKKTET (Tβ4 KLKKTET/7A ) resulted in enhanced cell migration and formation of tubular structures in Matrigel. Surprisingly, the increased cell motility caused by Tβ4 was not associated with the intracellular Ca 2+ elevation monitored with Fluo-4 NW or Fura-2 AM. Therefore, it is unlikely that externally added Tβ4 induces HUVEC migration via the surface membrane receptors known to generate Ca 2+ influx. Our data confirm the concept that externally added Tβ4 must be internalized to induce intracellular mechanisms supporting endothelial cell migration.

Influence of extra-cellular and intra-cellular acting thiol oxidants on the 45calcium uptake by the islets of Langerhans of the rat

International Nuclear Information System (INIS)

Haegele, R.G.

1981-01-01

The glucose-stimulated calcium uptake by the islets of Langerhans is dependent on the intra-cellular GSH/GSSG ratios. The inhibition of calcium uptake is not the consequence of a direct oxidation of membrane-fixed thiol groups. In contrast, direct oxidation of extra cellular thiols leads to an increase in calcium uptake when intra-cellular oxidation is simultaneously prevented. Since this effect only occurs at high intra-cellular GSH/GSSG ratios it can be assumed that the redox state of extra-cellular thiols is dependent on the redox state of the intra-cellular GSH/GSSG ratios. These findings support the theory that the oxidation of extra-cellular thiols by thiol oxidants leads to an increase in calcium uptake and that the extent of uptake is higher, the more the redox state of the extra-cellular thiols tends towards the reduced state prior to oxidation. (orig./MG) [de

Metallothioneins I and II: neuroprotective significance during CNS pathology

DEFF Research Database (Denmark)

Penkowa, Milena; Stankovic, Roger; Chung, Roger

2006-01-01

Metallothioneins (MTs) constitutes a superfamily of highly conserved, low molecular weight polypeptides, which are characterized by high contents of cysteine (sulphur) and metals. As intracellular metal-binding proteins they play a significant role in the regulation of essential metals. The major...

Mechanism of H. pylori intracellular entry: an in vitro study

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Hui eLiu

2012-03-01

Full Text Available The majority of H. pylori reside on gastric epithelial cell surfaces and in the overlying mucus, but a small fraction of H. pylori enter host epithelial and immune cells. To explore the role of the nudA invasin in host cell entry, a ΔnudA deletion derivative of strain J99 was constructed and transformants were verified by PCR and by fluorescence in situ hybridization. AGS cells were inoculated with either wild type (WT strain J99 or its ΔnudA mutant to determine the fraction of bacteria that were bound to the cells and inside these cells using the gentamicin protection assay. We observed no significant difference between either the density of H. pylori bound to AGS cell membranes or the density of intracellular H. pylori. To further explore this finding, separate chambers of each culture were fixed in glutaraldehyde for transmission electron microscopy (TEM and immunogold TEM. This addition to the classical gentamicin assay demonstrated that there were significantly more intracellular, and fewer membrane-bound, H. pylori in WT-infected AGS cells than in ΔnudA allele infected cells. Thus, the sum of intracellular and membrane-bound H. pylori was similar in the two groups. Since no other similar TEM study has been performed, it is at present unknown whether our observations can be reproduced by others Taken together however, our observations suggest that the classical gentamicin protection assay is not sufficiently sensitive to analyze H. pylori cell entry and that the addition of TEM to the test demonstrate that nudA plays a role in H. pylori entry into AGS cells in vitro. In addition, deletion of the invasin gene appears to limit H. pylori to the AGS cell surface, where it may be partly protected against gentamicin. In contrast, this specific environment may render H. pylori more vulnerable to host defense and therapeutic intervention, and less prone to trigger normal immune, carcinogenic, and other developmental response pathways.

Intracellular calcium mobilization in human lymphocytes in the presence of synthetic IgG Fc peptides

International Nuclear Information System (INIS)

Plummer, J.M.; Panahi, Y.P.; McClurg, M.R.; Hahn, G.S.; Naemura, J.R.

1986-01-01

Certain synthetic peptides derived from the Fc region of human IgG can suppress the mixed lymphocyte response. These peptides were tested for the ability to induce intracellular calcium mobilization in human lymphocytes using fura-2/calcium fluorescence. T cells were isolated by rosetting and were > 90% OKT3 positive. Lymphocytes were incubated with the acetoxymethyl ester of fura-2 (10 μM) for 60 minutes at 37 0 C. Fluorescence intensity changes at 505 nm were monitored at an excitation lambda of 340 nm. Fura-2 was not cytotoxic compared to quin-2 since fura-2 loaded mononuclear cells incorporated 3 H-thymidine when stimulated by PHA, succinyl Con A, PWM or LPS-STM whereas quin-2 loaded cells showed a dose dependent inhibition of proliferation. Those synthetic peptides (5 to 400 μg/ml) that suppressed the MLR induced a dose dependent increase in intracellular calcium in mononuclear cells, lymphocytes, non-T cells and T cells. The fura-2 calcium fluorescence time course response was similar for peptide, PHA and succinyl Con A. These results suggest that these immunoregulatory peptides suppress 3 H-thymidine incorporation at a point after intracellular calcium mobilization and that fura-2 has advantages over quin-2 in measuring intracellular calcium levels in lymphocytes

Control of the intracellular redox state by glucose participates in the insulin secretion mechanism.

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Eduardo Rebelato

Full Text Available BACKGROUND: Production of reactive oxygen species (ROS due to chronic exposure to glucose has been associated with impaired beta cell function and diabetes. However, physiologically, beta cells are well equipped to deal with episodic glucose loads, to which they respond with a fine tuned glucose-stimulated insulin secretion (GSIS. In the present study, a systematic investigation in rat pancreatic islets about the changes in the redox environment induced by acute exposure to glucose was carried out. METHODOLOGY/PRINCIPAL FINDINGS: Short term incubations were performed in isolated rat pancreatic islets. Glucose dose- and time-dependently reduced the intracellular ROS content in pancreatic islets as assayed by fluorescence in a confocal microscope. This decrease was due to activation of pentose-phosphate pathway (PPP. Inhibition of PPP blunted the redox control as well as GSIS in a dose-dependent manner. The addition of low doses of ROS scavengers at high glucose concentration acutely improved beta cell function. The ROS scavenger N-acetyl-L-cysteine increased the intracellular calcium response to glucose that was associated with a small decrease in ROS content. Additionally, the presence of the hydrogen peroxide-specific scavenger catalase, in its membrane-permeable form, nearly doubled glucose metabolism. Interestingly, though an increase in GSIS was also observed, this did not match the effect on glucose metabolism. CONCLUSIONS: The control of ROS content via PPP activation by glucose importantly contributes to the mechanisms that couple the glucose stimulus to insulin secretion. Moreover, we identified intracellular hydrogen peroxide as an inhibitor of glucose metabolism intrinsic to rat pancreatic islets. These findings suggest that the intracellular adjustment of the redox environment by glucose plays an important role in the mechanism of GSIS.

Medium pH in submerged cultivation modulates differences in the intracellular protein profile of Fusarium oxysporum.

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da Rosa-Garzon, Nathália Gonsales; Laure, Hélen Julie; Souza-Motta, Cristina Maria de; Rosa, José César; Cabral, Hamilton

2017-08-09

Fusarium oxysporum is a filamentous fungus that damages a wide range of plants and thus causes severe crop losses. In fungal pathogens, the genes and proteins involved in virulence are known to be controlled by environmental pH. Here, we report the influence of culture-medium pH (5, 6, 7, and 8) on the production of degradative enzymes involved in the pathogenesis of F. oxysporum URM 7401 and on the 2D-electrophoresis profile of intracellular proteins in this fungus. F. oxysporum URM 7401 was grown in acidic, neutral, and alkaline culture media in a submerged bioprocess. After 96 hr, the crude extract was processed to enzyme activity assays, while the intracellular proteins were obtained from mycelium and analyzed using 2D electrophoresis and mass spectrometry. We note that the diversity of secreted enzymes was changed quantitatively in different culture-medium pH. Also, the highest accumulated biomass and the intracellular protein profile of F. oxysporum URM 7401 indicate an increase in metabolism in neutral-alkaline conditions. The differential profiles of secreted enzymes and intracellular proteins under the evaluated conditions indicate that the global protein content in F. oxysporum URM 7401 is modulated by extracellular pH.

Effects of sodium on cell surface and intracellular 3H-naloxone binding sites

International Nuclear Information System (INIS)

Pollack, A.E.; Wooten, G.F.

1987-01-01

The binding of the opiate antagonist 3 H-naloxone was examined in rat whole brain homogenates and in crude subcellular fractions of these homogenates (nuclear, synaptosomal, and mitochondrial fractions) using buffers that approximated intra- (low sodium concentration) and extracellular (high sodium concentration) fluids. Saturation studies showed a two-fold decrease in the dissociation constant (Kd) in all subcellular fractions examined in extracellular buffer compared to intracellular buffer. In contrast, there was no significant effect of the buffers on the Bmax. Thus, 3 H-naloxone did not distinguish between binding sites present on cell surface and intracellular tissues in these two buffers. These results show that the sodium effect of opiate antagonist binding is probably not a function of altered selection of intra- and extracellular binding sites. 17 references, 2 tables

The interferon response to intracellular DNA: why so many receptors?

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Unterholzner, Leonie

2013-11-01

The detection of intracellular DNA has emerged to be a key event in the innate immune response to viruses and intracellular bacteria, and during conditions of sterile inflammation and autoimmunity. One of the consequences of the detection of DNA as a 'stranger' and a 'danger' signal is the production of type I interferons and pro-inflammatory cytokines. Much work has been dedicated to the elucidation of the signalling cascades that activate this DNA-induced gene expression programme. However, while many proteins have been proposed to act as sensors for intracellular DNA in recent years, none has been met with universal acceptance, and a theory linking all the recent observations is, as yet, lacking. This review presents the evidence for the various interferon-inducing DNA receptors proposed to date, and examines the hypotheses that might explain why so many different receptors appear to be involved in the innate immune recognition of intracellular DNA. Copyright © 2013 Elsevier GmbH. All rights reserved.

Intracellular vorinostat accumulation and its relationship to histone deacetylase activity in soft tissue sarcoma patients.

Science.gov (United States)

Burhenne, Jürgen; Liu, Lu; Heilig, Christoph E; Meid, Andreas D; Leisen, Margarete; Schmitt, Thomas; Kasper, Bernd; Haefeli, Walter E; Mikus, Gerd; Egerer, Gerlinde

2017-08-01

In the regulation of chromatin-structure and histone function, histone deacetylases (HDACs) are key enzymes and thus modulators of epigenetic regulation and gene expression. Accesses of the HDAC inhibitor vorinostat to intracellular compartments are essential to exert epigenetic effects. In ten sarcoma patients receiving oral Zolinza (400 mg qd) vorinostat concentrations in plasma and peripheral blood mononuclear cells (PBMCs) were quantified using validated LC/MS/MS assays to determine intracellular and extracellular pharmacokinetic data. Cellular HDAC activity was evaluated using a fluorogenic assay. Concentration-response relationships were established between intracellular and extracellular vorinostat concentrations and HDAC inhibition in PBMCs. Pharmacokinetics of vorinostat and its two main inactive metabolites were determined over 8 h in plasma and PBMCs. Steady state AUCs (±SD) and T 1/2 (±SD) were calculated to 4.61 ± 0.87 h µM and 1.73 ± 0.69 h (plasma) and 15.2 ± 9.03 h µM and 5.30 ± 4.27 h (PBMCs). Intracellular accumulation of vorinostat was determined together with prolonged vorinostat elimination in PBMCs. Cellular HDAC inhibition increased parallel with vorinostat concentrations in plasma and PBMCs. For effective inhibition of cellular HDACs (IC 50 ) vorinostat concentrations of 0.05 µM in plasma and 0.17 µM in PBMCs were necessary. HDAC inhibition closely followed intracellular vorinostat concentrations and was short-lasting, which may contribute to the limited efficacy seen with vorinostat in solid tumors so far.

Significance in the increase of women psychiatrists in Korea.

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Kim, Ha Kyoung; Kim, Soo In

2008-01-01

The number of female doctors has increased in Korea; 18.9% (13,083) of the total medical doctors registered (69,097) were women in 2006, compared to 13.6% (2,216) in 1975. The proportion of female doctors will jump up by 2010 considering that nearly 40% of the medical students are women as of today. This trend has had strong influence on the field of psychiatry; the percentage of women psychiatrists rose from 1.6 (6)% to 18% (453), from 1975 to 2006 and now women residents comprise 39% (206) of all. This is not only a reflection of a social phenomenon of the increase in professional women but also attributed to some specific characteristics of the psychiatry. Psychiatric practice may come more natural to women. While clinical activities of women psychiatrists are expanding, there are few women leaders and much less women are involving in academic activities in this field as yet. Though there is less sexual discrimination in the field of psychiatry, women psychiatrists are still having a lot of difficulties in balancing work and family matters. Many women psychiatrists also report they've ever felt an implied discrimination in their careers. In this study, we are to identify the characteristics of women psychiatrists and to explore the significance of the increase in women psychiatrists in Korea and the situation in which they are.

Metabolic responses of primary and transformed cells to intracellular Listeria monocytogenes.

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Nadine Gillmaier

Full Text Available The metabolic response of host cells, in particular of primary mammalian cells, to bacterial infections is poorly understood. Here, we compare the carbon metabolism of primary mouse macrophages and of established J774A.1 cells upon Listeria monocytogenes infection using (13C-labelled glucose or glutamine as carbon tracers. The (13C-profiles of protein-derived amino acids from labelled host cells and intracellular L. monocytogenes identified active metabolic pathways in the different cell types. In the primary cells, infection with live L. monocytogenes increased glycolytic activity and enhanced flux of pyruvate into the TCA cycle via pyruvate dehydrogenase and pyruvate carboxylase, while in J774A.1 cells the already high glycolytic and glutaminolytic activities hardly changed upon infection. The carbon metabolism of intracellular L. monocytogenes was similar in both host cells. Taken together, the data suggest that efficient listerial replication in the cytosol of the host cells mainly depends on the glycolytic activity of the hosts.

Chloroquine Increases Glucose Uptake via Enhancing GLUT4 Translocation and Fusion with the Plasma Membrane in L6 Cells

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Qi Zhou

2016-05-01

Full Text Available Background/Aims: Chloroquine can induce an increase in the cellular uptake of glucose; however, the underlying mechanism is unclear. Methods: In this study, translocation of GLUT4 and intracellular Ca2+ changes were simultaneously observed by confocal microscope in L6 cells stably over-expressing IRAP-mOrange. The GLUT4 fusion with the plasma membrane (PM was traced using HA-GLUT4-GFP. Glucose uptake was measured using a cell-based glucose uptake assay. GLUT4 protein was detected by Western blotting and mRNA level was detected by RT-PCR. Results: We found that chloroquine induced significant increases in glucose uptake, glucose transporter GLUT4 translocation to the plasma membrane (GTPM, GLUT4 fusion with the PM, and intracellular Ca2+ in L6 muscle cells. Chloroquine-induced increases of GTPM and intracellular Ca2+ were inhibited by Gallein (Gβγ inhibitor and U73122 (PLC inhibitor. However, 2-APB (IP3R blocker only blocked the increase in intracellular Ca2+ but did not inhibit GTPM increase. These results indicate that chloroquine, via the Gβγ-PLC-IP3-IP3R pathway, induces elevation of Ca2+, and this Ca2+ increase does not play a role in chloroqui-ne-evoked GTPM increase. However, GLUT4 fusion with the PM and glucose uptake were significantly inhibited with BAPTA-AM. This suggests that Ca2+ enhances GLUT4 fusion with the PM resulting in glucose uptake increase. Conclusion: Our data indicate that chloroquine via Gβγ-PLC-IP3-IP3R induces Ca2+ elevation, which in turn promotes GLUT4 fusion with the PM. Moreover, chloroquine can enhance GLUT4 trafficking to the PM. These mechanisms eventually result in glucose uptake increase in control and insulin-resistant L6 cells. These findings suggest that chloroquine might be a potential drug for improving insulin tolerance in diabetic patients.

Intracellular putrescine and spermidine deprivation induces increased uptake of the natural polyamines and methylglyoxal bis(guanylhydrazone).

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Alhonen-Hongisto, L; Seppänen, P; Jänne, J

1980-01-01

Inhibition of polyamine synthesis by alpha-difluoromethylornithine in cultured Ehrlich ascites-carcinoma cells rapidly enhanced the uptake of exogenous putrescine, spermidine and spermine from the culture medium. In tumour cells exposed to the drug for 2 days, the intracellular concentration of spermidine was decreased to less than 10% of that found in untreated cells. However, the strikingly stimulated transport system brought the concentration of spermidine to the control values in less than 2h after supplementation of the cells with micromolar concentrations of the polyamine. In the absence of polyamine deprivation, tumour cells did not accumulate extracellular polyamines to any appreciable extent. Ascites-tumour cells deprived of putrescine and spermidine likewise concentrated methylglyoxal bis(guanylhydrazone) [1,1'-[methylethanedylidine)dinitrilo]diguanidine] at a greatly enhanced rate. A previous "priming of tumour cells with difluoromethylornithine followed by an exposure of the cells to methylglyoxal bis(guanylhydrazone) resulted in a marked and rapid anti-proliferative effect. PMID:6786285

Intracellular disposition of chitosan nanoparticles in macrophages: intracellular uptake, exocytosis, and intercellular transport

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Jiang LQ

2017-08-01

Full Text Available Li Qun Jiang,1 Ting Yu Wang,1 Thomas J Webster,2 Hua-Jian Duan,1 Jing Ying Qiu,1 Zi Ming Zhao,1 Xiao Xing Yin,1,* Chun Li Zheng3,* 1Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, School of Pharmacy, Xuzhou Medical University, Xuzhou, People’s Republic of China; 2Department of Chemical Engineering, Northeastern University, Boston, MA, USA; 3School of Pharmacy, China Pharmaceutical University, Nanjing, People’s Republic of China *These authors contributed equally to this work Abstract: Biodegradable nanomaterials have been widely used in numerous medical fields. To further improve such efforts, this study focused on the intracellular disposition of chitosan nanoparticles (CsNPs in macrophages, a primary cell of the mononuclear phagocyte system (MPS. Such interactions with the MPS determine the nanoparticle retention time in the body and consequently play a significant role in their own clinical safety. In this study, various dye-labeled CsNPs (about 250 nm were prepared, and a murine macrophage cell line (RAW 264.7 was selected as a model macrophage. The results showed two mechanisms of macrophage incorporation of CsNPs, ie, a clathrin-mediated endocytosis pathway (the primary and phagocytosis. Following internalization, the particles partly dissociated in the cells, indicating cellular digestion of the nanoparticles. It was proved that, after intracellular uptake, a large proportion of CsNPs were exocytosed within 24 h; this excretion induced a decrease in fluorescence intensity in cells by 69%, with the remaining particles possessing difficulty being cleared. Exocytosis could be inhibited by both wortmannin and vacuolin-1, indicating that CsNP uptake was mediated by lysosomal and multivesicular body pathways, and after exocytosis, the reuptake of CsNPs by neighboring cells was verified by further experiments. This study, thus, elucidated the fate of CsNPs in macrophages as well as identified cellular disposition

Spectral phasor analysis of LAURDAN fluorescence in live A549 lung cells to study the hydration and time evolution of intracellular lamellar body-like structures

DEFF Research Database (Denmark)

Malacrida, Leonel; Astrada, Soledad; Briva, Arturo

2016-01-01

Using LAURDAN spectral imaging and spectral phasor analysis we concurrently studied the growth and hydration state of subcellular organelles (lamellar body-like, LB-like) from live A549 lung cancer cells at different post-confluence days. Our results reveal a time dependent two-step process...... governing the size and hydration of these intracellular LB-like structures. Specifically, a first step (days 1 to 7) is characterized by an increase in their size, followed by a second one (days 7 to 14) where the organelles display a decrease in their global hydration properties. Interestingly, our results...... also show that their hydration properties significantly differ from those observed in well-characterized artificial lamellar model membranes, challenging the notion that a pure lamellar membrane organization is present in these organelles at intracellular conditions. Finally, these LB-like structures...

Surveillance for Intracellular Antibody by Cytosolic Fc Receptor TRIM21

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William A. McEwan

2016-11-01

Full Text Available TRIM21 has emerged as an atypical Fc receptor that is broadly conserved and widely expressed in the cytoplasm of mammalian cells. Viruses that traffic surface-bound antibodies into the cell during infection recruit TRIM21 via a high affinity interaction between Fc and TRIM21 PRYSPRY domain. Following binding of intracellular antibody, TRIM21 acts as both antiviral effector and sensor for innate immune signalling. These activities serve to reduce viral replication by orders of magnitude in vitro and contribute to host survival during in vivo infection. Neutralization occurs rapidly after detection and requires the activity of the ubiquitin-proteasome system. The microbial targets of this arm of intracellular immunity are still being identified: TRIM21 activity has been reported following infection by several non-enveloped viruses and intracellular bacteria. These findings extend the sphere of influence of antibodies to the intracellular domain and have broad implications for immunity. TRIM21 has been implicated in the chronic auto-immune condition systemic lupus erythematosus and is itself an auto-antigen in Sjögren’s syndrome. This review summarises our current understanding of TRIM21’s role as a cytosolic Fc receptor and briefly discusses pathological circumstances where intracellular antibodies have been described, or are hypothesized to occur, and may benefit from further investigations of the role of TRIM21.

The encapsulation and intracellular delivery of trehalose using a thermally responsive nanocapsule

Energy Technology Data Exchange (ETDEWEB)

Zhang Wujie; He Xiaoming [Department of Mechanical Engineering, University of South Carolina, 300 Main Street, Columbia, SC 29208 (United States); Rong Jianhua; Wang Qian [Department of Chemistry and Biochemistry, University of South Carolina, 631 Sumter Street, Columbia, SC 29208 (United States)], E-mail: xmhe@sc.edu

2009-07-08

The thermally responsive wall permeability of an empty core-shell structured Pluronic nanocapsule (together with its temperature dependent size and surface charge) was successfully utilized for encapsulation, intracellular delivery, and controlled release of trehalose, a highly hydrophilic small (M{sub W} = 342 D) molecule (a disaccharide of glucose) that is exceptional for long-term stabilization of biologicals (particularly at ambient temperatures). It was found that trehalose can be physically encapsulated in the nanocapsule using a soaking-freeze-drying-heating procedure. The nanocapsule is capable of physically withholding trehalose with negligible release in hours for cellular uptake at 37 deg. C when its wall permeability is low. A quick release of the encapsulated sugar can be achieved by thermally cycling the nanocapsule between 37 and 22 deg. C (or lower). A significant amount of trehalose (up to 0.3 M) can be delivered into NIH 3T3 fibroblasts by incubating the cells with the trehalose-encapsulated nanocapsules at 37 deg. C for 40 min. Moreover, cytotoxicity of the nanocapsule for the purpose of intracellular delivery of trehalose was found to be negligible. Altogether, the thermally responsive nanocapsule is effective for intracellular delivery of trehalose, which is critical for the long-term stabilization of mammalian cells at ambient temperatures and the eventual success of modern cell-based medicine.

Is there a role for oligosaccharides in seed longevity? An assessment of intracellular glass stability

NARCIS (Netherlands)

Buitink, J.; Hemminga, M.A.; Hoekstra, F.A.

2000-01-01

We examined whether oligosaccharides extend seed longevity by increasing the intracellular glass stability. For that purpose, we used a spin probe technique to measure the molecular mobility and glass transition temperature of the cytoplasm of impatiens (Impatiens walleriana) and bell pepper

Production of intracellular selenium-enriched polysaccharides from thin stillage by Cordyceps sinensis and its bioactivities.

Science.gov (United States)

Yang, Shengli; Zhang, Hui

2016-01-01

Thin stillage was used as the substrate to produce intracellular selenium-enriched polysaccharides (ISPS) from Cordyceps sinensis to increase the value of agricultural coproducts. Fermentation parameters were optimized using response surface methodology (RSM) to improve the production of ISPS. Then, the effects of ISPS on the antioxidant activities in vitro, as well as the glycosylated serum protein concentration, malondialdehyde level, and total antioxidant capacity of streptozotocin-induced diabetic rats were studied. The optimized conditions were as follows: sodium selenite concentration, 33.78 µg/L; incubation time, 8.24 days; and incubation temperature, 26.69°C. A maximum yield of 197.35 mg/g ISPS was obtained from the validation experiments, which was quite close to the predicted maximum yield of 198.6839 mg/g. FT-IR spectra indicated that ISPS has been successfully selenylation modified with similar structure to polysaccharide of intracellular polysaccharides. The in vitro scavenging effects of 1.0 mg/mL ISPS on hydroxyl, superoxide, and 1,1-diphenyl-2-picrylhydrazyl radicals were 74.62±4.05, 71.45±3.63, and 79.48±4.75%, respectively. The reducing power of ISPS was 0.45±0.01 (absorbance at 700 nm). Fasting blood glucose and glycosylated serum protein of group C (rats with diabetes that received drinking water with ISPS) were significantly lower than those of group B (rats with diabetes) (P<0.01) after treatment was administered for 2 and 4 weeks. Serum malonaldehyde content of group C was significantly lower than that of group B at 4 weeks (P<0.01). At 4 weeks, malonaldehyde contents in heart, liver, and kidney tissues of group C were significantly lower than those of group B; however, malonaldehyde content in pancreas tissue of group C was not significantly different. Total antioxidant capacities in liver, pancreas and kidney tissues of group C were significantly higher than those of group B, but total antioxidant capacity in heart tissue was not

Production of intracellular selenium-enriched polysaccharides from thin stillage by Cordyceps sinensis and its bioactivities

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Shengli Yang

2016-02-01

Full Text Available Background: Thin stillage was used as the substrate to produce intracellular selenium-enriched polysaccharides (ISPS from Cordyceps sinensis to increase the value of agricultural coproducts. Methods: Fermentation parameters were optimized using response surface methodology (RSM to improve the production of ISPS. Then, the effects of ISPS on the antioxidant activities in vitro, as well as the glycosylated serum protein concentration, malondialdehyde level, and total antioxidant capacity of streptozotocin-induced diabetic rats were studied. Results: The optimized conditions were as follows: sodium selenite concentration, 33.78 µg/L; incubation time, 8.24 days; and incubation temperature, 26.69°C. A maximum yield of 197.35 mg/g ISPS was obtained from the validation experiments, which was quite close to the predicted maximum yield of 198.6839 mg/g. FT-IR spectra indicated that ISPS has been successfully selenylation modified with similar structure to polysaccharide of intracellular polysaccharides. The in vitro scavenging effects of 1.0 mg/mL ISPS on hydroxyl, superoxide, and 1,1-diphenyl-2-picrylhydrazyl radicals were 74.62±4.05, 71.45±3.63, and 79.48±4.75%, respectively. The reducing power of ISPS was 0.45±0.01 (absorbance at 700 nm. Fasting blood glucose and glycosylated serum protein of group C (rats with diabetes that received drinking water with ISPS were significantly lower than those of group B (rats with diabetes (P<0.01 after treatment was administered for 2 and 4 weeks. Serum malonaldehyde content of group C was significantly lower than that of group B at 4 weeks (P<0.01. At 4 weeks, malonaldehyde contents in heart, liver, and kidney tissues of group C were significantly lower than those of group B; however, malonaldehyde content in pancreas tissue of group C was not significantly different. Total antioxidant capacities in liver, pancreas and kidney tissues of group C were significantly higher than those of group B, but total

Identification of intracellular phospholipases A2 in the human eye: involvement in phagocytosis of photoreceptor outer segments

DEFF Research Database (Denmark)

Kolko, Miriam; Wang, Jinmei; Zhan, Chen

2007-01-01

PURPOSE: To identify intracellular phospholipases A(2) (PLA(2)) in the human retina and to explore the role of these enzymes in human retinal pigment epithelium (RPE) phagocytosis of photoreceptor outer segments (POS). METHODS: PCR amplification and Western blot analysis were used to identify m......)-VIA activity was found to be specifically increased 12 hours after ARPE-19 cells were fed with POS. Finally, RPE phagocytosis was inhibited by the iPLA(2)-VIA inhibitor bromoenol lactone. CONCLUSIONS: Various intracellular PLA(2) subtypes are present in the human retina. iPLA(2)-VIA may play...

Legionella pneumophila transcriptome during intracellular multiplication in human macrophages

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Sebastien P Faucher

2011-04-01

Full Text Available Legionella pneumophila is the causative agent of Legionnaires’ disease, an acute pulmonary infection. L. pneumophila is able to infect and multiply in both phagocytic protozoa, such as Acanthamoeba castellanii, and mammalian professional phagocytes. The best-known L. pneumophila virulence determinant is the Icm/Dot Type IVB secretion system (TFBSS, which is used to translocate more than 150 effector proteins to host cells. While the transcriptional response of Legionella to the intracellular environment of A. castellanii has been investigated, much less is known about the Legionella transcriptional response inside human macrophages. In this study, the transcriptome of L. pneumophila was monitored during exponential and post-exponential phase in rich AYE broth as well as during infection of human cultured macrophages. This was accomplished with microarrays and an RNA amplification procedure called SCOTS to detect small amounts of mRNA from low numbers of intracellular bacteria. Among the genes induced intracellularly are those involved in amino acid biosynthetic pathways leading to L-arginine, L-histidine and L-proline as well as many transport systems involved in amino acid and iron uptake. Gene involved in catabolism of glycerol is also induced during intracellular growth and could be used as a carbon source. The genes encoding the Icm/Dot system are not differentially expressed inside cells compared to control bacteria grown in rich broth, but the genes encoding several translocated effectors are strongly induced. Moreover, we used the transcriptome data to predict previously unrecognized Icm/Dot effector genes based on their expression pattern and confirmed translocation for three candidates. This study provides a comprehensive view of how L. pneumophila responds to the human macrophage intracellular environment.

DMS triggers apoptosis associated with the inhibition of SPHK1/NF-κB activation and increase in intracellular Ca2+ concentration in human cancer cells.

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Chen, Kan; Pan, Qiuwei; Gao, Ying; Yang, Xinyan; Wang, Shibing; Peppelenbosch, Maikel P; Kong, Xiangdong

2014-01-01

N,N-Dimethyl-D-erythro-sphingosine (DMS) is known to induce cell apoptosis by specifically inhibiting sphingosine kinase 1 (SPHK1) and modulating the activity of cellular ceramide levels. The present study investigated the effects and the mechanism(s) of action of DMS in human lung cancer cells. We found that DMS dose-dependently suppressed cell proliferation and induced cell apoptosis in the human lung cancer cell line, A549. Mechanistically, treatment with DMS suppressed the activation of SPHK1 and nuclear factor-κB (NF-κB) p65, but increased intracellular [Ca2+]i in A549 cells. This study demonstrates that DMS triggers the apoptosis of human lung cancer cells through the modulation of SPHK1, NF-κB and calcium signaling. These molecules may represent targets for anticancer drug design.

MicroRNA 128a increases intracellular ROS level by targeting Bmi-1 and inhibits medulloblastoma cancer cell growth by promoting senescence.

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Sujatha Venkataraman

Full Text Available BACKGROUND: MicroRNAs (miRNAs are a class of short non-coding RNAs that regulate cell homeostasis by inhibiting translation or degrading mRNA of target genes, and thereby can act as tumor suppressor genes or oncogenes. The role of microRNAs in medulloblastoma has only recently been addressed. We hypothesized that microRNAs differentially expressed during normal CNS development might be abnormally regulated in medulloblastoma and are functionally important for medulloblastoma cell growth. METHODOLOGY AND PRINCIPAL FINDINGS: We examined the expression of microRNAs in medulloblastoma and then investigated the functional role of one specific one, miR-128a, in regulating medulloblastoma cell growth. We found that many microRNAs associated with normal neuronal differentiation are significantly down regulated in medulloblastoma. One of these, miR-128a, inhibits growth of medulloblastoma cells by targeting the Bmi-1 oncogene. In addition, miR-128a alters the intracellular redox state of the tumor cells and promotes cellular senescence. CONCLUSIONS AND SIGNIFICANCE: Here we report the novel regulation of reactive oxygen species (ROS by microRNA 128a via the specific inhibition of the Bmi-1 oncogene. We demonstrate that miR-128a has growth suppressive activity in medulloblastoma and that this activity is partially mediated by targeting Bmi-1. This data has implications for the modulation of redox states in cancer stem cells, which are thought to be resistant to therapy due to their low ROS states.

Quantifying intracellular hydrogen peroxide perturbations in terms of concentration

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Beijing K. Huang

2014-01-01

Full Text Available Molecular level, mechanistic understanding of the roles of reactive oxygen species (ROS in a variety of pathological conditions is hindered by the difficulties associated with determining the concentration of various ROS species. Here, we present an approach that converts fold-change in the signal from an intracellular sensor of hydrogen peroxide into changes in absolute concentration. The method uses extracellular additions of peroxide and an improved biochemical measurement of the gradient between extracellular and intracellular peroxide concentrations to calibrate the intracellular sensor. By measuring peroxiredoxin activity, we found that this gradient is 650-fold rather than the 7–10-fold that is widely cited. The resulting calibration is important for understanding the mass-action kinetics of complex networks of redox reactions, and it enables meaningful characterization and comparison of outputs from endogenous peroxide generating tools and therapeutics across studies.

Caveats and limitations of plate reader-based high-throughput kinetic measurements of intracellular calcium levels

International Nuclear Information System (INIS)

Heusinkveld, Harm J.; Westerink, Remco H.S.

2011-01-01

Calcium plays a crucial role in virtually all cellular processes, including neurotransmission. The intracellular Ca 2+ concentration ([Ca 2+ ] i ) is therefore an important readout in neurotoxicological and neuropharmacological studies. Consequently, there is an increasing demand for high-throughput measurements of [Ca 2+ ] i , e.g. using multi-well microplate readers, in hazard characterization, human risk assessment and drug development. However, changes in [Ca 2+ ] i are highly dynamic, thereby creating challenges for high-throughput measurements. Nonetheless, several protocols are now available for real-time kinetic measurement of [Ca 2+ ] i in plate reader systems, though the results of such plate reader-based measurements have been questioned. In view of the increasing use of plate reader systems for measurements of [Ca 2+ ] i a careful evaluation of current technologies is warranted. We therefore performed an extensive set of experiments, using two cell lines (PC12 and B35) and two fluorescent calcium-sensitive dyes (Fluo-4 and Fura-2), for comparison of a linear plate reader system with single cell fluorescence microscopy. Our data demonstrate that the use of plate reader systems for high-throughput real-time kinetic measurements of [Ca 2+ ] i is associated with many pitfalls and limitations, including erroneous sustained increases in fluorescence, limited sensitivity and lack of single cell resolution. Additionally, our data demonstrate that probenecid, which is often used to prevent dye leakage, effectively inhibits the depolarization-evoked increase in [Ca 2+ ] i . Overall, the data indicate that the use of current plate reader-based strategies for high-throughput real-time kinetic measurements of [Ca 2+ ] i is associated with caveats and limitations that require further investigation. - Research highlights: → The use of plate readers for high-throughput screening of intracellular Ca 2+ is associated with many pitfalls and limitations. → Single cell

Antibody- and TRIM21-dependent intracellular restriction of Salmonella enterica.

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Rakebrandt, Nikolas; Lentes, Sabine; Neumann, Heinz; James, Leo C; Neumann-Staubitz, Petra

2014-11-01

TRIM21 ('tripartite motif-containing protein 21', Ro52) is a ubiquitously expressed cytosolic Fc receptor, which has a potent role in protective immunity against nonenveloped viruses. TRIM21 mediates intracellular neutralisation of antibody-coated viruses, a process called ADIN (antibody-dependent intracellular neutralisation). Our results reveal a similar mechanism to fight bacterial infections. TRIM21 is recruited to the intracellular pathogen Salmonella enterica in epithelial cells early in infection. TRIM21 does not bind directly to S. enterica, but to antibodies opsonising it. Most importantly, bacterial restriction is dependent on TRIM21 as well as on the opsonisation state of the bacteria. Finally, Salmonella and TRIM21 colocalise with the autophagosomal marker LC3, and intracellular defence is enhanced in starved cells suggesting an involvement of the autophagocytic pathway. Our data extend the protective role of TRIM21 from viruses to bacteria and thereby strengthening the general role of ADIN in cellular immunity. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Data for automated, high-throughput microscopy analysis of intracellular bacterial colonies using spot detection

DEFF Research Database (Denmark)

Ernstsen, Christina Lundgaard; Login, Frédéric H.; Jensen, Helene Halkjær

2017-01-01

Quantification of intracellular bacterial colonies is useful in strategies directed against bacterial attachment, subsequent cellular invasion and intracellular proliferation. An automated, high-throughput microscopy-method was established to quantify the number and size of intracellular bacteria...

Pursuing Intracellular Pathogens with Hyaluronan. From a 'Pro-Infection' Polymer to a Biomaterial for 'Trojan Horse' Systems.

Science.gov (United States)

Montanari, Elita; Di Meo, Chiara; Oates, Angela; Coviello, Tommasina; Matricardi, Pietro

2018-04-18

Hyaluronan (HA) is among the most important bioactive polymers in mammals, playing a key role in a number of biological functions. In the last decades, it has been increasingly studied as a biomaterial for drug delivery systems, thanks to its physico-chemical features and ability to target and enter certain cells. The most important receptor of HA is ‘Cluster of Differentiation 44’ (CD44), a cell surface glycoprotein over-expressed by a number of cancers and heavily involved in HA endocytosis. Moreover, CD44 is highly expressed by keratinocytes, activated macrophages and fibroblasts, all of which can act as ‘reservoirs’ for intracellular pathogens. Interestingly, both CD44 and HA appear to play a key role for the invasion and persistence of such microorganisms within the cells. As such, HA is increasingly recognised as a potential target for nano-carriers development, to pursuit and target intracellular pathogens, acting as a ‘Trojan Horse’. This review describes the biological relationship between HA, CD44 and the entry and survival of a number of pathogens within the cells and the subsequent development of HA-based nano-carriers for enhancing the intracellular activity of antimicrobials.

Feedback Regulation of Intracellular Hydrostatic Pressure in Surface Cells of the Lens

Science.gov (United States)

Gao, Junyuan; Sun, Xiurong; White, Thomas W.; Delamere, Nicholas A.; Mathias, Richard T.

2015-01-01

In wild-type lenses from various species, an intracellular hydrostatic pressure gradient goes from ∼340 mmHg in central fiber cells to 0 mmHg in surface cells. This gradient drives a center-to-surface flow of intracellular fluid. In lenses in which gap-junction coupling is increased, the central pressure is lower, whereas if gap-junction coupling is reduced, the central pressure is higher but surface pressure is always zero. Recently, we found that surface cell pressure was elevated in PTEN null lenses. This suggested disruption of a feedback control system that normally maintained zero surface cell pressure. Our purpose in this study was to investigate and characterize this feedback control system. We measured intracellular hydrostatic pressures in mouse lenses using a microelectrode/manometer-based system. We found that all feedback went through transport by the Na/K ATPase, which adjusted surface cell osmolarity such that pressure was maintained at zero. We traced the regulation of Na/K ATPase activity back to either TRPV4, which sensed positive pressure and stimulated activity, or TRPV1, which sensed negative pressure and inhibited activity. The inhibitory effect of TRPV1 on Na/K pumps was shown to signal through activation of the PI3K/AKT axis. The stimulatory effect of TRPV4 was shown in previous studies to go through a different signal transduction path. Thus, there is a local two-legged feedback control system for pressure in lens surface cells. The surface pressure provides a pedestal on which the pressure gradient sits, so surface pressure determines the absolute value of pressure at each radial location. We speculate that the absolute value of intracellular pressure may set the radial gradient in the refractive index, which is essential for visual acuity. PMID:26536260

Intracellular pH in increased after transformation of Chinese hamster embryo fibroblasts

International Nuclear Information System (INIS)

Ober, S.S.; Pardee, A.B.

1987-01-01

These studies reveal that a series of tumorigenic Chinese hamster embryo fibroblast (CHEF) cell lines maintain an internal pH (pH/sub i/) that is 0.12 +/- 0.04 pH unit above that of the nontumorigenic CHEF/18 parental line. pH measurements were made with [ 14 C]-benzoic acid. This increase of pH/sub i/ in the tumorigenic CHEF cells is not due to autocrine growth factor production or to the persistent activation of pathways previously shown to modulate Na + /H + -antiporter activity present in the CHEF/18 line. These findings suggest that the defect in pH/sub i/ regulation in the tumorigenic CHEF/18 derivatives lies in the Na + /H + antiporter itself. Further studies to determine the biological significance of an increased pH/sub i/ show that the external pH (pH 0 )-dependence curve for initiation of DNA synthesis in the tumorigenic CHEF lines is shifted by approximately 0.2 pH unit toward acidic values relative to that of the nontumorigenic CHEF/18 parent. These data show a critical role for pH/sub i/ in the regulation of DNA synthesis in Chinese hamster embryo fibroblasts and demonstrate that aberrations in pH/sub i/ can contribute to the acquisition of altered growth properties

Brucella abortus Induces a Warburg Shift in Host Metabolism That Is Linked to Enhanced Intracellular Survival of the Pathogen.

Science.gov (United States)

Czyż, Daniel M; Willett, Jonathan W; Crosson, Sean

2017-08-01

Intracellular bacterial pathogens exploit host cell resources to replicate and survive inside the host. Targeting these host systems is one promising approach to developing novel antimicrobials to treat intracellular infections. We show that human macrophage-like cells infected with Brucella abortus undergo a metabolic shift characterized by attenuated tricarboxylic acid cycle metabolism, reduced amino acid consumption, altered mitochondrial localization, and increased lactate production. This shift to an aerobic glycolytic state resembles the Warburg effect, a change in energy production that is well described in cancer cells and also occurs in activated inflammatory cells. B. abortus efficiently uses lactic acid as its sole carbon and energy source and requires the ability to metabolize lactate for normal survival in human macrophage-like cells. We demonstrate that chemical inhibitors of host glycolysis and lactate production do not affect in vitro growth of B. abortus in axenic culture but decrease its survival in the intracellular niche. Our data support a model in which infection shifts host metabolism to a Warburg-like state, and B. abortus uses this change in metabolism to promote intracellular survival. Pharmacological perturbation of these features of host cell metabolism may be a useful strategy to inhibit infection by intracellular pathogens. IMPORTANCE Brucella spp. are intracellular bacterial pathogens that cause disease in a range of mammals, including livestock. Transmission from livestock to humans is common and can lead to chronic human disease. Human macrophage-like cells infected with Brucella abortus undergo a Warburg-like metabolic shift to an aerobic glycolytic state where the host cells produce lactic acid and have reduced amino acid catabolism. We provide evidence that the pathogen can exploit this change in host metabolism to support growth and survival in the intracellular niche. Drugs that inhibit this shift in host cell metabolism

Evidence for an intracellular niche for Bordetella pertussis in broncho-alveolar lavage cells of mice

NARCIS (Netherlands)

Hellwig, SMM; Hazenbos, WLW; van de Winkel, JGJ; Mooi, FR

1999-01-01

Bordetella pertussis can attach, invade and survive intracellularly in human macrophages in vitro. To study the significance of this bacterial feature in vivo, we analyzed the presence of viable bacteria in broncho-alveolar lavage (BAL) cells of mice infected with B, pertussis. We found B. pertussis

THE SMALL BUT SIGNIFICANT AND NONTRANSITORY INCREASE IN PRICES (SSNIP TEST

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Liviana Niminet

2008-12-01

Full Text Available The Small but Significant Nontransitory Increase in Price Test was designed to define the relevant market by concepts of product, geographical area and time. This test, also called the ,,hypothetical monopolistic test” is the subject of many researches both economical and legal as it deals with economic concepts as well as with legally aspects.

Intracellular mechanisms of solar water disinfection

Science.gov (United States)

Castro-Alférez, María; Polo-López, María Inmaculada; Fernández-Ibáñez, Pilar

2016-12-01

Solar water disinfection (SODIS) is a zero-cost intervention measure to disinfect drinking water in areas of poor access to improved water sources, used by more than 6 million people in the world. The bactericidal action of solar radiation in water has been widely proven, nevertheless the causes for this remain still unclear. Scientific literature points out that generation of reactive oxygen species (ROS) inside microorganisms promoted by solar light absorption is the main reason. For the first time, this work reports on the experimental measurement of accumulated intracellular ROS in E. coli during solar irradiation. For this experimental achievement, a modified protocol based on the fluorescent probe dichlorodihydrofluorescein diacetate (DCFH-DA), widely used for oxidative stress in eukaryotic cells, has been tested and validated for E. coli. Our results demonstrate that ROS and their accumulated oxidative damages at intracellular level are key in solar water disinfection.

Intracellular pH in rat pancreatic ducts

DEFF Research Database (Denmark)

Novak, I; Hug, M; Greger, R

1997-01-01

In order to study the mechanism of H+ and HCO3- transport in a HCO3- secreting epithelium, pancreatic ducts, we have measured the intracellular pH (pHi) in this tissue using the pH sensitive probe BCECF. We found that exposures of ducts to solutions containing acetate/acetic acid or NH4+/NH3...... buffers (20 mmol/l) led to pHi changes in accordance with entry of lipid-soluble forms of the buffers, followed by back-regulation of pHi by duct cells. In another type of experiment, changes in extracellular pH of solutions containing HEPES or HCO3-/CO2 buffers led to significant changes in pHi that did....... Under some conditions, these exchangers can be invoked to regulate cell pH....

Subversion of the cytoskeleton by intracellular bacteria: lessons from Listeria, Salmonella, and Vibrio

Science.gov (United States)

de Souza Santos, Marcela; Orth, Kim

2018-01-01

Summary Entry into host cells and intracellular persistence by invasive bacteria are tightly coupled to the ability of the bacterium to disrupt the eukaryotic cytoskeletal machinery. Herein we review the main strategies used by three intracellular pathogens to harness key modulators of the cytoskeleton. Two of these bacteria, namely Listeria monocytogenes and Salmonella enterica serovar Typhimurium, exhibit quite distinct intracellular lifestyles, and therefore, provide a comprehensive panel for the understanding of the intricate bacteria-cytoskeleton interplay during infections. The emerging intracellular pathogen Vibrio parahaemolyticus is depicted as a developing model for the uncovering of novel mechanisms used to hijack the cytoskeleton. PMID:25440316

Increased frequency of retinopathy of prematurity over the last decade and significant regional differences.

Science.gov (United States)

Holmström, Gerd; Tornqvist, Kristina; Al-Hawasi, Abbas; Nilsson, Åsa; Wallin, Agneta; Hellström, Ann

2018-03-01

Retinopathy of prematurity (ROP) causes childhood blindness globally in prematurely born infants. Although increased levels of oxygen supply lead to increased survival and reduced frequency of cerebral palsy, increased incidence of ROP is reported. With the help of a Swedish register for ROP, SWEDROP, national and regional incidences of ROP and frequencies of treatment were evaluated from 2008 to 2015 (n = 5734), as well as before and after targets of provided oxygen changed from 85-89% to 91-95% in 2014. Retinopathy of prematurity (ROP) was found in 31.9% (1829/5734) of all infants with a gestational age (GA) of <31 weeks at birth and 5.7% of the infants (329/5734) had been treated for ROP. Analyses of the national data revealed an increased incidence of ROP during the 8-year study period (p = 0.003), but there was no significant increase in the frequency of treatment. There were significant differences between the seven health regions of Sweden, regarding both incidence of ROP and frequency of treatment (p < 0.001). Comparison of regional data before and after the new oxygen targets revealed a significant increase in treated ROP in one region [OR: 2.24 (CI: 1.11-4.49), p = 0.024] and a borderline increase in one other [OR: 3.08 (CI: 0.99-9.60), p = 0.052]. The Swedish national ROP register revealed an increased incidence of ROP during an 8-year period and significant regional differences regarding the incidence of ROP and frequency of treatment. © 2017 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

Tethering factors as organizers of intracellular vesicular traffic.

Science.gov (United States)

Yu, I-Mei; Hughson, Frederick M

2010-01-01

Intracellular trafficking entails the budding, transport, tethering, and fusion of transport vesicles and other membrane carriers. Here we review recent progress toward a mechanistic understanding of vesicle tethering. The known tethering factors are large complexes important for one or more intracellular trafficking pathways and are capable of interacting directly with many of the other principal components of the cellular trafficking machinery. Our review emphasizes recent developments in the in vitro reconstitution of vesicle tethering and the structural characterization of multisubunit tethering factors. The combination of these and other approaches has led to exciting progress toward understanding how these essential nanomachines work.

An intracellular motif of GLUT4 regulates fusion of GLUT4-containing vesicles.

Science.gov (United States)

Heyward, Catherine A; Pettitt, Trevor R; Leney, Sophie E; Welsh, Gavin I; Tavaré, Jeremy M; Wakelam, Michael J O

2008-05-20

Insulin stimulates glucose uptake by adipocytes through increasing translocation of the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane. Fusion of GLUT4-containing vesicles at the cell surface is thought to involve phospholipase D activity, generating the signalling lipid phosphatidic acid, although the mechanism of action is not yet clear. Here we report the identification of a putative phosphatidic acid-binding motif in a GLUT4 intracellular loop. Mutation of this motif causes a decrease in the insulin-induced exposure of GLUT4 at the cell surface of 3T3-L1 adipocytes via an effect on vesicle fusion. The potential phosphatidic acid-binding motif identified in this study is unique to GLUT4 among the sugar transporters, therefore this motif may provide a unique mechanism for regulating insulin-induced translocation by phospholipase D signalling.

Fluorescent probes and nanoparticles for intracellular sensing of pH values

Science.gov (United States)

Shi, Wen; Li, Xiaohua; Ma, Huimin

2014-12-01

Intracellular pH regulates a number of cell metabolism processes and its sensing is thus of great importance for cell studies. Among various methods, fluorescent probes have been widely used for sensing intracellular pH values because of their high sensitivity and spatiotemporal resolution capability. In this article, the development of fluorescent probes with good practicability in sensing intracellular pH values and pH variation during 2009 - 2014 is reviewed. These fluorescence probes are divided into two kinds: small molecules and nanoparticles. Photophysical properties, advantages/disadvantages and applications of the two kinds of probes are discussed in detail.

Intracellular insulin-receptor dissociation and segregation in a rat fibroblast cell line transfected with a human insulin receptor gene

International Nuclear Information System (INIS)

Levy, J.R.; Olefsky, J.M.

1988-01-01

The cellular processing of insulin and insulin receptors was studied using a rat fibroblast cell line that had been transfected with a normal human insulin receptor gene, expressing approximately 500 times the normal number of native fibroblasts insulin receptors. These cells bind and internalize insulin normally. Biochemically assays based on the selective precipitation by polyethylene glycol of intact insulin-receptor complexes but not of free intracellular insulin were developed to study the time course of intracellular insulin-receptor dissociation. Fibroblasts were incubated with radiolabeled insulin at 4 0 C, and internalization of insulin-receptor complexes was initiated by warming the cells to 37 0 C. Within 2 min, 90% of the internalized radioactivity was composed of intact insulin-receptor complexes. The dissociation of insulin from internalized insulin-receptor complexes was markedly inhibited by monensin and chloroquine. Furthermore, chloroquine markedly increased the number of cross-linkable intracellular insulin-receptor complexes, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. These findings suggest that acidification of intracellular vesicles is responsible for insulin-receptor dissociation. Physical segregation of dissociated intracellular insulin from its receptor was monitored. The results are consistent with the view that segregation of insulin and receptor occurs 5-10 min after initiation of dissociation. These studies demonstrate the intracellular itinerary of insulin-receptor complexes, including internalization, dissociation of insulin from the internalized receptor within an acidified compartment, segregation of insulin from the receptor, and subsequent ligand degradation

Yersinia pestis intracellular parasitism of macrophages from hosts exhibiting high and low severity of plague.

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Duraisamy Ponnusamy

Full Text Available BACKGROUND: Yersinia pestis causes severe disease in natural rodent hosts, but mild to inapparent disease in certain rodent predators such as dogs. Y. pestis initiates infection in susceptible hosts by parasitizing and multiplying intracellularly in local macrophages prior to systemic dissemination. Thus, we hypothesize that Y. pestis disease severity may depend on the degree to which intracellular Y. pestis overcomes the initial host macrophage imposed stress. METHODOLOGY/PRINCIPAL FINDINGS: To test this hypothesis, the progression of in vitro infection by Y. pestis KIM62053.1+ of mouse splenic and RAW264.7 tissue culture macrophages and dog peripheral blood-derived and DH82 tissue culture macrophages was studied using microscopy and various parameters of infection. The study showed that during the early stage of infection, intracellular Y. pestis assumed filamentous cellular morphology with multiple copies of the genome per bacterium in both mouse and dog macrophages. Later, in mouse macrophages, the infection elicited spacious vacuolar extension of Yersinia containing vacuoles (YCV, and the filamentous Y. pestis reverted to coccobacillary morphology with genomic equivalents approximately equaling colony forming units. In contrast, Y. pestis infected dog macrophages did not show noticeable extension of YCV, and intracellular Y. pestis retained the filamentous cellular morphology for the entire experiment in DH82 cells or were killed by blood-derived macrophages. In addition, during the later stage of infection, Y. pestis infected mouse macrophages exhibited cell lysis whereas dog macrophages did not. CONCLUSION/SIGNIFICANCE: Overall, these results support our hypothesis that Y. pestis in mouse macrophages can overcome the initial intracellular stress necessary for subsequent systemic infection. However, in dogs, failure of Y. pestis to overcome macrophage imposed stress may result in mild or in apparent disease in dogs.

Comparative Salt Stress Study on Intracellular Ion Concentration in Marine and Salt-adapted Freshwater Strains of Microalgae

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Ahmad Farhad TALEBI

2013-08-01

Full Text Available Salinity imposes significant stresses in various living organisms including microalgae. High extracellular concentration of Na+ directly influences ionic balance inside the cell and subsequently the cellular activities. In the present study, the effect of such stress on growth and intracellular ions concentration (IIC of Dunaliella salina and Chlorella Spp. was investigated. IIC was analyzed using Ion chromatography technique. D. salina showed the highest degree of resistance to increase in salinity as little changes occurred both in IIC and in growth parameters. D. salina could maintain the balance of K+ inside the cell and eject the excess Na+ even at NaCl concentrations above 1M. Moreover, D. salina accumulated β-carotene in order to protect its photosynthetic apparatus. Among Chlorella species, C. vulgaris showed signs of adaptation to high content of salinity, though it is a fresh water species by nature. Moreover, the response shown by C. vulgaris to rise in salinity was even stronger than that of C. salina, which is presumably a salt-water resistant species. In fact, C. vulgaris could maintain intracellular K+ better than C. salina in response to increasing salinity, and as a result, it could survive at NaCl concentrations as high as 0.75 M. Marine strains such as D. salina well cope with the fluctuations in salinity through the existing adaptation mechanisms i.e. maintaining the K+/N+ balance inside the cell, K+ accumulation and Na+ ejection, accumulation of photosynthetic pigments like β-carotene.

Exercise increases lactoferrin, but decreases lysozyme in salivary granulocytes.

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Gillum, Trevor; Kuennen, Matthew; McKenna, Zachary; Castillo, Micaela; Jordan-Patterson, Alex; Bohnert, Caitlin

2017-05-01

Intracellular lactoferrin (Lac) and lysozyme (Lys) content play an important role in regulating inflammation and promoting host protection. While exercise has demonstrated an increase in Lac and Lys concentration in exocrine solutions, little is known regarding intracellular concentration changes in response to exercise. To quantify intracellular Lac and Lys concentration before and after exercise in salivary CD45 + CD15 + cells. 11 males (20.3 ± 0.8 years, 57.2 ± 7.6 mL/kg/min V̇O 2pk , 11.1 ± 3.9% body fat) ran for 45 min at 75% of VO 2pk . 12 mL of stimulated saliva were collected pre and immediately post exercise. Saliva was filtered through a 30-µm filter before analysis of leukocytes (CD45 + ) and granulocytes (CD45 + CD15 + ) using flow cytometry. Median fluorescent intensity (MFI) of Lac increased from pre (64,268 ± 46,036 MFI) to post (117,134 ± 88,115 MFI) exercise (p exercise (pre: 16,933 ± 8249; post: 11,616 ± 6875) (p exercise. Conversely, the exercise-associated decrease of intracellular Lys content could be the cause of increased Lys in exocrine solutions.

Biomineralization Patterns of Intracellular Carbonatogenesis in Cyanobacteria: Molecular Hypotheses

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Jinhua Li

2016-02-01

Full Text Available The recent discovery of intracellular carbonatogenesis in several cyanobacteria species has challenged the traditional view that this process was extracellular and not controlled. However, a detailed analysis of the size distribution, chemical composition and 3-D-arrangement of carbonates in these cyanobacteria is lacking. Here, we characterized these features in Candidatus Gloeomargarita lithophora C7 and Candidatus Synechococcus calcipolaris G9 by conventional transmission electron microscopy, tomography, ultramicrotomy, and scanning transmission X-ray microscopy (STXM. Both Ca. G. lithophora C7 and Ca. S. calcipolaris G9 formed numerous polyphosphate granules adjacent or engulfing Ca-carbonate inclusions when grown in phosphate-rich solutions. Ca-carbonates were scattered within Ca. G. lithophora C7 cells under these conditions, but sometimes arranged in one or several chains. In contrast, Ca-carbonates formed at cell septa in Ca. S. calcipolaris G9 and were segregated equally between daughter cells after cell division, arranging as distorted disks at cell poles. The size distribution of carbonates evolved from a positively to a negatively skewed distribution as particles grew. Conventional ultramicrotomy did not preserve Ca-carbonates explaining partly why intracellular calcification has been overlooked in the past. All these new observations allow discussing with unprecedented insight some nucleation and growth processes occurring in intracellularly calcifying cyanobacteria with a particular emphasis on the possible involvement of intracellular compartments and cytoskeleton.

Luminal uptake and intracellular transport of insulin in renal proximal tubules

International Nuclear Information System (INIS)

Hellfritzsch, M.; Christensen, E.I.; Sonne, O.

1986-01-01

It is generally accepted that proteins taken up from the renal tubular fluid are transported into lysosomes in proximal tubule cells. Recently, however, it has been postulated that insulin in isolated perfused rat kidneys did not accumulate in lysosomes but to a certain degree in the Golgi region. The present study was undertaken to investigate the intracellular handling of biologically unaltered insulin in rat renal proximal tubule cells. Rats were prepared for in vivo micropuncture and either a colloidal gold insulin complex or insulin monoiodinated in the A-14 position ( 125 I-insulin) was microinfused into proximal tubules. After 5, 10, 25 or 60 min the tubules were fixed by microinfusion of glutaraldehyde and processed for electron microscopy or electron microscope autoradiography. A qualitative analysis of tubules infused with colloidal gold insulin or 125 I-insulin showed that insulin was taken up by endocytosis and transported to lysosomes, and a quantitative autoradiographic analysis of the 125 I-insulin microinfused tubules showed that the grain density after five min was significantly increased for endocytic vacuoles and for lysosomes. After 60 min the grain density was still significant over lysosomes. The accumulation of grains was non-significant over all other areas analyzed at any time. This study shows that insulin is taken up from the luminal side of the proximal tubule by endocytosis and transported to the lysosomes. There was no significant transport to the Golgi region

Evaluation of Significance of Diffusely Increased Bilateral Renal Uptake on Bone Scan

International Nuclear Information System (INIS)

Sung, Mi Sook; Yang, Woo Jin; Byun, Jae Young; Park, Jung Mi; Shinn, Kyung Sub; Bahk, Yong Whee

1990-01-01

Unexpected renal abnormality can be detected on bone scan using 99m Tc-MDP. The purpose of the study is to evaluate the diagnostic significance of diffusely increased bilateral renal uptake on bone scan. 1,500 bone scan were reviewed and 43 scans which showed diffusely increased bilateral renal uptake were selected for analysis. Laboratory findings for renal and liver function tests including routine urinalysis were reviewed in 43 patients. 26 of 43 case showed abnormality in urinalysis and renal function study. 20 of 43 cases showed abnormal liver function study and 3 of these cases were diagnosed as hepatorenal syndrome later. 13 of those 20 cases had liver cirrhosis with or without hepatoma. 12 of 43 cases showed abnormality both in renal and liver function studies. 2 of 43 cases showed diffusely increased bilateral renal uptake after chemotherapy for cancer but not on previous scans before chemotherapy. 2 of 43 cases showed hypercalcaemia and 8 of 43 cases had multifocal bone uptake due to metastasis or benign bone lesion. But the latter showed no hypercalcaemia at all. There was no significant correlation between increased renal uptake and MDP uptake in soft tissue other than kidneys. This study raised the possibility that the impaired liver and/or renal function may result in diffuse increase of bilateral renal uptake of MDP of unknown mechanism. It seems to need further study on this correlation.

Evaluation of Significance of Diffusely Increased Bilateral Renal Uptake on Bone Scan

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Sung, Mi Sook; Yang, Woo Jin; Byun, Jae Young; Park, Jung Mi; Shinn, Kyung Sub; Bahk, Yong Whee [Catholic University College of Medicine, Seoul (Korea, Republic of)

1990-03-15

Unexpected renal abnormality can be detected on bone scan using {sup 99m}Tc-MDP. The purpose of the study is to evaluate the diagnostic significance of diffusely increased bilateral renal uptake on bone scan. 1,500 bone scan were reviewed and 43 scans which showed diffusely increased bilateral renal uptake were selected for analysis. Laboratory findings for renal and liver function tests including routine urinalysis were reviewed in 43 patients. 26 of 43 case showed abnormality in urinalysis and renal function study. 20 of 43 cases showed abnormal liver function study and 3 of these cases were diagnosed as hepatorenal syndrome later. 13 of those 20 cases had liver cirrhosis with or without hepatoma. 12 of 43 cases showed abnormality both in renal and liver function studies. 2 of 43 cases showed diffusely increased bilateral renal uptake after chemotherapy for cancer but not on previous scans before chemotherapy. 2 of 43 cases showed hypercalcaemia and 8 of 43 cases had multifocal bone uptake due to metastasis or benign bone lesion. But the latter showed no hypercalcaemia at all. There was no significant correlation between increased renal uptake and MDP uptake in soft tissue other than kidneys. This study raised the possibility that the impaired liver and/or renal function may result in diffuse increase of bilateral renal uptake of MDP of unknown mechanism. It seems to need further study on this correlation.

St. John's wort significantly increased the systemic exposure and toxicity of methotrexate in rats

International Nuclear Information System (INIS)

Yang, Shih-Ying; Juang, Shin-Hun; Tsai, Shang-Yuan; Chao, Pei-Dawn Lee; Hou, Yu-Chi

2012-01-01

St. John's wort (SJW, Hypericum perforatum) is one of the popular nutraceuticals for treating depression. Methotrexate (MTX) is an immunosuppressant with narrow therapeutic window. This study investigated the effect of SJW on MTX pharmacokinetics in rats. Rats were orally given MTX alone and coadministered with 300 and 150 mg/kg of SJW, and 25 mg/kg of diclofenac, respectively. Blood was withdrawn at specific time points and serum MTX concentrations were assayed by a specific monoclonal fluorescence polarization immunoassay method. The results showed that 300 mg/kg of SJW significantly increased the AUC 0−t and C max of MTX by 163% and 60%, respectively, and 150 mg/kg of SJW significantly increased the AUC 0−t of MTX by 55%. In addition, diclofenac enhanced the C max of MTX by 110%. The mortality of rats treated with SJW was higher than that of controls. In conclusion, coadministration of SJW significantly increased the systemic exposure and toxicity of MTX. The combined use of MTX with SJW would need to be with caution. -- Highlights: ► St. John's wort significantly increased the AUC 0−t and C max of methotrexate. ► Coadministration of St. John's wort increased the exposure and toxicity of methotrexate. ► The combined use of methotrexate with St. John's wort will need to be with caution.

Detection and quantification of intracellular bacterial colonies by automated, high-throughput microscopy.

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Ernstsen, Christina L; Login, Frédéric H; Jensen, Helene H; Nørregaard, Rikke; Møller-Jensen, Jakob; Nejsum, Lene N

2017-08-01

To target bacterial pathogens that invade and proliferate inside host cells, it is necessary to design intervention strategies directed against bacterial attachment, cellular invasion and intracellular proliferation. We present an automated microscopy-based, fast, high-throughput method for analyzing size and number of intracellular bacterial colonies in infected tissue culture cells. Cells are seeded in 48-well plates and infected with a GFP-expressing bacterial pathogen. Following gentamicin treatment to remove extracellular pathogens, cells are fixed and cell nuclei stained. This is followed by automated microscopy and subsequent semi-automated spot detection to determine the number of intracellular bacterial colonies, their size distribution, and the average number per host cell. Multiple 48-well plates can be processed sequentially and the procedure can be completed in one working day. As a model we quantified intracellular bacterial colonies formed by uropathogenic Escherichia coli (UPEC) during infection of human kidney cells (HKC-8). Urinary tract infections caused by UPEC are among the most common bacterial infectious diseases in humans. UPEC can colonize tissues of the urinary tract and is responsible for acute, chronic, and recurrent infections. In the bladder, UPEC can form intracellular quiescent reservoirs, thought to be responsible for recurrent infections. In the kidney, UPEC can colonize renal epithelial cells and pass to the blood stream, either via epithelial cell disruption or transcellular passage, to cause sepsis. Intracellular colonies are known to be clonal, originating from single invading UPEC. In our experimental setup, we found UPEC CFT073 intracellular bacterial colonies to be heterogeneous in size and present in nearly one third of the HKC-8 cells. This high-throughput experimental format substantially reduces experimental time and enables fast screening of the intracellular bacterial load and cellular distribution of multiple

Intracellular sodium hydrogen exchange inhibition and clinical myocardial protection.

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Mentzer, Robert M; Lasley, Robert D; Jessel, Andreas; Karmazyn, Morris

2003-02-01

Although the mechanisms underlying ischemia/reperfusion injury remain elusive, evidence supports the etiologic role of intracellular calcium overload and oxidative stress induced by reactive oxygen species. Activation of the sodium hydrogen exchanger (NHE) is associated with intracellular calcium accumulation. Inhibition of the NHE-1 isoform may attenuate the consequences of this injury. Although there is strong preclinical and early clinical evidence that NHE inhibitors may be cardioprotective, definitive proof of this concept in humans awaits the results of ongoing clinical trials.

Regulation of intracellular pH in LLC-PK1 cells by Na+/H+ exchange.

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Montrose, M H; Murer, H

1986-01-01

Suspensions of LLC-PK1 cells (a continuous epitheliod cell line with renal characteristics) are examined for mechanisms of intracellular pH regulation using the fluorescent probe BCECF. Initial experiments determine suitable calibration procedures for use of the BCECF fluorescent signal. They also determine that the cell suspension contains cells which (after 4 hr in suspension) have Na+ and K+ gradients comparable to those of cells in monolayer culture. The steady-state intracellular pH (7.05 +/- 0.01, n = 5) of cells which have recovered in (pH 7.4) Na+-containing medium is not affected over several minutes by addition of 100 microM amiloride or removal of extracellular Na+ (Na+o less than 1 mM). In contrast, when the cells recover from an acid load (caused by NH4 preincubation and removal), the recovery is largely Na+ dependent and is sensitive to 100 microM amiloride. These results suggest that with resting pH near neutrality, both Na+o/H+i and Na+i/H+o exchange reactions are functionally inactive (compared to cellular buffering capacity). In contrast, Na+o/H+i exchange is activated by an increased cellular acid load. This activation may be observed directly either as a stimulation of net H+ efflux or net Na+ influx with decreasing intracellular pH. The extrapolation of this latter data suggests a "set point" of Na+/H+ exchange of approximately pH 7.0, consistent with the observed resting intracellular pH of approximately 7.05.

Fluorescent nanoparticles for intracellular sensing: A review

International Nuclear Information System (INIS)

Ruedas-Rama, Maria J.; Walters, Jamie D.; Orte, Angel; Hall, Elizabeth A.H.

2012-01-01

Highlights: ► Analytical applications of fluorescent nanoparticles (NPs) in intracellular sensing. ► Critical review on performance of QDots, metal NPs, silica NPs, and polymer NPs. ► Highlighted potential of fluorescence lifetime imaging microscopy (FLIM). - Abstract: Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy.

Intracellular mediators of potassium-induced aldosterone secretion

International Nuclear Information System (INIS)

Ganguly, A.; Chiou, S.; Davis, J.S.

1990-01-01

We have investigated the intracellular messengers of potassium in eliciting aldosterone secretion in calf adrenal glomerulosa cells since there were unresolved issues relating to the role of phosphoinositides, cAMP and protein kinases. We observed no evidence of hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) in 3 H-inositol labeled alf adrenal cells or increase of cAMP in response to potassium. Addition of calcium channel blocker, nitrendipine after stimulating adrenal glomerulosa cells with potassium, markedly inhibited aldosterone secretion. A calmodulin inhibitor (W-7) produced greater reduction of aldosterone secretion than an inhibitor of protein kinase C (H-7). These results suggest that a rise in cytosolic free calcium concentration through voltage-dependent calcium channel and calmodulin are the critical determinants of aldosterone secretion stimulated by potassium

Fatty Acid Signaling: The New Function of Intracellular Lipases

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Zuzana Papackova

2015-02-01

Full Text Available Until recently, intracellular triacylglycerols (TAG stored in the form of cytoplasmic lipid droplets have been considered to be only passive “energy conserves”. Nevertheless, degradation of TAG gives rise to a pleiotropic spectrum of bioactive intermediates, which may function as potent co-factors of transcription factors or enzymes and contribute to the regulation of numerous cellular processes. From this point of view, the process of lipolysis not only provides energy-rich equivalents but also acquires a new regulatory function. In this review, we will concentrate on the role that fatty acids liberated from intracellular TAG stores play as signaling molecules. The first part provides an overview of the transcription factors, which are regulated by fatty acids derived from intracellular stores. The second part is devoted to the role of fatty acid signaling in different organs/tissues. The specific contribution of free fatty acids released by particular lipases, hormone-sensitive lipase, adipose triacylglycerol lipase and lysosomal lipase will also be discussed.

Knock-down of hypoxia-induced carbonic anhydrases IX and XII radiosensitizes tumor cells by increasing intracellular acidosis

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Doyen, Jérome [Institute for Research on Cancer and Aging of Nice, CNRS UMR 7284, University of Nice Sophia-Antipolis,, Nice (France); Department of Radiation Oncology, Centre Antoine-Lacassagne, Nice (France); Parks, Scott K. [Institute for Research on Cancer and Aging of Nice, CNRS UMR 7284, University of Nice Sophia-Antipolis,, Nice (France); Marcié, Serge [Department of Radiation Oncology, Centre Antoine-Lacassagne, Nice (France); Pouysségur, Jacques [Institute for Research on Cancer and Aging of Nice, CNRS UMR 7284, University of Nice Sophia-Antipolis,, Nice (France); Centre Scientifique de Monaco (Monaco); Chiche, Johanna, E-mail: chiche@unice.fr [Institute for Research on Cancer and Aging of Nice, CNRS UMR 7284, University of Nice Sophia-Antipolis,, Nice (France)

2013-01-07

The relationship between acidosis within the tumor microenvironment and radioresistance of hypoxic tumor cells remains unclear. Previously we reported that hypoxia-induced carbonic anhydrases (CA) IX and CAXII constitute a robust intracellular pH (pH{sub i})-regulating system that confers a survival advantage on hypoxic human colon carcinoma LS174Tr cells in acidic microenvironments. Here we investigate the role of acidosis, CAIX and CAXII knock-down in combination with ionizing radiation. Fibroblasts cells (-/+ CAIX) and LS174Tr cells (inducible knock-down for ca9/ca12) were analyzed for cell cycle phase distribution and survival after irradiation in extracellular pH{sub o} manipulations and hypoxia (1% O{sub 2}) exposure. Radiotherapy was used to target ca9/ca12-silenced LS174Tr tumors grown in nude mice. We found that diminishing the pH{sub i}-regulating capacity of fibroblasts through inhibition of Na{sup +}/H{sup +} exchanger 1 sensitize cells to radiation-induced cell death. Secondly, the pH{sub i}-regulating function of CAIX plays a key protective role in irradiated fibroblasts in an acidic environment as accompanied by a reduced number of cells in the radiosensitive phases of the cell cycle. Thirdly, we demonstrate that irradiation of LS174Tr spheroids, silenced for either ca9 or both ca9/ca12, showed a respective 50 and 75% increase in cell death as a result of a decrease in cell number in the radioresistant S phase and a disruption of CA-mediated pH{sub i} regulation. Finally, LS174Tr tumor progression was strongly decreased when ca9/ca12 silencing was combined with irradiation in vivo. These findings highlight the combinatory use of radiotherapy with targeting of the pH{sub i}-regulating CAs as an anti-cancer strategy.

Data for automated, high-throughput microscopy analysis of intracellular bacterial colonies using spot detection.

Science.gov (United States)

Ernstsen, Christina L; Login, Frédéric H; Jensen, Helene H; Nørregaard, Rikke; Møller-Jensen, Jakob; Nejsum, Lene N

2017-10-01

Quantification of intracellular bacterial colonies is useful in strategies directed against bacterial attachment, subsequent cellular invasion and intracellular proliferation. An automated, high-throughput microscopy-method was established to quantify the number and size of intracellular bacterial colonies in infected host cells (Detection and quantification of intracellular bacterial colonies by automated, high-throughput microscopy, Ernstsen et al., 2017 [1]). The infected cells were imaged with a 10× objective and number of intracellular bacterial colonies, their size distribution and the number of cell nuclei were automatically quantified using a spot detection-tool. The spot detection-output was exported to Excel, where data analysis was performed. In this article, micrographs and spot detection data are made available to facilitate implementation of the method.

Quantification and isotopic analysis of intracellular sulfur metabolites in the dissimilatory sulfate reduction pathway

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Sim, Min Sub; Paris, Guillaume; Adkins, Jess F.; Orphan, Victoria J.; Sessions, Alex L.

2017-06-01

Microbial sulfate reduction exhibits a normal isotope effect, leaving unreacted sulfate enriched in 34S and producing sulfide that is depleted in 34S. However, the magnitude of sulfur isotope fractionation is quite variable. The resulting changes in sulfur isotope abundance have been used to trace microbial sulfate reduction in modern and ancient ecosystems, but the intracellular mechanism(s) underlying the wide range of fractionations remains unclear. Here we report the concentrations and isotopic ratios of sulfur metabolites in the dissimilatory sulfate reduction pathway of Desulfovibrio alaskensis. Intracellular sulfate and APS levels change depending on the growth phase, peaking at the end of exponential phase, while sulfite accumulates in the cell during stationary phase. During exponential growth, intracellular sulfate and APS are strongly enriched in 34S. The fractionation between internal and external sulfate is up to 49‰, while at the same time that between external sulfate and sulfide is just a few permil. We interpret this pattern to indicate that enzymatic fractionations remain large but the net fractionation between sulfate and sulfide is muted by the closed-system limitation of intracellular sulfate. This 'reservoir effect' diminishes upon cessation of exponential phase growth, allowing the expression of larger net sulfur isotope fractionations. Thus, the relative rates of sulfate exchange across the membrane versus intracellular sulfate reduction should govern the overall (net) fractionation that is expressed. A strong reservoir effect due to vigorous sulfate reduction might be responsible for the well-established inverse correlation between sulfur isotope fractionation and the cell-specific rate of sulfate reduction, while at the same time intraspecies differences in sulfate uptake and/or exchange rates could account for the significant scatter in this relationship. Our approach, together with ongoing investigations of the kinetic isotope

Fluorescent probes and nanoparticles for intracellular sensing of pH values

International Nuclear Information System (INIS)

Shi, Wen; Li, Xiaohua; Ma, Huimin

2014-01-01

Intracellular pH regulates a number of cell metabolism processes and its sensing is thus of great importance for cell studies. Among various methods, fluorescent probes have been widely used for sensing intracellular pH values because of their high sensitivity and spatiotemporal resolution capability. In this article, the development of fluorescent probes with good practicability in sensing intracellular pH values and pH variation during 2009 − 2014 is reviewed. These fluorescence probes are divided into two kinds: small molecules and nanoparticles. Photophysical properties, advantages/disadvantages and applications of the two kinds of probes are discussed in detail. (topical review)

Intracellular sphingosine releases calcium from lysosomes.

Science.gov (United States)

Höglinger, Doris; Haberkant, Per; Aguilera-Romero, Auxiliadora; Riezman, Howard; Porter, Forbes D; Platt, Frances M; Galione, Antony; Schultz, Carsten

2015-11-27

To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC.

Thymosin {beta}4 promotes the migration of endothelial cells without intracellular Ca{sup 2+} elevation

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Selmi, Anna [Department of Molecular and Medical Biophysics, Medical University of Lodz, 92-215 Lodz (Poland); Malinowski, Mariusz [Institute of Medical Biology, Polish Academy of Sciences, Lodz (Poland); Brutkowski, Wojciech [Nencki Institute of Experimental Biology, Polish Academy of Sciences, 02-093 Warsaw (Poland); Bednarek, Radoslaw [Department of Molecular and Medical Biophysics, Medical University of Lodz, 92-215 Lodz (Poland); Cierniewski, Czeslaw S., E-mail: czeslaw.cierniewski@umed.lodz.pl [Department of Molecular and Medical Biophysics, Medical University of Lodz, 92-215 Lodz (Poland); Institute of Medical Biology, Polish Academy of Sciences, Lodz (Poland)

2012-08-15

Numerous studies have demonstrated the effects of T{beta}4 on cell migration, proliferation, apoptosis and inflammation after exogenous treatment, but the mechanism by which T{beta}4 functions is still unclear. Previously, we demonstrated that incubation of endothelial cells with T{beta}4 induced synthesis and secretion of various proteins, including plasminogen activator inhibitor type 1 and matrix metaloproteinases. We also showed that T{beta}4 interacts with Ku80, which may operate as a novel receptor for T{beta}4 and mediates its intracellular activity. In this paper, we provide evidence that T{beta}4 induces cellular processes without changes in the intracellular Ca{sup 2+} concentration. External treatment of HUVECs with T{beta}4 and its mutants deprived of the N-terminal tetrapeptide AcSDKP (T{beta}4{sub AcSDKPT/4A}) or the actin-binding sequence KLKKTET (T{beta}4{sub KLKKTET/7A}) resulted in enhanced cell migration and formation of tubular structures in Matrigel. Surprisingly, the increased cell motility caused by T{beta}4 was not associated with the intracellular Ca{sup 2+} elevation monitored with Fluo-4 NW or Fura-2 AM. Therefore, it is unlikely that externally added T{beta}4 induces HUVEC migration via the surface membrane receptors known to generate Ca{sup 2+} influx. Our data confirm the concept that externally added T{beta}4 must be internalized to induce intracellular mechanisms supporting endothelial cell migration.

Metabolic states following accumulation of intracellular aggregates: implications for neurodegenerative diseases.

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Alexei Vazquez

Full Text Available The formation of intracellular aggregates is a common etiology of several neurodegenerative diseases. Mitochondrial defects and oxidative stress has been pointed as the major mechanistic links between the accumulation of intracellular aggregates and cell death. In this work we propose a "metabolic cell death by overcrowding" as an alternative hypothesis. Using a model of neuron metabolism, we predict that as the concentration of protein aggregates increases the neurons transit through three different metabolic phases. The first phase (0-6 mM corresponds with the normal neuron state, where the neuronal activity is sustained by the oxidative phosphorylation of lactate. The second phase (6-8.6 mM is characterized by a mixed utilization of lactate and glucose as energy substrates and a switch from ammonia uptake to ammonia release by neurons. In the third phase (8.6-9.3 mM neurons are predicted to support their energy demands from glycolysis and an alternative pathway for energy generation, involving reactions from serine synthesis, one carbon metabolism and the glycine cleavage system. The model also predicts a decrease in the maximum neuronal capacity for energy generation with increasing the concentration of protein aggregates. Ultimately this maximum capacity becomes zero when the protein aggregates reach a concentration of about 9.3 mM, predicting the cessation of neuronal activity.

Overexpression of the transcription factor Yap1 modifies intracellular redox conditions and enhances recombinant protein secretion

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Marizela Delic

2014-10-01

Full Text Available Oxidative folding of secretory proteins in the endoplasmic reticulum (ER is a redox active process, which also impacts the redox conditions in the cytosol. As the transcription factor Yap1 is involved in the transcriptional response to oxidative stress, we investigate its role upon the production of secretory proteins, using the yeast Pichia pastoris as model, and report a novel important role of Yap1 during oxidative protein folding. Yap1 is needed for the detoxification of reactive oxygen species (ROS caused by increased oxidative protein folding. Constitutive co-overexpression of PpYAP1 leads to increased levels of secreted recombinant protein, while a lowered Yap1 function leads to accumulation of ROS and strong flocculation. Transcriptional analysis revealed that more than 150 genes were affected by overexpression of YAP1, in particular genes coding for antioxidant enzymes or involved in oxidation-reduction processes. By monitoring intracellular redox conditions within the cytosol and the ER using redox-sensitive roGFP1 variants, we could show that overexpression of YAP1 restores cellular redox conditions of protein-secreting P. pastoris by reoxidizing the cytosolic redox state to the levels of the wild type. These alterations are also reflected by increased levels of oxidized intracellular glutathione (GSSG in the YAP1 co-overexpressing strain. Taken together, these data indicate a strong impact of intracellular redox balance on the secretion of (recombinant proteins without affecting protein folding per se. Re-establishing suitable redox conditions by tuning the antioxidant capacity of the cell reduces metabolic load and cell stress caused by high oxidative protein folding load, thereby increasing the secretion capacity.

The non-peptidic part determines the internalization mechanism and intracellular trafficking of peptide amphiphiles.

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Dimitris Missirlis

Full Text Available BACKGROUND: Peptide amphiphiles (PAs are a class of amphiphilic molecules able to self-assemble into nanomaterials that have shown efficient in vivo targeted delivery. Understanding the interactions of PAs with cells and the mechanisms of their internalization and intracellular trafficking is critical in their further development for therapeutic delivery applications. METHODOLOGY/PRINCIPAL FINDINGS: PAs of a novel, cell- and tissue-penetrating peptide were synthesized possessing two different lipophilic tail architectures and their interactions with prostate cancer cells were studied in vitro. Cell uptake of peptides was greatly enhanced post-modification. Internalization occurred via lipid-raft mediated endocytosis and was common for the two analogs studied. On the contrary, we identified the non-peptidic part as the determining factor of differences between intracellular trafficking and retention of PAs. PAs composed of di-stearyl lipid tails linked through poly(ethylene glycol to the peptide exhibited higher exocytosis rates and employed different recycling pathways compared to ones consisting of di-palmitic-coupled peptides. As a result, cell association of the former PAs decreased with time. CONCLUSIONS/SIGNIFICANCE: Control over peptide intracellular localization and retention is possible by appropriate modification with synthetic hydrophobic tails. We propose this as a strategy to design improved peptide-based delivery systems.

A method for functional trans-complementation of intracellular Francisella tularensis.

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Shaun Steele

Full Text Available Francisella tularensis is a highly infectious bacterial pathogen that invades and replicates within numerous host cell types. After uptake, F. tularensis bacteria escape the phagosome, replicate within the cytosol, and suppress cytokine responses. However, the mechanisms employed by F. tularensis to thrive within host cells are mostly unknown. Potential F. tularensis mutants involved in host-pathogen interactions are typically discovered by negative selection screens for intracellular replication or virulence. Mutants that fulfill these criteria fall into two categories: mutants with intrinsic intracellular growth defects and mutants that fail to modify detrimental host cell processes. It is often difficult and time consuming to discriminate between these two possibilities. We devised a method to functionally trans-complement and thus identify mutants that fail to modify the host response. In this assay, host cells are consistently and reproducibly infected with two different F. tularensis strains by physically tethering the bacteria to antibody-coated beads. To examine the efficacy of this protocol, we tested phagosomal escape, cytokine suppression, and intracellular replication for F. tularensis ΔripA and ΔpdpC. ΔripA has an intracellular growth defect that is likely due to an intrinsic defect and fails to suppress IL-1β secretion. In the co-infection model, ΔripA was unable to replicate in the host cell when wild-type bacteria infected the same cell, but cytokine suppression was rescued. Therefore, ΔripA intracellular growth is due to an intrinsic bacterial defect while cytokine secretion results from a failed host-pathogen interaction. Likewise, ΔpdpC is deficient for phagosomal escape, intracellular survival and suppression of IL-1β secretion. Wild-type bacteria that entered through the same phagosome as ΔpdpC rescued all of these phenotypes, indicating that ΔpdpC failed to properly manipulate the host. In summary, functional

Mechanically induced intracellular calcium waves in osteoblasts demonstrate calcium fingerprints in bone cell mechanotransduction.

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Godin, Lindsay M; Suzuki, Sakiko; Jacobs, Christopher R; Donahue, Henry J; Donahue, Seth W

2007-11-01

An early response to mechanical stimulation of bone cells in vitro is an increase in intracellular calcium concentration ([Ca (2+)](i)). This study analyzed the [Ca (2+)](i) wave area, magnitude, duration, rise time, fall time, and time to onset in individual osteoblasts for two identical bouts of mechanical stimulation separated by a 30-min rest period. The area under the [Ca (2+)](i) wave increased in the second loading bout compared to the first. This suggests that rest periods may potentiate mechanically induced intracellular calcium signals. Furthermore, many of the [Ca (2+)](i) wave parameters were strongly, positively correlated between the two bouts of mechanical stimulation. For example, in individual primary osteoblasts, if a cell had a large [Ca (2+)](i) wave area in the first bout it was likely to have a large [Ca (2+)](i) wave area in the second bout (r (2) = 0.933). These findings support the idea that individual bone cells have "calcium fingerprints" (i.e., a unique [Ca (2+)](i) wave profile that is reproducible for repeated exposure to a given stimulus).

Lead perturbs epidermal growth factor (EGF) modulation of intracellular calcium metabolism in clonal rat osteoblastic (ROS 17/2.8) cells

International Nuclear Information System (INIS)

Long, G.J.; Rosen, J.F.

1991-01-01

EGF, a single chain polypeptide growth factor important for many cellular functions including glycolysis and protein phophorylation, is known to modulate calcium metabolism in several cell systems. It has been shown that EGF causes an increase in Ca 2+ influx and accumulation of inositol triphosphate, and probably exhibits many, if not all, of its effects via the calcium messenger system. Lead is known to interact with and perturb normal calcium signaling pathways; hence, the purpose of this work was to determine if lead perturbs EGF modulation of calcium metabolism in ROS 17/2.8 cells and if cell functions controlled by EGF were impaired. Cells were labelled with 45 Ca (1.87 mM Ca) for 20 hr in the presence of 5 μM Pb, 50 ng/ml EGF or μM Pb and 50 ng/ml EGF. Following an EGTA rinse, kinetic parameters were determined from 45 Ca efflux curves. Three kinetic compartments described the intracellular metabolism of 45 Ca. 5 μM Pb significantly altered the effect of EGF on intracellular calcium metabolism. Calcium distribution was shifted from the fast exchanging, quantitatively small calcium pools, S 1 and S 2 to the slow exchanging, quantitatively large S 2 . There was also a 50% increase in total cell calcium in cells treated with 5 μM Pb and 50 ng/ml EGF over cells treated with 50 ng/ml EGF alone. There was also a 25% decrease in the half-time for calcium exchange from S 3 to S 1 was also decreased. These data show that Pb impairs the normal modulation of intracellular calcium homeostasis by EGF and may therefore perturb functions that are modulated by EGF via the calcium messenger system

Leishmania hijacking of the macrophage intracellular compartments.

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Liévin-Le Moal, Vanessa; Loiseau, Philippe M

2016-02-01

Leishmania spp., transmitted to humans by the bite of the sandfly vector, are responsible for the three major forms of leishmaniasis, cutaneous, diffuse mucocutaneous and visceral. Leishmania spp. interact with membrane receptors of neutrophils and macrophages. In macrophages, the parasite is internalized within a parasitophorous vacuole and engages in a particular intracellular lifestyle in which the flagellated, motile Leishmania promastigote metacyclic form differentiates into non-motile, metacyclic amastigote form. This phenomenon is induced by Leishmania-triggered events leading to the fusion of the parasitophorous vacuole with vesicular members of the host cell endocytic pathway including recycling endosomes, late endosomes and the endoplasmic reticulum. Maturation of the parasitophorous vacuole leads to the intracellular proliferation of the Leishmania amastigote forms by acquisition of host cell nutrients while escaping host defense responses. © 2015 FEBS.

pH-sensitive intracellular photoluminescence of carbon nanotube-fluorescein conjugates in human ovarian cancer cells

International Nuclear Information System (INIS)

Chen, M T; Ishikawa, F N; Gundersen, M A; Gomez, L M; Vernier, P T; Zhou, C

2009-01-01

To add to the understanding of the properties of functionalized carbon nanotubes in biological applications, we report a monotonic pH sensitivity of the intracellular fluorescence emission of single-walled carbon nanotube-fluorescein carbazide (SWCNT-FC) conjugates in human ovarian cancer cells. Light-stimulated intracellular hydrolysis of the amide linkage and localized intracellular pH changes are proposed as mechanisms. SWCNT-FC conjugates may serve as intracellular pH sensors.

An intracellular motif of GLUT4 regulates fusion of GLUT4-containing vesicles

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Welsh Gavin I

2008-05-01

Full Text Available Abstract Background Insulin stimulates glucose uptake by adipocytes through increasing translocation of the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane. Fusion of GLUT4-containing vesicles at the cell surface is thought to involve phospholipase D activity, generating the signalling lipid phosphatidic acid, although the mechanism of action is not yet clear. Results Here we report the identification of a putative phosphatidic acid-binding motif in a GLUT4 intracellular loop. Mutation of this motif causes a decrease in the insulin-induced exposure of GLUT4 at the cell surface of 3T3-L1 adipocytes via an effect on vesicle fusion. Conclusion The potential phosphatidic acid-binding motif identified in this study is unique to GLUT4 among the sugar transporters, therefore this motif may provide a unique mechanism for regulating insulin-induced translocation by phospholipase D signalling.

Memantine Can Reduce Ethanol-Induced Caspase-3 Activity and Apoptosis in H4 Cells by Decreasing Intracellular Calcium.

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Wang, Xiaolong; Chen, Jiajun; Wang, Hongbo; Yu, Hao; Wang, Changliang; You, Jiabin; Wang, Pengfei; Feng, Chunmei; Xu, Guohui; Wu, Xu; Zhao, Rui; Zhang, Guohua

2017-08-01

Caspase-3 activation and apoptosis are associated with various neurodegenerative disorders. Calcium activation is an important factor in promoting apoptosis. We, therefore, assessed the role of intracellular calcium in ethanol-induced activation of caspase-3 in H4 human neuroglioma cells and the protective effect of the NMDA receptor antagonist, memantine, on ethanol-induced apoptosis in H4 cells. H4 cells were treated with 100 mM EtOH (in culture medium) for 2 days. For interaction studies, cells were treated with memantine (4 μM), EDTA (1 mM), or BAPTA-AM (10 μM) before treatment with EtOH. Knockdown of the gene encoding the NR1 subunit of the NMDA receptor was performed using RNAi. Apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry. Cell viability was detected using an MTS cell proliferation kit. Fluorescence dual wavelength spectrophotometry was used to determine the intracellular calcium concentration. The levels of NR1, caspase-3, IP3R1, and SERCA1 proteins were detected by western blotting. NR1, IP3R1, and SERCA1 mRNA levels were detected by qPCR. We observed increased expression of NR1, IP3R1, SERCA1, and increased intracellular levels of calcium ions in H4 cells exposed to ethanol. In addition, the calcium chelators, EDTA and BAPTA, and RNAi disruption of the NMDA receptor reduced ethanol-induced caspase-3 activation in H4 cells. Memantine treatment reduced the ethanol-induced increase of intracellular calcium, caspase-3 activation, apoptosis, and the ethanol-induced decrease in cell viability. Our results indicate that ethanol-induced caspase-3 activation and apoptosis are likely to be dependent on cytosolic calcium levels and that they can be reduced by memantine treatment.

Cannabidiol induces intracellular calcium elevation and cytotoxicity in oligodendrocytes.

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Mato, Susana; Victoria Sánchez-Gómez, María; Matute, Carlos

2010-11-01

Heavy marijuana use has been linked to white matter histological alterations. However, the impact of cannabis constituents on oligodendroglial pathophysiology remains poorly understood. Here, we investigated the in vitro effects of cannabidiol, the main nonpsychoactive marijuana component, on oligodendrocytes. Exposure to cannabidiol induced an intracellular Ca(2+) rise in optic nerve oligodendrocytes that was not primarily mediated by entry from the extracellular space, nor by interactions with ryanodine or IP(3) receptors. Application of the mitochondrial protonophore carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP; 1 μM) completely prevented subsequent cannabidiol-induced Ca(2+) responses. Conversely, the increase in cytosolic Ca(2+) levels elicited by FCCP was reduced after previous exposure to cannabidiol, further suggesting that the mitochondria acts as the source of cannabidiol-evoked Ca(2+) rise in oligodendrocytes. n addition, brief exposure to cannabidiol (100 nM-10 μM) led to a concentration-dependent decrease of oligodendroglial viability that was not prevented by antagonists of CB(1), CB(2), vanilloid, A(2A) or PPARγ receptors, but was instead reduced in the absence of extracellular Ca(2+). The oligodendrotoxic effect of cannabidiol was partially blocked by inhibitors of caspase-3, -8 and -9, PARP-1 and calpains, suggesting the activation of caspase-dependent and -independent death pathways. Cannabidiol also elicited a concentration-dependent alteration of mitochondrial membrane potential, and an increase in reactive oxygen species (ROS) that was reduced in the absence of extracellular Ca(2+). Finally, cannabidiol-induced cytotoxicity was partially prevented by the ROS scavenger trolox. Together, these results suggest that cannabidiol causes intracellular Ca(2+) dysregulation which can lead to oligodendrocytes demise.

Intracellular S1P Generation Is Essential for S1P-Induced Motility of Human Lung Endothelial Cells: Role of Sphingosine Kinase 1 and S1P Lyase

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Berdyshev, Evgeny V.; Gorshkova, Irina; Usatyuk, Peter; Kalari, Satish; Zhao, Yutong; Pyne, Nigel J.; Pyne, Susan; Sabbadini, Roger A.; Garcia, Joe G. N.; Natarajan, Viswanathan

2011-01-01

Background Earlier we have shown that extracellular sphingosine-1-phosphate (S1P) induces migration of human pulmonary artery endothelial cells (HPAECs) through the activation of S1P1 receptor, PKCε, and PLD2-PKCζ-Rac1 signaling cascade. As endothelial cells generate intracellular S1P, here we have investigated the role of sphingosine kinases (SphKs) and S1P lyase (S1PL), that regulate intracellular S1P accumulation, in HPAEC motility. Methodology/Principal Findings Inhibition of SphK activity with a SphK inhibitor 2-(p-Hydroxyanilino)-4-(p-Chlorophenyl) Thiazole or down-regulation of Sphk1, but not SphK2, with siRNA decreased S1Pint, and attenuated S1Pext or serum-induced motility of HPAECs. On the contrary, inhibition of S1PL with 4-deoxypyridoxine or knockdown of S1PL with siRNA increased S1Pint and potentiated motility of HPAECs to S1Pext or serum. S1Pext mediates cell motility through activation of Rac1 and IQGAP1 signal transduction in HPAECs. Silencing of SphK1 by siRNA attenuated Rac1 and IQGAP1 translocation to the cell periphery; however, knockdown of S1PL with siRNA or 4-deoxypyridoxine augmented activated Rac1 and stimulated Rac1 and IQGAP1 translocation to cell periphery. The increased cell motility mediated by down-regulation was S1PL was pertussis toxin sensitive suggesting “inside-out” signaling of intracellularly generated S1P. Although S1P did not accumulate significantly in media under basal or S1PL knockdown conditions, addition of sodium vanadate increased S1P levels in the medium and inside the cells most likely by blocking phosphatases including lipid phosphate phosphatases (LPPs). Furthermore, addition of anti-S1P mAb to the incubation medium blocked S1Pext or 4-deoxypyridoxine-dependent endothelial cell motility. Conclusions/Significance These results suggest S1Pext mediated endothelial cell motility is dependent on intracellular S1P production, which is regulated, in part, by SphK1 and S1PL. PMID:21304987

Intracellular S1P generation is essential for S1P-induced motility of human lung endothelial cells: role of sphingosine kinase 1 and S1P lyase.

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Evgeny V Berdyshev

Full Text Available BACKGROUND: Earlier we have shown that extracellular sphingosine-1-phosphate (S1P induces migration of human pulmonary artery endothelial cells (HPAECs through the activation of S1P(1 receptor, PKCε, and PLD2-PKCζ-Rac1 signaling cascade. As endothelial cells generate intracellular S1P, here we have investigated the role of sphingosine kinases (SphKs and S1P lyase (S1PL, that regulate intracellular S1P accumulation, in HPAEC motility. METHODOLOGY/PRINCIPAL FINDINGS: Inhibition of SphK activity with a SphK inhibitor 2-(p-Hydroxyanilino-4-(p-Chlorophenyl Thiazole or down-regulation of Sphk1, but not SphK2, with siRNA decreased S1P(int, and attenuated S1P(ext or serum-induced motility of HPAECs. On the contrary, inhibition of S1PL with 4-deoxypyridoxine or knockdown of S1PL with siRNA increased S1P(int and potentiated motility of HPAECs to S1P(ext or serum. S1P(ext mediates cell motility through activation of Rac1 and IQGAP1 signal transduction in HPAECs. Silencing of SphK1 by siRNA attenuated Rac1 and IQGAP1 translocation to the cell periphery; however, knockdown of S1PL with siRNA or 4-deoxypyridoxine augmented activated Rac1 and stimulated Rac1 and IQGAP1 translocation to cell periphery. The increased cell motility mediated by down-regulation was S1PL was pertussis toxin sensitive suggesting "inside-out" signaling of intracellularly generated S1P. Although S1P did not accumulate significantly in media under basal or S1PL knockdown conditions, addition of sodium vanadate increased S1P levels in the medium and inside the cells most likely by blocking phosphatases including lipid phosphate phosphatases (LPPs. Furthermore, addition of anti-S1P mAb to the incubation medium blocked S1P(ext or 4-deoxypyridoxine-dependent endothelial cell motility. CONCLUSIONS/SIGNIFICANCE: These results suggest S1P(ext mediated endothelial cell motility is dependent on intracellular S1P production, which is regulated, in part, by SphK1 and S1PL.

Trichloroethylene-mediated cytotoxicity in human epidermal keratinocytes is mediated by the rapid accumulation of intracellular calcium: Interception by naringenin.

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Ali, F; Khan, A Q; Khan, R; Sultana, S

2016-02-01

Industrial solvents pose a significant threat to the humankind. The mechanisms of their toxicity still remain in debate. Trichloroethylene (TCE) is a widespread industrial solvent responsible for severe liver dysfunction, cutaneous toxicity in occupationally exposed humans. We utilized an in vitro system of human epidermal keratinocyte (HaCaT) cells in this study to avoid complex cell and extracellular interactions. We report the cytotoxicity of organic solvent TCE in HaCaT and its reversal by a natural flavanone, naringenin (Nar). The cytotoxicity was attributed to the rapid intracellular free calcium (Ca(2+)) release, which might lead to the elevation of protein kinase C along with robust free radical generation, instability due to energy depletion, and sensitization of intracellular stress signal transducer nuclear factor κB. These effects were actually seen to induce significant amount of genomic DNA fragmentation. Furthermore, all these effects of TCE were effectively reversed by the treatment of Nar, a natural flavanone. Our studies identify intracellular Ca as a unique target used by organic solvents in the cytotoxicity and highlight the Ca(2+) ion stabilizer properties of Nar. © The Author(s) 2015.

Depletion of intracellular calcium stores facilitates the influx of extracellular calcium in platelet derived growth factor stimulated A172 glioblastoma cells.

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Vereb, G; Szöllösi, J; Mátyus, L; Balázs, M; Hyun, W C; Feuerstein, B G

1996-05-01

Calcium signaling in non-excitable cells is the consequence of calcium release from intracellular stores, at times followed by entry of extracellular calcium through the plasma membrane. To study whether entry of calcium depends upon the level of saturation of intracellular stores, we measured calcium channel opening in the plasma membrane of single confluent A172 glioblastoma cells stimulated with platelet derived growth factor (PDGF) and/or bradykinin (BK). We monitored the entry of extracellular calcium by measuring manganese quenching of Indo-1 fluorescence. PDGF raised intracellular calcium concentration ([Ca2+]i) after a dose-dependent delay (tdel) and then opened calcium channels after a dose-independent delay (tch). At higher doses (> 3 nM), BK increased [Ca2+]i after a tdel approximately 0 s, and tch decreased inversely with both dose and peak [Ca2+]i. Experiments with thapsigargin (TG), BK, and PDGF indicated that BK and PDGF share intracellular Ca2+ pools that are sensitive to TG. When these stores were depleted by treatment with BK and intracellular BAPTA, tdel did not change, but tch fell to almost 0 s in PDGF stimulated cells, indicating that depletion of calcium stores affects calcium channel opening in the plasma membrane. Our data support the capacitative model for calcium channel opening and the steady-state model describing quantal Ca2+ release from intracellular stores.

Lactoferricin Peptides Increase Macrophages' Capacity To Kill Mycobacterium avium.

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Silva, Tânia; Moreira, Ana C; Nazmi, Kamran; Moniz, Tânia; Vale, Nuno; Rangel, Maria; Gomes, Paula; Bolscher, Jan G M; Rodrigues, Pedro N; Bastos, Margarida; Gomes, Maria Salomé

2017-01-01

Mycobacterial infections cause a significant burden of disease and death worldwide. Their treatment is long, toxic, costly, and increasingly prone to failure due to bacterial resistance to currently available antibiotics. New therapeutic options are thus clearly needed. Antimicrobial peptides represent an important source of new antimicrobial molecules, both for their direct activity and for their immunomodulatory potential. We have previously reported that a short version of the bovine antimicrobial peptide lactoferricin with amino acids 17 to 30 (LFcin17-30), along with its variants obtained by specific amino acid substitutions, killed Mycobacterium avium in broth culture. In the present work, those peptides were tested against M. avium living inside its natural host cell, the macrophage. We found that the peptides increased the antimicrobial action of the conventional antibiotic ethambutol inside macrophages. Moreover, the d-enantiomer of the lactoferricin peptide (d-LFcin17-30) was more stable and induced significant killing of intracellular mycobacteria by itself. Interestingly, d-LFcin17-30 did not localize to M. avium -harboring phagosomes but induced the production of proinflammatory cytokines and increased the formation of lysosomes and autophagosome-like vesicles. These results lead us to conclude that d-LFcin17-30 primes macrophages for intracellular microbial digestion through phagosomal maturation and/or autophagy, culminating in mycobacterial killing. IMPORTANCE The genus Mycobacterium comprises several pathogenic species, including M. tuberculosis , M. leprae , M. avium , etc. Infections caused by these bacteria are particularly difficult to treat due to their intrinsic impermeability, low growth rate, and intracellular localization. Antimicrobial peptides are increasingly acknowledged as potential treatment tools, as they have a high spectrum of activity, low tendency to induce bacterial resistance, and immunomodulatory properties. In this study, we

PKC-η-MARCKS Signaling Promotes Intracellular Survival of Unopsonized Burkholderia thailandensis.

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Micheva-Viteva, Sofiya N; Shou, Yulin; Ganguly, Kumkum; Wu, Terry H; Hong-Geller, Elizabeth

2017-01-01

Pathogenic Burkholderia rely on host factors for efficient intracellular replication and are highly refractory to antibiotic treatment. To identify host genes that are required by Burkholderia spp. during infection, we performed a RNA interference (RNAi) screen of the human kinome and identified 35 host kinases that facilitated Burkholderia thailandensis intracellular survival in human monocytic THP-1 cells. We validated a selection of host kinases using imaging flow cytometry to assess efficiency of B. thailandensis survival in the host upon siRNA-mediated knockdown. We focused on the role of the novel protein kinase C isoform, PKC-η, in Burkholderia infection and characterized PKC-η/MARCKS signaling as a key event that promotes the survival of unopsonized B. thailandensis CDC2721121 within host cells. While infection of lung epithelial cells with unopsonized Gram-negative bacteria stimulated phosphorylation of Ser175/160 in the MARCKS effector domain, siRNA-mediated knockdown of PKC-η expression reduced the levels of phosphorylated MARCKS by >3-fold in response to infection with Bt CDC2721121. We compared the effect of the conventional PKC-α and novel PKC-η isoforms on the growth of B. thailandensis CDC2721121 within monocytic THP-1 cells and found that ≥75% knock-down of PRKCH transcript levels reduced intracellular bacterial load 100% more efficiently when compared to growth in cells siRNA-depleted of the classical PKC-α, suggesting that the PKC-η isoform can specifically mediate Burkholderia intracellular survival. Based on imaging studies of intracellular B. thailandensis , we found that PKC-η function stimulates phagocytic pathways that promote B. thailandensis escape into the cytoplasm leading to activation of autophagosome flux. Identification of host kinases that are targeted by Burkholderia during infection provides valuable molecular insights in understanding Burkholderia pathogenesis, and ultimately, in designing effective host

Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors

International Nuclear Information System (INIS)

Zhao Weihong; Zhong Li; Wu Jianqing; Chen Linyuan; Qing Keyun; Weigel-Kelley, Kirsten A.; Larsen, Steven H.; Shou Weinian; Warrington, Kenneth H.; Srivastava, Arun

2006-01-01

We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increased the transduction efficiency of conventional AAV vectors by ∼25-fold in WT MEFs, but only by ∼4-fold in KO MEFs. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency ∼23-fold in WT MEFs, but only ∼4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, ∼59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only ∼28% in KO MEFs, indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increase in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of recombinant AAV vectors in human gene

Microsporidia are natural intracellular parasites of the nematode Caenorhabditis elegans.

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Troemel, Emily R; Félix, Marie-Anne; Whiteman, Noah K; Barrière, Antoine; Ausubel, Frederick M

2008-12-09

For decades the soil nematode Caenorhabditis elegans has been an important model system for biology, but little is known about its natural ecology. Recently, C. elegans has become the focus of studies of innate immunity and several pathogens have been shown to cause lethal intestinal infections in C. elegans. However none of these pathogens has been shown to invade nematode intestinal cells, and no pathogen has been isolated from wild-caught C. elegans. Here we describe an intracellular pathogen isolated from wild-caught C. elegans that we show is a new species of microsporidia. Microsporidia comprise a large class of eukaryotic intracellular parasites that are medically and agriculturally important, but poorly understood. We show that microsporidian infection of the C. elegans intestine proceeds through distinct stages and is transmitted horizontally. Disruption of a conserved cytoskeletal structure in the intestine called the terminal web correlates with the release of microsporidian spores from infected cells, and appears to be part of a novel mechanism by which intracellular pathogens exit from infected cells. Unlike in bacterial intestinal infections, the p38 MAPK and insulin/insulin-like growth factor (IGF) signaling pathways do not appear to play substantial roles in resistance to microsporidian infection in C. elegans. We found microsporidia in multiple wild-caught isolates of Caenorhabditis nematodes from diverse geographic locations. These results indicate that microsporidia are common parasites of C. elegans in the wild. In addition, the interaction between C. elegans and its natural microsporidian parasites provides a system in which to dissect intracellular intestinal infection in vivo and insight into the diversity of pathogenic mechanisms used by intracellular microbes.

Ghrelin increases intracellular Ca²⁺ concentration in the various hormone-producing cell types of the rat pituitary gland.

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Yamazaki, Mami; Aizawa, Sayaka; Tanaka, Toru; Sakai, Takafumi; Sakata, Ichiro

2012-09-20

Ghrelin, isolated from the stomach as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), has potent growth hormone release ability in vivo and in vitro. Although GHS-R is abundantly expressed in the pituitary gland, there is no direct evidence of a relationship between hormone-producing cells and functional GHS-R in the pituitary gland. The aim of this study was to determine which anterior pituitary cells respond to ghrelin stimulation in male rats. We performed Fura-2 Ca(2+) imaging analysis using isolated pituitary cells, and performed immunocytochemistry to identify the type of pituitary hormone-producing cells. In Fura-2 Ca(2+) imaging analysis, ghrelin administration increased the intracellular Ca(2+) concentration in approximately 50% of total isolated anterior pituitary cells, and 20% of these cells strongly responded to ghrelin. Immunocytochemical analysis revealed that 82.9 ± 1.3% of cells that responded to ghrelin stimulation were GH-immunopositive. On the other hand, PRL-, LH-, and ACTH-immunopositive cells constituted 2.0 ± 0.3%, 12.6 ± 0.3%, and 2.5 ± 0.8% of ghrelin-responding pituitary cells, respectively. TSH-immunopositive cells did not respond to ghrelin treatment. These results suggest that ghrelin directly acts not only on somatotrophs, but also on mammotrophs, gonadotrophs, and corticotrophs in the rat pituitary gland. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

DMPD: NOD-like receptors (NLRs): bona fide intracellular microbial sensors. [Dynamic Macrophage Pathway CSML Database

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Full Text Available 18585455 NOD-like receptors (NLRs): bona fide intracellular microbial sensors. Shaw...tml) (.csml) Show NOD-like receptors (NLRs): bona fide intracellular microbial sensors. PubmedID 18585455 Ti...tle NOD-like receptors (NLRs): bona fide intracellular microbial sensors. Authors

Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel

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Milos B. Rokic

2018-04-01

Full Text Available P2X2 receptors (P2X2R exhibit a slow desensitization during the initial ATP application and a progressive, calcium-dependent increase in rates of desensitization during repetitive stimulation. This pattern is observed in whole-cell recordings from cells expressing recombinant and native P2X2R. However, desensitization is not observed in perforated-patched cells and in two-electrode voltage clamped oocytes. Addition of ATP, but not ATPγS or GTP, in the pipette solution also abolishes progressive desensitization, whereas intracellular injection of apyrase facilitates receptor desensitization. Experiments with injection of alkaline phosphatase or addition of staurosporine and ATP in the intracellular solution suggest a role for a phosphorylation-dephosphorylation in receptor desensitization. Mutation of residues that are potential phosphorylation sites identified a critical role of the S363 residue in the intracellular ATP action. These findings indicate that intracellular calcium and ATP have opposing effects on P2X2R gating: calcium allosterically facilitates receptor desensitization and ATP covalently prevents the action of calcium. Single cell measurements further revealed that intracellular calcium stays elevated after washout in P2X2R-expressing cells and the blockade of mitochondrial sodium/calcium exchanger lowers calcium concentrations during washout periods to basal levels, suggesting a role of mitochondria in this process. Therefore, the metabolic state of the cell can influence P2X2R gating.

14-3-3 Proteins Buffer Intracellular Calcium Sensing Receptors to Constrain Signaling.

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Michael P Grant

Full Text Available Calcium sensing receptors (CaSR interact with 14-3-3 binding proteins at a carboxyl terminal arginine-rich motif. Mutations identified in patients with familial hypocalciuric hypercalcemia, autosomal dominant hypocalcemia, pancreatitis or idiopathic epilepsy support the functional importance of this motif. We combined total internal reflection fluorescence microscopy and biochemical approaches to determine the mechanism of 14-3-3 protein regulation of CaSR signaling. Loss of 14-3-3 binding caused increased basal CaSR signaling and plasma membrane levels, and a significantly larger signaling-evoked increase in plasma membrane receptors. Block of core glycosylation with tunicamycin demonstrated that changes in plasma membrane CaSR levels were due to differences in exocytic rate. Western blotting to quantify time-dependent changes in maturation of expressed wt CaSR and a 14-3-3 protein binding-defective mutant demonstrated that signaling increases synthesis to maintain constant levels of the immaturely and maturely glycosylated forms. CaSR thus operates by a feed-forward mechanism, whereby signaling not only induces anterograde trafficking of nascent receptors but also increases biosynthesis to maintain steady state levels of net cellular CaSR. Overall, these studies suggest that 14-3-3 binding at the carboxyl terminus provides an important buffering mechanism to increase the intracellular pool of CaSR available for signaling-evoked trafficking, but attenuates trafficking to control the dynamic range of responses to extracellular calcium.

Intracellular response to process optimization and impact on productivity and product aggregates for a high-titer CHO cell process.

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Handlogten, Michael W; Lee-O'Brien, Allison; Roy, Gargi; Levitskaya, Sophia V; Venkat, Raghavan; Singh, Shailendra; Ahuja, Sanjeev

2018-01-01

A key goal in process development for antibodies is to increase productivity while maintaining or improving product quality. During process development of an antibody, titers were increased from 4 to 10 g/L while simultaneously decreasing aggregates. Process development involved optimization of media and feed formulations, feed strategy, and process parameters including pH and temperature. To better understand how CHO cells respond to process changes, the changes were implemented in a stepwise manner. The first change was an optimization of the feed formulation, the second was an optimization of the medium, and the third was an optimization of process parameters. Multiple process outputs were evaluated including cell growth, osmolality, lactate production, ammonium concentration, antibody production, and aggregate levels. Additionally, detailed assessment of oxygen uptake, nutrient and amino acid consumption, extracellular and intracellular redox environment, oxidative stress, activation of the unfolded protein response (UPR) pathway, protein disulfide isomerase (PDI) expression, and heavy and light chain mRNA expression provided an in-depth understanding of the cellular response to process changes. The results demonstrate that mRNA expression and UPR activation were unaffected by process changes, and that increased PDI expression and optimized nutrient supplementation are required for higher productivity processes. Furthermore, our findings demonstrate the role of extra- and intracellular redox environment on productivity and antibody aggregation. Processes using the optimized medium, with increased concentrations of redox modifying agents, had the highest overall specific productivity, reduced aggregate levels, and helped cells better withstand the high levels of oxidative stress associated with increased productivity. Specific productivities of different processes positively correlated to average intracellular values of total glutathione. Additionally

Fluorescent nanoparticles for intracellular sensing: A review

Energy Technology Data Exchange (ETDEWEB)

Ruedas-Rama, Maria J., E-mail: mjruedas@ugr.esmailto [Department of Physical Chemistry, Faculty of Pharmacy, University of Granada, Campus Cartuja, 18071, Granada (Spain); Walters, Jamie D. [Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, UK CB2 1QT (United Kingdom); Orte, Angel [Department of Physical Chemistry, Faculty of Pharmacy, University of Granada, Campus Cartuja, 18071, Granada (Spain); Hall, Elizabeth A.H., E-mail: lisa.hall@biotech.cam.ac.uk [Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT (United Kingdom)

2012-11-02

Highlights: Black-Right-Pointing-Pointer Analytical applications of fluorescent nanoparticles (NPs) in intracellular sensing. Black-Right-Pointing-Pointer Critical review on performance of QDots, metal NPs, silica NPs, and polymer NPs. Black-Right-Pointing-Pointer Highlighted potential of fluorescence lifetime imaging microscopy (FLIM). - Abstract: Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy.

Bioavailable Concentrations of Delphinidin and Its Metabolite, Gallic Acid, Induce Antioxidant Protection Associated with Increased Intracellular Glutathione in Cultured Endothelial Cells

Science.gov (United States)

Goszcz, Katarzyna; Deakin, Sherine J.; Duthie, Garry G.; Stewart, Derek

2017-01-01

Despite limited bioavailability and rapid degradation, dietary anthocyanins are antioxidants with cardiovascular benefits. This study tested the hypothesis that the antioxidant protection conferred by the anthocyanin, delphinidin, is mediated by modulation of endogenous antioxidant defences, driven by its degradation product, gallic acid. Delphinidin was found to degrade rapidly (t1/2 ~ 30 min), generating gallic acid as a major degradation product. Both delphinidin and gallic acid generated oxygen-centred radicals at high (100 μM) concentrations in vitro. In a cultured human umbilical vein endothelial cell model of oxidative stress, the antioxidant protective effects of both delphinidin and gallic acid displayed a hormesic profile; 100 μM concentrations of both were cytotoxic, but relatively low concentrations (100 nM–1 μM) protected the cells and were associated with increased intracellular glutathione. We conclude that delphinidin is intrinsically unstable and unlikely to confer any direct antioxidant activity in vivo yet it offered antioxidant protection to cells at low concentrations. This paradox might be explained by the ability of the degradation product, gallic acid, to confer benefit. The findings are important in understanding the mode of protection conferred by anthocyanins and reinforce the necessity to conduct in vitro experiments at biologically relevant concentrations. PMID:29081896

Soluble Moringa oleifera leaf extract reduces intracellular cadmium accumulation and oxidative stress in Saccharomyces cerevisiae.

Science.gov (United States)

Kerdsomboon, Kittikhun; Tatip, Supinda; Kosasih, Sattawat; Auesukaree, Choowong

2016-05-01

Moringa oleifera leaves are a well-known source of antioxidants and traditionally used for medicinal applications. In the present study, the protective action of soluble M. oleifera leaf extract (MOLE) against cadmium toxicity was investigated in the model eukaryote Saccharomyces cerevisiae. The results showed that this extract exhibited a protective effect against oxidative stress induced by cadmium and H2O2 through the reduction of intracellular reactive oxygen species. Interestingly, not only the co-exposure of soluble MOLE with cadmium but also pretreatment of this extract prior to cadmium exposure significantly reduced the cadmium uptake through an inhibition of Fet4p, a low-affinity iron(II) transporter. In addition, the supplementation of soluble MOLE significantly reduced intracellular iron accumulation in a Fet4p-independent manner. Our findings suggest the potential use of soluble extract from M. oleifera leaves as a dietary supplement for protection against cadmium accumulation and oxidative stress. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Imaging intracellular pH in a reef coral and symbiotic anemone.

Science.gov (United States)

Venn, A A; Tambutté, E; Lotto, S; Zoccola, D; Allemand, D; Tambutté, S

2009-09-29

The challenges corals and symbiotic cnidarians face from global environmental change brings new urgency to understanding fundamental elements of their physiology. Intracellular pH (pHi) influences almost all aspects of cellular physiology but has never been described in anthozoans or symbiotic cnidarians, despite its pivotal role in carbon concentration for photosynthesis and calcification. Using confocal microscopy and the pH sensitive probe carboxy SNARF-1, we mapped pHi in short-term light and dark-incubated cells of the reef coral Stylophora pistillata and the symbiotic anemone Anemonia viridis. In all cells isolated from both species, pHi was markedly lower than the surrounding seawater pH of 8.1. In cells that contained symbiotic algae, mean values of pHi were significantly higher in light treated cells than dark treated cells (7.41 +/- 0.22 versus 7.13 +/- 0.24 for S. pistillata; and 7.29 +/- 0.15 versus 7.01 +/- 0.27 for A. viridis). In contrast, there was no significant difference in pHi in light and dark treated cells without algal symbionts. Close inspection of the interface between host cytoplasm and algal symbionts revealed a distinct area of lower pH adjacent to the symbionts in both light and dark treated cells, possibly associated with the symbiosome membrane complex. These findings are significant developments for the elucidation of models of inorganic carbon transport for photosynthesis and calcification and also provide a cell imaging procedure for future investigations into how pHi and other fundamental intracellular parameters in corals respond to changes in the external environment such as reductions in seawater pH.

Reduction of intracellular glutathione content and radiosensitivity

International Nuclear Information System (INIS)

Vos, O.; Schans, G.P. van der; Roos-Verheij, W.S.D.

1986-05-01

The intracellular glutathione (GSH) content in HeLa, CHO and V79 cells was reduced by incubating the cells in growth medium containing buthionine sulfoximine (BSO) or diethyl maleate (DEM). Clonogenicity, single strand DNA breaks (ssb) and double strand DNA breaks (dsb) were used as criteria for radiation induced damage after X- or γ irradiation. In survival experiments DEM gave a slightly larger sensitization although it gave a smaller reduction of the intracellular GSH. In general, sensitization was larger for dsb than for ssb, also the reduction of the OER was generally larger for dsb than for ssb. This may be due to the higher dose rate in case of dsb experiments resulting in a higher rate of radiochemical oxygen consumption. In general, no effect was found on post-irradiation repair of ssb and dsb. (Auth.)

Reduction of intracellular glutathione content and radiosensitivity

International Nuclear Information System (INIS)

Vos, O.; Schans, G.P. van der; Roos-Verheij, W.S.D.

1986-01-01

The intracellular glutathione (GSH) content of HeLa, CHO and V79 cells was reduced by incubating the cells in growth medium containing buthionine sulphoximine or diethyl maleate (DEM). Clonogenicity, single-strand DNA breaks (ssb) and double-strand DNA breaks (dsb) were used as criteria for radiation-induced damage after X- or γ-irradiation. In survival experiments, DEM gave a slightly larger sensitization although it gave a smaller reduction of the intracellular GSH. In general, sensitization was larger for dsb than for ssb, also the reduction of the o.e.r. was generally larger for dsb than for ssb. This may be due to the higher dose rate in case of dsb experiments resulting in a higher rate of radiochemical oxygen consumption. In general, no effect was found on post-irradiation repair of ssb and dsb. (author)

HFE mRNA expression is responsive to intracellular and extracellular iron loading: short communication.

Science.gov (United States)

Mehta, Kosha J; Farnaud, Sebastien; Patel, Vinood B

2017-10-01

In liver hepatocytes, the HFE gene regulates cellular and systemic iron homeostasis by modulating cellular iron-uptake and producing the iron-hormone hepcidin in response to systemic iron elevation. However, the mechanism of iron-sensing in hepatocytes remain enigmatic. Therefore, to study the effect of iron on HFE and hepcidin (HAMP) expressions under distinct extracellular and intracellular iron-loading, we examined the effect of holotransferrin treatment (1, 2, 5 and 8 g/L for 6 h) on intracellular iron levels, and mRNA expressions of HFE and HAMP in wild-type HepG2 and previously characterized iron-loaded recombinant-TfR1 HepG2 cells. Gene expression was analyzed by real-time PCR and intracellular iron was measured by ferrozine assay. Data showed that in the wild-type cells, where intracellular iron content remained unchanged, HFE expression remained unaltered at low holotransferrin treatments but was upregulated upon 5 g/L (p HFE and HAMP expressions were elevated only at low 1 g/L treatment (p HFE (p HFE mRNA was independently elevated by extracellular and intracellular iron-excess. Thus, it may be involved in sensing both, extracellular and intracellular iron. Repression of HAMP expression under simultaneous intracellular and extracellular iron-loading resembles non-hereditary iron-excess pathologies.

Multielemental accumulation and its intracellular distribution in tissues of some aquatic birds

International Nuclear Information System (INIS)

Nam, Dong-Ha; Anan, Yasumi; Ikemoto, Tokutaka; Tanabe, Shinsuke

2005-01-01

This study was aimed at determining multielemental concentration and its intracellular distribution in selected tissues of cormorant and waterfowl species. Non-essential elements such as Hg, Tl, Cd, Pb and V in tissues were generally consistent with those in ingested items, indicating the significance of food sources of non-essential metal accumulation in great cormorants and mallards. Great cormorants and four waterfowl species examined reflected natural background levels of toxic metals such as Cd, Hg and Pb as well as some essential elements, indicating no specific metal exposure from local sources. Most of Cu, Zn, Se, Rb, Ag, Cd, Cs, and Hg contents were present in the hepatocytosolic fraction, whereas a large percentage of V and Mo were present in insoluble fraction in great cormorant, mallard, and spot-billed duck. The major role of these subcelluar fractions in elemental regulation accounts for the high percentage contribution of each cellular fraction to the total metal contents. Cadmium and Cu are chiefly sequestered through binding to metallothioneins (MTs) of hepatocytosolic fraction in these three avian species. Both MTs and high-molecular-weight substance (HMWS) for Zn and low-molecular-weight substance (LMWS) for Rb were also involved in their sequestration in cytosolic fractions. Relatively different species-specific cytosolic substances were responsible for varying degrees of Ag, Mn, and Co accumulation. It is worth noting that these intracellular metal levels in birds are closely regulated by metal-associated cellular constituents. Therefore, risk assessment studies of metal accumulation in such wild birds should take intracellular metal distribution and specific cellular constituents into account

Jinlida granule inhibits palmitic acid induced-intracellular lipid accumulation and enhances autophagy in NIT-1 pancreatic β cells through AMPK activation.

Science.gov (United States)

Wang, Dingkun; Tian, Min; Qi, Yuan; Chen, Guang; Xu, Lijun; Zou, Xin; Wang, Kaifu; Dong, Hui; Lu, Fuer

2015-02-23

Jinlida granule (JLDG), composed of seventeen Chinese medical herbs, is a widely used Chinese herbal prescription for treating diabetes mellitus. However, the mechanism underlying this effect remains unclear. To determine the main components in JLDG and to explore the effect of JLDG on autophagy and lipid accumulation in NIT-1 pancreatic β cells exposed to politic acid (PA) through AMP activated protein kinase (AMPK) signaling pathway. JLDG was prepared and the main components contained in the granules were identified by ultra performance liquid chromatography (UPLC) fingerprint. Intracellular lipid accumulation in NIT-1 cells was induced by culturing with medium containing PA. Intracellular lipid droplets were observed by Oil Red O staining and triglyceride (TG) content was measured by colorimetric assay. The formation of autophagosomes was observed under transmission electron microscope. The expression of AMPK and phospho-AMPK (pAMPK) proteins as well as its downstream fatty acid metabolism-related proteins (fatty acid synthase, FAS; acetyl-coA carboxylase, ACC; carnitine acyltransferase 1, CPT-1) and autophagy-related genes (mammal target of rapamycin, mTOR; tuberous sclerosis complex 1, TSC1; microtubule-associated protein 1 light chain 3, LC3-II) were determined by Western blot. The expression of sterol regulating element binding protein 1c (SREBP-1c) mRNA was examined by real time PCR (RT-PCR). Our data showed that JLDG could significantly reduce PA-induced intracellular lipid accumulation in NIT-1 pancreatic β cells. This effect was associated with increased protein expression of pAMPK and AMPK in NIT-1 cells. Treatment with JLDG also decreased the expression of AMPK downstream lipogenic genes (SREBP-1c mRNA, FAS and ACC proteins) whereas increased the expression of fatty acid oxidation gene (CPT-1 protein). Additionally, JLDG-treated cells displayed a markedly increase in the number of autophagosomes which was accompanied by the down-regulation of m

The β-hemolysin and intracellular survival of Streptococcus agalactiae in human macrophages.

Science.gov (United States)

Sagar, Anubha; Klemm, Carolin; Hartjes, Lara; Mauerer, Stefanie; van Zandbergen, Ger; Spellerberg, Barbara

2013-01-01

S. agalactiae (group B streptococci, GBS) is a major microbial pathogen in human neonates and causes invasive infections in pregnant women and immunocompromised individuals. The S. agalactiae β-hemolysin is regarded as an important virulence factor for the development of invasive disease. To examine the role of β-hemolysin in the interaction with professional phagocytes, the THP-1 monocytic cell line and human granulocytes were infected with a serotype Ia S. agalactiae wild type strain and its isogenic nonhemolytic mutant. We could show that the nonhemolytic mutants were able to survive in significantly higher numbers than the hemolytic wild type strain, in THP-1 macrophage-like cells and in assays with human granulocytes. Intracellular bacterial multiplication, however, could not be observed. The hemolytic wild type strain stimulated a significantly higher release of Tumor Necrosis Factor-α than the nonhemolytic mutant in THP-1 cells, while similar levels of the chemokine Interleukin-8 were induced. In order to investigate bacterial mediators of IL-8 release in this setting, purified cell wall preparations from both strains were tested and found to exert a potent proinflammatory stimulus on THP-1 cells. In conclusion, our results indicate that the β-hemolysin has a strong influence on the intracellular survival of S. agalactiae and that a tightly controlled regulation of β-hemolysin expression is required for the successful establishment of S. agalactiae in different host niches.

Glutathione provides a source of cysteine essential for intracellular multiplication of Francisella tularensis.

Directory of Open Access Journals (Sweden)

Khaled Alkhuder

2009-01-01

Full Text Available Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularemia. Its ability to multiply and survive in macrophages is critical for its virulence. By screening a bank of HimarFT transposon mutants of the F. tularensis live vaccine strain (LVS to isolate intracellular growth-deficient mutants, we selected one mutant in a gene encoding a putative gamma-glutamyl transpeptidase (GGT. This gene (FTL_0766 was hence designated ggt. The mutant strain showed impaired intracellular multiplication and was strongly attenuated for virulence in mice. Here we present evidence that the GGT activity of F. tularensis allows utilization of glutathione (GSH, gamma-glutamyl-cysteinyl-glycine and gamma-glutamyl-cysteine dipeptide as cysteine sources to ensure intracellular growth. This is the first demonstration of the essential role of a nutrient acquisition system in the intracellular multiplication of F. tularensis. GSH is the most abundant source of cysteine in the host cytosol. Thus, the capacity this intracellular bacterial pathogen has evolved to utilize the available GSH, as a source of cysteine in the host cytosol, constitutes a paradigm of bacteria-host adaptation.

Delivery of rifampicin-chitin nanoparticles into the intracellular compartment of polymorphonuclear leukocytes.

Science.gov (United States)

Smitha, K T; Nisha, N; Maya, S; Biswas, Raja; Jayakumar, R

2015-03-01

Polymorphonuclear leukocytes (PMNs) provide the primary host defence against invading pathogens by producing reactive oxygen species (ROS) and microbicidal products. However, few pathogens can survive for a prolonged period of time within the PMNs. Additionally their intracellular lifestyle within the PMNs protect themselves from the additional lethal action of host immune systems such as antibodies and complements. Antibiotic delivery into the intracellular compartments of PMNs is a major challenge in the field of infectious diseases. In order to deliver antibiotics within the PMNs and for the better treatment of intracellular bacterial infections we synthesized rifampicin (RIF) loaded amorphous chitin nanoparticles (RIF-ACNPs) of 350±50 nm in diameter. RIF-ACNPs nanoparticles are found to be non-hemolytic and non-toxic against a variety of host cells. The release of rifampicin from the prepared nanoparticles was ∼60% in 24 h, followed by a sustained pattern till 72 h. The RIF-ACNPs nanoparticles showed 5-6 fold enhanced delivery of RIF into the intracellular compartments of PMNs. The RIF-ACNPs showed anti-microbial activity against Escherichia coli, Staphylococcus aureus and a variety of other bacteria. In summary, our results suggest that RIF-ACNPs could be used to treat a variety of intracellular bacterial infections. Copyright © 2014 Elsevier B.V. All rights reserved.

Intracellular phase for an extracellular bacterial pathogen: MgtC shows the way

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Audrey Bernut

2015-08-01

Full Text Available Pseudomonas aeruginosa is an extracellular pathogen known to impair host phagocytic functions. However, our recent results identify MgtC as a novel actor in P. aeruginosa virulence, which plays a role in an intramacrophage phase of this pathogen. In agreement with its intracellular function, P. aeruginosa mgtC gene expression is strongly induced when the bacteria reside within macrophages. MgtC was previously known as a horizontally-acquired virulence factor important for multiplication inside macrophages in several intracellular bacterial pathogens. MgtC thus provides a singular example of a virulence determinant that subverts macrophages both in intracellular and extracellular pathogens. Moreover, we demonstrate that P. aeru-ginosa MgtC is required for optimal growth in Mg2+ deprived medium, a property shared by MgtC factors from intracellular pathogens and, under Mg2+ limitation, P. aeruginosaMgtC prevents biofilm formation. We propose that MgtC has a similar function in intracellular and extracellular pathogens, which contributes to macrophage resistance and fine-tune adaptation to the host in relation to the different bacterial lifestyles. MgtC thus appears as an attractive target for antivirulence strategies and our work provides a natural peptide as MgtC antagonist, which paves the way for the development of MgtC inhibitors.

Modeling nanoparticle uptake and intracellular distribution using stochastic process algebras

Energy Technology Data Exchange (ETDEWEB)

Dobay, M. P. D., E-mail: maria.pamela.david@physik.uni-muenchen.de; Alberola, A. Piera; Mendoza, E. R.; Raedler, J. O., E-mail: joachim.raedler@physik.uni-muenchen.de [Ludwig-Maximilians University, Faculty of Physics, Center for NanoScience (Germany)

2012-03-15

Computational modeling is increasingly important to help understand the interaction and movement of nanoparticles (NPs) within living cells, and to come to terms with the wealth of data that microscopy imaging yields. A quantitative description of the spatio-temporal distribution of NPs inside cells; however, it is challenging due to the complexity of multiple compartments such as endosomes and nuclei, which themselves are dynamic and can undergo fusion and fission and exchange their content. Here, we show that stochastic pi calculus, a widely-used process algebra, is well suited for mapping surface and intracellular NP interactions and distributions. In stochastic pi calculus, each NP is represented as a process, which can adopt various states such as bound or aggregated, as well as be passed between processes representing location, as a function of predefined stochastic channels. We created a pi calculus model of gold NP uptake and intracellular movement and compared the evolution of surface-bound, cytosolic, endosomal, and nuclear NP densities with electron microscopy data. We demonstrate that the computational approach can be extended to include specific molecular binding and potential interaction with signaling cascades as characteristic for NP-cell interactions in a wide range of applications such as nanotoxicity, viral infection, and drug delivery.

Modeling nanoparticle uptake and intracellular distribution using stochastic process algebras

International Nuclear Information System (INIS)

Dobay, M. P. D.; Alberola, A. Piera; Mendoza, E. R.; Rädler, J. O.

2012-01-01

Computational modeling is increasingly important to help understand the interaction and movement of nanoparticles (NPs) within living cells, and to come to terms with the wealth of data that microscopy imaging yields. A quantitative description of the spatio-temporal distribution of NPs inside cells; however, it is challenging due to the complexity of multiple compartments such as endosomes and nuclei, which themselves are dynamic and can undergo fusion and fission and exchange their content. Here, we show that stochastic pi calculus, a widely-used process algebra, is well suited for mapping surface and intracellular NP interactions and distributions. In stochastic pi calculus, each NP is represented as a process, which can adopt various states such as bound or aggregated, as well as be passed between processes representing location, as a function of predefined stochastic channels. We created a pi calculus model of gold NP uptake and intracellular movement and compared the evolution of surface-bound, cytosolic, endosomal, and nuclear NP densities with electron microscopy data. We demonstrate that the computational approach can be extended to include specific molecular binding and potential interaction with signaling cascades as characteristic for NP-cell interactions in a wide range of applications such as nanotoxicity, viral infection, and drug delivery.

Modeling nanoparticle uptake and intracellular distribution using stochastic process algebras

Science.gov (United States)

Dobay, M. P. D.; Alberola, A. Piera; Mendoza, E. R.; Rädler, J. O.

2012-03-01

Computational modeling is increasingly important to help understand the interaction and movement of nanoparticles (NPs) within living cells, and to come to terms with the wealth of data that microscopy imaging yields. A quantitative description of the spatio-temporal distribution of NPs inside cells; however, it is challenging due to the complexity of multiple compartments such as endosomes and nuclei, which themselves are dynamic and can undergo fusion and fission and exchange their content. Here, we show that stochastic pi calculus, a widely-used process algebra, is well suited for mapping surface and intracellular NP interactions and distributions. In stochastic pi calculus, each NP is represented as a process, which can adopt various states such as bound or aggregated, as well as be passed between processes representing location, as a function of predefined stochastic channels. We created a pi calculus model of gold NP uptake and intracellular movement and compared the evolution of surface-bound, cytosolic, endosomal, and nuclear NP densities with electron microscopy data. We demonstrate that the computational approach can be extended to include specific molecular binding and potential interaction with signaling cascades as characteristic for NP-cell interactions in a wide range of applications such as nanotoxicity, viral infection, and drug delivery.

Pharmacokinetic-Pharmacodynamic modelling of intracellular Mycobacterium tuberculosis growth and kill rates is predictive of clinical treatment duration.

Science.gov (United States)

Aljayyoussi, Ghaith; Jenkins, Victoria A; Sharma, Raman; Ardrey, Alison; Donnellan, Samantha; Ward, Stephen A; Biagini, Giancarlo A

2017-03-29

Tuberculosis (TB) treatment is long and complex, typically involving a combination of drugs taken for 6 months. Improved drug regimens to shorten and simplify treatment are urgently required, however a major challenge to TB drug development is the lack of predictive pre-clinical tools. To address this deficiency, we have adopted a new high-content imaging-based approach capable of defining the killing kinetics of first line anti-TB drugs against intracellular Mycobacterium tuberculosis (Mtb) residing inside macrophages. Through use of this pharmacokinetic-pharmacodynamic (PK-PD) approach we demonstrate that the killing dynamics of the intracellular Mtb sub-population is critical to predicting clinical TB treatment duration. Integrated modelling of intracellular Mtb killing alongside conventional extracellular Mtb killing data, generates the biphasic responses typical of those described clinically. Our model supports the hypothesis that the use of higher doses of rifampicin (35 mg/kg) will significantly reduce treatment duration. Our described PK-PD approach offers a much needed decision making tool for the identification and prioritisation of new therapies which have the potential to reduce TB treatment duration.

Molecular detection and characterization of sustainable intracellular ...

African Journals Online (AJOL)

3Centre for Biopolymer and Bio-Molecular Research, Athlone College of Technology, Republic of Ireland. ... cells was associated with the elongation of micro-villar extension that ... Keywords: Intracellular contaminants, cell cultures, bacteria culture, pre-clinical studies. ... production work involving culture technology.

Cationic polymers for intracellular delivery of proteins

NARCIS (Netherlands)

Coué, G.M.J.P.C.; Engbersen, Johannes F.J.; Samal, Sangram; Dubruel, Peter

2015-01-01

Many therapeutic proteins exert their pharmaceutical action inside the cytoplasm or onto individual organelles inside the cell. Intracellular protein delivery is considered to be the most direct, fastest and safest approach for curing gene-deficiency diseases, enhancing vaccination and triggering

Biological synthesis and characterization of intracellular gold ...

Indian Academy of Sciences (India)

thods of reduction of metal ions using plants or microorganisms are often ... have several advantages over bacteria, they are often pre- ferred. ... in static condition for a period of 7 days. ... work was focused on the production of intracellular gold.

Size-Dependent Regulation of Intracellular Trafficking of Polystyrene Nanoparticle-Based Drug-Delivery Systems.

Science.gov (United States)

Wang, Ting; Wang, Lu; Li, Xiaoming; Hu, Xingjie; Han, Yuping; Luo, Yao; Wang, Zejun; Li, Qian; Aldalbahi, Ali; Wang, Lihua; Song, Shiping; Fan, Chunhai; Zhao, Yun; Wang, Maolin; Chen, Nan

2017-06-07

Nanoparticles (NPs) have shown great promise as intracellular imaging probes or nanocarriers and are increasingly being used in biomedical applications. A detailed understanding of how NPs get "in and out" of cells is important for developing new nanomaterials with improved selectivity and less cytotoxicity. Both physical and chemical characteristics have been proven to regulate the cellular uptake of NPs. However, the exocytosis process and its regulation are less explored. Herein, we investigated the size-regulated endocytosis and exocytosis of carboxylated polystyrene (PS) NPs. PS NPs with a smaller size were endocytosed mainly through the clathrin-dependent pathway, whereas PS NPs with a larger size preferred caveolae-mediated endocytosis. Furthermore, our results revealed exocytosis of larger PS NPs and tracked the dynamic process at the single-particle level. These results indicate that particle size is a key factor for the regulation of intracellular trafficking of NPs and provide new insight into the development of more effective cellular nanocarriers.

Getting Across the Plasma Membrane and Beyond: Intracellular Uses of Colloidal Semiconductor Nanocrystals

Directory of Open Access Journals (Sweden)

Camilla Luccardini

2007-01-01

Full Text Available Semiconductor nanocrystals (NCs are increasingly being used as photoluminescen markers in biological imaging. Their brightness, large Stokes shift, and high photostability compared to organic fluorophores permit the exploration of biological phenomena at the single-molecule scale with superior temporal resolution and spatial precision. NCs have predominantly been used as extracellular markers for tagging and tracking membrane proteins. Successful internalization and intracellular labelling with NCs have been demonstrated for both fixed immunolabelled and live cells. However, the precise localization and subcellular compartment labelled are less clear. Generally, live cell studies are limited by the requirement of fairly invasive protocols for loading NCs and the relatively large size of NCs compared to the cellular machinery, along with the subsequent sequestration of NCs in endosomal/lysosomal compartments. For long-period observation the potential cytotoxicity of cytoplasmically loaded NCs must be evaluated. This review focuses on the challenges of intracellular uses of NCs.

Intracellular Ca2+ Regulation in Calcium Sensitive Phenotype of Saccharomyces cerevisiae

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HERMANSYAH

2010-03-01

Full Text Available Intracellular cytosolic Ca2+ concentration accumulation plays an essential information in Saccharomyces cerevisiae i.e. to explain cellular mechanism of Ca2+ sensitive phenotype. Disruption both S. cerevisiae PPase PTP2 and MSG5 genes showed an inhibited growth in the presence of Ca2+. On the other hand, by using Luminocounter with apoaequorin system, a method based upon luminescent photoprotein aequorin, intracellular Ca2+ concentration was accumulated as a consequence of calcium sensitive phenotype of S. cerevisiae. This fact indicated that PPase ptp2Δ and msg5Δ were involved in intracellular Ca2+ transport in addition their already known pathways i.e Mitogen Activated Protein Kinase cell wall integrity pathway, high osmolarity glycerol (HOG pathway, and pheromone response FUS3 pathway.

Mycobacterium avium-intracellulare cellulitis occurring with septic arthritis after joint injection: a case report

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Murdoch David M

2007-02-01

Full Text Available Abstract Background Cellulitis caused by Mycobacterium avium-intracellulare has rarely been described. Mycobacterium avium-intracellulare is a rare cause of septic arthritis after intra-articular injection, though the causative role of injection is difficult to ascertain in such cases. Case presentation A 57-year-old with rheumatoid arthritis treated with prednisone and azathioprine developed bilateral painful degenerative shoulder arthritis. After corticosteroid injections into both acromioclavicular joints, he developed bilateral cellulitis centered over the injection sites. Skin biopsy showed non-caseating granulomas, and culture grew Mycobacterium avium-intracellulare. Joint aspiration also revealed Mycobacterium avium-intracellulare infection. Conclusion Although rare, skin and joint infections caused by Mycobacterium avium-intracellulare should be considered in any immunocompromised host, particularly after intra-articular injection. Stains for acid-fast bacilli may be negative in pathologic samples even in the presence of infection; cultures of tissue specimens should always be obtained.

Intracellular nitrate of marine diatoms as a driver of anaerobic nitrogen cycling in sinking aggregates

DEFF Research Database (Denmark)

Kamp, Anja; Stief, Peter; Bristow, Laura A.

2016-01-01

% was recovered as nitrite. Hence, aggregate-associated diatoms accumulate nitrate from the surrounding water and sustain complex nitrogen transformations, including loss of fixed nitrogen, in anoxic, pelagic microniches. Additionally, it may be expected that intracellular nitrate not converted before...... store nitrate intracellularly, we explored the fate of intracellular nitrate and its availability for microbial metabolism within anoxic diatom-bacteria aggregates. The ubiquitous nitrate-storing diatom Skeletonema marinoi was studied as both axenic cultures and laboratory-produced diatom......-bacteria aggregates. Stable 15N isotope incubations under dark and anoxic conditions revealed that axenic S. marinoi is able to reduce intracellular nitrate to ammonium that is immediately excreted by the cells. When exposed to a light:dark cycle and oxic conditions, S. marinoi stored nitrate intracellularly...

Effect of metabolic and respiratory acidosis on intracellular calcium in osteoblasts.

Science.gov (United States)

Frick, Kevin K; Bushinsky, David A

2010-08-01

In vivo, metabolic acidosis {decreased pH from decreased bicarbonate concentration ([HCO(3)(-)])} increases urine calcium (Ca) without increased intestinal Ca absorption, resulting in a loss of bone Ca. Conversely, respiratory acidosis [decreased pH from increased partial pressure of carbon dioxide (Pco(2))] does not appreciably alter Ca homeostasis. In cultured bone, chronic metabolic acidosis (Met) significantly increases cell-mediated net Ca efflux while isohydric respiratory acidosis (Resp) does not. The proton receptor, OGR1, appears critical for cell-mediated, metabolic acid-induced bone resorption. Perfusion of primary bone cells or OGR1-transfected Chinese hamster ovary (CHO) cells with Met induces transient peaks of intracellular Ca (Ca(i)). To determine whether Resp increases Ca(i), as does Met, we imaged Ca(i) in primary cultures of bone cells. pH for Met = 7.07 ([HCO(3)(-)] = 11.8 mM) and for Resp = 7.13 (Pco(2) = 88.4 mmHg) were similar and lower than neutral (7.41). Both Met and Resp induced a marked, transient increase in Ca(i) in individual bone cells; however, Met stimulated Ca(i) to a greater extent than Resp. We used OGR1-transfected CHO cells to determine whether OGR1 was responsible for the greater increase in Ca(i) in Met than Resp. Both Met and Resp induced a marked, transient increase in Ca(i) in OGR1-transfected CHO cells; however, in these cells Met was not different than Resp. Thus, the greater induction of Ca(i) by Met in primary bone cells is not a function of OGR1 alone, but must involve H(+) receptors other than OGR1, or pathways sensitive to Pco(2), HCO(3)(-), or total CO(2) that modify the effect of H(+) in primary bone cells.

Intracellular Nitrate of Marine Diatoms as a Driver of Anaerobic Nitrogen Cycling in Sinking Aggregates

Directory of Open Access Journals (Sweden)

Anja Kamp

2016-11-01

Full Text Available Diatom-bacteria aggregates are key for the vertical transport of organic carbon in the ocean. Sinking aggregates also represent pelagic microniches with intensified microbial activity, oxygen depletion in the center, and anaerobic nitrogen cycling. Since some of the aggregate-forming diatom species store nitrate intracellularly, we explored the fate of intracellular nitrate and its availability for microbial metabolism within anoxic diatom-bacteria aggregates. The ubiquitous nitrate-storing diatom Skeletonema marinoi was studied as both axenic cultures and laboratory-produced diatom-bacteria aggregates. Stable 15N isotope incubations under dark and anoxic conditions revealed that axenic S. marinoi is able to reduce intracellular nitrate to ammonium that is immediately excreted by the cells. When exposed to a light:dark cycle and oxic conditions, S. marinoi stored nitrate intracellularly in concentrations > 60 mmol L-1 both as free-living cells and associated to aggregates. Intracellular nitrate concentrations exceeded extracellular concentrations by three orders of magnitude. Intracellular nitrate was used up within 2-3 days after shifting diatom-bacteria aggregates to dark and anoxic conditions. Thirty-one percent of the diatom-derived nitrate was converted to nitrogen gas, indicating that a substantial fraction of the intracellular nitrate pool of S. marinoi becomes available to the aggregate-associated bacterial community. Only 5% of the intracellular nitrate was reduced to ammonium, while 59% was recovered as nitrite. Hence, aggregate-associated diatoms accumulate nitrate from the surrounding water and sustain complex nitrogen transformations, including loss of fixed nitrogen, in anoxic, pelagic microniches. Additionally, it may be expected that intracellular nitrate not converted before the aggregates have settled onto the seafloor could fuel benthic nitrogen transformations.

The influence of statins on the free intracellular calcium concentration in human umbilical vein endothelial cells

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Figulla Hans R

2004-05-01

Full Text Available Abstract Background Statins are cholesterol-lowering drugs that are widely used to reduce the risk of cardiac infarction. Their beneficial clinical effects, however, are not restricted to their influence on cholesterol production. As several studies have shown that they have a potency of relaxing blood vessels. Methods We measured the effects of statins on the intracellular free calcium concentration ([Ca2+]i in human umbilical vein endothelial cells (HUVEC after acute application and 24-h-preincubation of statins. Results Incubation of the cells for 24 h with cerivastatin or fluvastatin significantly increased the resting [Ca2+]i. For cerivastatin this effect manifested at a concentration of 1 μM. Increase of resting [Ca2+]i in the presence of cerivastatin also occurred when the nitric oxide synthase was inhibited. Transient Ca2+ release induced by histamine was not affected. Conclusions The increase of resting [Ca2+]i after incubation with cerivastatin or fluvastatin may provide an explanation for the direct effects of statins on the endothelial-dependent vasodilatation and restoration of endothelial activity in vivo.

Kinetic identification of an intracellular calcium compartment sensitive to phosphate and dinitrophenol in intact isolated rabbit aorta

International Nuclear Information System (INIS)

Hai, C.M.; Phair, R.D.

1986-01-01

Previous work from this laboratory revealed the presence of at least three distinct intracellular calcium compartments in intact segments of rabbit aorta. In this study one of these intracellular compartments is shown to be sensitive to dinitrophenol and to increased extracellular phosphate. Intact aortic segments were loaded with 45 Ca in bicarbonate-buffered physiologic salt solution for 1 hour, and then transferred to a flow-through chamber perfused with physiologic salt solution. Effluent from the chamber was collected for 8 hours, and 45 Ca efflux curves were analyzed using compartmental analysis. When aortic segments were loaded and washed out in dinitrophenol, the slowest component of the efflux curve was less prominent; in high phosphate it was more prominent. The rate constant changes required to account for these data were primarily in the exchange between the cytosolic and slowest intracellular calcium compartment, suggesting that the slowest calcium compartment resolved during the 8-hour washout was mitochondrial. This compartment contained 5.4 +/- 3.2 nmol calcium/g wet wt. tissue. The calcium flux across its membranes was 0.32 +/- 0.04 nmol min-1g-1. Because this flux is much smaller than the plasma-membrane calcium flux, we suggest that, in normal physiological circumstances, plasma-membrane extrusion is more important for the removal of Ca from the smooth muscle cytosol than is uptake into this slow intracellular compartment

Influence of docosahexaenoic acid in vitro on intracellular adriamycin concentration in lymphocytes and human adriamycin-sensitive and -resistant small-cell lung cancer cell lines, and on cytotoxicity in the tumor cell lines

NARCIS (Netherlands)

Zijlstra, J G; de Vries, E G; Muskiet, F A; Martini, I A; Timmer-Bosscha, H; Mulder, N H

1987-01-01

An increase in the therapeutic effects of cancer chemotherapeutic agents and circumvention of drug resistance in cancer cells might result from an increase in the intracellular drug level. Alteration of the lipid domain of the cell membrane can result in a higher intracellular drug level. This

Cellular chloride and bicarbonate retention alters intracellular pH regulation in Cftr KO crypt epithelium.

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Walker, Nancy M; Liu, Jinghua; Stein, Sydney R; Stefanski, Casey D; Strubberg, Ashlee M; Clarke, Lane L

2016-01-15

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), an anion channel providing a major pathway for Cl(-) and HCO3 (-) efflux across the apical membrane of the epithelium. In the intestine, CF manifests as obstructive syndromes, dysbiosis, inflammation, and an increased risk for gastrointestinal cancer. Cftr knockout (KO) mice recapitulate CF intestinal disease, including intestinal hyperproliferation. Previous studies using Cftr KO intestinal organoids (enteroids) indicate that crypt epithelium maintains an alkaline intracellular pH (pHi). We hypothesized that Cftr has a cell-autonomous role in downregulating pHi that is incompletely compensated by acid-base regulation in its absence. Here, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein microfluorimetry of enteroids showed that Cftr KO crypt epithelium sustains an alkaline pHi and resistance to cell acidification relative to wild-type. Quantitative real-time PCR revealed that Cftr KO enteroids exhibit downregulated transcription of base (HCO3 (-))-loading proteins and upregulation of the basolateral membrane HCO3 (-)-unloader anion exchanger 2 (Ae2). Although Cftr KO crypt epithelium had increased Ae2 expression and Ae2-mediated Cl(-)/HCO3 (-) exchange with maximized gradients, it also had increased intracellular Cl(-) concentration relative to wild-type. Pharmacological reduction of intracellular Cl(-) concentration in Cftr KO crypt epithelium normalized pHi, which was largely Ae2-dependent. We conclude that Cftr KO crypt epithelium maintains an alkaline pHi as a consequence of losing both Cl(-) and HCO3 (-) efflux, which impairs pHi regulation by Ae2. Retention of Cl(-) and an alkaline pHi in crypt epithelium may alter several cellular processes in the proliferative compartment of Cftr KO intestine. Copyright © 2016 the American Physiological Society.

Cellular chloride and bicarbonate retention alters intracellular pH regulation in Cftr KO crypt epithelium

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Walker, Nancy M.; Liu, Jinghua; Stein, Sydney R.; Stefanski, Casey D.; Strubberg, Ashlee M.

2015-01-01

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), an anion channel providing a major pathway for Cl− and HCO3− efflux across the apical membrane of the epithelium. In the intestine, CF manifests as obstructive syndromes, dysbiosis, inflammation, and an increased risk for gastrointestinal cancer. Cftr knockout (KO) mice recapitulate CF intestinal disease, including intestinal hyperproliferation. Previous studies using Cftr KO intestinal organoids (enteroids) indicate that crypt epithelium maintains an alkaline intracellular pH (pHi). We hypothesized that Cftr has a cell-autonomous role in downregulating pHi that is incompletely compensated by acid-base regulation in its absence. Here, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein microfluorimetry of enteroids showed that Cftr KO crypt epithelium sustains an alkaline pHi and resistance to cell acidification relative to wild-type. Quantitative real-time PCR revealed that Cftr KO enteroids exhibit downregulated transcription of base (HCO3−)-loading proteins and upregulation of the basolateral membrane HCO3−-unloader anion exchanger 2 (Ae2). Although Cftr KO crypt epithelium had increased Ae2 expression and Ae2-mediated Cl−/HCO3− exchange with maximized gradients, it also had increased intracellular Cl− concentration relative to wild-type. Pharmacological reduction of intracellular Cl− concentration in Cftr KO crypt epithelium normalized pHi, which was largely Ae2-dependent. We conclude that Cftr KO crypt epithelium maintains an alkaline pHi as a consequence of losing both Cl− and HCO3− efflux, which impairs pHi regulation by Ae2. Retention of Cl− and an alkaline pHi in crypt epithelium may alter several cellular processes in the proliferative compartment of Cftr KO intestine. PMID:26542396

Decreased intracellular [Ca2+ ] coincides with reduced expression of Dhprα1s, RyR1, and diaphragmatic dysfunction in a rat model of sepsis.

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Wang, Meng-Meng; Hao, Li-Ying; Guo, Feng; Zhong, Bin; Zhong, Xiao-Mei; Yuan, Jing; Hao, Yi-Fei; Zhao, Shuang; Sun, Xue-Fei; Lei, Ming; Jiao, Guang-Yu

2017-12-01

Sepsis can cause decreased diaphragmatic contractility. Intracellular calcium as a second messenger is central to diaphragmatic contractility. However, changes in intracellular calcium concentration ([Ca 2+ ]) and the distribution and co-localization of relevant calcium channels [dihydropyridine receptors, (DHPRα1s) and ryanodine receptors (RyR1)] remain unclear during sepsis. In this study we investigated the effect of changed intracellular [Ca 2+ ] and expression and distribution of DHPRα1s and RyR1 on diaphragm function during sepsis. We measured diaphragm contractility and isolated diaphragm muscle cells in a rat model of sepsis. The distribution and co-localization of DHPRα1s and RyR1 were determined using immunohistochemistry and immunofluorescence, whereas intracellular [Ca 2+ ] was measured by confocal microscopy and fluorescence spectrophotometry. Septic rat diaphragm contractility, expression of DHPRα1s and RyR1, and intracellular [Ca 2+ ] were significantly decreased in the rat sepsis model compared with controls. Decreased intracellular [Ca 2+ ] coincides with diaphragmatic contractility and decreased expression of DHPRα1s and RyR1 in sepsis. Muscle Nerve 56: 1128-1136, 2017. © 2017 Wiley Periodicals, Inc.

Killing of intracellular Mycobacterium tuberculosis by receptor-mediated drug delivery

International Nuclear Information System (INIS)

Majumdar, S.; Basu, S.K.

1991-01-01

p-Aminosalicylic acid (PAS) conjugated to maleylated bovine serum albumin (MBSA) was taken up efficiently through high-affinity MBSA-binding sites on macrophages. Binding of the radiolabeled conjugate to cultured mouse peritoneal macrophages at 4 degrees C was competed for by MBSA but not by PAS. At 37 degrees C, the radiolabeled conjugate was rapidly degraded by the macrophages, leading to release of acid-soluble degradation products in the medium. The drug conjugate was nearly 100 times as effective as free PAS in killing the intracellular mycobacteria in mouse peritoneal macrophages infected in culture with Mycobacterium tuberculosis. The killing of intracellular mycobacteria mediated by the drug conjugate was effectively prevented by simultaneous addition of excess MBSA (100 micrograms/ml) or chloroquine (3 microM) to the medium, whereas these agents did not affect the microbicidal action of free PAS. These results suggest that (i) uptake of the PAS-MBSA conjugate was mediated by cell surface receptors on macrophages which recognize MBSA and (ii) lysosomal hydrolysis of the internalized conjugate resulted in intracellular release of a pharmacologically active form of the drug, which led to selective killing of the M. tuberculosis harbored by mouse macrophages infected in culture. This receptor-mediated modality of delivering drugs to macrophages could contribute to greater therapeutic efficacy and minimization of toxic side effects in the management of tuberculosis and other intracellular mycobacterial infections

[Establishment of oxygen and glucose deprive model of in vitro cultured hippocampal neuron and effect of ligustrazine on intracellular Ca+ level in model neurons].

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Wan, Hai-tong; Wang, Yu; Yang, Jie-hong

2007-03-01

To establish the oxygen and glucose deprive (OGD) model in cultured hippocampal neuron and study the effect of ligustrazine on intracellular Ca2+ level in the model neurons. The OGD model was established in cultured hippocampal neuron, and the intracellular Ca2+ level in it was detected by laser scanning confocal microscope (LSCM). The OGD model was successfully established in cultured hippocampal neurons; the intracellular Ca2+ level in the OGD model group was significantly higher than that in the blank control group (P neuron, which could be antagonized by ligustrazine, indicating that ligustrazine has a protective effect on hippocampal neuron from hypoxic-ischemic injury.

Intracellular Glutathione Depletion by Oridonin Leads to Apoptosis in Hepatic Stellate Cells

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Liang-Mou Kuo

2014-03-01

Full Text Available Proliferation of hepatic stellate cells (HSCs plays a key role in the pathogenesis of liver fibrosis. Induction of HSC apoptosis by natural products is considered an effective strategy for treating liver fibrosis. Herein, the apoptotic effects of 7,20-epoxy-ent-kaurane (oridonin, a diterpenoid isolated from Rabdosia rubescens, and its underlying mechanisms were investigated in rat HSC cell line, HSC-T6. We found that oridonin inhibited cell viability of HSC-T6 in a concentration-dependent manner. Oridonin induced a reduction in mitochondrial membrane potential and increases in caspase 3 activation, subG1 phase, and DNA fragmentation. These apoptotic effects of oridonin were completely reversed by thiol antioxidants, N-acetylcysteine (NAC and glutathione monoethyl ester. Moreover, oridonin increased production of reactive oxygen species (ROS, which was also inhibited by NAC. Significantly, oridonin reduced intracellular glutathione (GSH level in a concentration- and time-dependent fashion. Additionally, oridonin induced phosphorylations of extracellular signal-regulated kinase (ERK, c-Jun N-terminal kinase (JNK, and p38 mitogen-activated protein kinase (MAPK. NAC prevented the activation of MAPKs in oridonin-induced cells. However, selective inhibitors of MAPKs failed to alter oridonin-induced cell death. In summary, these results demonstrate that induction of apoptosis in HSC-T6 by oridonin is associated with a decrease in cellular GSH level and increase in ROS production.

Complexation of intracellular cyanide by hydroxocobalamin using a human cellular model.

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Astier, A; Baud, F J

1996-01-01

1. The rational for administering hydroxocobalamin (OHCbl) as an antidote to cyanide poisoning is based on the high affinity of CN ion for cobalt compounds. However, only few data are available on the influence of OHCbl on the intracellular cyanide pool. 2. In human fibroblasts incubated for 10 min with 500 microM of [14C] cyanide, the accumulation ratio was 25 at 37 degrees C (10.45 +/- 1.51 mM) and 11.9 at 4 degrees C. 3. Using the monoblastic U-937 cell line, a rapid uptake of radioactive cyanide was observed with a maximum accumulation ratio of 1.97 at 5 min. 4. A linear relationship between cyanide uptake by U-937 cells and cyanide concentration in incubation medium (10-500 microM; 5 min) was found suggesting a first order process (k = 0.25 min-1). 5. After incubation of fibroblasts with 500 microM of OHCbl, a 75% decrease of intracellular cyanide was observed, with concomittant formation of intracellular cyanocobalamin CNCbl (intracellular/extracellular ratio: 158). 6. These findings suggest that OHCbl is able to penetrate into heavily cyanide loaded cells and to complex cyanide to the non-toxic CNCbl form.

Continuous background light significantly increases flashing-light enhancement of photosynthesis and growth of microalgae.

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Abu-Ghosh, Said; Fixler, Dror; Dubinsky, Zvy; Iluz, David

2015-01-01

Under specific conditions, flashing light enhances the photosynthesis rate in comparison to continuous illumination. Here we show that a combination of flashing light and continuous background light with the same integrated photon dose as continuous or flashing light alone can be used to significantly enhance photosynthesis and increase microalgae growth. To test this hypothesis, the green microalga Dunaliella salina was exposed to three different light regimes: continuous light, flashing light, and concomitant application of both. Algal growth was compared under three different integrated light quantities; low, intermediate, and moderately high. Under the combined light regime, there was a substantial increase in all algal growth parameters, with an enhanced photosynthesis rate, within 3days. Our strategy demonstrates a hitherto undescribed significant increase in photosynthesis and algal growth rates, which is beyond the increase by flashing light alone. Copyright © 2015 Elsevier Ltd. All rights reserved.

Breast-cancer-associated metastasis is significantly increased in a model of autoimmune arthritis.

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Das Roy, Lopamudra; Pathangey, Latha B; Tinder, Teresa L; Schettini, Jorge L; Gruber, Helen E; Mukherjee, Pinku

2009-01-01

Sites of chronic inflammation are often associated with the establishment and growth of various malignancies including breast cancer. A common inflammatory condition in humans is autoimmune arthritis (AA) that causes inflammation and deformity of the joints. Other systemic effects associated with arthritis include increased cellular infiltration and inflammation of the lungs. Several studies have reported statistically significant risk ratios between AA and breast cancer. Despite this knowledge, available for a decade, it has never been questioned if the site of chronic inflammation linked to AA creates a milieu that attracts tumor cells to home and grow in the inflamed bones and lungs which are frequent sites of breast cancer metastasis. To determine if chronic inflammation induced by autoimmune arthritis contributes to increased breast cancer-associated metastasis, we generated mammary gland tumors in SKG mice that were genetically prone to develop AA. Two breast cancer cell lines, one highly metastatic (4T1) and the other non-metastatic (TUBO) were used to generate the tumors in the mammary fat pad. Lung and bone metastasis and the associated inflammatory milieu were evaluated in the arthritic versus the non-arthritic mice. We report a three-fold increase in lung metastasis and a significant increase in the incidence of bone metastasis in the pro-arthritic and arthritic mice compared to non-arthritic control mice. We also report that the metastatic breast cancer cells augment the severity of arthritis resulting in a vicious cycle that increases both bone destruction and metastasis. Enhanced neutrophilic and granulocytic infiltration in lungs and bone of the pro-arthritic and arthritic mice and subsequent increase in circulating levels of proinflammatory cytokines, such as macrophage colony stimulating factor (M-CSF), interleukin-17 (IL-17), interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), and tumor necrosis factor-alpha (TNF-alpha) may contribute

Breast cancer-associated metastasis is significantly increased in a model of autoimmune arthritis

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Das Roy, Lopamudra; Pathangey, Latha B; Tinder, Teresa L; Schettini, Jorge L; Gruber, Helen E; Mukherjee, Pinku

2009-01-01

Introduction Sites of chronic inflammation are often associated with the establishment and growth of various malignancies including breast cancer. A common inflammatory condition in humans is autoimmune arthritis (AA) that causes inflammation and deformity of the joints. Other systemic effects associated with arthritis include increased cellular infiltration and inflammation of the lungs. Several studies have reported statistically significant risk ratios between AA and breast cancer. Despite this knowledge, available for a decade, it has never been questioned if the site of chronic inflammation linked to AA creates a milieu that attracts tumor cells to home and grow in the inflamed bones and lungs which are frequent sites of breast cancer metastasis. Methods To determine if chronic inflammation induced by autoimmune arthritis contributes to increased breast cancer-associated metastasis, we generated mammary gland tumors in SKG mice that were genetically prone to develop AA. Two breast cancer cell lines, one highly metastatic (4T1) and the other non-metastatic (TUBO) were used to generate the tumors in the mammary fat pad. Lung and bone metastasis and the associated inflammatory milieu were evaluated in the arthritic versus the non-arthritic mice. Results We report a three-fold increase in lung metastasis and a significant increase in the incidence of bone metastasis in the pro-arthritic and arthritic mice compared to non-arthritic control mice. We also report that the metastatic breast cancer cells augment the severity of arthritis resulting in a vicious cycle that increases both bone destruction and metastasis. Enhanced neutrophilic and granulocytic infiltration in lungs and bone of the pro-arthritic and arthritic mice and subsequent increase in circulating levels of proinflammatory cytokines, such as macrophage colony stimulating factor (M-CSF), interleukin-17 (IL-17), interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), and tumor necrosis factor

Early effects of gliadin on enterocyte intracellular signalling involved in intestinal barrier function.

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Clemente, M G; De Virgiliis, S; Kang, J S; Macatagney, R; Musu, M P; Di Pierro, M R; Drago, S; Congia, M; Fasano, A

2003-02-01

Despite the progress made in understanding the immunological aspects of the pathogenesis of coeliac disease (CD), the early steps that allow gliadin to cross the intestinal barrier are still largely unknown. The aim of this study was to establish whether gliadin activates a zonulin dependent enterocyte intracellular signalling pathway(s) leading to increased intestinal permeability. The effect of gliadin on the enterocyte actin cytoskeleton was studied on rat intestinal epithelial (IEC-6) cell cultures by fluorescence microscopy and spectrofluorimetry. Zonulin concentration was measured on cell culture supernatants by enzyme linked immunosorbent assay. Transepithelial intestinal resistance (Rt) was measured on ex vivo intestinal tissues mounted in Ussing chambers. Incubation of cells with gliadin led to a reversible protein kinase C (PKC) mediated actin polymerisation temporarily coincident with zonulin release. A significant reduction in Rt was observed after gliadin addition on rabbit intestinal mucosa mounted in Ussing chambers. Pretreatment with the zonulin inhibitor FZI/0 abolished the gliadin induced actin polymerisation and Rt reduction but not zonulin release. Gliadin induces zonulin release in intestinal epithelial cells in vitro. Activation of the zonulin pathway by PKC mediated cytoskeleton reorganisation and tight junction opening leads to a rapid increase in intestinal permeability.

Mechanical strain stimulates vasculogenesis and expression of angiogenesis guidance molecules of embryonic stem cells through elevation of intracellular calcium, reactive oxygen species and nitric oxide generation.

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Sharifpanah, Fatemeh; Behr, Sascha; Wartenberg, Maria; Sauer, Heinrich

2016-12-01

Differentiation of embryonic stem (ES) cells may be regulated by mechanical strain. Herein, signaling molecules underlying mechanical stimulation of vasculogenesis and expression of angiogenesis guidance cues were investigated in ES cell-derived embryoid bodies. Treatment of embryoid bodies with 10% static mechanical strain using a Flexercell strain system significantly increased CD31-positive vascular structures and the angiogenesis guidance molecules plexinB1, ephrin B2, neuropilin1 (NRP1), semaphorin 4D (sem4D) and robo4 as well as vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor-BB (PDGF-BB) as evaluated by Western blot and real time RT-PCR. In contrast ephrin type 4 receptor B (EphB4) expression was down-regulated upon mechanical strain, indicating an arterial-type differentiation. Robo1 protein expression was modestly increased with no change in mRNA expression. Mechanical strain increased intracellular calcium as well as reactive oxygen species (ROS) and nitric oxide (NO). Mechanical strain-induced vasculogenesis was abolished by the NOS inhibitor L-NAME, the NADPH oxidase inhibitor VAS2870, upon chelation of intracellular calcium by BAPTA as well as upon siRNA inactivation of ephrin B2, NRP1 and robo4. BAPTA blunted the strain-induced expression of angiogenic growth factors, the increase in NO and ROS as well as the expression of NRP1, sem4D and plexinB1, whereas ephrin B2, EphB4 as well as robo1 and robo4 expression were not impaired. Mechanical strain stimulates vasculogenesis of ES cells by the intracellular messengers ROS, NO and calcium as well as by upregulation of angiogenesis guidance molecules and the angiogenic growth factors VEGF, FGF-2 and PDGF-BB. Copyright © 2016 Elsevier B.V. All rights reserved.

Dynamics of gradient formation by intracellular shuttling

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Berezhkovskii, Alexander M. [Mathematical and Statistical Computing Laboratory, Division of Computational Bioscience, Center for Information Technology, National Institutes of Health, Bethesda, Maryland 20892 (United States); Shvartsman, Stanislav Y. [Department of Chemical and Biological Engineering and Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey 08544 (United States)

2015-08-21

A number of important cellular functions rely on the formation of intracellular protein concentration gradients. Experimental studies discovered a number of mechanisms for the formation of such gradients. One of the mechanisms relies on the intracellular shuttling of a protein that interconverts between the two states with different diffusivities, under the action of two enzymes, one of which is localized to the plasma membrane, whereas the second is uniformly distributed in the cytoplasm. Recent work reported an analytical solution for the steady state gradient in this mechanism, obtained in the framework of a one-dimensional reaction-diffusion model. Here, we study the dynamics in this model and derive analytical expressions for the Laplace transforms of the time-dependent concentration profiles in terms of elementary transcendental functions. Inverting these transforms numerically, one can obtain time-dependent concentration profiles of the two forms of the protein.

Insulin-Like Growth Factor I (IGF-1) Deficiency Ameliorates Sex Difference in Cardiac Contractile Function and Intracellular Ca2+ Homeostasis

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Ceylan-Isik, Asli F.; Li, Qun; Ren, Jun

2011-01-01

Sex difference in cardiac contractile function exists which may contribute to the different prevalence in cardiovascular diseases between genders. However, the precise mechanisms of action behind sex difference in cardiac function are still elusive. Given that sex difference exists in insulin-like growth factor I (IGF-1) cascade, this study is designed to evaluate the impact of severe liver IGF-1 deficiency (LID) on sex difference in cardiac function. Echocardiographic, cardiomyocyte contractile and intracellular Ca2+ properties were evaluated including ventricular geometry, fractional shortening, peak shortening, maximal velocity of shortening/relengthening (± dL/dt), time-to-peak shortening (TPS), time-to-90% relengthening (TR90), fura-fluorescence intensity (FFI) and intracellular Ca2+ clearance. Female C57 mice exhibited significantly higher plasma IGF-1 levels than their male counterpart. LID mice possessed comparably low IGF-1 levels in both sexes. Female C57 and LID mice displayed lower body, heart and liver weights compared to male counterparts. Echocardiographic analysis revealed larger LV mass in female C57 but not LID mice without sex difference in other cardiac geometric indices. Myocytes from female C57 mice exhibited reduced peak shortening, ± dL/dt, longer TPS, TR90 and intracellular Ca2+ clearance compared with males. Interestingly, this sex difference was greatly attenuated or abolished by IGF-1 deficiency. Female C57 mice displayed significantly decreased mRNA and protein levels of Na+-Ca2+ exchanger, SERCA2a and phosphorylated phospholamban as well as SERCA activity compared with male C57 mice. These sex differences in Ca2+ regulatory proteins were abolished or overtly attenuated by IGF-1 deficiency. In summary, our data suggested that IGF-1 deficiency may significantly attenuated or mitigate the sex difference in cardiomyocyte contractile function associated with intracellular Ca2+ regulation. PMID:21763763

Insulin-like growth factor I (IGF-1) deficiency ameliorates sex difference in cardiac contractile function and intracellular Ca(2+) homeostasis.

Science.gov (United States)

Ceylan-Isik, Asli F; Li, Qun; Ren, Jun

2011-10-10

Sex difference in cardiac contractile function exists which may contribute to the different prevalence in cardiovascular diseases between genders. However, the precise mechanisms of action behind sex difference in cardiac function are still elusive. Given that sex difference exists in insulin-like growth factor I (IGF-1) cascade, this study is designed to evaluate the impact of severe liver IGF-1 deficiency (LID) on sex difference in cardiac function. Echocardiographic, cardiomyocyte contractile and intracellular Ca(2+) properties were evaluated including ventricular geometry, fractional shortening, peak shortening, maximal velocity of shortening/relengthening (±dL/dt), time-to-peak shortening (TPS), time-to-90% relengthening (TR(90)), fura-fluorescence intensity (FFI) and intracellular Ca(2+) clearance. Female C57 mice exhibited significantly higher plasma IGF-1 levels than their male counterpart. LID mice possessed comparably low IGF-1 levels in both sexes. Female C57 and LID mice displayed lower body, heart and liver weights compared to male counterparts. Echocardiographic analysis revealed larger LV mass in female C57 but not LID mice without sex difference in other cardiac geometric indices. Myocytes from female C57 mice exhibited reduced peak shortening, ±dL/dt, longer TPS, TR(90) and intracellular Ca(2+) clearance compared with males. Interestingly, this sex difference was greatly attenuated or abolished by IGF-1 deficiency. Female C57 mice displayed significantly decreased mRNA and protein levels of Na(+)-Ca(2+) exchanger, SERCA2a and phosphorylated phospholamban as well as SERCA activity compared with male C57 mice. These sex differences in Ca(2+) regulatory proteins were abolished or overtly attenuated by IGF-1 deficiency. In summary, our data suggested that IGF-1 deficiency may significantly attenuated or mitigate the sex difference in cardiomyocyte contractile function associated with intracellular Ca(2+) regulation. Copyright © 2011 Elsevier Ireland

Emerging Paradigm of Intracellular Targeting of G Protein-Coupled Receptors.

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Chaturvedi, Madhu; Schilling, Justin; Beautrait, Alexandre; Bouvier, Michel; Benovic, Jeffrey L; Shukla, Arun K

2018-05-04

G protein-coupled receptors (GPCRs) recognize a diverse array of extracellular stimuli, and they mediate a broad repertoire of signaling events involved in human physiology. Although the major effort on targeting GPCRs has typically been focused on their extracellular surface, a series of recent developments now unfold the possibility of targeting them from the intracellular side as well. Allosteric modulators binding to the cytoplasmic surface of GPCRs have now been described, and their structural mechanisms are elucidated by high-resolution crystal structures. Furthermore, pepducins, aptamers, and intrabodies targeting the intracellular face of GPCRs have also been successfully utilized to modulate receptor signaling. Moreover, small molecule compounds, aptamers, and synthetic intrabodies targeting β-arrestins have also been discovered to modulate GPCR endocytosis and signaling. Here, we discuss the emerging paradigm of intracellular targeting of GPCRs, and outline the current challenges, potential opportunities, and future outlook in this particular area of GPCR biology. Copyright © 2018 Elsevier Ltd. All rights reserved.

Functional and molecular characterization of transmembrane intracellular pH regulators in human dental pulp stem cells.

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Chen, Gunng-Shinng; Lee, Shiao-Pieng; Huang, Shu-Fu; Chao, Shih-Chi; Chang, Chung-Yi; Wu, Gwo-Jang; Li, Chung-Hsing; Loh, Shih-Hurng

2018-06-01

Homeostasis of intracellular pH (pH i ) plays vital roles in many cell functions, such as proliferation, apoptosis, differentiation and metastasis. Thus far, Na + -H + exchanger (NHE), Na + -HCO 3 - co-transporter (NBC), Cl - /HCO 3 - exchanger (AE) and Cl - /OH - exchanger (CHE) have been identified to co-regulate pH i homeostasis. However, functional and biological pH i -regulators in human dental pulp stem cells (hDPSCs) have yet to be identified. Microspectrofluorimetry technique with pH-sensitive fluorescent dye, BCECF, was used to detect pH i changes. NH 4 Cl and Na + -acetate pre-pulse were used to induce intracellular acidosis and alkalosis, respectively. Isoforms of pH i -regulators were detected by Western blot technique. The resting pH i was no significant difference between that in HEPES-buffered (nominal HCO 3 - -free) solution or CO 2 /HCO 3 -buffered system (7.42 and 7.46, respectively). The pH i recovery following the induced-intracellular acidosis was blocked completely by removing [Na + ] o , while only slowed (-63%) by adding HOE694 (a NHE1 specific inhibitor) in HEPES-buffered solution. The pH i recovery was inhibited entirely by removing [Na + ] o , while adding HOE 694 pulse DIDS (an anion-transporter inhibitor) only slowed (-55%) the acid extrusion. Both in HEPES-buffered and CO 2 /HCO 3 -buffered system solution, the pH i recovery after induced-intracellular alkalosis was entirely blocked by removing [Cl - ] o . Western blot analysis showed the isoforms of pH i regulators, including NHE1/2, NBCe1/n1, AE1/2/3/4 and CHE in the hDPSCs. We demonstrate for the first time that resting pH i is significantly higher than 7.2 and meditates functionally by two Na + -dependent acid extruders (NHE and NBC), two Cl - -dependent acid loaders (CHE and AE) and one Na + -independent acid extruder(s) in hDPSCs. These findings provide novel insight for basic and clinical treatment of dentistry. Copyright © 2018 Elsevier Ltd. All rights reserved.

Synthesis and Characterization of a Gd-DOTA-D-Permeation Peptide for Magnetic Resonance Relaxation Enhancement of Intracellular Targets

Directory of Open Access Journals (Sweden)

Andrew M. Prantner

2003-10-01

Full Text Available Many MR contrast agents have been developed and proven effective for extracellular nontargeted applications, but exploitation of intracellular MR contrast agents has been elusive due to the permeability barrier of the plasma membrane. Peptide transduction domains can circumvent this permeability barrier and deliver cargo molecules to the cell interior. Based upon enhanced cellular uptake of permeation peptides with D-amino acid residues, an all-D Tat basic domain peptide was conjugated to DOTA and chelated to gadolinium. Gd-DOTA-D-Tat peptide in serum at room temperature showed a relaxivity of 7.94 ± 0.11 mM−1 sec−1 at 4.7 T. The peptide complex displayed no significant binding to serum proteins, was efficiently internalized by human Jurkat leukemia cells resulting in intracellular T1 relaxation enhancement, and in preliminary T1-weighted MRI experiments, significantly enhanced liver, kidney, and mesenteric signals.

Ontogenic and sexual differences in pituitary GnRH receptors and intracellular Ca2+ mobilization induced by GnRH.

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Lacau-Mengido, I M; González Iglesias, A; Lux-Lantos, V; Libertun, C; Becú-Villalobos, D

1998-04-01

The present experiments were designed in order to elucidate the participation of the developing hypophysis in determining the changing sensitivity of gonadotrophins to gonadotropin-releasing hormone (GnRH) during ontogeny in the rat. To that end, we chose two well defined developmental ages that differ markedly in sexual and ontogenic characteristics of hypophyseal sensitivity to GnRH, 15 and 30 d. In order to study sex differences and the role of early sexual organization of the hypothalamus, experiments were carried out in males, females, and neonatally androgenized females (TP females). We evaluated (1) the characteristics of pituitary GnRH receptors, and (2) associated changes in GnRH-induced mobilization of intracellular Ca2+ (a second messenger involved in gonadotropins exocytosis). We measured binding characteristics of the GnRH analog D-Ser(TBu)6-des-Gly10-GnRH ethylamide in pituitary homogenates. We found that Kds did not vary among the different sex groups. Total number and concentration of receptors decreased in the female rat from 15-30 d of age, whereas in the male and TP female, receptors/pituitary increased, and the concentration/mg tissue did not change. Also, at 30 days of age, males presented higher content and concentration of receptors than females, and higher content than TP females. In order to evaluate if developmental and sexual differences in pituitary sensitivity to GnRH might be expressed through variations in the intracellular Ca2+ signal, we studied the mobilization of intracellular Ca2+ induced by GnRH (1 x 10(-8) to 1 x 10(-11) M) in a suspension of dispersed pituitary cells in the six groups. In cells from 15-d-old females, Ca2+ response was greater than in 30-d-old females at the doses of 10(-8) to 10(-10) M, indicating that in the infantile female rat activation of highly concentrated GnRH receptors is reflected in an increase in signal transduction mediated by Ca2+. In males and in female rats androgenized at birth, there was also

Recent advances in intracellular and in vivo ROS sensing: focus on nanoparticle and nanotube applications.

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Uusitalo, Larissa M; Hempel, Nadine

2012-01-01

Reactive oxygen species (ROS) are increasingly being implicated in the regulation of cellular signaling cascades. Intracellular ROS fluxes are associated with cellular function ranging from proliferation to cell death. Moreover, the importance of subtle, spatio-temporal shifts in ROS during localized cellular signaling events is being realized. Understanding the biochemical nature of the ROS involved will enhance our knowledge of redox-signaling. An ideal intracellular sensor should therefore resolve real-time, localized ROS changes, be highly sensitive to physiologically relevant shifts in ROS and provide specificity towards a particular molecule. For in vivo applications issues such as bioavailability of the probe, tissue penetrance of the signal and signal-to-noise ratio also need to be considered. In the past researchers have heavily relied on the use of ROS-sensitive fluorescent probes and, more recently, genetically engineered ROS sensors. However, there is a great need to improve on current methods to address the above issues. Recently, the field of molecular sensing and imaging has begun to take advantage of the unique physico-chemical properties of nanoparticles and nanotubes. Here we discuss the recent advances in the use of these nanostructures as alternative platforms for ROS sensing, with particular emphasis on intracellular and in vivo ROS detection and quantification.

Ultrafine particles cause cytoskeletal dysfunctions in macrophages: role of intracellular calcium

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Brown David M

2005-10-01

Full Text Available Abstract Background Particulate air pollution is reported to cause adverse health effects in susceptible individuals. Since most of these particles are derived form combustion processes, the primary composition product is carbon with a very small diameter (ultrafine, less than 100 nm in diameter. Besides the induction of reactive oxygen species and inflammation, ultrafine particles (UFP can cause intracellular calcium transients and suppression of defense mechanisms of alveolar macrophages, such as impaired migration or phagocytosis. Methods In this study the role of intracellular calcium transients caused by UFP was studied on cytoskeleton related functions in J774A.1 macrophages. Different types of fine and ultrafine carbon black particles (CB and ufCB, respectively, such as elemental carbon (EC90, commercial carbon (Printex 90, diesel particulate matter (DEP and urban dust (UD, were investigated. Phagosome transport mechanisms and mechanical cytoskeletal integrity were studied by cytomagnetometry and cell viability was studied by fluorescence microscopy. Macrophages were exposed in vitro with 100 and 320 μg UFP/ml/million cells for 4 hours in serum free medium. Calcium antagonists Verapamil, BAPTA-AM and W-7 were used to block calcium channels in the membrane, to chelate intracellular calcium or to inhibit the calmodulin signaling pathways, respectively. Results Impaired phagosome transport and increased cytoskeletal stiffness occurred at EC90 and P90 concentrations of 100 μg/ml/million cells and above, but not with DEP or UD. Verapamil and W-7, but not BAPTA-AM inhibited the cytoskeletal dysfunctions caused by EC90 or P90. Additionally the presence of 5% serum or 1% bovine serum albumin (BSA suppressed the cytoskeletal dysfunctions. Cell viability showed similar results, where co-culture of ufCB together with Verapamil, W-7, FCS or BSA produced less cell dead compared to the particles only.

Cystatins - Extra- and intracellular cysteine protease inhibitors: High-level secretion and uptake of cystatin C in human neuroblastoma cells

DEFF Research Database (Denmark)

Wallin, Hanna; Bjarnadottir, Maria; Vogel, Lotte

2010-01-01

signal peptides) for cellular export following translation. Results indicating existence of systems for significant internalisation of type 2 cystatins from the extracellular to intracellular compartments are reviewed. Data showing that human neuroblastoma cell lines generally secrete high levels...

Budesonide Inhibits Intracellular Infection with Non-Typeable Haemophilus influenzae Despite Its Anti-Inflammatory Effects in Respiratory Cells and Human Lung Tissue: A Role for p38 MAP Kinase.

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Wagner, Christopher; Goldmann, Torsten; Rohmann, Kristina; Rupp, Jan; Marwitz, Sebastian; Rotta Detto Loria, Johannes; Limmer, Stefan; Zabel, Peter; Dalhoff, Klaus; Drömann, Daniel

2015-01-01

Inhaled corticosteroids (ICS) are widely used in the treatment of obstructive lung diseases. Recent data suggest a higher pneumonia risk in chronic obstructive pulmonary disease (COPD) patients treated with ICS. Since non-typeable Haemophilus influenzae (NTHi) is the most common pathogen associated with acute exacerbations of COPD, we investigated the effects of budesonide (BUD) on NTHi-induced inflammation and invasive infection. The alveolar epithelial cell line A549 and specimens of human lung tissue (HLT) were used in our experiments. Intracellular infection was determined by a lysis/culture assay of infected cells. Activated p38 mitogen-associated protein kinase (MAPK) was assessed using Western blotting and immunohistochemistry, expression of toll-like receptor 2 (TLR2) was determined by PCR, and CXCL-8 levels were measured using ELISA. Immunohistochemistry was used for detection of CXCL-8, platelet-activating factor receptor (PAF-R) and NTHi. BUD significantly reduced CXCL-8 secretion in A549 cells and lung tissue infected with NTHi. Furthermore, BUD decreased the expression of PAF-R in HLT and A549 cells. In A549 cells and HLT, BUD inhibited intracellular infection and - synergistically with NTHi - increased the expression of TLR2 (in A549 cells). TLR2 stimulation did not influence the intracellular infection of A549 cells, but p38 MAPK inhibition resulted in a significant reduction of infection. The present study adds new insights into the effects of glucocorticoids on pulmonary host defence after NTHi infection. Although the inflammatory response to infection is suppressed by BUD, interestingly, the intracellular infection is also inhibited. This effect seems to depend on the inhibition of p38 MAPK - a key enzyme in many pro-inflammatory pathways - as well as of PAF-R expression. © 2015 S. Karger AG, Basel.

Neurodegeneration in Alzheimer Disease: Role of Amyloid Precursor Protein and Presenilin 1 Intracellular Signaling

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Mario Nizzari

2012-01-01

Full Text Available Alzheimer disease (AD is a heterogeneous neurodegenerative disorder characterized by (1 progressive loss of synapses and neurons, (2 intracellular neurofibrillary tangles, composed of hyperphosphorylated Tau protein, and (3 amyloid plaques. Genetically, AD is linked to mutations in few proteins amyloid precursor protein (APP and presenilin 1 and 2 (PS1 and PS2. The molecular mechanisms underlying neurodegeneration in AD as well as the physiological function of APP are not yet known. A recent theory has proposed that APP and PS1 modulate intracellular signals to induce cell-cycle abnormalities responsible for neuronal death and possibly amyloid deposition. This hypothesis is supported by the presence of a complex network of proteins, clearly involved in the regulation of signal transduction mechanisms that interact with both APP and PS1. In this review we discuss the significance of novel finding related to cell-signaling events modulated by APP and PS1 in the development of neurodegeneration.

Expression of the potential therapeutic target CXXC5 in primary acute myeloid leukemia cells - high expression is associated with adverse prognosis as well as altered intracellular signaling and transcriptional regulation.

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Bruserud, Øystein; Reikvam, Håkon; Fredly, Hanne; Skavland, Jørn; Hagen, Karen-Marie; van Hoang, Tuyen Thy; Brenner, Annette K; Kadi, Amir; Astori, Audrey; Gjertsen, Bjørn Tore; Pendino, Frederic

2015-02-20

The CXXC5 gene encodes a transcriptional activator with a zinc-finger domain, and high expression in human acute myeloid leukemia (AML) cells is associated with adverse prognosis. We now characterized the biological context of CXXC5 expression in primary human AML cells. The global gene expression profile of AML cells derived from 48 consecutive patients was analyzed; cells with high and low CXXC5 expression then showed major differences with regard to extracellular communication and intracellular signaling. We observed significant differences in the phosphorylation status of several intracellular signaling mediators (CREB, PDK1, SRC, STAT1, p38, STAT3, rpS6) that are important for PI3K-Akt-mTOR signaling and/or transcriptional regulation. High CXXC5 expression was also associated with high mRNA expression of several stem cell-associated transcriptional regulators, the strongest associations being with WT1, GATA2, RUNX1, LYL1, DNMT3, SPI1, and MYB. Finally, CXXC5 knockdown in human AML cell lines caused significantly increased expression of the potential tumor suppressor gene TSC22 and genes encoding the growth factor receptor KIT, the cytokine Angiopoietin 1 and the selenium-containing glycoprotein Selenoprotein P. Thus, high CXXC5 expression seems to affect several steps in human leukemogenesis, including intracellular events as well as extracellular communication.

Ebselen increases cytosolic free Ca2+ concentration, stimulates glutamate release and increases GFAP content in rat hippocampal astrocytes

International Nuclear Information System (INIS)

Salazar, Miguel; Pariente, Jose Antonio; Salido, Gines Maria; Gonzalez, Antonio

2008-01-01

We have investigated the effect of the seleno-organic compound and radical scavenger ebselen on rat hippocampal astrocytes in culture. Throughout our study we carried out determinations of [Ca 2+ ] c in fura-2-loaded cells by single cell imaging, glutamate secretion employing an enzymatic-based assay and GFAP expression, which was monitorized by immunocytochemistry and confocal microscopy. Our results show that ebselen (1-20 μM) dose dependently increases [Ca 2+ ] c , stimulates glutamate release and increases GFAP content, a hallmark of astrocyte reactivity. Ebselen did not alter significantly cell viability as assayed by determination of LDH release into the extracellular medium. Ebselen-evoked glutamate release and increase in GFAP content were Ca 2+ -dependent, because incubation of astrocytes in the absence of extracellular Ca 2+ (medium containing 0.5 mM EGTA) and in the presence of the intracellular Ca 2+ chelator BAPTA (10 μM) significantly reduced ebselen-evoked changes in these parameters. The effects of ebselen we have observed may underline various signalling pathways which are important for cell proliferation, differentiation and function. However, aberrations in astroglial physiology could significantly compromise brain function, due to their role as modulators of neuron activity. Therefore, we consider that careful attention should be paid when employing ebselen as a prophylactic agent against brain damage

DMPD: Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 16982211 Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. Wullaer...vg) (.html) (.csml) Show Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. PubmedID 1698221...1 Title Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. Author

Intracellular pH and inorganic phosphate content of heart in vivo: A 31P-NMR study

International Nuclear Information System (INIS)

Katz, L.A.; Swain, J.A.; Portman, M.A.; Balaban, R.S.

1988-01-01

Studies were performed to determine the contribution of red blood cells to the 31 P-nuclear magnetic resonance (NMR) spectrum of the canine heart in vivo and the feasibility of measuring myocardial intracellular phosphate and pH. This was accomplished by replacing whole blood with a perfluorochemical perfusion emulsion blood substitute, Oxypherol, and noting the difference in the 31 P-NMR spectrum of the heart. NMR data were collected with a NMR transmitter-receiver coil on the surface of the distal portion of the left ventricle. These studies demonstrated that a small contribution from 2,3-diphosphoglycerate (2,3-DPG) and phosphodiesters in the blood could be detected. The magnitude and shift of these blood-borne signals permitted the relative quantification of intracellular inorganic phosphate (P i ) content as well as intracellular pH. Under resting conditions, the intracellular ATP/P i was 7.0 ± 0.08. This corresponds to a free intracellular P 1 content of ∼ 0.8 μmol./g wet wt. The intracellular pH was 7.10 ± 0.01. Acute respiratory alkalosis and acidosis, with the arterial pH ranging from ∼7.0 to 7.7, resulted in only small changes in the intracellular pH. These latter results demonstrate an effective myocardial intracellular proton-buffering mechanism in vivo

Stress-induced dissociations between intracellular calcium signaling and insulin secretion in pancreatic islets.

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Qureshi, Farhan M; Dejene, Eden A; Corbin, Kathryn L; Nunemaker, Craig S

2015-05-01

In healthy pancreatic islets, glucose-stimulated changes in intracellular calcium ([Ca(2+)]i) provide a reasonable reflection of the patterns and relative amounts of insulin secretion. We report that [Ca(2+)]i in islets under stress, however, dissociates with insulin release in different ways for different stressors. Islets were exposed for 48h to a variety of stressors: cytokines (low-grade inflammation), 28mM glucose (28G, glucotoxicity), free fatty acids (FFAs, lipotoxicity), thapsigargin (ER stress), or rotenone (mitochondrial stress). We then measured [Ca(2+)]i and insulin release in parallel studies. Islets exposed to all stressors except rotenone displayed significantly elevated [Ca(2+)]i in low glucose, however, increased insulin secretion was only observed for 28G due to increased nifedipine-sensitive calcium-channel flux. Following 3-11mM glucose stimulation, all stressors substantially reduced the peak glucose-stimulated [Ca(2+)]i response (first phase). Thapsigargin and cytokines also substantially impacted aspects of calcium influx and ER calcium handling. Stressors did not significantly impact insulin secretion in 11mM glucose for any stressor, although FFAs showed a borderline reduction, which contributed to a significant decrease in the stimulation index (11:3mM glucose) observed for FFAs and also for 28G. We also clamped [Ca(2+)]i using 30mM KCl+250μM diazoxide to test the amplifying pathway. Only rotenone-treated islets showed a robust increase in 3-11mM glucose-stimulated insulin secretion under clamped conditions, suggesting that low-level mitochondrial stress might activate the metabolic amplifying pathway. We conclude that different stressors dissociate [Ca(2+)]i from insulin secretion differently: ER stressors (thapsigargin, cytokines) primarily affect [Ca(2+)]i but not conventional insulin secretion and 'metabolic' stressors (FFAs, 28G, rotenone) impacted insulin secretion. Copyright © 2015 Elsevier Ltd. All rights reserved.

Chelation of intracellular calcium blocks insulin action in the adipocyte

International Nuclear Information System (INIS)

Pershadsingh, H.A.; Shade, D.L.; Delfert, D.M.; McDonald, J.M.

1987-01-01

The hypothesis that intracellular Ca 2+ is an essential component of the intracellular mechanism of insulin action in the adipocyte was evaluated. Cells were loaded with the Ca 2+ chelator quin-2, by preincubating them with quin-2 AM, the tetrakis(acetoxymethyl) ester of quin-2. Quin-2 loading inhibited insulin-stimulated glucose transport without affecting basal activity. The ability of insulin to stimulate glucose uptake in quin-2-loaded cells could be partially restored by preincubating cells with buffer supplemented with 1.2 mM CaCl 2 and the Ca 2+ ionophore A23187. These conditions had no effect on basal activity and omission of CaCl 2 from the buffer prevented the restoration of insulin-stimulated glucose uptake by A23187. Quin-2 loading also inhibited insulin-stimulated glucose oxidation and the ability of insulin to inhibit cAMP-stimulated lipolysis without affecting their basal activities. Incubation of cells with 100 μM quin-2 or quin-2 AM had no effect on intracellular ATP concentration or the specific binding of 125 I=labeled insulin to adipocytes. These findings suggest that intracellular Ca 2+ is an essential component in the coupling of the insulin-activated receptor complex to cellular physiological/metabolic machinery. Furthermore, differing quin-2 AM dose-response profiles suggest the presence of dual Ca 2+ -dependent pathways in the adipocyte. One involves insulin stimulation of glucose transport and oxidation, whereas the other involves the antilipolytic action of insulin

Multiple-Targeted Graphene-based Nanocarrier for Intracellular Imaging of mRNAs

Energy Technology Data Exchange (ETDEWEB)

Wang, Ying; Li, Zhaohui; Liu, Misha; Hu, Dehong; Lin, Yuehe; Li, Jinghong

2017-08-29

Simultaneous detection and imaging of multiple intracellular messenger RNA (mRNAs) hold great significant for early cancer diagnostics and preventive medicine development. Herein, we propose a multiple-targeted graphene oxide (GO) nanocarrier that can simultaneously detect and image different type mRNAs in living cells. First of all, in vitro detection of multiple targets have been realized successfully based on the multiple-targeted GO nanocarrier with linear relationship ranging from 3 nM to 200 nM, as well as sensitive detection limit of 1.84 nM for manganese superoxide dismutase (Mn-SOD) mRNA and 2.45 nM for β-actin mRNA. Additionally, this nanosensing platform composed of fluorescent labeled single strand DNA probes and GO nanocarrier can identify Mn-SOD mRNA and endogenous mRNA of β-actin in living cancer cells, showing rapid response, high specificity, nuclease stability, and good biocompatibility during the cell imaging. Thirdly, changes of the expression levels of mRNA in living cells before or after the drug treatment can be monitored successfully. By using multiple ssDNA as probes and GO nanocarrier as the cellular delivery cargo, the proposed simultaneous multiple-targeted sensing platform will be of great potential as a powerful tool for intracellular trafficking process from basic research to clinical diagnosis.

The β-Hemolysin and Intracellular Survival of Streptococcus agalactiae in Human Macrophages

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Sagar, Anubha; Klemm, Carolin; Hartjes, Lara; Mauerer, Stefanie; van Zandbergen, Ger; Spellerberg, Barbara

2013-01-01

S. agalactiae (group B streptococci, GBS) is a major microbial pathogen in human neonates and causes invasive infections in pregnant women and immunocompromised individuals. The S. agalactiae β-hemolysin is regarded as an important virulence factor for the development of invasive disease. To examine the role of β-hemolysin in the interaction with professional phagocytes, the THP-1 monocytic cell line and human granulocytes were infected with a serotype Ia S. agalactiae wild type strain and its isogenic nonhemolytic mutant. We could show that the nonhemolytic mutants were able to survive in significantly higher numbers than the hemolytic wild type strain, in THP-1 macrophage-like cells and in assays with human granulocytes. Intracellular bacterial multiplication, however, could not be observed. The hemolytic wild type strain stimulated a significantly higher release of Tumor Necrosis Factor-α than the nonhemolytic mutant in THP-1 cells, while similar levels of the chemokine Interleukin-8 were induced. In order to investigate bacterial mediators of IL-8 release in this setting, purified cell wall preparations from both strains were tested and found to exert a potent proinflammatory stimulus on THP-1 cells. In conclusion, our results indicate that the β-hemolysin has a strong influence on the intracellular survival of S. agalactiae and that a tightly controlled regulation of β-hemolysin expression is required for the successful establishment of S. agalactiae in different host niches. PMID:23593170

The β-hemolysin and intracellular survival of Streptococcus agalactiae in human macrophages.

Directory of Open Access Journals (Sweden)

Anubha Sagar

Full Text Available S. agalactiae (group B streptococci, GBS is a major microbial pathogen in human neonates and causes invasive infections in pregnant women and immunocompromised individuals. The S. agalactiae β-hemolysin is regarded as an important virulence factor for the development of invasive disease. To examine the role of β-hemolysin in the interaction with professional phagocytes, the THP-1 monocytic cell line and human granulocytes were infected with a serotype Ia S. agalactiae wild type strain and its isogenic nonhemolytic mutant. We could show that the nonhemolytic mutants were able to survive in significantly higher numbers than the hemolytic wild type strain, in THP-1 macrophage-like cells and in assays with human granulocytes. Intracellular bacterial multiplication, however, could not be observed. The hemolytic wild type strain stimulated a significantly higher release of Tumor Necrosis Factor-α than the nonhemolytic mutant in THP-1 cells, while similar levels of the chemokine Interleukin-8 were induced. In order to investigate bacterial mediators of IL-8 release in this setting, purified cell wall preparations from both strains were tested and found to exert a potent proinflammatory stimulus on THP-1 cells. In conclusion, our results indicate that the β-hemolysin has a strong influence on the intracellular survival of S. agalactiae and that a tightly controlled regulation of β-hemolysin expression is required for the successful establishment of S. agalactiae in different host niches.

Khz-cp (crude polysaccharide extract obtained from the fusion of Ganoderma lucidum and Polyporus umbellatus mycelia) induces apoptosis by increasing intracellular calcium levels and activating P38 and NADPH oxidase-dependent generation of reactive oxygen species in SNU-1 cells.

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Kim, Tae Hwan; Kim, Ju Sung; Kim, Zoo Haye; Huang, Ren Bin; Chae, Young Lye; Wang, Ren Sheng

2014-07-10

Khz-cp is a crude polysaccharide extract that is obtained after nuclear fusion in Ganoderma lucidum and Polyporus umbellatus mycelia (Khz). It inhibits the growth of cancer cells. Khz-cp was extracted by solvent extraction. The anti-proliferative activity of Khz-cp was confirmed by using Annexin-V/PI-flow cytometry analysis. Intracellular calcium increase and measurement of intracellular reactive oxygen species (ROS) were performed by using flow cytometry and inverted microscope. SNU-1 cells were treated with p38, Bcl-2 and Nox family siRNA. siRNA transfected cells was employed to investigate the expression of apoptotic, growth and survival genes in SNU-1 cells. Western blot analysis was performed to confirm the expression of the genes. In the present study, Khz-cp induced apoptosis preferentially in transformed cells and had only minimal effects on non-transformed cells. Furthermore, Khz-cp was found to induce apoptosis by increasing the intracellular Ca2+ concentration ([Ca2+]i) and activating P38 to generate reactive oxygen species (ROS) via NADPH oxidase and the mitochondria. Khz-cp-induced apoptosis was caspase dependent and occurred via a mitochondrial pathway. ROS generation by NADPH oxidase was critical for Khz-cp-induced apoptosis, and although mitochondrial ROS production was also required, it appeared to occur secondary to ROS generation by NADPH oxidase. Activation of NADPH oxidase was shown by the translocation of the regulatory subunits p47phox and p67phox to the cell membrane and was necessary for ROS generation by Khz-cp. Khz-cp triggered a rapid and sustained increase in [Ca2+]i that activated P38. P38 was considered to play a key role in the activation of NADPH oxidase because inhibition of its expression or activity abrogated membrane translocation of the p47phox and p67phox subunits and ROS generation. In summary, these data indicate that Khz-cp preferentially induces apoptosis in cancer cells and that the signaling mechanisms involve an

Intracellular pH gradients in migrating cells

DEFF Research Database (Denmark)

Martin, Christine; Pedersen, Stine Helene Falsig; Schwab, Albrecht

2011-01-01

might function as such unevenly distributed regulators as they modulate the interaction of focal adhesion proteins and components of the cytoskeleton in vitro. However, an intracellular pH (pH(i)) gradient reflecting a spatial asymmetry of protons has not been shown so far. One major regulator of p...

Biological synthesis and characterization of intracellular gold ...

Indian Academy of Sciences (India)

In the present study, Aspergillus fumigatus was used for the intracellular synthesis of gold nanoparticles. Stable nanoparticles were produced when an aqueous solution of chloroauric acid (HAuCl4) was reduced by A. fumigatus biomass as the reducing agent. Production of nanoparticles was confirmed by the colour ...

Modulating and Measuring Intracellular H2O2 Using Genetically Encoded Tools to Study Its Toxicity to Human Cells.

Science.gov (United States)

Huang, Beijing K; Stein, Kassi T; Sikes, Hadley D

2016-12-16

Reactive oxygen species (ROS) such as H 2 O 2 play paradoxical roles in mammalian physiology. It is hypothesized that low, baseline levels of H 2 O 2 are necessary for growth and differentiation, while increased intracellular H 2 O 2 concentrations are associated with pathological phenotypes and genetic instability, eventually reaching a toxic threshold that causes cell death. However, the quantities of intracellular H 2 O 2 that lead to these different responses remain an unanswered question in the field. To address this question, we used genetically encoded constructs that both generate and quantify H 2 O 2 in a dose-response study of H 2 O 2 -mediated toxicity. We found that, rather than a simple concentration-response relationship, a combination of intracellular concentration and the cumulative metric of H 2 O 2 concentration multiplied by time (i.e., the area under the curve) determined the occurrence and level of cell death. Establishing the quantitative relationship between H 2 O 2 and cell toxicity promotes a deeper understanding of the intracellular effects of H 2 O 2 specifically as an individual reactive oxygen species, and it contributes to an understanding of its role in various redox-related diseases.

In vivo assessment of intracellular redox state in rat liver using hyperpolarized [1-13 C]Alanine.

Science.gov (United States)

Park, Jae Mo; Khemtong, Chalermchai; Liu, Shie-Chau; Hurd, Ralph E; Spielman, Daniel M

2017-05-01

The intracellular lactate to pyruvate concentration ratio is a commonly used tissue assay biomarker of redox, being proportional to free cytosolic [NADH]/[NAD + ]. In this study, we assessed the use of hyperpolarized [1- 13 C]alanine and the subsequent detection of the intracellular products of [1- 13 C]pyruvate and [1- 13 C]lactate as a useful substrate for assessing redox levels in the liver in vivo. Animal experiments were conducted to measure in vivo metabolism at baseline and after ethanol infusion. A solution of 80-mM hyperpolarized [1- 13 C]alanine was injected intravenously at baseline (n = 8) and 45 min after ethanol infusion (n = 4), immediately followed by the dynamic acquisition of 13 C MRS spectra. In vivo rat liver spectra showed peaks from [1- 13 C] alanine and the products of [1- 13 C]lactate, [1- 13 C]pyruvate, and 13 C-bicarbonate. A significantly increased 13 C-lactate/ 13 C-pyruvate ratio was observed after ethanol infusion (8.46 ± 0.58 at baseline versus 13.58 ± 0.69 after ethanol infusion; P alanine is presented, with the validity of the proposed 13 C-pyruvate/ 13 C-lactate metric tested using an ethanol challenge to alter liver redox state. Magn Reson Med 77:1741-1748, 2017. © 2017 International Society for Magnetic Resonance in Medicine. © 2017 International Society for Magnetic Resonance in Medicine.

Pulsed magneto-motive ultrasound imaging to detect intracellular accumulation of magnetic nanoparticles

International Nuclear Information System (INIS)

Mehrmohammadi, Mohammad; Qu Min; Sokolov, Konstantin V; Emelianov, Stanislav Y; Ma, Li L; Johnston, Keith P; Romanovicz, Dwight K

2011-01-01

As applications of nanoparticles in medical imaging and biomedicine rapidly expand, the interactions of nanoparticles with living cells have become an area of active interest. For example, intracellular accumulation of nanoparticles-an important part of cell-nanoparticle interaction-has been well studied using plasmonic nanoparticles and optical or optics-based techniques due to the change in optical properties of the nanoparticle aggregates. However, magnetic nanoparticles, despite their wide range of clinical applications, do not exhibit plasmonic-resonant properties and therefore their intracellular aggregation cannot be detected by optics-based imaging techniques. In this study, we investigated the feasibility of a novel imaging technique-pulsed magneto-motive ultrasound (pMMUS)-to identify intracellular accumulation of endocytosed magnetic nanoparticles. In pMMUS imaging a focused, high intensity, pulsed magnetic field is used to excite the cells labeled with magnetic nanoparticles, and ultrasound imaging is then used to monitor the mechanical response of the tissue. We demonstrated previously that clusters of magnetic nanoparticles amplify the pMMUS signal in comparison to the signal from individual nanoparticles. Here we further demonstrate that pMMUS imaging can identify interaction between magnetic nanoparticles and living cells, i.e. intracellular accumulation of nanoparticles within the cells. The results of our study suggest that pMMUS imaging can not only detect the presence of magnetic nanoparticles but also provides information about their intracellular accumulation non-invasively and in real-time.

Intracellular Methamphetamine Prevents the Dopamine-induced Enhancement of Neuronal Firing*

Science.gov (United States)

Saha, Kaustuv; Sambo, Danielle; Richardson, Ben D.; Lin, Landon M.; Butler, Brittany; Villarroel, Laura; Khoshbouei, Habibeh

2014-01-01

The dysregulation of the dopaminergic system is implicated in multiple neurological and neuropsychiatric disorders such as Parkinson disease and drug addiction. The primary target of psychostimulants such as amphetamine and methamphetamine is the dopamine transporter (DAT), the major regulator of extracellular dopamine levels in the brain. However, the behavioral and neurophysiological correlates of methamphetamine and amphetamine administration are unique from one another, thereby suggesting these two compounds impact dopaminergic neurotransmission differentially. We further examined the unique mechanisms by which amphetamine and methamphetamine regulate DAT function and dopamine neurotransmission; in the present study we examined the impact of extracellular and intracellular amphetamine and methamphetamine on the spontaneous firing of cultured midbrain dopaminergic neurons and isolated DAT-mediated current. In dopaminergic neurons the spontaneous firing rate was enhanced by extracellular application of amphetamine > dopamine > methamphetamine and was DAT-dependent. Amphetamine > methamphetamine similarly enhanced DAT-mediated inward current, which was sensitive to isosmotic substitution of Na+ or Cl− ion. Although isosmotic substitution of extracellular Na+ ions blocked amphetamine and methamphetamine-induced DAT-mediated inward current similarly, the removal of extracellular Cl− ions preferentially blocked amphetamine-induced inward current. The intracellular application of methamphetamine, but not amphetamine, prevented the dopamine-induced increase in the spontaneous firing of dopaminergic neurons and the corresponding DAT-mediated inward current. The results reveal a new mechanism for methamphetamine-induced dysregulation of dopaminergic neurons. PMID:24962577

F NMR measurement of intracellular free calcium in human red blood cells

International Nuclear Information System (INIS)

Gupta, R.K.; Schanne, F.A.X.

1986-01-01

Optical techniques for the measurement of intracellular Ca are not readily applicable to the human red cell because of the intense absorption of hemoglobin. The authors have therefore examined the use of 19 F NMR of 5,5'-difluoro-1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetra acetic acid (5FBAPTA) introduced non-disruptively by intracellular hydrolysis of the membrane-permeant acetoxymethyl ester derivative. 19 F NMR spectra of 5FBAPTA-containing erythrocytes at 188 MHz displayed two well resolved resonances corresponding to the free and Ca-bound forms of the chelator, the resonance of the free form being ten-fold larger than that of the Ca-bound form. Addition of the ionophore A23187 resulted in the disappearance of the resonance of the free anion and a quantitative increase in the intensity of the resonance of the Ca-complex. From these data, and a K/sub D/ of 708 nM for the Ca-5FBAPTA complex, the authors estimate red cell free Ca to be 70 nM, which is in the range of values obtained for other cells, despite the fact that the human red cell, which lacks intracellular organelles for storing Ca, possesses only 1 μmol total Ca/1. cells in comparison to mmols of total Ca found in other cells. The authors ability to use 19 F NMR to measure free Ca in the red blood cell paves the way for future NMR studies of red cell free Ca concentrations in human essential hypertension as well as in other diseases states in which alterations in cellular Ca homeostasis may be involved

Brucella abortus nicotinamidase (PncA) contributes to its intracellular replication and infectivity in mice.

Science.gov (United States)

Kim, Suk; Kurokawa, Daisuke; Watanabe, Kenta; Makino, Sou-Ichi; Shirahata, Toshikazu; Watarai, Masahisa

2004-05-15

Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and non-professional phagocytes, and cause abortion in domestic animals and undulant fever in humans. The mechanism and factors of virulence are not fully understood. Nicotinamidase/pyrazinamidase mutant (pncA mutant) of Brucella abortus failed to replicate in HeLa cells, and showed a lower rate of intracellular replication than that of wild-type strain in macrophages. Addition of nicotinic acid, but not nicotinamide, into medium supported intracellular replication of pncA mutant in HeLa cells and macrophages. The pncA mutant was not co-localizing with either late endosomes or lysosomes. The B. abortus virB4 mutant was completely cleared from the spleens of mice after 4 weeks, while the pncA mutant showed a 1.5-log reduction of the number of bacteria isolated from spleens after 10 weeks. Although pncA mutant showed reduced virulence in mice and defective intracellular replication, its ability to confer protection against the virulent B. abortus strain 544 was fully retained. These results suggest that PncA does not contribute to intracellular trafficking of B. abortus, but contributes to utilization of nutrients required for intracellular growth. Our results indicate that detailed characterizations of the pncA mutant may help the improvement of currently available live vaccines. Copyright 2004 Federation of European Microbiological Societies

Getting “Inside” Type I IFNs: Type I IFNs in Intracellular Bacterial Infections

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Deann T. Snyder

2017-01-01

Full Text Available Type I interferons represent a unique and complex group of cytokines, serving many purposes during innate and adaptive immunity. Discovered in the context of viral infections, type I IFNs are now known to have myriad effects in infectious and autoimmune disease settings. Type I IFN signaling during bacterial infections is dependent on many factors including whether the infecting bacterium is intracellular or extracellular, as different signaling pathways are activated. As such, the repercussions of type I IFN induction can positively or negatively impact the disease outcome. This review focuses on type I IFN induction and downstream consequences during infection with the following intracellular bacteria: Chlamydia trachomatis, Listeria monocytogenes, Mycobacterium tuberculosis, Salmonella enterica serovar Typhimurium, Francisella tularensis, Brucella abortus, Legionella pneumophila, and Coxiella burnetii. Intracellular bacterial infections are unique because the bacteria must avoid, circumvent, and even co-opt microbial “sensing” mechanisms in order to reside and replicate within a host cell. Furthermore, life inside a host cell makes intracellular bacteria more difficult to target with antibiotics. Because type I IFNs are important immune effectors, modulating this pathway may improve disease outcomes. But first, it is critical to understand the context-dependent effects of the type I IFN pathway in intracellular bacterial infections.

Involvement of intracellular free Ca2+ in enhanced release of herpes simplex virus by hydrogen peroxide

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Ogawa Yuzo

2006-08-01

Full Text Available Abstract Background It was reported that elevation of the intracellular concentration of free Ca2+ ([Ca2+]i by a calcium ionophore increased the release of herpes simplex virus type 1 (HSV-1. Freely diffusible hydrogen peroxide (H2O2 is implied to alter Ca2+ homeostasis, which further enhances abnormal cellular activity, causing changes in signal transduction, and cellular dysfunction. Whether H2O2 could affect [Ca2+]i in HSV-1-infected cells had not been investigated. Results H2O2 treatment increased the amount of cell-free virus and decreased the proportion of viable cells. After the treatment, an elevation in [Ca2+]i was observed and the increase in [Ca2+]i was suppressed when intracellular and cytosolic Ca2+ were buffered by Ca2+ chelators. In the presence of Ca2+ chelators, H2O2-mediated increases of cell-free virus and cell death were also diminished. Electron microscopic analysis revealed enlarged cell junctions and a focal disintegration of the plasma membrane in H2O2-treated cells. Conclusion These results indicate that H2O2 can elevate [Ca2+]i and induces non-apoptotic cell death with membrane lesions, which is responsible for the increased release of HSV-1 from epithelial cells.

Regulation of intracellular glucose and polyol pathway by thiamine and benfotiamine in vascular cells cultured in high glucose.

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Berrone, Elena; Beltramo, Elena; Solimine, Carmela; Ape, Alessandro Ubertalli; Porta, Massimo

2006-04-07

Hyperglycemia is a causal factor in the development of the vascular complications of diabetes. One of the biochemical mechanisms activated by excess glucose is the polyol pathway, the key enzyme of which, aldose reductase, transforms d-glucose into d-sorbitol, leading to imbalances of intracellular homeostasis. We aimed at verifying the effects of thiamine and benfotiamine on the polyol pathway, transketolase activity, and intracellular glucose in endothelial cells and pericytes under high ambient glucose. Human umbilical vein endothelial cells and bovine retinal pericytes were cultured in normal (5.6 mmol/liter) or high (28 mmol/liter) glucose, with or without thiamine or benfotiamine 50 or 100 mumol/liter. Transketolase and aldose reductase mRNA expression was determined by reverse transcription-PCR, and their activity was measured spectrophotometrically; sorbitol concentrations were quantified by gas chromatography-mass spectrometry and intracellular glucose concentrations by fluorescent enzyme-linked immunosorbent assay method. Thiamine and benfotiamine reduce aldose reductase mRNA expression, activity, sorbitol concentrations, and intracellular glucose while increasing the expression and activity of transketolase, for which it is a coenzyme, in human endothelial cells and bovine retinal pericytes cultured in high glucose. Thiamine and benfotiamine correct polyol pathway activation induced by high glucose in vascular cells. Activation of transketolase may shift excess glycolytic metabolites into the pentose phosphate cycle, accelerate the glycolytic flux, and reduce intracellular free glucose, thereby preventing its conversion to sorbitol. This effect on the polyol pathway, together with other beneficial effects reported for thiamine in high glucose, could justify testing thiamine as a potential approach to the prevention and/or treatment of diabetic complications.

Mycobacterium intracellulare Pleurisy Identified on Liquid Cultures of the Pleural Fluid and Pleural Biopsy.

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Lim, Jong Gu; O, Sei Won; Lee, Ki Dong; Suk, Dong Keun; Jung, Tae Young; Shim, Tae Sun; Chon, Gyu Rak

2013-03-01

Pleural effusion is a rare complication in non-tuberculous mycobacterial infection. We report a case of Mycobacterium intracellulare pleuritis with idiopathic pulmonary fibrosis in a 69-year-old man presenting with dyspnea. Pleural effusion revealed lymphocyte dominant exudate. M. intracellulare was identified using a polymerase chain reaction-restriction fragment length polymorphism method and liquid cultures of pleural effusion and pleural biopsy. After combination therapy for M. intracellulare pulmonary disease, the patient was clinically well at a 1-month follow-up.

New intracellular activities of matrix metalloproteinases shine in the moonlight.

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Jobin, Parker G; Butler, Georgina S; Overall, Christopher M

2017-11-01

Adaption of a single protein to perform multiple independent functions facilitates functional plasticity of the proteome allowing a limited number of protein-coding genes to perform a multitude of cellular processes. Multifunctionality is achievable by post-translational modifications and by modulating subcellular localization. Matrix metalloproteinases (MMPs), classically viewed as degraders of the extracellular matrix (ECM) responsible for matrix protein turnover, are more recently recognized as regulators of a range of extracellular bioactive molecules including chemokines, cytokines, and their binders. However, growing evidence has convincingly identified select MMPs in intracellular compartments with unexpected physiological and pathological roles. Intracellular MMPs have both proteolytic and non-proteolytic functions, including signal transduction and transcription factor activity thereby challenging their traditional designation as extracellular proteases. This review highlights current knowledge of subcellular location and activity of these "moonlighting" MMPs. Intracellular roles herald a new era of MMP research, rejuvenating interest in targeting these proteases in therapeutic strategies. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of nitro-heterocyclic derivatives against Leishmania (Leishmania) infantum promastigotes and intracellular amastigotes.

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Petri e Silva, Simone Carolina Soares; Palace-Berl, Fanny; Tavares, Leoberto Costa; Soares, Sandra Regina Castro; Lindoso, José Angelo Lauletta

2016-04-01

Leishmaniasis is an overlooked tropical disease affecting approximately 1 million people in several countries. Clinical manifestation depends on the interaction between Leishmania and the host's immune response. Currently available treatment options for leishmaniasis are limited and induce severe side effects. In this research, we tested nitro-heterocyclic compounds (BSF series) as a new alternative against Leishmania. Its activity was measured in Leishmania (Leishmania) infantum promastigotes and intracellular amastigotes using MTT colorimetric assay. Additionally, we assessed the phosphatidylserine exposure by promastigotes, measured by flow cytometry, as well as nitric oxide production, measured by Griess' method. The nitro-heterocyclic compounds (BSF series) showed activity against L. (L.) infantum promastigotes, inducting the phosphatidylserine exposition by promastigotes, decreasing intracellular amastigotes and increasing oxide nitric production. The selectivity index was more prominent to Leishmania than to macrophages. Compared to amphotericin b, our compounds presented higher IC50, however the selectivity index was more specific to parasite than to amphotericin b. In conclusion, these nitro-heterocyclic compounds showed to be promising as an anti-Leishmania drug, in in vitro studies. Copyright © 2016 Elsevier Inc. All rights reserved.

Real-time monitoring of intracellular redox changes in Methylococcus capsulatus (Bath) for efficient bioconversion of methane to methanol.

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Ishikawa, Masahito; Tanaka, Yuya; Suzuki, Risa; Kimura, Kota; Tanaka, Kenya; Kamiya, Kazuhide; Ito, Hidehiro; Kato, Souichiro; Kamachi, Toshiaki; Hori, Katsutoshi; Nakanishi, Shuji

2017-10-01

This study aimed to develop a novel method for real-time monitoring of the intracellular redox states in a methanotroph Methylococcus capsulatus, using Peredox as a genetically encoded fluorescent sensor of the NADH:NAD + ratio. As expected, the fluorescence derived from the Peredox-expressing M. capsulatus transformant increased by supplementation of electron donor compounds (methane and formate), while it decreased by specifically inhibiting the methanol oxidation reaction. Electrochemical measurements confirmed that the Peredox fluorescence reliably represents the intracellular redox changes. This study is the first to construct a reliable redox-monitoring method for methanotrophs, which will facilitate to develop more efficient methane-to-methanol bioconversion processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

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Unzueta, Ugutz; Céspedes, María Virtudes; Ferrer-Miralles, Neus; Casanova, Isolda; Cedano, Juan; Corchero, José Luis; Domingo-Espín, Joan; Villaverde, Antonio; Mangues, Ramón; Vázquez, Esther

2012-01-01

Background Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4) is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing. Results Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4+ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer. Conclusion Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles, and imaging agents. PMID:22923991

Intracellular Transport and Kinesin Superfamily Proteins: Structure, Function and Dynamics

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Hirokawa, N.; Takemura, R.

Using various molecular cell biological and molecular genetic approaches, we identified kinesin superfamily proteins (KIFs) and characterized their significant functions in intracellular transport, which is fundamental for cellular morphogenesis, functioning, and survival. We showed that KIFs not only transport various membranous organelles, proteins complexes and mRNAs fundamental for cellular functions but also play significant roles in higher brain functions such as memory and learning, determination of important developmental processes such as left-right asymmetry formation and brain wiring. We also elucidated that KIFs recognize and bind to their specific cargoes using scaffolding or adaptor protein complexes. Concerning the mechanism of motility, we discovered the simplest unique monomeric motor KIF1A and determined by molecular biophysics, cryoelectron microscopy and X-ray crystallography that KIF1A can move on a microtubule processively as a monomer by biased Brownian motion and by hydolyzing ATP.

Intracellular cholesterol-binding proteins enhance HDL-mediated cholesterol uptake in cultured primary mouse hepatocytes.

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Storey, Stephen M; McIntosh, Avery L; Huang, Huan; Landrock, Kerstin K; Martin, Gregory G; Landrock, Danilo; Payne, H Ross; Atshaves, Barbara P; Kier, Ann B; Schroeder, Friedhelm

2012-04-15

A major gap in our knowledge of rapid hepatic HDL cholesterol clearance is the role of key intracellular factors that influence this process. Although the reverse cholesterol transport pathway targets HDL to the liver for net elimination of free cholesterol from the body, molecular details governing cholesterol uptake into hepatocytes are not completely understood. Therefore, the effects of sterol carrier protein (SCP)-2 and liver fatty acid-binding protein (L-FABP), high-affinity cholesterol-binding proteins present in hepatocyte cytosol, on HDL-mediated free cholesterol uptake were examined using gene-targeted mouse models, cultured primary hepatocytes, and 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol). While SCP-2 overexpression enhanced NBD-cholesterol uptake, counterintuitively, SCP-2/SCP-x gene ablation also 1) enhanced the rapid molecular phase of free sterol uptake detectable in rate and maximal uptake of HDL free cholesterol and 2) differentially enhanced free cholesterol uptake mediated by the HDL3, rather than the HDL2, subfraction. The increased HDL free cholesterol uptake was not due to increased expression or distribution of the HDL receptor [scavenger receptor B1 (SRB1)], proteins regulating SRB1 [postsynaptic density protein (PSD-95)/Drosophila disk large tumor suppressor (dlg)/tight junction protein (ZO1) and 17-kDa membrane-associated protein], or other intracellular cholesterol trafficking proteins (steroidogenic acute response protein D, Niemann Pick C, and oxysterol-binding protein-related proteins). However, expression of L-FABP, the single most prevalent hepatic cytosolic protein that binds cholesterol, was upregulated twofold in SCP-2/SCP-x null hepatocytes. Double-immunogold electron microscopy detected L-FABP sufficiently close to SRB1 for direct interaction, similar to SCP-2. These data suggest a role for L-FABP in HDL cholesterol uptake, a finding confirmed with SCP-2/SCP-x/L-FABP null

Modulation of Acid-sensing Ion Channel 1a by Intracellular pH and Its Role in Ischemic Stroke.

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Li, Ming-Hua; Leng, Tian-Dong; Feng, Xue-Chao; Yang, Tao; Simon, Roger P; Xiong, Zhi-Gang

2016-08-26

An important contributor to brain ischemia is known to be extracellular acidosis, which activates acid-sensing ion channels (ASICs), a family of proton-gated sodium channels. Lines of evidence suggest that targeting ASICs may lead to novel therapeutic strategies for stroke. Investigations of the role of ASICs in ischemic brain injury have naturally focused on the role of extracellular pH in ASIC activation. By contrast, intracellular pH (pHi) has received little attention. This is a significant gap in our understanding because the ASIC response to extracellular pH is modulated by pHi, and activation of ASICs by extracellular protons is paradoxically enhanced by intracellular alkalosis. Our previous studies show that acidosis-induced cell injury in in vitro models is attenuated by intracellular acidification. However, whether pHi affects ischemic brain injury in vivo is completely unknown. Furthermore, whereas ASICs in native neurons are composed of different subunits characterized by distinct electrophysiological/pharmacological properties, the subunit-dependent modulation of ASIC activity by pHi has not been investigated. Using a combination of in vitro and in vivo ischemic brain injury models, electrophysiological, biochemical, and molecular biological approaches, we show that the intracellular alkalizing agent quinine potentiates, whereas the intracellular acidifying agent propionate inhibits, oxygen-glucose deprivation-induced cell injury in vitro and brain ischemia-induced infarct volume in vivo Moreover, we find that the potentiation of ASICs by quinine depends on the presence of the ASIC1a, ASIC2a subunits, but not ASIC1b, ASIC3 subunits. Furthermore, we have determined the amino acids in ASIC1a that are involved in the modulation of ASICs by pHi. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Significant increase of surface ozone at a rural site, north of eastern China

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Z. Ma

2016-03-01

Full Text Available Ozone pollution in eastern China has become one of the top environmental issues. Quantifying the temporal trend of surface ozone helps to assess the impacts of the anthropogenic precursor reductions and the likely effects of emission control strategies implemented. In this paper, ozone data collected at the Shangdianzi (SDZ regional atmospheric background station from 2003 to 2015 are presented and analyzed to obtain the variation in the trend of surface ozone in the most polluted region of China, north of eastern China or the North China Plain. A modified Kolmogorov–Zurbenko (KZ filter method was performed on the maximum daily average 8 h (MDA8 concentrations of ozone to separate the contributions of different factors from the variation of surface ozone and remove the influence of meteorological fluctuations on surface ozone. Results reveal that the short-term, seasonal and long-term components of ozone account for 36.4, 57.6 and 2.2 % of the total variance, respectively. The long-term trend indicates that the MDA8 has undergone a significant increase in the period of 2003–2015, with an average rate of 1.13 ± 0.01 ppb year−1 (R2 = 0.92. It is found that meteorological factors did not significantly influence the long-term variation of ozone and the increase may be completely attributed to changes in emissions. Furthermore, there is no significant correlation between the long-term O3 and NO2 trends. This study suggests that emission changes in VOCs might have played a more important role in the observed increase of surface ozone at SDZ.

Effects of intracellular iron overload on cell death and identification of potent cell death inhibitors.

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Fang, Shenglin; Yu, Xiaonan; Ding, Haoxuan; Han, Jianan; Feng, Jie

2018-06-11

Iron overload causes many diseases, while the underlying etiologies of these diseases are unclear. Cell death processes including apoptosis, necroptosis, cyclophilin D-(CypD)-dependent necrosis and a recently described additional form of regulated cell death called ferroptosis, are dependent on iron or iron-dependent reactive oxygen species (ROS). However, whether the accumulation of intracellular iron itself induces ferroptosis or other forms of cell death is largely elusive. In present study, we study the role of intracellular iron overload itself-induced cell death mechanisms by using ferric ammonium citrate (FAC) and a membrane-permeable Ferric 8-hydroxyquinoline complex (Fe-8HQ) respectively. We show that FAC-induced intracellular iron overload causes ferroptosis. We also identify 3-phosphoinositide-dependent kinase 1 (PDK1) inhibitor GSK2334470 as a potent ferroptosis inhibitor. Whereas, Fe-8HQ-induced intracellular iron overload causes unregulated necrosis, but partially activates PARP-1 dependent parthanatos. Interestingly, we identify many phenolic compounds as potent inhibitors of Fe-8HQ-induced cell death. In conclusion, intracellular iron overload-induced cell death form might be dependent on the intracellular iron accumulation rate, newly identified cell death inhibitors in our study that target ferroptosis and unregulated oxidative cell death represent potential therapeutic strategies against iron overload related diseases. Copyright © 2018 Elsevier Inc. All rights reserved.

Enhanced NMDA receptor-mediated intracellular calcium signaling in magnocellular neurosecretory neurons in heart failure rats.

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Stern, Javier E; Potapenko, Evgeniy S

2013-08-15

An enhanced glutamate excitatory function within the hypothalamic supraoptic and paraventricluar nuclei is known to contribute to increased neurosecretory and presympathetic neuronal activity, and hence, neurohumoral activation, during heart failure (HF). Still, the precise mechanisms underlying enhanced glutamate-driven neuronal activity in HF remain to be elucidated. Here, we performed simultaneous electrophysiology and fast confocal Ca²⁺ imaging to determine whether altered N-methyl-d-aspartate (NMDA) receptor-mediated changes in intracellular Ca²⁺ levels (NMDA-ΔCa²⁺) occurred in hypothalamic magnocellular neurosecretory cells (MNCs) in HF rats. We found that activation of NMDA receptors resulted in a larger ΔCa²⁺ in MNCs from HF when compared with sham rats. The enhanced NMDA-ΔCa²⁺ was neither dependent on the magnitude of the NMDA-mediated current (voltage clamp) nor on the degree of membrane depolarization or firing activity evoked by NMDA (current clamp). Differently from NMDA receptor activation, firing activity evoked by direct membrane depolarization resulted in similar changes in intracellular Ca²⁺ in sham and HF rats. Taken together, our results support a relatively selective alteration of intracellular Ca²⁺ homeostasis and signaling following activation of NMDA receptors in MNCs during HF. The downstream functional consequences of such altered ΔCa²⁺ signaling during HF are discussed.

Intracellular crowding effects on the self-association of the bacterial cell division protein FtsZ.

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Naddaf, Lamis; Sayyed-Ahmad, Abdallah

2014-12-15

The dimerization rate of the bacterial cell division protein FtsZ is strongly affected by the intracellular crowding. Yet the complexity of the intracellular environment makes it difficult to investigate via all-atom molecular dynamics or other detailed theoretical methods. We study the crowding effect on FtsZ dimerization which is the first step of an oligomerization process that results in more elaborate supramolecular structures. In particular, we consider the effect of intracellular crowding on the reaction rates, and their dependence on the different concentrations of crowding agents. We achieved this goal by using Brownian dynamics (BD) simulation techniques and a modified post-processing approach in which we decompose the rate constant in crowded media as a product of the rate constant in the dilute solution times a factor that incorporates the crowding effect. The latter factor accounts for the diffusion reduction and crowder induced energy. In addition we include the crowding effects on water viscosity in the BD simulations of crowded media. We finally show that biomolecular crowding has a considerable effect on the FtsZ dimerization by increasing the dimerization rate constant from 2.6×10(7)M(-1)s(-1) in the absence of crowders to 1.0×10(8)M(-1)s(-1) at crowding level of 0.30. Copyright © 2014 Elsevier Inc. All rights reserved.

A shell-formation related carbonic anhydrase in Crassostrea gigas modulates intracellular calcium against CO2 exposure: Implication for impacts of ocean acidification on mollusk calcification.

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Wang, Xiudan; Wang, Mengqiang; Jia, Zhihao; Song, Xiaorui; Wang, Lingling; Song, Linsheng

2017-08-01

Ocean acidification (OA) could decrease the shells and skeletons formation of mollusk by reducing the availability of carbonate ions at calcification sites. Carbonic anhydrases (CAs) convert CO 2 to HCO 3 - and play important roles in biomineralization process from invertebrate to vertebrate. In the present study, a CA (designated as CgCA) was identified and characterized in Pacific oyster C. gigas. The cDNA of CgCA was of 927bp encoding a predicted polypeptide of 308 amino acids with a signal peptide and a CA catalytic function domain. The mRNA transcripts of CgCA were constitutively expressed in all tested tissues with the highest levels in mantle and hemocytes. During the early development period, the mRNA transcripts of CgCA could be detected in all the stages with the highest level in D-veliger larvae. Elevated CO 2 increased the mRNA transcripts of CgCA in muscle, mantle, hepatopancreas, gill and hemocytes significantly (p<0.05) and induced the translocation of CgCA in hemocytes and mantle. Moreover, elevated CO 2 also caused the decrease of intracellular Ca 2+ in hemocytes (p<0.05). The inhibition of CA by acetazolamide and suppression of CgCA gene via RNA interference could increase the intracellular Ca 2+ in hemocytes (p<0.05). Besides, the decrease of intracellular Ca 2+ content caused by Ca 2+ reagent ionomycin could affect localization of CgCA in mantle tissue. The results indicated CgCA played essential roles in calcification and elevated CO 2 accelerated the mutual modulation between calcium and CgCA, implying reduced calcification rate and dissolved shells under OA. Copyright © 2017 Elsevier B.V. All rights reserved.

Engineering the Intracellular Micro- and Nano-environment via Magnetic Nanoparticles

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Tseng, Peter

generate user-controllable (time-varying and localizable), massively parallel forces on arrays of cells mediated by coalesced ensembles of magnetic nanoparticles. The above process is simplified and adapted for single cell analysis by precisely aligning fibronectin patterned cells to a single flanking micromagnet. The cells are loaded with magnetic-fluorescent nanoparticles, which are then localized to uniform positions at the internal edge of the cell membrane over huge arrays of cells using large external fields, allowing us to conduct composed studies on cellular response to force. By applying forces approaching the yield tension (5 nN / mum) of single cells, we are able to generate highly coordinated responses in cellular behavior. We discover that increasing tension generates highly directed, PAK-dependent leading-edge type filopodia that increase in intensity with rising tension. In addition, we find that our generated forces can simulate cues created during cellular mitosis, as we are consistently able to generate significant (45 to 90 degree) biasing of the metaphase plate during cell division. Large sample size and rapid sample generation also allow us to analyze cells at an unprecedented rate---a single sample can simultaneously stimulate thousands of cells for high statistical accuracy in measurements. We believe these approaches have potential not just as a tool to study single-cell response, but as a means of cell control, potentially through modifying cell movement, division, or differentiation. More generally, once approaches to release nanoparticles from endosomes are implemented, the technique provides a platform to dynamically apply a range of localized stimuli arbitrarily within cells. Through the bioconjugation of proteins, nucleic acids, small molecules, or whole organelles a broad range of questions should be accessible concerning molecular localization and its importance in cell function.

Intracellular bacteria: the origin of dinoflagellate toxicity.

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Silva, E S

1990-01-01

Dinoflagellate blooms of the same species have been registered either as toxic or nontoxic and, in the latter case, toxicity may be of different types. A hypothesis has been formulated according to which the bacteria having in some way taken part in the toxin formation are either inside the dinoflagellate cell or in the nutritive liquid. The presence of intracellular bacteria in those microorganisms has been studied mainly in material from cultures, a few from the sea, and several strains were isolated from different species. Experiments with crossed inoculations have shown that the bacterial strain from Gonyaulax tamarensis caused the cells of some other species to become toxic. From nontoxic clonal cultures of Prorocentrum balticum, Glenodinium foliaceum, and Gyrodinium instriatum, after inoculation of that bacterial strain, cultures were obtained whose cell extracts showed the same kind of toxicity as G. tamarensis. No toxic action could be found in the extracts of the bacterial cells form the assayed strains. The interference of intracellular bacteria in the metabolism of dinoflagellates must be the main cause of their toxicity.

Intracellular calcium levels determine differential modulation of allosteric interactions within G protein-coupled receptor heteromers.

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Navarro, Gemma; Aguinaga, David; Moreno, Estefania; Hradsky, Johannes; Reddy, Pasham P; Cortés, Antoni; Mallol, Josefa; Casadó, Vicent; Mikhaylova, Marina; Kreutz, Michael R; Lluís, Carme; Canela, Enric I; McCormick, Peter J; Ferré, Sergi

2014-11-20

The pharmacological significance of the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer is well established and it is being considered as an important target for the treatment of Parkinson’s disease and other neuropsychiatric disorders. However, the physiological factors that control its distinctive biochemical properties are still unknown. We demonstrate that different intracellular Ca2+ levels exert a differential modulation of A2AR-D2R heteromer-mediated adenylyl-cyclase and MAPK signaling in striatal cells. This depends on the ability of low and high Ca2+ levels to promote a selective interaction of the heteromer with the neuronal Ca2+-binding proteins NCS-1 and calneuron-1, respectively. These Ca2+-binding proteins differentially modulate allosteric interactions within the A2AR-D2R heteromer, which constitutes a unique cellular device that integrates extracellular (adenosine and dopamine) and intracellular (Ca+2) signals to produce a specific functional response.

Intracellular Chemistry: Integrating Molecular Inorganic Catalysts with Living Systems.

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Ngo, Anh H; Bose, Sohini; Do, Loi H

2018-03-23

This concept article focuses on the rapid growth of intracellular chemistry dedicated to the integration of small-molecule metal catalysts with living cells and organisms. Although biological systems contain a plethora of biomolecules that can deactivate inorganic species, researchers have shown that small-molecule metal catalysts could be engineered to operate in heterogeneous aqueous environments. Synthetic intracellular reactions have recently been reported for olefin hydrogenation, hydrolysis/oxidative cleavage, azide-alkyne cycloaddition, allylcarbamate cleavage, C-C bond cross coupling, and transfer hydrogenation. Other promising targets for new biocompatible reaction discovery will also be discussed, with a special emphasis on how such innovations could lead to the development of novel technologies and chemical tools. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structure-activity relationships of dimethylsphingosine (DMS) derivatives and their effects on intracellular pH and Ca2+ in the U937 monocyte cell line.

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Chang, Young-Ja; Lee, Yun-Kyung; Lee, Eun-Hee; Park, Jeong-Ju; Chung, Sung-Kee; Im, Dong-Soon

2006-08-01

We recently reported that dimethylsphingosine (DMS), a metabolite of sphingolipids, increased intracellular pH and Ca2+ concentration in U937 human monocytes. In the present study, we found that dimethylphytosphingosine (DMPH) induced the above responses more robustly than DMS. However, phytosphingosine, monomethylphytosphingosine or trimethylsphingosine showed little or no activity. Synthetic C3 deoxy analogues of sphingosine did show similar activities, with the C16 analogue more so than C18. The following structure-activity relationships were observed between DMS derivatives and the intracellular pH and Ca2+ concentrations in U937 monocytes; 1) dimethyl modification is important for the DMS-induced increase of intracellular pH and Ca2+, 2) the addition of an OH group on C4 enhances both activities, 3) the deletion of the OH group on C3 has a negligible effect on the activities, and 4) C16 appears to be more effective than C18. We also found that W-7, a calmodulin inhibitor, blocked the DMS-induced pH increase, whereas, KN-62, ML9, and MMPX, specific inhibitors for calmodulin-dependent kinase II, myosin light chain kinase, and Ca(2+)-calmodulin-dependent phosphodiesterase, respectively, did not affect DMS-induced increases of pH in the U937 monocytes.

Radiolabeled Cu-ATSM as a novel indicator of overreduced intracellular state due to mitochondrial dysfunction: studies with mitochondrial DNA-less ρ0 cells and cybrids carrying MELAS mitochondrial DNA mutation

International Nuclear Information System (INIS)

Yoshii, Yukie; Yoneda, Makoto; Ikawa, Masamichi; Furukawa, Takako; Kiyono, Yasushi; Mori, Tetsuya; Yoshii, Hiroshi; Oyama, Nobuyuki; Okazawa, Hidehiko; Saga, Tsuneo; Fujibayashi, Yasuhisa

2012-01-01

Objectives: Radiolabeled Cu-diacetyl-bis (N 4 -methylthiosemicarbazone) ( ⁎ Cu-ATSM), including 60/62/64 Cu-ATSM, is a potential imaging agent of hypoxic tumors for positron emission tomography (PET). We have reported that ⁎ Cu-ATSM is trapped in tumor cells under intracellular overreduced states, e.g., hypoxia. Here we evaluated ⁎ Cu-ATSM as an indicator of intracellular overreduced states in mitochondrial disorders using cell lines with mitochondrial dysfunction. Methods: Mitochondrial DNA-less ρ 0 206 cells; the parental 143B human osteosarcoma cells; the cybrids carrying mutated mitochondria from a patient of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) (2SD); and that carrying wild-type one (2SA) were used. Cells were treated under normoxia or hypoxia, and 64 Cu-ATSM uptake was examined to compare it with levels of biological reductant NADH and NADPH. Results: ρ 0 206 cells showed higher 64 Cu-ATSM uptake than control 143B cells under normoxia, whereas 64 Cu-ATSM uptake was not significantly increased under hypoxia in ρ 0 206 cells. Additionally, 64 Cu-ATSM uptake showed correlate change to the NADH and NADPH levels, but not oxygenic conditions. 2SD cells showed increased 64 Cu-ATSM uptake under normoxia as compared with the control 2SA, and 64 Cu-ATSM uptake followed NADH and NADPH levels, but not oxygenic conditions. Conclusions: 64 Cu-ATSM accumulated in cells with overreduced states due to mitochondrial dysfunction, even under normoxia. We recently reported that 62 Cu-ATSM-PET can visualize stroke-like episodes maintaining oxygen supply in MELAS patients. Taken together, our data indicate that ⁎ Cu-ATSM uptake reflects overreduced intracellular states, despite oxygenic conditions; thus, ⁎ Cu-ATSM would be a promising marker of intracellular overreduced states for disorders with mitochondrial dysfunction, such as MELAS, Parkinson's disease and Alzheimer's disease.

Intracellular signaling by diffusion: can waves of hydrogen peroxide transmit intracellular information in plant cells?

DEFF Research Database (Denmark)

Vestergaard, Christian L.; Flyvbjerg, Henrik; Møller, Ian Max

2012-01-01

of the physical and biochemical conditions in plant cells. As model system, we use a H(2)O(2) signal originating at the plasma membrane (PM) and spreading through the cytosol. We consider two maximally simple types of signals, isolated pulses and harmonic oscillations. First we consider the basic limits......Amplitude- and frequency-modulated waves of Ca(2+) ions transmit information inside cells. Reactive Oxygen Species (ROS), specifically hydrogen peroxide, have been proposed to have a similar role in plant cells. We consider the feasibility of such an intracellular communication system in view...

Regulation of dopamine transporter trafficking by intracellular amphetamine

DEFF Research Database (Denmark)

Kahlig, Kristopher M; Lute, Brandon J; Wei, Yuqiang

2006-01-01

-induced cell surface DAT redistribution may result in long-lasting changes in DA homeostasis. The molecular mechanism by which AMPH induces trafficking is not clear. Because AMPH is a substrate, we do not know whether extracellular AMPH stimulates trafficking through its interaction with DAT and subsequent...... alteration in DAT function, thereby triggering intracellular signaling or whether AMPH must be transported and then act intracellularly. In agreement with our previous studies, extracellular AMPH caused cytosolic redistribution of the wild-type human DAT (WT-hDAT). However, AMPH did not induce cytosolic...... redistribution in an uptake-impaired hDAT (Y335A-hDAT) that still binds AMPH. The divalent cation zinc (Zn(2+)) inhibits WT-hDAT activity, but it restores Y335A-hDAT uptake. Coadministration of Zn(2+) and AMPH consistently reduced WT-hDAT trafficking but stimulated cytosolic redistribution of Y335A...

Intracellular Na(+) and metabolic modulation of Na/K pump and excitability in the rat suprachiasmatic nucleus neurons.

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Wang, Yi-Chi; Yang, Jyh-Jeen; Huang, Rong-Chi

2012-10-01

Na/K pump activity and metabolic rate are both higher during the day in the suprachiasmatic nucleus (SCN) that houses the circadian clock. Here we investigated the role of intracellular Na(+) and energy metabolism in regulating Na/K pump activity and neuronal excitability. Removal of extracellular K(+) to block the Na/K pump excited SCN neurons to fire at higher rates and return to normal K(+) to reactivate the pump produced rebound hyperpolarization to inhibit firing. In the presence of tetrodotoxin to block the action potentials, both zero K(+)-induced depolarization and rebound hyperpolarization were blocked by the cardiac glycoside strophanthidin. Ratiometric Na(+) imaging with a Na(+)-sensitive fluorescent dye indicated saturating accumulation of intracellular Na(+) in response to pump blockade with zero K(+). The Na(+) ionophore monensin also induced Na(+) loading and hyperpolarized the membrane potential, with the hyperpolarizing effect of monensin abolished in zero Na(+) or by pump blockade. Conversely, Na(+) depletion with Na(+)-free pipette solution depolarized membrane potential but retained residual Na/K pump activity. Cyanide inhibition of oxidative phosphorylation blocked the Na/K pump to depolarize resting potential and increase spontaneous firing in most cells, and to raise intracellular Na(+) levels in all cells. Nonetheless, the Na/K pump was incompletely blocked by cyanide but completely blocked by iodoacetate to inhibit glycolysis, indicating the involvement of both oxidative phosphorylation and glycolysis in fueling the Na/K pump. Together, the results indicate the importance of intracellular Na(+) and energy metabolism in regulating Na/K pump activity as well as neuronal excitability in the SCN neurons.

Phg1/TM9 proteins control intracellular killing of bacteria by determining cellular levels of the Kil1 sulfotransferase in Dictyostelium.

Directory of Open Access Journals (Sweden)

Marion Le Coadic

Full Text Available Dictyostelium discoideum has largely been used to study phagocytosis and intracellular killing of bacteria. Previous studies have shown that Phg1A, Kil1 and Kil2 proteins are necessary for efficient intracellular killing of Klebsiella bacteria. Here we show that in phg1a KO cells, cellular levels of lysosomal glycosidases and lysozyme are decreased, and lysosomal pH is increased. Surprisingly, overexpression of Kil1 restores efficient killing in phg1a KO cells without correcting these lysosomal anomalies. Conversely, kil1 KO cells are defective for killing, but their enzymatic content and lysosomal pH are indistinguishable from WT cells. The killing defect of phg1a KO cells can be accounted for by the observation that in these cells the stability and the cellular amount of Kil1 are markedly reduced. Since Kil1 is the only sulfotransferase characterized in Dictyostelium, an (unidentified sulfated factor, defective in both phg1a and kil1 KO cells, may play a key role in intracellular killing of Klebsiella bacteria. In addition, Phg1B plays a redundant role with Phg1A in controlling cellular amounts of Kil1 and intracellular killing. Finally, cellular levels of Kil1 are unaffected in kil2 KO cells, and Kil1 overexpression does not correct the killing defect of kil2 KO cells, suggesting that Kil2 plays a distinct role in intracellular killing.

Biological macromolecules based targeted nanodrug delivery systems for the treatment of intracellular infections.

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Aparna, V; Shiva, M; Biswas, Raja; Jayakumar, R

2018-04-15

Intracellular infections are tricky to treat, the reason being the poor penetration of antibiotics/antimycotics into the microbial niche (host cell). Macrophages are primary targets of facultative and obligate intracellular bacteria/fungi to be abused as host cells. The need for drugs with better intracellular penetration led to the development of endocytosable drug carriers, which can cross the cell membrane of the host cells (macrophages) by imitating the entry path of the pathogens. Therefore, the drugs can be targeted to macrophages ensuring enhanced therapeutic effect. This review discusses the exploitation of various nanocarriers for targeted delivery of drugs to the macrophages in the last two decades. Copyright © 2018 Elsevier B.V. All rights reserved.

The calcium feedback loop and T cell activation: how cytoskeleton networks control intracellular calcium flux.

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Joseph, Noah; Reicher, Barak; Barda-Saad, Mira

2014-02-01

During T cell activation, the engagement of a T cell with an antigen-presenting cell (APC) results in rapid cytoskeletal rearrangements and a dramatic increase of intracellular calcium (Ca(2+)) concentration, downstream to T cell antigen receptor (TCR) ligation. These events facilitate the organization of an immunological synapse (IS), which supports the redistribution of receptors, signaling molecules and organelles towards the T cell-APC interface to induce downstream signaling events, ultimately supporting T cell effector functions. Thus, Ca(2+) signaling and cytoskeleton rearrangements are essential for T cell activation and T cell-dependent immune response. Rapid release of Ca(2+) from intracellular stores, e.g. the endoplasmic reticulum (ER), triggers the opening of Ca(2+) release-activated Ca(2+) (CRAC) channels, residing in the plasma membrane. These channels facilitate a sustained influx of extracellular Ca(2+) across the plasma membrane in a process termed store-operated Ca(2+) entry (SOCE). Because CRAC channels are themselves inhibited by Ca(2+) ions, additional factors are suggested to enable the sustained Ca(2+) influx required for T cell function. Among these factors, we focus here on the contribution of the actin and microtubule cytoskeleton. The TCR-mediated increase in intracellular Ca(2+) evokes a rapid cytoskeleton-dependent polarization, which involves actin cytoskeleton rearrangements and microtubule-organizing center (MTOC) reorientation. Here, we review the molecular mechanisms of Ca(2+) flux and cytoskeletal rearrangements, and further describe the way by which the cytoskeletal networks feedback to Ca(2+) signaling by controlling the spatial and temporal distribution of Ca(2+) sources and sinks, modulating TCR-dependent Ca(2+) signals, which are required for an appropriate T cell response. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters

Estimating the biophysical properties of neurons with intracellular calcium dynamics.

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Ye, Jingxin; Rozdeba, Paul J; Morone, Uriel I; Daou, Arij; Abarbanel, Henry D I

2014-06-01

We investigate the dynamics of a conductance-based neuron model coupled to a model of intracellular calcium uptake and release by the endoplasmic reticulum. The intracellular calcium dynamics occur on a time scale that is orders of magnitude slower than voltage spiking behavior. Coupling these mechanisms sets the stage for the appearance of chaotic dynamics, which we observe within certain ranges of model parameter values. We then explore the question of whether one can, using observed voltage data alone, estimate the states and parameters of the voltage plus calcium (V+Ca) dynamics model. We find the answer is negative. Indeed, we show that voltage plus another observed quantity must be known to allow the estimation to be accurate. We show that observing both the voltage time course V(t) and the intracellular Ca time course will permit accurate estimation, and from the estimated model state, accurate prediction after observations are completed. This sets the stage for how one will be able to use a more detailed model of V+Ca dynamics in neuron activity in the analysis of experimental data on individual neurons as well as functional networks in which the nodes (neurons) have these biophysical properties.

IP3-dependent intracellular Ca2+ release is required for cAMP-induced c-fos expression in hippocampal neurons

International Nuclear Information System (INIS)

Zhang, Wenting; Tingare, Asmita; Ng, David Chi-Heng; Johnson, Hong W.; Schell, Michael J.; Lord, Rebecca L.; Chawla, Sangeeta

2012-01-01

Highlights: ► cAMP-induced c-fos expression in hippocampal neurons requires a submembraneous Ca 2+ pool. ► The submembraneous Ca 2+ pool derives from intracellular ER stores. ► Expression of IP 3 -metabolizing enzymes inhibits cAMP-induced c-fos expression. ► SRE-mediated and CRE-mediated gene expression is sensitive to IP 3 -metabolizing enzymes. ► Intracellular Ca 2+ release is required for cAMP-induced nuclear translocation of TORC1. -- Abstract: Ca 2+ and cAMP are widely used in concert by neurons to relay signals from the synapse to the nucleus, where synaptic activity modulates gene expression required for synaptic plasticity. Neurons utilize different transcriptional regulators to integrate information encoded in the spatiotemporal dynamics and magnitude of Ca 2+ and cAMP signals, including some that are Ca 2+ -responsive, some that are cAMP-responsive and some that detect coincident Ca 2+ and cAMP signals. Because Ca 2+ and cAMP can influence each other’s amplitude and spatiotemporal characteristics, we investigated how cAMP acts to regulate gene expression when increases in intracellular Ca 2+ are buffered. We show here that cAMP-mobilizing stimuli are unable to induce expression of the immediate early gene c-fos in hippocampal neurons in the presence of the intracellular Ca 2+ buffer BAPTA-AM. Expression of enzymes that attenuate intracellular IP 3 levels also inhibited cAMP-dependent c-fos induction. Synaptic activity induces c-fos transcription through two cis regulatory DNA elements – the CRE and the SRE. We show here that in response to cAMP both CRE-mediated and SRE-mediated induction of a luciferase reporter gene is attenuated by IP 3 metabolizing enzymes. Furthermore, cAMP-induced nuclear translocation of the CREB coactivator TORC1 was inhibited by depletion of intracellular Ca 2+ stores. Our data indicate that Ca 2+ release from IP 3 -sensitive pools is required for cAMP-induced transcription in hippocampal neurons.

Intracellular uptake and degradation of extracellular tracers in mouse skeletal muscle in vitro: the effect of denervation

International Nuclear Information System (INIS)

Libelius, R.; Lundquist, I.; Templeton, W.; Thesleff, S.

1978-01-01

Innervated and chronically denervated mouse skeletal muscles have been incubated under various conditions in a Ringer solution containing one of the three macromolecules [ 3 H] α-neurotoxin, [ 3 H]inulin and horseradish peroxidase. Following extensive wash-out for 4 h of the extracellular compartment, the amount of each macromolecule retained intracellularly was obtained. Intracellular uptake of a [ 3 H]monoacetylated α-neurotoxin in vitro at 37 C was found to be increased in denervated mouse extensor digitorum longus muscles compared to innervated control muscles. Similarly, the uptake in vitro at 37 C of [ 3 H] inulin and horseradish peroxidase was also increased in denervated muscles. At 4 C the uptake of [ 3 H]inulin and horseradish peroxidase was markedly reduced. Protamine was found to stimulate the uptake of [ 3 H]inulin at 37 C, but not at 4 C. Reduction in specific activity by addition of 50-fold excess of unlabelled inulin failed to affect the uptake of [ 3 H]inulin suggesting that this uptake process obeyed bulk kinetics. Furthermore, the endocytized [ 3 H]inulin was found to be strongly retained in the muscles since prolonged washing or addition of unlabelled inulin to the washing solution did not reduce the uptake. Characterization of [ 3 H]inulin taken up by the muscles was performed by gel chromatography on Sephadex G-25. Using a purified [ 3 H]inulin solution it was observed that about 45% of the total radioactivity remaining in the muscles was eluted as [ 3 H]inulin. Additional radioactivity consisted of lower molecular weight compounds. These degradation products of [ 3 H]inulin were only present in the muscle homogenate and were not detected in the incubation solution. The results suggest that intracellular uptake of different macromolecules by endocytosis in skeletal muscles increases following denervation, and that following uptake, degradation of the endocytized material may occur. (author)

Activation of PKA and Epac proteins by cyclic AMP depletes intracellular calcium stores and reduces calcium availability for vasoconstriction.

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Cuíñas, Andrea; García-Morales, Verónica; Viña, Dolores; Gil-Longo, José; Campos-Toimil, Manuel

2016-06-15

We investigated the implication of PKA and Epac proteins in the endothelium-independent vasorelaxant effects of cyclic AMP (cAMP). Cytosolic Ca(2+) concentration ([Ca(2+)]c) was measured by fura-2 imaging in rat aortic smooth muscle cells (RASMC). Contraction-relaxation experiments were performed in rat aortic rings deprived of endothelium. In extracellular Ca(2+)-free solution, cAMP-elevating agents induced an increase in [Ca(2+)]c in RASMC that was reproduced by PKA and Epac activation and reduced after depletion of intracellular Ca(2+) reservoirs. Arginine-vasopressin (AVP)-evoked increase of [Ca(2+)]c and store-operated Ca(2+) entry (SOCE) were inhibited by cAMP-elevating agents, PKA or Epac activation in these cells. In aortic rings, the contractions induced by phenylephrine in absence of extracellular Ca(2+) were inhibited by cAMP-elevating agents, PKA or Epac activation. In these conditions, reintroduction of Ca(2+) induced a contraction that was inhibited by cAMP-elevating agents, an effect reduced by PKA inhibition and reproduced by PKA or Epac activators. Our results suggest that increased cAMP depletes intracellular, thapsigargin-sensitive Ca(2+) stores through activation of PKA and Epac in RASMC, thus reducing the amount of Ca(2+) released by IP3-generating agonists during the contraction of rat aorta. cAMP rise also inhibits the contraction induced by depletion of intracellular Ca(2+), an effect mediated by reduction of SOCE after PKA or Epac activation. Both effects participate in the cAMP-induced endothelium-independent vasorelaxation. Copyright © 2016 Elsevier Inc. All rights reserved.

Study on the inactivation of intracellular enzyme molecules by X-ray irradiation

International Nuclear Information System (INIS)

Lee, S.B.

1977-01-01

Inactivation of the glutamic acid dehydrogenase and glucose-6-phosphate dehydrogenase enzyme molecules in the Ehrlich ascites tumor cells of the mouse were studied. The above mentioned intracellular enzyme molecules were irradiated by the X-ray radiation under the condition of 65 kV, 1 Amp under the atmosphere of nitrogen gases and by 4 0 C. Thereby, irradiation doses were 580 KR/min(error: +-3%). After irradiation, the cell homogentes were prepared through liquid air techniques. There after, the activities of the enzymes were measured with photometric method given by O. Warburg and W. Christian. The dose effect curves of the activities of the two enzymes by the X-ray irradiation showed both exponential and the inactivation doses were 6.5x10 6 and 5.0x10 6 R respectively. These results showed one side that the inactivation process of the intracellular enzyme molecules was one hit reaction after target theory, and the other side that this inactivation process could not be the primary causes of the death through X-ray irradiation of the vertebrate animals, because of the high resistance of the intracellular protein molecules against X-ray irradiation. The one hit reaction by the inactivation process of the irradiated intracellular enzyme molecules was discussed. (author)

Intracellular Hg(0) Oxidation in Desulfovibrio desulfuricans ND132.

Science.gov (United States)

Wang, Yuwei; Schaefer, Jeffra K; Mishra, Bhoopesh; Yee, Nathan

2016-10-03

The disposal of elemental mercury (Hg(0)) wastes in mining and manufacturing areas has caused serious soil and groundwater contamination issues. Under anoxic conditions, certain anaerobic bacteria can oxidize dissolved elemental mercury and convert the oxidized Hg to neurotoxic methylmercury. In this study, we conducted experiments with the Hg-methylating bacterium Desulfovibrio desulfuricans ND132 to elucidate the role of cellular thiols in anaerobic Hg(0) oxidation. The concentrations of cell-surface and intracellular thiols were measured, and specific fractions of D. desulfuricans ND132 were examined for Hg(0) oxidation activity and analyzed with extended X-ray absorption fine structure (EXAFS) spectroscopy. The experimental data indicate that intracellular thiol concentrations are approximately six times higher than those of the cell wall. Cells reacted with a thiol-blocking reagent were severely impaired in Hg(0) oxidation activity. Spheroplasts lacking cell walls rapidly oxidized Hg(0) to Hg(II), while cell wall fragments exhibited low reactivity toward Hg(0). EXAFS analysis of spheroplast samples revealed that multiple different forms of Hg-thiols are produced by the Hg(0) oxidation reaction and that the local coordination environment of the oxidized Hg changes with reaction time. The results of this study indicate that Hg(0) oxidation in D. desulfuricans ND132 is an intracellular process that occurs by reaction with thiol-containing molecules.

Autophagic clearance of bacterial pathogens: molecular recognition of intracellular microorganisms.

Science.gov (United States)

Pareja, Maria Eugenia Mansilla; Colombo, Maria I

2013-01-01

Autophagy is involved in several physiological and pathological processes. One of the key roles of the autophagic pathway is to participate in the first line of defense against the invasion of pathogens, as part of the innate immune response. Targeting of intracellular bacteria by the autophagic machinery, either in the cytoplasm or within vacuolar compartments, helps to control bacterial proliferation in the host cell, controlling also the spreading of the infection. In this review we will describe the means used by diverse bacterial pathogens to survive intracellularly and how they are recognized by the autophagic molecular machinery, as well as the mechanisms used to avoid autophagic clearance.

A Gold Nanoparticle Bio-Optical Transponder to Dynamically Monitor Intracellular pH.

Science.gov (United States)

Carnevale, Kate J F; Riskowski, Ryan A; Strouse, Geoffrey F

2018-06-13

A pH-sensitive bio-optical transponder (pH-BOT) capable of simultaneously reporting the timing of intracellular DNA cargo release from a gold nanoparticle (AuNP) and the evolving intracellular pH (pH i) during endosomal maturation is demonstrated. The pH-BOT is designed with a triple-dye-labeled duplex DNA appended to a 6.6 nm AuNP, utilizing pH-responsive fluorescein paired with DyLight405 as a surface energy transfer (SET) coupled dye pair to ratiometrically report the pH at and after cargo release. A non-SET-coupled dye, DyLight 700, is used to provide dynamic tracking throughout the experiment. The pH-BOT beacon of the cargo uptake, release, and processing was visualized using live-cell confocal fluorescent microscopy in Chinese hamster ovary cells, and it was observed that while maturation of endosomes carrying pH-BOT is slowed significantly, the pH-BOT is distributed throughout the endolysosomal system while remaining at pH ∼6. This observed decoupling of endosomal maturation from acidification lends support to those models that propose that pH alone is not sufficient to explain endosomal maturation and may enable greater insight into our understanding of the fundamental processes of biology.

Horseradish Peroxidase-Encapsulated Hollow Silica Nanospheres for Intracellular Sensing of Reactive Oxygen Species

Science.gov (United States)

Chen, Hsin-Yi; Wu, Si-Han; Chen, Chien-Tsu; Chen, Yi-Ping; Chang, Feng-Peng; Chien, Fan-Ching; Mou, Chung-Yuan

2018-04-01

Reactive oxygen species (ROS) have crucial roles in cell signaling and homeostasis. Overproduction of ROS can induce oxidative damage to various biomolecules and cellular structures. Therefore, developing an approach capable of monitoring and quantifying ROS in living cells is significant for physiology and clinical diagnoses. Some cell-permeable fluorogenic probes developed are useful for the detection of ROS while in conjunction with horseradish peroxidase (HRP). Their intracellular scenario is however hindered by the membrane-impermeable property of enzymes. Herein, a new approach for intracellular sensing of ROS by using horseradish peroxidase-encapsulated hollow silica nanospheres (designated HRP@HSNs), with satisfactory catalytic activity, cell membrane permeability, and biocompatibility, was prepared via a microemulsion method. These HRP@HSNs, combined with selective probes or targeting ligands, could be foreseen as ROS-detecting tools in specific organelles or cell types. As such, dihydrorhodamine 123-coupled HRP@HSNs were used for the qualitative and semi-quantitative analysis of physiological H2O2 levels in activated RAW 264.7 macrophages. We envision that this HSNs encapsulating active enzymes can be conjugated with selective probes and targeting ligands to detect ROS in specific organelles or cell types of interest.

Metabolic Characterization of Intact Cells Reveals Intracellular Amyloid Beta but Not Its Precursor Protein to Reduce Mitochondrial Respiration

Science.gov (United States)

Schaefer, Patrick M.; von Einem, Bjoern; Walther, Paul; Calzia, Enrico; von Arnim, Christine A. F.

2016-01-01

One hallmark of Alzheimer´s disease are senile plaques consisting of amyloid beta (Aβ), which derives from the processing of the amyloid precursor protein (APP). Mitochondrial dysfunction has been linked to the pathogenesis of Alzheimer´s disease and both Aβ and APP have been reported to affect mitochondrial function in isolated systems. However, in intact cells, considering a physiological localization of APP and Aβ, it is pending what triggers the mitochondrial defect. Thus, the aim of this study was to dissect the impact of APP versus Aβ in inducing mitochondrial alterations with respect to their subcellular localization. We performed an overexpression of APP or beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), increasing APP and Aβ levels or Aβ alone, respectively. Conducting a comprehensive metabolic characterization we demonstrate that only APP overexpression reduced mitochondrial respiration, despite lower extracellular Aβ levels compared to BACE overexpression. Surprisingly, this could be rescued by a gamma secretase inhibitor, oppositionally indicating an Aβ-mediated mitochondrial toxicity. Analyzing Aβ localization revealed that intracellular levels of Aβ and an increased spatial association of APP/Aβ with mitochondria are associated with reduced mitochondrial respiration. Thus, our data provide marked evidence for a prominent role of intracellular Aβ accumulation in Alzheimer´s disease associated mitochondrial dysfunction. Thereby it highlights the importance of the localization of APP processing and intracellular transport as a decisive factor for mitochondrial function, linking two prominent hallmarks of neurodegenerative diseases. PMID:28005987

Salmonella Intracellular Lifestyles and Their Impact on Host-to-Host Transmission.

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Pucciarelli, M Graciela; García-Del Portillo, Francisco

2017-07-01

More than a century ago, infections by Salmonella were already associated with foodborne enteric diseases with high morbidity in humans and cattle. Intestinal inflammation and diarrhea are hallmarks of infections caused by nontyphoidal Salmonella serovars, and these pathologies facilitate pathogen transmission to the environment. In those early times, physicians and microbiologists also realized that typhoid and paratyphoid fever caused by some Salmonella serovars could be transmitted by "carriers," individuals outwardly healthy or at most suffering from some minor chronic complaint. In his pioneering study of the nontyphoidal serovar Typhimurium in 1967, Takeuchi published the first images of intracellular bacteria enclosed by membrane-bound vacuoles in the initial stages of the intestinal epithelium penetration. These compartments, called Salmonella -containing vacuoles, are highly dynamic phagosomes with differing biogenesis depending on the host cell type. Single-cell studies involving real-time imaging and gene expression profiling, together with new approaches based on genetic reporters sensitive to growth rate, have uncovered unprecedented heterogeneous responses in intracellular bacteria. Subpopulations of intracellular bacteria displaying fast, reduced, or no growth, as well as cytosolic and intravacuolar bacteria, have been reported in both in vitro and in vivo infection models. Recent investigations, most of them focused on the serovar Typhimurium, point to the selection of persisting bacteria inside macrophages or following an autophagy attack in fibroblasts. Here, we discuss these heterogeneous intracellular lifestyles and speculate on how these disparate behaviors may impact host-to-host transmissibility of Salmonella serovars.

High Prevalence, Genetic Diversity and Intracellular Growth Ability of Legionella in Hot Spring Environments

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Zhou, Haijian; Wang, Huanxin; Xu, Ying; Zhao, Mingqiang; Guan, Hong; Li, Machao; Shao, Zhujun

2013-01-01

Background Legionella is the causative agent of Legionnaires' disease, and hot springs are a major source of outbreaks of this disease. It is important from a public health perspective to survey hot spring environments for the presence of Legionella. Methods Prospective surveillance of the extent of Legionella pollution was conducted at three hot spring recreational areas in Beijing, China in 2011. Pulsed-field gel electrophoresis (PFGE) and sequence-based typing (SBT) were used to describe the genetic polymorphism of isolates. The intracellular growth ability of the isolates was determined by interacting with J774 cells and plating the dilutions onto BCYE agar plates. Results Overall, 51.9% of spring water samples showed Legionella-positive, and their concentrations ranged from 1 CFU/liter to 2,218 CFU/liter. The positive rates of Legionella were significantly associated with a free chlorine concentration of ≥0.2 mg/L, urea concentration of ≥0.05 mg/L, total microbial counts of ≥400 CFU/ml and total coliform of ≥3 MPN/L (pLegionella concentrations were significantly associated with sample temperature, pH, total microbial counts and total coliform (pLegionella pneumophila was the most frequently isolated species (98.9%), and the isolated serogroups included serogroups 3 (25.3%), 6 (23.4%), 5 (19.2%), 1 (18.5%), 2 (10.2%), 8 (0.4%), 10 (0.8%), 9 (1.9%) and 12 (0.4%). Two hundred and twenty-eight isolates were analyzed by PFGE and 62 different patterns were obtained. Fifty-seven L. pneumophila isolates were selected for SBT analysis and divided into 35 different sequence types with 5 main clonal groups. All the 57 isolates had high intracellular growth ability. Conclusions Our results demonstrated high prevalence and genetic polymorphism of Legionella in springs in Beijing, China, and the SBT and intracellular growth assay results suggested that the Legionella isolates of hot spring environments were pathogenic. Improved control and prevention strategies are

High prevalence, genetic diversity and intracellular growth ability of Legionella in hot spring environments.

Directory of Open Access Journals (Sweden)

Tian Qin

Full Text Available BACKGROUND: Legionella is the causative agent of Legionnaires' disease, and hot springs are a major source of outbreaks of this disease. It is important from a public health perspective to survey hot spring environments for the presence of Legionella. METHODS: Prospective surveillance of the extent of Legionella pollution was conducted at three hot spring recreational areas in Beijing, China in 2011. Pulsed-field gel electrophoresis (PFGE and sequence-based typing (SBT were used to describe the genetic polymorphism of isolates. The intracellular growth ability of the isolates was determined by interacting with J774 cells and plating the dilutions onto BCYE agar plates. RESULTS: Overall, 51.9% of spring water samples showed Legionella-positive, and their concentrations ranged from 1 CFU/liter to 2,218 CFU/liter. The positive rates of Legionella were significantly associated with a free chlorine concentration of ≥0.2 mg/L, urea concentration of ≥0.05 mg/L, total microbial counts of ≥400 CFU/ml and total coliform of ≥3 MPN/L (p<0.01. The Legionella concentrations were significantly associated with sample temperature, pH, total microbial counts and total coliform (p<0.01. Legionella pneumophila was the most frequently isolated species (98.9%, and the isolated serogroups included serogroups 3 (25.3%, 6 (23.4%, 5 (19.2%, 1 (18.5%, 2 (10.2%, 8 (0.4%, 10 (0.8%, 9 (1.9% and 12 (0.4%. Two hundred and twenty-eight isolates were analyzed by PFGE and 62 different patterns were obtained. Fifty-seven L. pneumophila isolates were selected for SBT analysis and divided into 35 different sequence types with 5 main clonal groups. All the 57 isolates had high intracellular growth ability. CONCLUSIONS: Our results demonstrated high prevalence and genetic polymorphism of Legionella in springs in Beijing, China, and the SBT and intracellular growth assay results suggested that the Legionella isolates of hot spring environments were pathogenic. Improved control

Intracellular bimodal nanoparticles based on quantum dots for high-field MRI at 21.1 T.

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Rosenberg, Jens T; Kogot, Joshua M; Lovingood, Derek D; Strouse, Geoffrey F; Grant, Samuel C

2010-09-01

Multimodal, biocompatible contrast agents for high magnetic field applications represent a new class of nanomaterials with significant potential for tracking of fluorescence and MR in vitro and vivo. Optimized for high-field MR applications-including biomedical imaging at 21.1 T, the highest magnetic field available for MRI-these nanoparticles capitalize on the improved performance of chelated Dy(3+) with increasing magnetic field coupled to a noncytotoxic Indium Phosphide/Zinc Sulfide (InP/ZnS) quantum dot that provides fluorescence detection, MR responsiveness, and payload delivery. By surface modifying the quantum dot with a cell-penetrating peptide sequence coupled to an MR contrast agent, the bimodal nanomaterial functions as a self-transfecting high-field MR/optical contrast agent for nonspecific intracellular labeling. Fluorescent images confirm sequestration in perinuclear vesicles of labeled cells, with no apparent cytotoxicity. These techniques can be extended to impart cell selectivity or act as a delivery vehicle for genetic or pharmaceutical interventions. 2010 Wiley-Liss, Inc.

Patient-derived acute myeloid leukemia (AML) bone marrow cells display distinct intracellular kinase phosphorylation patterns

International Nuclear Information System (INIS)

Shults, Keith; Flye, Leanne; Green, Lisa; Daly, Thomas; Manro, Jason R; Lahn, Michael

2009-01-01

Multiparametric analyses of phospho-protein activation in patients with acute myeloid leukemia (AML) offers a quantitative measure to monitor the activity of novel intracellular kinase (IK) inhibitors. As recent clinical investigation with FMS-like tyrosine-3 inhibitors demonstrated, targeting IK with selective inhibitors can have a modest clinical benefit. Because multiple IKs are active in patients with AML, multikinase inhibitors may provide the necessary inhibition profile to achieve a more sustained clinical benefit. We here describe a method of assessing the activation of several IKs by flow cytometry. In 40 different samples of patients with AML we observed hyper-activated phospho-proteins at baseline, which is modestly increased by adding stem cell factor to AML cells. Finally, AML cells had a significantly different phospho-protein profile compared with cells of the lymphocyte gate. In conclusion, our method offers a way to determine the activation status of multiple kinases in AML and hence is a reliable assay to evaluate the pharmacodynamic activity of novel multikinase inhibitors

Intracellular serpins, firewalls and tissue necrosis.

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