scholarly journals Regulation of V-ATPase recycling via a RhoA- and ROCKII-dependent pathway in epididymal clear cells

2011 ◽  
Vol 301 (1) ◽  
pp. C31-C43 ◽  
Author(s):  
Winnie Waichi Shum ◽  
Nicolas Da Silva ◽  
Clémence Belleannée ◽  
Mary McKee ◽  
Dennis Brown ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Apical Membrane ◽  
Green Fluorescence Protein ◽  
Cortical Actin ◽  
Clear Cells ◽  
Luminal Perfusion ◽  
Intracellular Signaling Cascade ◽  
Fluorescence Protein ◽  
And Storage

Luminal acidification in the epididymis is critical for sperm maturation and storage. Clear cells express the vacuolar H+-ATPase (V-ATPase) in their apical membrane and are major contributors to proton secretion. We showed that this process is regulated via recycling of V-ATPase-containing vesicles. We now report that RhoA and its effector ROCKII are enriched in rat epididymal clear cells. In addition, cortical F-actin was detected beneath the apical membrane and along the lateral membrane of “resting” clear cells using a pan-actin antibody or phalloidin-TRITC. In vivo luminal perfusion of the cauda epididymal tubule with the ROCK inhibitors Y27632 (10–30 μM) and HA1077 (30 μM) or with the cell-permeable Rho inhibitor Clostridium botulinum C3 transferase (3.75 μg/ml) induced the apical membrane accumulation of V-ATPase and extension of V-ATPase-labeled microvilli in clear cells. However, these newly formed microvilli were devoid of ROCKII. In addition, Y27632 (30 μM) or HA1077 (30 μM) decreased the ratio of F-actin to G-actin detected by Western blot analysis in epididymal epithelial cells, and Y27632 also decreased the ratio of F-actin to G-actin in clear cells isolated by fluorescence activated cell sorting from B1-enhanced green fluorescence protein (EGFP) transgenic mice. These results provide evidence that depolymerization of the cortical actin cytoskeleton via inhibition of RhoA or its effector ROCKII favors the recruitment of V-ATPase from the cytosolic compartment into the apical membrane in clear cells. In addition, our data suggest that the RhoA-ROCKII pathway is not locally involved in the elongation of apical microvilli. We propose that inhibition of RhoA-ROCKII might be part of the intracellular signaling cascade that is triggered upon agonist-induced apical membrane V-ATPase accumulation.

scholarly journals A cytokine network involving IL-36γ, IL-23, and IL-22 promotes antimicrobial defense and recovery from intestinal barrier damage

2018 ◽  
Vol 115 (22) ◽  
pp. E5076-E5085 ◽  
Author(s):  
Vu L. Ngo ◽  
Hirohito Abo ◽  
Estera Maxim ◽  
Akihito Harusato ◽  
Duke Geem ◽  
...  
Keyword(s):  
Host Defense ◽  
Immune Cells ◽  
Intracellular Signaling ◽  
Intestinal Barrier ◽  
Intestinal Damage ◽  
Cytokine Network ◽  
Potent Inducer ◽  
Intracellular Signaling Cascade

The gut epithelium acts to separate host immune cells from unrestricted interactions with the microbiota and other environmental stimuli. In response to epithelial damage or dysfunction, immune cells are activated to produce interleukin (IL)-22, which is involved in repair and protection of barrier surfaces. However, the specific pathways leading to IL-22 and associated antimicrobial peptide (AMP) production in response to intestinal tissue damage remain incompletely understood. Here, we define a critical IL-36/IL-23/IL-22 cytokine network that is instrumental for AMP production and host defense. Using a murine model of intestinal damage and repair, we show that IL-36γ is a potent inducer of IL-23 both in vitro and in vivo. IL-36γ–induced IL-23 required Notch2-dependent (CD11b+CD103+) dendritic cells (DCs), but not Batf3-dependent (CD11b−CD103+) DCs or CSF1R-dependent macrophages. The intracellular signaling cascade linking IL-36 receptor (IL-36R) to IL-23 production by DCs involved MyD88 and the NF-κB subunits c-Rel and p50. Consistent with in vitro observations, IL-36R– and IL-36γ–deficient mice exhibited dramatically reduced IL-23, IL-22, and AMP levels, and consequently failed to recover from acute intestinal damage. Interestingly, impaired recovery of mice deficient in IL-36R or IL-36γ could be rescued by treatment with exogenous IL-23. This recovery was accompanied by a restoration of IL-22 and AMP expression in the colon. Collectively, these data define a cytokine network involving IL-36γ, IL-23, and IL-22 that is activated in response to intestinal barrier damage and involved in providing critical host defense.

scholarly journals Generation of a GFP Reporter Akabane Virus with Enhanced Fluorescence Intensity by Modification of Artificial Ambisense S Genome

Viruses ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 634
Author(s):  
Akiko Takenaka-Uema ◽  
Shin Murakami ◽  
Nanako Ushio ◽  
Tomoya Kobayashi-Kitamura ◽  
Masashi Uema ◽  
...  
Keyword(s):  
Imaging Study ◽  
Green Fluorescence Protein ◽  
Histopathological Study ◽  
Akabane Virus ◽  
Gfp Reporter ◽  
The Central Nervous System ◽  
Infected Cells ◽  
Fluorescence Protein

We previously generated a recombinant reporter Akabane virus expressing enhanced green fluorescence protein (eGFP-AKAV), with an artificial S genome encoding eGFP in the ambisense RNA. Although the eGFP-AKAV was able to detect infected cells in in vivo histopathological study, its fluorescent signal was too weak to apply to in vivo imaging study. Here, we successfully generated a modified reporter, eGFP/38-AKAV, with 38-nucleotide deletion of the internal region of the 5′ untranslated region of S RNA. The eGFP/38-AKAV expressed higher intensity of eGFP fluorescence both in vitro and in vivo than the original eGFP-AKAV did. In addition, eGFP/38-AKAV was pathogenic in mice at a comparable level to that in wild-type AKAV. In the mice infected with eGFP/38-AKAV, the fluorescent signals, i.e., the virus-infected cells, were detected in the central nervous system using the whole-organ imaging. Our findings indicate that eGFP/38-AKAV could be used as a powerful tool to help elucidate the dynamics of AKAV in vivo.

Tissue hypoxygenation activates the adrenomedullin system in vivo

2000 ◽  
Vol 278 (2) ◽  
pp. R513-R519 ◽  
Author(s):  
Karl-Heinz Hofbauer ◽  
Boye L. Jensen ◽  
Armin Kurtz ◽  
Peter Sandner
Keyword(s):  
Intracellular Signaling ◽  
Messenger Rna ◽  
Rnase Protection Assay ◽  
Tissue Hypoxia ◽  
Mrna Abundance ◽  
Sprague Dawley ◽  
Rnase Protection ◽  
Sprague Dawley Rats ◽  
Intracellular Signaling Cascade

Our study aimed to investigate the influence of tissue hypoxygenation on the adrenomedullin (ADM) system in vivo. For this purpose, male Sprague-Dawley rats were exposed to normobaric hypoxia (8% oxygen) or to functional anemia [0.1% carbon monoxide (CO)] or to cobalt chloride (60 mg/kg) for 6 h. Messenger RNA levels for ADM and its receptor (ADM-R) were assessed in diverse organs by RNase protection assay. Additionally, ADM protein concentrations in these organs, as in plasma, were determined by a RIA. We found that ADM mRNA abundance increased in response to hypoxia and to CO inhalation up to 15-fold in all organs examined. Similarly, ADM-R mRNA abundance increased during hypoxia and CO inhalation in all organs examined with exception of the liver. The effects of hypoxia and of CO inhalation on ADM and ADM-R mRNAs were mimicked by injection of cobaltous chloride. Hypoxia also significantly increased ADM protein content in all organs, and plasma levels of ADM rose twofold in response to hypoxia and CO inhalation. These findings indicate that tissue hypoxia leads to a widespread activation of the ADM system, which comprises a parallel stimulation of ADM and ADM receptor mRNA as enhanced ADM protein synthesis and secretion. The ADM system may, therefore, play a significant role in the physiological response to tissue hypoxia. It appears that ADM and ADM-R belong to the family of classic oxygen-regulated genes, which are activated by a decrease of the pericellular oxygen tension through the same intracellular signaling cascade.

scholarly journals Chronic release of tailless phage particles from Lactococcus lactis

2021 ◽  
Author(s):  
Yue Liu ◽  
Svetlana Alexeeva ◽  
Herwig Bachmann ◽  
Jesús Adrián Guerra Martínez ◽  
Nataliya Yeremenko ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Lipid Membranes ◽  
Lipid Analysis ◽  
Starter Culture ◽  
Green Fluorescence Protein ◽  
Bacterial Cells ◽  
Host Interaction ◽  
Phage Production ◽  
Fluorescence Protein

Lactococcus lactis strains residing in the microbial community of a complex dairy starter culture named 'Ur' are hosts to prophages belonging to the family Siphoviridae. L. lactis strains (TIFN1 to TIFN7) showed detectable spontaneous phage production and release (109-1010 phage particles/mL) and up to 10-fold increases upon prophage induction, while in both cases we observed no obvious cell lysis, typically described for the lytic life cycle of Siphoviridae phages. Intrigued by this phenomenon, we investigated the host-phage interaction using strain TIFN1 (harboring prophage proPhi1) as a representative. We confirmed that during the massive phage release, all bacterial cells remain viable. Further, by monitoring phage replication in vivo, using a green fluorescence protein reporter combined with flow cytometry, we demonstrated that the majority of the bacterial population (over 80%) is actively producing phage particles when induced with mitomycin C. The released tailless phage particles were found to be engulfed in lipid membranes, as evidenced by electron microscopy and lipid staining combined with chemical lipid analysis. Based on the collective observations, we propose a model of phage-host interaction in L. lactis TIFN1, where the phage particles are engulfed in membranes upon release, thereby leaving the producing host intact. Moreover, we discuss possible mechanisms of chronic, or non-lytic release of LAB Siphoviridae phages and its impact on the bacterial host.

scholarly journals Expressional Analysis of GFP-Tagged Cells in an In Vivo Mouse Model of Giant Cell Tumor of Bone

2013 ◽  
Vol 7 (1) ◽  
pp. 109-113 ◽  
Author(s):  
S Singh ◽  
M Singh ◽  
I Mak ◽  
M Ghert
Keyword(s):  
Giant Cell Tumor ◽  
Giant Cell ◽  
Stromal Cells ◽  
Green Fluorescence Protein ◽  
Cell Tumor ◽  
Green Fluorescence ◽  
Continuous Growth ◽  
Stromal Component ◽  
Fluorescence Protein

Giant cell tumor of bone in a neoplastic stromal cell which survives for multiple passages in primary cell culture with a stable phenotype. In the pathological environment of GCT, the neoplastic nature of the mesenchymal stromal component drives local hematopoietic precursors to undergo fusion and form multinucleated osteoclast like giant cells. There is currently very limited knowledge about the pathogenesis of GCT due to the lack of suitable in vivo models for this tumor. Here we report stable gene transfer of Green fluorescence protein (GFP) in GCT stromal cells. In the present study, we have used GCT stromal cells that stably express enhanced green fluorescence protein (GFP) that are used in a new in vivo culture model. Our results show the utility of the GFP tagged cell lines that stably express GFP signals up to 52 weeks of continuous growth. The in vivo model described herein can serve as an excellent system for in vivo therapeutic and mechanistic evaluation of existing and novel targets for GCT.

scholarly journals A Bioassay-Based Approach for the Batch-To-Batch Consistency Evaluation of Xuesaitong Injection on a Zebrafish Thrombosis Model

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiangwei Ma ◽  
Yanyu Chen ◽  
Shumin Jiang ◽  
Xiaoping Zhao
Keyword(s):  
Quality Control ◽  
High Performance ◽  
Green Fluorescence Protein ◽  
Coagulation Cascade ◽  
Dependent Manner ◽  
Zebrafish Model ◽  
Ginsenoside Rd ◽  
Thrombosis Model ◽  
Fluorescence Protein

Quality control of Chinese medicine (CM) is mainly based on chemical testing, which sometimes shows weak correlation to pharmacological effects. Thus, there is a great demand to establish bioactivity-based assays to ensure the quality of CM. The aim of the present study was to establish a bioassay-based approach to evaluate the biological activity of Xuesaitong injection (XST) based on an in vivo zebrafish model. Zebrafish larvae with arachidonic acid (AA)-induced thrombus were applied to evaluate anti-thrombosis effects of XST and explore the potential mechanism of XST. Analysis of major components in normal and abnormal XST samples was performed by high performance liquid chromatography (HPLC). The results indicate that XST could significantly restore heart red blood cells (RBCs) intensity of thrombotic zebrafish in a dose-dependent manner, whilst decreasing RBCs accumulation in the caudal vein. The results were confirmed using a green fluorescence protein (GFP)-labeled zebrafish thrombosis model. Moreover, we could show that XST downregulates the expression of the fibrinogen alpha chain (fga) gene to inhibit the coagulation cascade during the process of thrombosis in zebrafish. Notoginsenoside R1, ginsenoside Rg1, ginsenoside Rb1 and ginsenoside Rd, which were considered to be the major components of XST, also showed moderate anti-thrombosis efficacy. Further results showed that the zebrafish thrombosis model could efficiently distinguish five abnormal batches of XST from 24 normal batches. Furthermore, the inhibition rates of different batches were correlated with the content level of major components. Our results suggested that the proposed zebrafish thrombosis model could be successfully used to evaluate the batch-to-batch consistency of XST, which provided an alternative way for the quality control of CM.

Rho-dependent transfer of Citron-kinase to the cleavage furrow of dividing cells

2001 ◽  
Vol 114 (18) ◽  
pp. 3273-3284 ◽  
Author(s):  
Masatoshi Eda ◽  
Shigenobu Yonemura ◽  
Takayuki Kato ◽  
Naoki Watanabe ◽  
Toshimasa Ishizaki ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Green Fluorescence Protein ◽  
Cell Cortex ◽  
Cleavage Furrow ◽  
Cortical Actin ◽  
Interphase Cells ◽  
C3 Exoenzyme ◽  
Dividing Cells ◽  
Fluorescence Protein ◽  
Citron Kinase

Citron-kinase (Citron-K) is a Rho effector working in cytokinesis. It is enriched in cleavage furrow, but how Rho mobilizes Citron-K remains unknown. Using anti-Citron antibody and a Citron-K Green Fluorescence Protein (GFP)-fusion, we monitored its localization in cell cycle. We have found: (1) Citron-K is present as aggregates in interphase cells, disperses throughout the cytoplasm in prometaphase, translocates to cell cortex in anaphase and accumulates in cleavage furrow in telophase; (2) Rho colocalizes with Citron-K in the cortex of ana- to telophase cells and the two proteins are concentrated in the cleavage furrow and to the midbody; (3) inactivation of Rho by C3 exoenzyme does not affect the dispersion of Citron-K in prometaphase, but prevented its transfer to the cell cortex, and Citron-K stays in association with the midzone spindles of C3 exoenzyme-treated cells. To clarify further the mechanism of the Rho-mediated transfer and concentration of Citron-K in cleavage furrow, we expressed active Val14RhoA in interphase cells expressing GFP-Citron-K. Val14RhoA expression transferred Citron-K to the ventral cortex of interphase cells, where it formed band-like structures in a complex with Rho. This structure was localized at the same plane as actin stress fibers, and they exclude each other. Disruption of F-actin abolished the band and dispersed the Citron-K-Rho-containing patches throughout the cell cortex. Similarly, in dividing cells, a structure composed of Rho and Citron-K in cleavage furrow excludes cortical actin cytoskeleton, and disruption of F-actin disperses Citron-K throughout the cell cortex. These results suggest that Citron-K is a novel type of a passenger protein, which is dispersed to the cytoplasm in prometaphase and associated with midzone spindles by a Rho-independent signal. Rho is then activated, binds to Citron-K and translocates it to cell cortex, where the complex is then concentrated in the cleavage furrow by the action of actin cytoskeleton beneath the equator of dividing cells.

Use of blood outgrowth endothelial cells for gene therapy for hemophilia A

Blood ◽  
2002 ◽  
Vol 99 (2) ◽  
pp. 457-462 ◽  
Author(s):  
Yi Lin ◽  
Liming Chang ◽  
Anna Solovey ◽  
John F. Healey ◽  
Pete Lollar ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Endothelial Cells ◽  
Hemophilia A ◽  
Scale Up ◽  
Coagulation Factor ◽  
Green Fluorescence Protein ◽  
Cell Dose ◽  
Effective Therapeutic Strategy ◽  
Fluorescence Protein

Abstract A culture of human blood outgrowth endothelial cells (BOECs) was established from a sample of peripheral blood and was transfected using a nonviral plasmid carrying complementary DNA for modified human coagulation factor VIII (B domain deleted and replaced with green fluorescence protein). BOECs were then chemically selected, expanded, cryopreserved, and re-expanded in culture. Stably transfected BOECs were administered intravenously daily for 3 days to NOD/SCID mice at 4 cell dose levels (from 5 × 104 to 40 × 104 cells per injection). In 156 days of observation, mice showed levels of human FVIII that increased with cell dose and time. Mice in all cell dose groups achieved therapeutic levels (more than 10 ng/mL) of human FVIII, and mice in the 3 highest dose groups acquired levels that were normal (100-200 ng/mL) or even above the normal range (highest observed value, 1174 ng/mL). These levels indicate that the BOECs expanded in vivo after administration. When the mice were killed, it was found that BOEC accumulated only in bone marrow and spleen and that these cells retained endothelial phenotype and transgene expression. Cell doses used here would make scale-up to humans feasible. Thus, the use of engineered autologous BOECs, which here resulted in sustained and therapeutic levels of FVIII, may comprise an effective therapeutic strategy for use in gene therapy for hemophilia A.

scholarly journals Action Potential–Enhanced ATP Release From Taste Cells Through Hemichannels

2010 ◽  
Vol 104 (2) ◽  
pp. 896-901 ◽  
Author(s):  
Yoshihiro Murata ◽  
Toshiaki Yasuo ◽  
Ryusuke Yoshida ◽  
Kunihiko Obata ◽  
Yuchio Yanagawa ◽  
...  
Keyword(s):  
Action Potentials ◽  
Apical Membrane ◽  
Atp Release ◽  
Green Fluorescence Protein ◽  
Luciferase Assay ◽  
Type Ii ◽  
Green Fluorescence ◽  
Pannexin 1 ◽  
Taste Cells ◽  
Fluorescence Protein

Only some taste cells fire action potentials in response to sapid stimuli. Type II taste cells express many taste transduction molecules but lack well-elaborated synapses, bringing into question the functional significance of action potentials in these cells. We examined the dependence of adenosine triphosphate (ATP) transmitter release from taste cells on action potentials. To identify type II taste cells we used mice expressing a green fluorescence protein (GFP) transgene from the α-gustducin promoter. Action potentials were recorded by an electrode basolaterally attached to a single GFP-positive taste cell. We monitored ATP release from gustducin-expressing taste cells by collecting the electrode solution immediately after tastant-stimulated action potentials and using a luciferase assay to quantify ATP. Stimulation of gustducin-expressing taste cells with saccharin, quinine, or glutamate on the apical membrane increased ATP levels in the electrode solution; the amount of ATP depended on the firing rate. Increased spontaneous firing rates also induced ATP release from gustducin-expressing taste cells. ATP release from gustducin-expressing taste cells was depressed by tetrodotoxin and inhibited below the detection limit by carbenoxolone. Our data support the hypothesis that action potentials in taste cells responsive to sweet, bitter, or umami tastants enhance ATP release through pannexin 1, not connexin-based hemichannels.

scholarly journals The Feasibility of Pressurised Intraperitoneal Aerosolised Virotherapy (PIPAV) to Administer Oncolytic Adenoviruses

Pharmaceutics ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2043
Author(s):  
Sophia J. Tate ◽  
Leen Van de Sande ◽  
Wim P. Ceelen ◽  
Jared Torkington ◽  
Alan L. Parker
Keyword(s):  
Anticancer Agents ◽  
Treatment Options ◽  
Intraperitoneal Administration ◽  
Green Fluorescence Protein ◽  
Firefly Luciferase ◽  
Oncolytic Adenoviruses ◽  
Poor Treatment ◽  
Fluorescence Protein

Background: The prognosis of patients with peritoneal metastases is poor. Treatment options are limited because systemically delivered chemotherapy is not usually effective in this type of disease. Pressurised intraperitoneal aerosolised chemotherapy (PIPAC) is a recently developed alternative technology for delivering intraperitoneal chemotherapy, potentially enhancing treatment efficacy. Here, we assess the feasibility of pressurised intraperitoneal aerosolised virotherapy (PIPAV) to deliver a different class of anticancer agents, oncolytic adenoviruses, in vitro and in vivo. Methods: Adenoviral vectors expressing reporter genes green fluorescence protein (Ad5.GFP) or firefly luciferase (Ad5.Luc) were subject to pressurised aerosolisation. The ability of the virus to survive PIPAV was assessed in vitro and in vivo by monitoring reporter gene activity. Wistar rats subjected to PIPAV were assessed for any adverse procedure related events. Results: In vitro transduction assays demonstrated that Ad5 retained viability following pressurised aerosolisation and could transduce permissive cells equally effectively as non-aerosolised control vector. PIPAV was well tolerated in rats, although minimal transduction was observed following intraperitoneal administration. Conclusions: PIPAV appears viable and well tolerated, though in vivo efficacy requires further optimisation.

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