scholarly journals In vivo evidence for a regulatory role of phosphorylation ofArabidopsisRubisco activase at the Thr78 site

2019 ◽  
Vol 116 (37) ◽  
pp. 18723-18731 ◽  
Author(s):  
Sang Yeol Kim ◽  
Christopher M. Harvey ◽  
Jonas Giese ◽  
Ines Lassowskat ◽  
Vijayata Singh ◽  
...  
Keyword(s):  
Redox Regulation ◽  
Rubisco Activase ◽  
Regulatory Role ◽  
Wild Type ◽  
Low Light ◽  
Induction Kinetics ◽  
Potential Regulatory Role ◽  
Wild Type Control

ArabidopsisRubisco activase (Rca) is phosphorylated at threonine-78 (Thr78) in low light and in the dark, suggesting a potential regulatory role in photosynthesis, but this has not been directly tested. To do so, we transformed anrca-knockdown mutant largely lacking redox regulation with wild-type Rca-β or Rca-β with Thr78-to-Ala (T78A) or Thr78-to-Ser (T78S) site–directed mutations. Interestingly, the T78S mutant was hyperphosphorylated at the Ser78 site relative to Thr78 of the Rca-β wild-type control, as evidenced by immunoblotting with custom antibodies and quantitative mass spectrometry. Moreover, plants expressing the T78S mutation had reduced photosynthesis and quantum efficiency of photosystem II (ϕPSII) and reduced growth relative to control plants expressing wild-type Rca-β under all conditions tested. Gene expression was also altered in a manner consistent with reduced growth. In contrast, plants expressing Rca-β with the phospho-null T78A mutation had faster photosynthetic induction kinetics and increased ϕPSIIrelative to Rca-β controls. While expression of the wild-type Rca-β or the T78A mutant fully rescued the slow-growth phenotype of therca-knockdown mutant grown in a square-wave light regime, the T78A mutants grew faster than the Rca-β control plants at low light (30 µmol photons m−2s−1) and in a fluctuating low-light/high-light environment. Collectively, these results suggest that phosphorylation of Thr78 (or Ser78 in the T78S mutant) plays a negative regulatory role in vivo and provides an explanation for the absence of Ser at position 78 in terrestrial plant species.

scholarly journals Expression of lysophosphatidic acid receptor 5 is necessary for the regulation of intestinal Na+/H+ exchanger 3 by lysophosphatidic acid in vivo

2018 ◽  
Vol 315 (4) ◽  
pp. G433-G442 ◽  
Author(s):  
Kayte A. Jenkin ◽  
Peijian He ◽  
C. Chris Yun
Keyword(s):  
Epithelial Cells ◽  
Lysophosphatidic Acid ◽  
Direct Evidence ◽  
Intestinal Epithelial Cells ◽  
Regulatory Role ◽  
Intestinal Epithelial ◽  
Fluid Absorption ◽  
Wild Type

Lysophosphatidic acid (LPA) is a bioactive lipid molecule, which regulates a broad range of pathophysiological processes. Recent studies have demonstrated that LPA modulates electrolyte flux in the intestine, and its potential as an antidiarrheal agent has been suggested. Of six LPA receptors, LPA5 is highly expressed in the intestine. Recent studies by our group have demonstrated activation of Na+/H+ exchanger 3 (NHE3) by LPA5. However, much of what has been elucidated was achieved using colonic cell lines that were transfected to express LPA5. In the current study, we engineered a mouse that lacks LPA5 in intestinal epithelial cells, Lpar5ΔIEC, and investigated the role of LPA5 in NHE3 regulation and fluid absorption in vivo. The intestine of Lpar5ΔIEC mice appeared morphologically normal, and the stool frequency and fecal water content were unchanged compared with wild-type mice. Basal rates of NHE3 activity and fluid absorption and total NHE3 expression were not changed in Lpar5ΔIEC mice. However, LPA did not activate NHE3 activity or fluid absorption in Lpar5ΔIEC mice, providing direct evidence for the regulatory role of LPA5. NHE3 activation involves trafficking of NHE3 from the terminal web to microvilli, and this mobilization of NHE3 by LPA was abolished in Lpar5ΔIEC mice. Dysregulation of NHE3 was specific to LPA, and insulin and cholera toxin were able to stimulate and inhibit NHE3, respectively, in both wild-type and Lpar5ΔIEC mice. The current study for the first time demonstrates the necessity of LPA5 in LPA-mediated stimulation of NHE3 in vivo. NEW & NOTEWORTHY This study is the first to assess the role of LPA5 in NHE3 regulation and fluid absorption in vivo using a mouse that lacks LPA5 in intestinal epithelial cells, Lpar5ΔIEC. Basal rates of NHE3 activity and fluid absorption, and total NHE3 expression were not changed in Lpar5ΔIEC mice. However, LPA did not activate NHE3 activity or fluid absorption in Lpar5ΔIEC mice, providing direct evidence for the regulatory role of LPA5.

scholarly journals Characterization of the Role of the Divalent Metal Ion-Dependent Transcriptional Repressor MntR in the Virulence of Staphylococcus aureus

2003 ◽  
Vol 71 (5) ◽  
pp. 2584-2590 ◽  
Author(s):  
Masaru Ando ◽  
Yukari C. Manabe ◽  
Paul J. Converse ◽  
Eishi Miyazaki ◽  
Robert Harrison ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Metal Ion ◽  
Mouse Skin ◽  
Wild Type ◽  
Divalent Metal Ion ◽  
Gel Shift Assay ◽  
Wild Type Control

ABSTRACT DtxR-type metal ion-dependent repressors, present in many bacterial pathogens, may regulate expression of virulence genes such as that encoding diphtheria toxin. SirR, a DtxR homologue initially identified in Staphylococcus epidermidis, governs the expression of the adjacent sitABC operon encoding a putative metal ion ABC transporter system. We identified a sirR homologue, mntR, in Staphylococcus aureus and demonstrated by gel shift assay that the corynebacterial repressor DtxR binds to the S. aureus mntABC operator in the presence of Fe2+ or Mn2+. Since a mutant DtxR, DtxR(E175K), functions as an iron-independent hyperrepressor in certain settings, we constructed a heterodiploid S. aureus strain expressing dtxR(E175K) from the native mntR promoter. Transcription of the S. aureus mntABC operon was repressed in the presence of Fe2+ or Mn2+ in wild-type and heterodiploid S. aureus strains. Under metal ion-limiting conditions, mntABC transcription was reduced but not abolished in S. aureus isolates expressing dtxR(E175K) compared with an isogenic control, suggesting that DtxR(E175K) binds the S. aureus MntR box in vivo. Under all conditions tested, mntABC transcription in the dtxR(E175K)-expressing strain was reduced relative to the isogenic control, indicating that DtxR(E175K) function was constitutively active. In the mouse skin abscess model, dtxR(E175K)-expressing S. aureus recombinants showed significantly reduced CFU levels compared with the isogenic wild-type control. We conclude that the S. aureus MntR box is recognized by corynebacterial DtxR proteins and thus belongs to the DtxR family of metal-dependent operator sites. Moreover, constitutive repression by DtxR(E175K) reduces the virulence of S. aureus in the mouse skin abscess model.

scholarly journals Monocarboxylate Transporter 6-Mediated Interactions with Prostaglandin F2α: In Vitro and In Vivo Evidence Utilizing a Knockout Mouse Model

Pharmaceutics ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 201
Author(s):  
Robert S. Jones ◽  
Mark D. Parker ◽  
Marilyn E. Morris
Keyword(s):  
Prostaglandin F2α ◽  
Monocarboxylate Transporter ◽  
Drug Transporter ◽  
Wild Type ◽  
Knockout Mouse Model ◽  
Overnight Fasting ◽  
Potential Regulatory Role

Monocarboxylate transporter 6 (MCT6; SLC16A5) is a recently studied drug transporter that currently has no annotated endogenous function. Currently, only a handful of compounds have been characterized as substrates for MCT6 (e.g., bumetanide, nateglinide, probenecid, and prostaglandin F2α (PGF2α)). The objective of our research was to characterize the MCT6-specific transporter kinetic parameters and MCT6-specific in vitro and in vivo interactions of PGF2α. Murine and human MCT6-mediated transport of PGF2α was assessed in MCT6-transfected oocytes. Additionally, endogenous PGF2α and a primary PGF2α metabolite (PGFM) were measured in plasma and urine in Mct6 knockout (Mct6−/−) and wild-type (Mct6+/+) mice. Results demonstrated that the affinity was approximately 40.1 and 246 µM respectively, for mouse and human, at pH 7.4. In vivo, plasma PGF2α concentrations in Mct6−/− mice were significantly decreased, compared to Mct6+/+ mice (3.3-fold). Mct6-/- mice demonstrated a significant increase in urinary PGF2α concentrations (1.7-fold). A similar trend was observed with plasma PGFM concentrations. However, overnight fasting resulted in significantly increased plasma PGF2α concentrations, suggesting a diet-dependent role of Mct6 regulation on the homeostasis of systemic PGF2α. Overall, these results are the first to suggest the potential regulatory role of MCT6 in PGF2α homeostasis, and potentially other PGs, in distribution and metabolism.

scholarly journals Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

2021 ◽  
Vol 11 (15) ◽  
pp. 6865
Author(s):  
Eun Seon Lee ◽  
Joung Hun Park ◽  
Seong Dong Wi ◽  
Ho Byoung Chae ◽  
Seol Ki Paeng ◽  
...  
Keyword(s):  
Cold Stress ◽  
Wild Type ◽  
Rna Chaperone ◽  
E Coli ◽  
Cold Sensitive ◽  
Thioredoxin H ◽  
Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

scholarly journals Essential role of glucose transporter GLUT3 for post-implantation embryonic development

2008 ◽  
Vol 200 (1) ◽  
pp. 23-33 ◽  
Author(s):  
S Schmidt ◽  
A Hommel ◽  
V Gawlik ◽  
R Augustin ◽  
N Junicke ◽  
...  
Keyword(s):  
Essential Role ◽  
Glucose Transporter ◽  
Growth Arrest ◽  
Complete Loss ◽  
Transporter Gene ◽  
Wild Type ◽  
Post Implantation ◽  
Time Point

Deletion of glucose transporter geneSlc2a3(GLUT3) has previously been reported to result in embryonic lethality. Here, we define the exact time point of growth arrest and subsequent death of the embryo.Slc2a3−/−morulae and blastocysts developed normally, implantedin vivo, and formed egg-cylinder-stage embryos that appeared normal until day 6.0. At day 6.5, apoptosis was detected in the ectodermal cells ofSlc2a3−/−embryos resulting in severe disorganization and growth retardation at day 7.5 and complete loss of embryos at day 12.5. GLUT3 was detected in placental cone, in the visceral ectoderm and in the mesoderm of 7.5-day-old wild-type embryos. Our data indicate that GLUT3 is essential for the development of early post-implanted embryos.

scholarly journals A reassessment of the role of B7-1 expression in tumor rejection.

1995 ◽  
Vol 182 (5) ◽  
pp. 1415-1421 ◽  
Author(s):  
T C Wu ◽  
A Y Huang ◽  
E M Jaffee ◽  
H I Levitsky ◽  
D M Pardoll
Keyword(s):  
T Cells ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Tumor Rejection ◽  
Type B ◽  
Wild Type ◽  
Systemic Immunity ◽  
Murine Tumor

Introduction of the B7-1 gene into murine tumor cells can result in rejection of the B7-1 transductants and, in some cases, systemic immunity to subsequent challenge with the nontransduced tumor cells. These effects have been largely attributed to the function of B7-1 as a costimulator in directly activating tumor specific, major histocompatibility class I-restricted CD8+ T cells. We examined the role of B7-1 expression in the direct rejection as well as in the induction of systemic immunity to a nonimmunogenic murine tumor. B-16 melanoma cells with high levels of B7-1 expression did not grow in C57BL/6 recipient mice, while wild-type B-16 cells and cells with low B7-1 expression grew progressively within 21 d. In mixing experiments with B7-1hi and wild-type B-16 cells, tumors grew out in vivo even when a minority of cells were B7-1-. Furthermore, the occasional tumors that grew out after injection of 100% B-16 B7-1hi cells showed markedly decreased B7-1 expression. In vivo antibody depletions showed that NK1.1 and CD8+ T cells, but not CD4+ T cells, were essential for the in vivo rejection of tumors. Animals that rejected B-16 B7-1hi tumors did not develop enhanced systemic immunity against challenge with wild-type B-16 cells. These results suggest that a major role of B7-1 expression by tumors is to mediate direct recognition and killing by natural killer cells. With an intrinsically nonimmunogenic tumor, this direct killing does not lead to enhanced systemic immunity.

scholarly journals Characterization of TNF receptor subtype expression and signaling on pulmonary endothelial cells in mice

2011 ◽  
Vol 300 (5) ◽  
pp. L781-L789 ◽  
Author(s):  
Szabolcs Bertok ◽  
Michael R. Wilson ◽  
Anthony D. Dorr ◽  
Justina O. Dokpesi ◽  
Kieran P. O'Dea ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Adhesion Molecules ◽  
Surface Expression ◽  
Tnf Receptor ◽  
Wild Type ◽  
Tnf Receptors ◽  
Pulmonary Endothelial Cells ◽  
Microvascular Inflammation

TNF plays a crucial role in the pathogenesis of acute lung injury. However, the expression profile of its two receptors, p55 and p75, on pulmonary endothelium and their influence on TNF signaling during lung microvascular inflammation remain uncertain. Using flow cytometry, we characterized the expression profile of TNF receptors on the surface of freshly harvested pulmonary endothelial cells (PECs) from mice and found expression of both receptors with dominance of p55. To investigate the impact of stimulating individual TNF receptors, we treated wild-type and TNF receptor knockout mice with intravenous TNF and determined surface expression of adhesion molecules (E-selectin, VCAM-1, ICAM-1) on PECs by flow cytometry. TNF-induced upregulation of all adhesion molecules was substantially attenuated by absence of p55, whereas lack of p75 had a similar but smaller effect that varied between adhesion molecules. Selective blockade of individual TNF receptors by specific antibodies in wild-type primary PEC culture confirmed that the in vivo findings were due to direct effects of TNF receptor inhibition on endothelium and not other cells (e.g., circulating leukocytes). Finally, we found that PEC surface expression of p55 dramatically decreased in the early stages of endotoxemia following intravenous LPS, while no change in p75 expression was detected. These data demonstrate a crucial in vivo role of p55 and an auxiliary role of p75 in TNF-mediated adhesion molecule upregulation on PECs. It is possible that the importance of the individual receptors varies at different stages of pulmonary microvascular inflammation following changes in their relative expression.

scholarly journals Systems Modeling of the Role of Interleukin-21 in the Maintenance of Effector CD4+ T Cell Responses during Chronic Helicobacter pylori Infection

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Adria Carbo ◽  
Danyvid Olivares-Villagómez ◽  
Raquel Hontecillas ◽  
Josep Bassaganya-Riera ◽  
Rupesh Chaturvedi ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Helicobacter Pylori ◽  
T Cell ◽  
Wild Type ◽  
Content Type ◽  
Interleukin 21 ◽  
H Pylori ◽  
Deficient Mice

ABSTRACTThe development of gastritis duringHelicobacter pyloriinfection is dependent on an activated adaptive immune response orchestrated by T helper (Th) cells. However, the relative contributions of the Th1 and Th17 subsets to gastritis and control of infection are still under investigation. To investigate the role of interleukin-21 (IL-21) in the gastric mucosa duringH. pyloriinfection, we combined mathematical modeling of CD4+T cell differentiation within vivomechanistic studies. We infected IL-21-deficient and wild-type mice withH. pyloristrain SS1 and assessed colonization, gastric inflammation, cellular infiltration, and cytokine profiles. ChronicallyH. pylori-infected IL-21-deficient mice had higherH. pyloricolonization, significantly less gastritis, and reduced expression of proinflammatory cytokines and chemokines compared to these parameters in infected wild-type littermates. Thesein vivodata were used to calibrate anH. pyloriinfection-dependent, CD4+T cell-specific computational model, which then described the mechanism by which IL-21 activates the production of interferon gamma (IFN-γ) and IL-17 during chronicH. pyloriinfection. The model predicted activated expression of T-bet and RORγt and the phosphorylation of STAT3 and STAT1 and suggested a potential role of IL-21 in the modulation of IL-10. Driven by our modeling-derived predictions, we found reduced levels of CD4+splenocyte-specifictbx21androrcexpression, reduced phosphorylation of STAT1 and STAT3, and an increase in CD4+T cell-specific IL-10 expression inH. pylori-infected IL-21-deficient mice. Our results indicate that IL-21 regulates Th1 and Th17 effector responses during chronicH. pyloriinfection in a STAT1- and STAT3-dependent manner, therefore playing a major role controllingH. pyloriinfection and gastritis.IMPORTANCEHelicobacter pyloriis the dominant member of the gastric microbiota in more than 50% of the world’s population.H. pyloricolonization has been implicated in gastritis and gastric cancer, as infection withH. pyloriis the single most common risk factor for gastric cancer. Current data suggest that, in addition to bacterial virulence factors, the magnitude and types of immune responses influence the outcome of colonization and chronic infection. This study uses a combined computational and experimental approach to investigate how IL-21, a proinflammatory T cell-derived cytokine, maintains the chronic proinflammatory T cell immune response driving chronic gastritis duringH. pyloriinfection. This research will also provide insight into a myriad of other infectious and immune disorders in which IL-21 is increasingly recognized to play a central role. The use of IL-21-related therapies may provide treatment options for individuals chronically colonized withH. pylorias an alternative to aggressive antibiotics.

Heparan sulfate side chains have a critical role in the inhibitory effects of perlecan on vascular smooth muscle cell response to arterial injury

2014 ◽  
Vol 307 (3) ◽  
pp. H337-H345 ◽  
Author(s):  
Lara Gotha ◽  
Sang Yup Lim ◽  
Azriel B. Osherov ◽  
Rafael Wolff ◽  
Beiping Qiang ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Critical Role ◽  
Arterial Injury ◽  
Side Chains ◽  
Wild Type ◽  
Injury Response ◽  
Migratory Response

Perlecan is a proteoglycan composed of a 470-kDa core protein linked to three heparan sulfate (HS) glycosaminoglycan chains. The intact proteoglycan inhibits the smooth muscle cell (SMC) response to vascular injury. Hspg2Δ3/Δ3 (MΔ3/Δ3) mice produce a mutant perlecan lacking the HS side chains. The objective of this study was to determine differences between these two types of perlecan in modifying SMC activities to the arterial injury response, in order to define the specific role of the HS side chains. In vitro proliferative and migratory activities were compared in SMC isolated from MΔ3/Δ3 and wild-type mice. Proliferation of MΔ3/Δ3 SMC was 1.5× greater than in wild type ( P < 0.001), increased by addition of growth factors, and showed a 42% greater migratory response than wild-type cells to PDGF-BB ( P < 0.001). In MΔ3/Δ3 SMC adhesion to fibronectin, and collagen types I and IV was significantly greater than wild type. Addition of DRL-12582, an inducer of perlecan expression, decreased proliferation and migratory response to PDGF-BB stimulation in wild-type SMC compared with MΔ3/Δ3. In an in vivo carotid artery wire injury model, the medial thickness, medial area/lumen ratio, and macrophage infiltration were significantly increased in the MΔ3/Δ3 mice, indicating a prominent role of the HS side chain in limiting vascular injury response. Mutant perlecan that lacks HS side chains had a marked reduction in the inhibition of in vitro SMC function and the in vivo arterial response to injury, indicating the critical role of HS side chains in perlecan function in the vessel wall.

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