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1.
Previous work has shown that the maximum fluorescence yield from PS 2 of Synechococcus PCC 7942 occurs when the cells are at the CO 2 compensation point. The addition of inorganic carbon (C i), as CO 2 or HCO 3
–, causes a lowering of the fluorescence yield due to both photochemical (q p) and non-photochemical (q N) quenching. In this paper, we characterize the q N that is induced by C i addition to cells grown at high light intensities (500 mol photons m –2 s –1). The C i-induced q N was considerably greater in these cells than in cells grown at low light intensities (50 mol photons m –2 s –1), when assayed at a white light (WL) intensity of 250 mol photons m –2 s –1. In high-light grown cells we measured q N values as high as 70%, while in low-light grown cells the q N was about 16%. The q N was relieved when cells regained the CO 2 compensation point, when cells were illuminated by supplemental far-red light (FRL) absorbed mainly by PS 1, or when cells were illuminated with increased WL intensities. These characteristics indicate that the q N was not a form of energy quenching (q E). Supplemental FRL illumination caused significant enhancement of photosynthetic O 2 evolution that could be correlated with the changes in q p and q N. The increases in q p induced by C i addition represent increases in the effective quantum yield of PS 2 due to increased levels of oxidized Q A. The increase in q N induced by C i represents a decrease in PS 2 activity related to decreases in the potential quantum yield. The lack of diagnostic changes in the 77 K fluorescence emission spectrum argue against q N being related to classical state transitions, in which the decrease in potential quantum yield of PS 2 is due either to a decrease in absorption cross-section or by increased spill-over of excitation energy to PS 1. Both the C i-induced q p ( t
0.5<0.5 s) and q N ( t
0.51.6 s) were rapidly relieved by the addition of DCMU. The two time constants give further support for two separate quenching mechanisms. We have thus characterized a novel form of q N in cyanobacteria, not related to state transitions or energy quenching, which is induced by the addition of C i to cells at the CO 2-compensation point.Abbreviations BTP-
1,3-bis[tris(hydroxymethyl)-methylaminopropane]
- Chl-
chlorophyll
- C i-
inorganic carbon (CO 2+HCO 3
–+CO 3
2–)
- DCMU-
3-(3,4-dichlorophenyl)-, 1-dimethylurea)
- F-
chlorophyll fluorescence measured at any time in the absence of a saturating flash
- F o-
chlorophyll fluorescence with only the weak modulated measuring beam on
- F M'-
chlorophyll fluorescence during a saturating flash
- F M-
maximum chlorophyll fluorescence, measured in the presence of WL and FRL at the CO 2-compensation point or in the presence of DCMU
- F V-
variable fluorescence (= F M'–F 0)
- FRL-
supplemental illumination with far red light
- MB-
modulated measuring beam of the PAM fluorometer
- MV-
methyl viologen
- PAM-
pulse amplitude modulation
- PFD-
incident photon flux density
- PS 1, 2-
Photosystems 1 and 2
- Q A-
primary electron-accepting plastoquinione of PS 2
- q N-
non-photochemical quenching of chlorophyll fluorescence
- q p-
photochemical quenching of chlorophyll fluorescence; rubisco-ribulose bisphosphate carboxylase/oxygenase
- SF-
saturating flash (600 ms duration)
- WL-
white light illumination 相似文献
2.
Intestinal homeostasis and the coordinated actions of digestion, absorption and excretion are tightly regulated by a number of gastrointestinal hormones. Most of them exert their actions through G-protein-coupled receptors. Recently, we showed that the absence of Gα q/Gα 11 signaling impaired the maturation of Paneth cells, induced their differentiation toward goblet cells, and affected the regeneration of the colonic mucosa in an experimental model of colitis. Although an immunohistochemical study showed that Gα q/Gα 11 were highly expressed in enterocytes, it seemed that enterocytes were not affected in Int-G q/G 11 double knock-out intestine. Thus, we used an intestinal epithelial cell line to examine the role of signaling through Gα q/Gα 11 in enterocytes and manipulated the expression level of Gα q and/or Gα 11. The proliferation was inhibited in IEC-6 cells that overexpressed Gα q/Gα 11 and enhanced in IEC-6 cells in which Gα q/Gα 11 was downregulated. The expression of T-cell factor 1 was increased according to the overexpression of Gα q/Gα 11. The expression of Notch1 intracellular cytoplasmic domain was decreased by the overexpression of Gα q/Gα 11 and increased by the downregulation of Gα q/Gα 11. The relative mRNA expression of Muc2, a goblet cell marker, was elevated in a Gα q/Gα 11 knock-down experiment. Our findings suggest that Gα q/Gα 11-mediated signaling inhibits proliferation and may support a physiological function, such as absorption or secretion, in terminally differentiated enterocytes. 相似文献
5.
Phosphatidic acid (PA) is interactive with Gα q-linked agonists to stimulate GPCR signaling via phospholipase C-β 1 (PLC-β 1). Phorbol 12-myristate 13-acetate (PMA) increases cellular levels of PA and phospholipase D activity (PLD). This study evaluated whether PMA can stimulate PLC-β 1 activity via PA, independent of GPCR input in transfected COS 7 cells. PMA alone had little effect on PLC activity in cells co-transfected with PLC-β 1 and Gα q. Activated Gα q, induced by co-transfecting muscarinic cholinergic receptor (m1R), was necessary for stimulation of PLC-β 1 activity by PMA. Stimulation by PMA was dependent on the PA-regulatory motif of PLC-β 1 implicating PA in this mechanism. PLD1 knockdown by antisense decreased responsiveness of PLC-β 1 to both PMA and carbachol. PA alone thus has little effect on PLC-β 1 activity, but PA and PLD1 synergize with activated Gα q to stimulate PLC-β 1 signaling. Coordinate interaction with activated Gα q may serve as an important mechanism to fine tune response to ligands while preventing spurious initiation of PLC-β signaling by PA in cells. 相似文献
6.
Phosphatidic acid (PA), generated downstream of monomeric Rho GTPases via phospholipase D (PLD) and additionally by diacylglycerol kinases (DGK), both stimulates phospholipase C-β 1 (PLC-β 1) and potentiates stimulation of PLC-β 1 activity by Gα q in vitro. PA is a potential candidate for integrating signaling by monomeric and heterotrimeric G proteins to regulate signal output by G protein coupled receptors (GPCR), and we have sought to understand the mechanisms involved. We previously identified the region spanning residues 944–957, lying within the PLC-β 1 C-terminus αA helix and flexible loop of the Gα q binding domain, as required for stimulation of lipase activity by PA in vitro. Regulation by PA does not require residues essential for stimulation by Gα q or GTPase activating activity. The present studies evaluated shorter alanine/glycine replacement mutants and finally point mutations to identify Tyr 952 and Ile 955 as key determinants for regulation by PA, assessed by both in vitro enzymatic and cell-based co-transfection assays. Replacement of Tyr 952 and Ile 955, PLC-β 1 (Y952G/I955G), results in an 85% loss in stimulation by PA relative to WT-PLC-β 1 in vitro. COS 7 cells co-transfected with PLC-β 1 (Y952G/I955G) demonstrate a 10-fold increase in the EC 50 for stimulation and a 60% decrease in maximum stimulation by carbachol via Gα q linked m1 muscarinic receptors, relative to cells co-transfected with WT-PLC-β 1 but otherwise similar conditions. Residues required for regulation by PA are not essential for stimulation by G protein subunits. WT-PLC-β 1 and PLC-β 1(Y952G/I955G) activity is increased comparably by co-transfection with Gα q and neither is markedly affected by co-transfection with Gβ 1γ 2. Inhibiting PLD-generated PA production by 1-butanol has little effect on maximum stimulation, but shifts the EC 50 for agonist stimulation of WT-PLC-β 1 by 10-fold, producing a phenotype similar to PLC-β 1 (Y952G/I955G) with respect to agonist potency. 1-Butanol is without effect on carbachol stimulated PLC activity in cells co-transfected with either PLC-β 1(Y952G/I955G) or on endogenous PLC activity, indicating that regulation by PA requires direct interaction with the PLC-β 1 PA-binding region. These data show that endogenous PA regulates signal output by Gα q-linked GPCRs in transfected cells directly through PLC-β 1. Gα q and PA may co-ordinate to regulate signaling. Regulation by PA may constitute part of a mechanism that routes receptor signaling to specific PLC isoforms. 相似文献
7.
Sphingosine-1-phosphate (S1P), formed by sphingosine kinases (SphKs), regulates cellular proliferation and migration by acting as an agonist at specific receptors or intracellularly. Since S1P's effects are probably dependent on subcellular localization of its formation and degradation, we have studied the influence of G protein-coupled receptors on the localization of SphK1. Activation of G q-coupled receptors induced a profound, rapid (half-life 3–5 s) and long-lasting (> 2 h) translocation of SphK1 to the plasma membrane. This was mimicked by expression of constitutively active G protein α-subunits specifically of the G q family. Classical G q signalling pathways, or phosphorylation at Ser 225, phospholipase D and Ca 2+/calmodulin were not involved in M 3 receptor-induced SphK1 translocation in HEK-293 cells. Translocation was associated with S1P receptor internalization, which was dependent on catalytic activity of SphK1 and S1P receptor binding and thus resulted from S1P receptor cross-activation. It is concluded that SphK1 is an important effector of G q-coupled receptors, linking them via cross-activation of S1P receptors to G i and G 12/13 signalling pathways. 相似文献
8.
Sphingosine kinases (SphK) catalyse the formation of sphingosine-1-phosphate (S1P) and play important roles in the cardiovascular, nervous and immune systems. We have shown before that G q-coupled receptors induce a rapid and long-lasting translocation of SphK1 to the plasma membrane and cross-activation of S1P receptors. Here, we further addressed G q regulation of SphK1 by analysing the influence of the WD40 repeat protein, WDR36. WDR36 has been described as a scaffold tethering Gα q to phospholipase C (PLC)-β and the thromboxane A 2 receptor-β (TPβ receptor). Overexpression of WDR36 in HEK-293 cells enhanced TPβ receptor-induced inositol phosphate production, as reported (Cartier et al. 2011), but significantly attenuated inositol phosphate production induced by muscarinic M 3 and bradykinin B 2 receptors. In agreement with its effect on PLCβ, WDR36 augmented TPβ receptor-induced [Ca 2+] i increases. Surprisingly, WDR36 also augmented M 3 receptor-induced [Ca 2+] i increases, which was due to increased Ca 2+ mobilization while the Ca 2+ content of thapsigargin-sensitive stores remained unaltered. Interestingly, overexpression of WDR36 significantly delayed SphK1 translocation by G q-coupled M 3, B 2 and H 1 receptors in HEK-293 cells, while TPβ receptor-induced SphK1 translocation was generally slow and not altered by WDR36 in these cells. Finally, in C2C12 myoblasts, overexpression of WDR36 delayed SphK1 translocation induced by B 2 receptors. It is concluded that WDR36 reduces signalling of G q-coupled receptors other than TPβ towards PLC and SphK1, most likely by scavenging Gα q and PLCβ. Our results support a role of WDR36 in orchestration of G q signalling complexes, and might help to functionally unravel its genetic association with asthma and allergy. 相似文献
9.
Many bacterial toxins covalently modify components of eukaryotic signalling pathways in a highly specific manner, and can be used as powerful tools to decipher the function of their molecular target(s). The Pasteurella multocida toxin (PMT) mediates its cellular effects through the activation of members of three of the four heterotrimeric G-protein families, G q, G 12 and G i. PMT has been shown by others to lead to the deamidation of recombinant Gα i at Gln-205 to inhibit its intrinsic GTPase activity. We have investigated modification of native Gα subunits mediated by PMT in Swiss 3T3 cells using 2-D gel electrophoresis and antibody detection. An acidic change in the isoelectric point was observed for the Gα subunit of the G q and G i families following PMT treatment of Swiss 3T3 cells, which is consistent with the deamidation of these Gα subunits. Surprisingly, PMT also induced a similar modification of Gα 11, a member of the G q family of G-proteins that is not activated by PMT. Furthermore, an alkaline change in the isoelectric point of Gα 13 was observed following PMT treatment of cells, suggesting differential modification of this Gα subunit by PMT. G s was not affected by PMT treatment. Prolonged treatment with PMT led to a reduction in membrane-associated Gα i, but not Gα q. We also show that PMT inhibits the GTPase activity of G q. 相似文献
10.
Caveolae are membrane invaginations that can sequester various signaling proteins. Caveolae have been shown to provide mechanical strength to cells by flattening to accommodate increased volume when cells are subjected to hypo-osmotic stress. We have previously found that caveolin, the main structural component of caveolae, specifically binds Gα q and stabilizes its activation state resulting in an enhanced Ca 2+ signal upon activation. Here, we show that osmotic stress caused by decreasing the osmolarity in half reversibly changes the configuration of caveolae without releasing a significant portion of caveolin molecules. This change in configuration due to flattening leads to a loss in Cav1-Gα q association. This loss in Gα q/Cav1 association due to osmotic stress results in a significant reduction of Gα q/phospholipase Cβ-mediated Ca 2+ signals. This reduced Ca 2+ response is also seen when caveolae are reduced by treatment with siRNA(Cav1) or by dissolving them by methyl-β-cyclodextran. No change in Ca 2+ release with osmotic swelling can be seen when growth factor pathways are activated. Taken together, these results connect the mechanical deformation of caveolae to Gα q-mediated Ca 2+ signals. 相似文献
11.
In an attempt to determine the relationship between the Epstein–Barr virus nuclear antigen-1 (EBNA-1) expression level and specific foreign protein productivity ( qp), EBNA-1-amplifed HEK293 cells, which achieved a higher EBNA-1 expression level than that achieved by HEK293E cells, were established using dihydrofolate reductase ( dhfr)-mediated gene amplification. Compared with a control culture in a null pool, Fc-fusion protein production by transient transfection in the EBNA-1-amplified pool showed a significant improvement. qp was linearly correlated with the EBNA-1 expression level in the transient transfection of EBNA-1-amplified clones, as indicated by the correlation coefficient (R 2 = 0.7407). The Fc-fusion protein production and qp in a transient gene expression-based culture with EBNA-1-amplified HEK293 cells, E-amp-68, were approximately 2.0 and 3.2 times, respectively, higher than those in a culture with HEK293E cells. The increase in qp by EBNA-1 amplification mainly resulted from an enhancement in the amount of replicated DNA and level of mRNA expression but not an improved transfection efficiency. Taken together, it was found that EBNA-1 amplification could improve the therapeutic protein production in an HEK293 cell-based transient gene expression system. 相似文献
13.
A variety of approaches has been published to enhance specific productivity ( qp) of recombinant Chinese hamster ovary (CHO) cells. Changes in culture conditions, e. g. temperature shifts, sodium butyrate treatment and hyperosmolality, were shown to improve q p. To contribute to a better understanding of the correlation between hyperosmolality and enhanced qp, we analyzed cellular kinetics and intracellular adenine nucleotide pools during osmotic shift periods. Known phenotypes like increased formation rates for lactate and product (anti‐IL‐8 antibody; qlactate, qp) as well as increased cell specific uptake rate for glucose ( qglucose) were found—besides inhibition of cell growth and G1‐arrest occurred during batch cultivations with osmotic shift. The analysis of intracellular AXP pools revealed enlarged ATP amounts for cells as response to hyperosmolality while energy charges remained unchanged. Enhanced ATP‐pools coincided with severely increased ATP formation rates ( qATP) which outweighed by far the putative requirements attributed to regulatory volume increase. Therefore elevated qATP mirrored an increased cellular demand for energy while experiencing hyperosmotic shift. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1212–1216, 2015 相似文献
14.
Much research has been conducted about different types of fermentation at high temperature, but only a few of them have studied cell viability changes during high-temperature fermentation. In this study, Acetobacter senegalensis, a thermo-tolerant strain, was used for gluconic acid production at 38 °C. The influences of different carbon sources and physicochemical conditions on cell viability and the resuscitation of viable but nonculturable (VBNC) cells formed during fermentation were studied. Based on the obtained results, A. senegalensis could oxidize 95 g l− 1 glucose to gluconate at 38 °C (pH 5.5, yield 83%). However, despite the availability of carbon and nitrogen sources, the specific rates of glucose consumption (qs) and gluconate production (qp) reduced progressively. Interestingly, gradual qs and qp reduction coincided with gradual decrease in cellular dehydrogenase activity, cell envelope integrity, and cell culturability as well as with the formation of VBNC cells. Entry of cells into VBNC state during stationary phase partly stemmed from high fermentation temperature and long-term oxidation of glucose, because just about 48% of VBNC cells formed during stationary phase were resuscitated by supplementing the culture medium with an alternative favorite carbon source (low concentration of ethanol) and/or reducing incubation temperature to 30 °C. This indicates that ethanol, as a favorable carbon source, supports the repair of stressed cells. Since formation of VBNC cells is often inevitable during high-temperature fermentation, using an alternative carbon source together with changing physicochemical conditions may enable the resuscitation of VBNC cells and their use for several production cycles. 相似文献
15.
In order to study G q-tubulin interaction in the cytosol, GH 3 and AtT-20 cells (stably expressing TRH receptor) were transiently transfected with G qα cDNA. Forty-eight hours after transfection, thyrotropin-releasing hormone (TRH)-stimulated prolactin (PRL) secretion by G qα-transfected GH 3 cells increased by 90% compared to mock-transfected cells. In addition, using immunocytochemistry it was observed that G qα-specific staining was much more prominent in G qα-transfected GH 3 and AtT-20 cells (also transfected with G qα) compared to mock-transfected cells. Thus, transfection resulted in successful overexpression of functional G qα. Forty-eight hours after transfection, cells were processed to obtain soluble and polymerized tubulin fractions. Tubulin levels were determined in these fractions by immunoblotting using polyclonal anti-tubulin antibodies. Compared to mock-transfected cells soluble tubulin levels decreased in G qα-transfected GH 1 and AtT-20 cells, by 33 and 52%, respectively. Moreover, compared to mock-transfected cells a 50% reduction in the ratio (an index of the flux between tubulin pools) of soluble and polymerized tubulin levels was observed in G qα-transfected GH 3 and AtT-20 cells. To determine whether these effects on tubulin were mediated by G q directly, we examined the influence of purified G q on tubulin polymerization. G q (0.5 μM) inhibited polymerization of crude tubulin (present in GH 3 cell cytosol) by 53%. In contrast to its effects on GH 3 cell cytosol tubulin, G q stimulated purified tubulin polymerization by 160%. These results suggest that G q modulates the polymerization and depolymerization cycles of tubulin and that this modulation is in turn influenced by other unknown cellular components. © 1996 Wiley-Liss, Inc. 相似文献
16.
Many G q‐coupled receptors mediate mitogenic signals by stimulating extracellular signal‐regulated protein kinases (ERKs) that are typically regulated by the small GTPase Ras. Recent studies have revealed that members of the Gα q family may possess the ability to activate Ras/ERK by interacting with the adaptor protein tetratricopeptide repeat 1 (TPR1). Within the Gα q family, the highly promiscuous Gα 14 can relay signals from numerous receptors. Here, we examined if Gα 14 interacts with TPR1 to stimulate Ras signaling pathways. Expression of the constitutively active Gα 14QL mutant in HEK293 cells led to the formation of GTP‐bound Ras as well as increased phosphorylations of downstream signaling molecules including ERK and IκB kinase. Stimulation of endogenous G 14‐coupled somatostatin type 2 and α 2‐adrenergic receptors produced similar responses in human hepatocellular HepG2 carcinoma cells. Co‐immunoprecipitation assays using HEK293 cells demonstrated a stronger association of TPR1 for Gα 14QL than Gα 14, suggesting that TPR1 preferentially binds to the GTP‐bound form of Gα 14. Activated Gα 14 also interacted with the Ras guanine nucleotide exchange factors SOS1 and SOS2. Expression of a dominant negative mutant of TPR1 or siRNA‐mediated knockdown of TPR1 effectively abolished the ability of Gα 14 to induce Ras signaling in native HepG2 or transfected HEK293 cells. Although expression of the dominant negative mutant of TPR1 suppressed Gα 14QL‐induced phosphorylations of ERK and IκB kinase, it did not affect Gα 14QL‐induced stimulation of phospholipase Cβ or c‐Jun N‐terminal kinase. Our results suggest that TPR1 is required for Gα 14 to stimulate Ras‐dependent signaling pathways, but not for the propagation of signals along Ras‐independent pathways. J. Cell. Biochem. 113: 3486–3497, 2012. © 2012 Wiley Periodicals, Inc. 相似文献
17.
During signal transduction, the G protein, Gα q, binds and activates phospholipase C-β isozymes. Several diseases have been shown to manifest upon constitutively activating mutation of Gα q, such as uveal melanoma. Therefore, methods are needed to directly inhibit Gα q. Previously, we demonstrated that a peptide derived from a helix-turn-helix (HTH) region of PLC-β3 (residues 852–878) binds Gα q with low micromolar affinity and inhibits Gα q by competing with full-length PLC-β isozymes for binding. Since the HTH peptide is unstructured in the absence of Gα q, we hypothesized that embedding the HTH in a folded protein might stabilize the binding-competent conformation and further improve the potency of inhibition. Using the molecular modeling software Rosetta, we searched the Protein Data Bank for proteins with similar HTH structures near their surface. The candidate proteins were computationally docked against Gα q, and their surfaces were redesigned to stabilize this interaction. We then used yeast surface display to affinity mature the designs. The most potent design bound Gα q/i with high affinity in vitro (K D = 18 nM) and inhibited activation of PLC-β isozymes in HEK293 cells. We anticipate that our genetically encoded inhibitor will help interrogate the role of Gα q in healthy and disease model systems. Our work demonstrates that grafting interaction motifs into folded proteins is a powerful approach for generating inhibitors of protein–protein interactions. 相似文献
19.
A series of novel multipotent 2-piperidone derivatives were designed, synthesized and biologically evaluated as chemical agents for the treatment of Alzheimer’s disease (AD). The results showed that most of the target compounds displayed significant potency to inhibit Aβ 1–42 self-aggregation. Among them, compound 7q exhibited the best inhibition of Aβ 1–42 self-aggregation (59.11% at 20 μM) in a concentration-dependent manner. Additionally, the compounds 6b, 7p and 7q as representatives were found to present anti-inflammation properties in lipopolysaccharide (LPS)-induced microglial BV-2 cells. They could effectively suppress the production of pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6. Meanwhile, compound 7q could prevent the neuronal cell SH-SY5Y death by LPS-stimulated microglia cell activation mediated neurotoxicity. The molecular modeling studies demonstrated that compounds matched the pharmacophore well and had good predicted physicochemical properties and estimated IC 50 values. Moreover, compound 7q exerted a good binding to the active site of myeloid differentiation factor 88 (MyD88) through the docking analysis and could interfere with its homodimerization or heterodimerization. Consequently, these compounds emerged as promising candidates for further development of novel multifunctional agents for AD treatment. 相似文献
20.
Summary Biosorption of heavy metals by gram-positive, non-pathogenic and non-toxicogenic Paenibacillus polymyxa P13 was evaluated. Copper was chosen as a model element because it is a pollutant originated from several industries. An
EPS (exopolysaccharide)-producing phenotype exhibited significant Cu(II) biosorption capacity. Under optimal assay conditions
(pH 6 and 25 °C), the adsorption isotherm for Cu(II) in aqueous solutions obeyed the Langmuir model. A high q value (biosorption capacity) was observed with whole cells ( qmax=112 mgCu g −1). EPS production was associated with hyperosmotic stress by high salt (1 M NaCl), which led to a significant increase in
the biosorption capacity of whole cells ( qmax=150 mgCu g −1). Biosorption capacity for Cu(II) of the purified EPS was investigated. The maximum biosorption value ( q) of 1602 mg g −1 observed with purified EPS at 0.1 mg ml −1 was particularly promising for use in field applications. 相似文献
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