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1.
We have investigated the possibility that adenylyl cyclase (AC) activity and membrane protein levels of the -subunits of the stimulatory and inhibitory G-proteins of AC (Gs and Gi−2) in cultured prolactin-producing rat pituitary adenoma cells (GH3 cells) are modulated by phospholipase C (PLC)-generated second messengers. Pretreatment of cells (6–48 h) with ionomycin (1 μM) or 1-oleoyl-2-acetylglycerol (OAG; 1μM) showed that ionomycin regulated Gs levels in a time-dependent, biphasic manner; a two-fold increase followed a 40% initial reduction, while OAG lowered Gs levels by more than 50% at all time-points. Gi−2 levels remained unchanged by both pretreatments. OAG, but not ionomycin, increased basal AC activity without increasing enzyme protein levels. Alterations in AC responsiveness to peptide hormones (e.g. thyroliberin and vasoactive intestinal peptide) correlated to membrane Gs protein -subunit content. These results demonstrate the involvement of G-protein translation regulation as one mechanism of ‘cross-talk’ between the PLC- and AC-dependent signalling pathways.  相似文献   

2.
Dimethyl sulfoxide (DMSO)-induced erythroid differentiation of Friend mouse erythroleukemia (MEL) cells is associated with a marked transient modulation of catecholamine sensitivity. Within 24 h after induction and well before the onset of hemoglobin synthesis, we observed a 3-fold increase in β-receptor density and a more than 10-fold increase in receptor-coupled cAMP formation. During the following 4 days, in parallel with the development of normoblast-like cells, receptor numbers returned to preinduction levels while catecholamine-dependent cAMP formation remained significantly elevated. Simultaneously, the apparent potency of the β-adrenoceptor agonist isoprenaline increased 10-fold. Improved receptor—cyclase coupling is probably due to a major shift in the expression of Gi and Gs regulatory proteins. Bacterial toxin-mediated ADP-ribosylation of membrane proteins suggests that the dominating species in native cells is Gi (G:G = 1:7). By contrast, Gs predominates in differentiated cells (G:G = 1.8:1). Receptor-independent forskolin-stimulated cAMP formation showed a pronounced, albeit transient, decrease during differentiation. We suggest that these changes in cellular cAMP responses may be important for transient positive or negative cooperative interactions between hormones and growth factors in the course of erythroid cell development.  相似文献   

3.
Heterotrimeric G proteins relay signals from G protein-coupled receptors (GPCRs) to the interior of the cell. The signaling cascades induced by G protein activation control a wide range of cellular processes. The α subunit is believed to determine which G protein couples to each GPCR, and is the primary determinant of the type of signal transmitted. Several members of the Gα family have been expressed in active form in Escherichia coli. However, production levels of these proteins are limited: in most cases only 10% of total Gα protein expressed is active; the rest accumulates in inclusion bodies. Although G has been readily expressed in soluble form (to 10 mg/L), other α subunits are minimally soluble, and many are exclusively expressed to inclusion bodies. Previous efforts to solubilize and refold Gα from inclusion bodies have not been successful. Here we did a thorough study of the characteristics of Gα subunits (human Giα(1), human Gsα(short), human G11α and human Gtα(cone)), solubilized and purified from inclusion bodies. We find that we can obtain soluble protein both by on-column and rapid-dilution techniques. Comparison to native, soluble G expressed from E. coli showed that although the refolded Gα subunits were soluble and retained partial α-helicity characteristic of the native, folded Gα subunit, they did not bind GDP or GTP as effectively as native protein. We conclude that the refolded G protein has a native-like secondary structure, but is predominately in a molten globular state.  相似文献   

4.
Extra-large stimulatory Gα (XLαs) is a large variant of G protein αs subunit (Gαs) that uses an alternative promoter and thus differs from Gαs at the first exon. XLαs activation by G protein–coupled receptors mediates cAMP generation, similarly to Gαs; however, Gαs and XLαs have been shown to have distinct cellular and physiological functions. For example, previous work suggests that XLαs can stimulate inositol phosphate production in renal proximal tubules and thereby regulate serum phosphate levels. In this study, we show that XLαs directly and specifically stimulates a specific isoform of phospholipase Cβ (PLCβ), PLCβ4, both in transfected cells and with purified protein components. We demonstrate that neither the ability of XLαs to activate cAMP generation nor the canonical G protein switch II regions are required for PLCβ stimulation. Furthermore, this activation is nucleotide independent but is inhibited by Gβγ, suggesting a mechanism of activation that relies on Gβγ subunit dissociation. Surprisingly, our results indicate that enhanced membrane targeting of XLαs relative to Gαs confers the ability to activate PLCβ4. We also show that PLCβ4 is required for isoproterenol-induced inositol phosphate accumulation in osteocyte-like Ocy454 cells. Taken together, we demonstrate a novel mechanism for activation of phosphoinositide turnover downstream of Gs-coupled receptors that may have a critical role in endocrine physiology.  相似文献   

5.
G proteins are heterotrimeric GTPases that play a key role in signal transduction. The α subunit of Gs bound to GTP is capable of activating adenylyl cyclase. The amino acid sequences derived from two X. laevis cDNA clones that apparently code for Gsα subunits are 92% identical to those found in the short form of human Gsα. Despite this high homology, the X. laevis Gsα clones expressed in vitro, yielded a protein that are not able to activate the adenylyl cyclase present in S49 cyc membranes in contrast with human Gsα similarly expressed. This finding suggested that the few amino acid substitutions found in the amphibian subunit are important in defining the functionality of the human Gsα. The construction of chimeras composed of different fractions of the cDNAs of the two species was adopted as an approach in determining the regions of the molecule important in its functionality in this assay. Four pairs of chimeras were constructed using reciprocal combinations of the cDNAs coding for human and Xenopus Gsα. These eight constructs were expressed in vitro and equivalent amounts of the resulting proteins were assayed in the activation of adenylyl cyclase with GTPγs and isoproterenol. The results obtained here clearly indicate that the Gα sequence that extends from amino acid 70 to 140, is important for the functionality of human Gsα in activating adenylyl cyclase.  相似文献   

6.
Nacre formation is an ideal model to study biomineralization processes. Although much has been done about biomineralization mechanism of nacre, little is known as to how cellular signaling regulates this process. We are interested in whether G protein signaling plays a role in mineralization. Degenerate primers against conserved amino acid regions of G proteins were employed to amplify cDNA from the pearl oyster Pinctada fucata. As a result, the cDNA encoding a novel Gsα (pfGsα) from the pearl oyster was isolated. The Gsα cDNA encodes a polypeptide of 377 amino acid residues, which shares high similarity to the octopus (Octopus vulgaris) Gsα. The well-conserved A, C, G (switch I), switch II functional domains and the carboxyl terminus that is a critical site for interaction with receptors are completely identical to those from other mollusks. However, pfGsα has a unique amino acid sequence, which encodes switch III and interaction sites of adenylyl cyclase respectively. In situ hybridization and Northern blotting analysis revealed that the oyster Gsα mRNA is widely expressed in a variety of tissues, with highest levels in the outer fold of mantle and epithelia of gill, the regions essential for biomineralization. We also show that overexpression of the pfGsα in mammalian MC3T3-E1 cells resulted in increased cAMP levels. Mutant pfGsα that has impaired CTX substrate diminished its ability to induce cAMP production. Furthermore, the alkaline phosphatase (ALP) activity, an indicator for mineralization, is induced by the Gsα in MC3T3-E1 cells. These results indicated that Gsα may be involved in regulation of physiological function, particularly in biological biomineralization.  相似文献   

7.
Abnormal activation of the Wnt/β-catenin signaling pathway and subsequent upregulation of β-catenin driven downstream targets—c-Myc and cyclin D1 is associated with development of breast cancer. The objective of our study was to determine if curcumin could modulate the key elements of Wnt pathway in breast cancer cells; an effect that might underscore its usefulness for chemoprevention/treatment of this malignancy. Curcumin showed a cytotoxic effect on MCF-7 cells with 50% inhibitory concentration (IC50) of 35 μM; while IC50 for MDA-MB-231 cells was 30 μM. Treatment with low cytostatic dose of 20 μM curcumin showed G2/M arrest in both breast cancer cells. The effect of curcumin (20 μM) treatment on expression of Wnt/β-catenin pathway components in breast cancer cells (MCF-7 and MDA-MB-231) was analyzed by immunofluorescence and Western blotting. Curcumin was found to effectively inhibit the expression of several Wnt/β-catenin pathway components—disheveled, β-catenin, cyclin D1 and slug in both MCF-7 and MDA-MB-231. Immunofluorescence analysis showed that curcumin markedly reduced the nuclear expression of disheveled and β-catenin proteins. Further, the protein levels of the positively regulated β-catenin targets—cyclin D1 and slug, were downregulated by curcumin treatment. The expression levels of two integral proteins of Wnt signaling, GSK3β and E-cadherin were also altered by curcumin treatment. In conclusion, our data demonstrated that the efficacy of curcumin in inhibition of cell proliferation and induction of apoptosis might occur through modulation of β-catenin pathway in human breast cancer cells.  相似文献   

8.
In Albright hereditary osteodystrophy, a monogenic obesity disorder linked to heterozygous mutations of Gsα, the G protein that mediates receptor-stimulated cAMP generation, obesity develops only when the mutation is on the maternal allele. Likewise, mice with maternal (but not paternal) germline Gsα mutation develop obesity, insulin resistance, and diabetes. These parent-of-origin effects are due to Gsα imprinting, with preferential expression from the maternal allele in some tissues. As Gsα is ubiquitously expressed, the tissue involved in this metabolic imprinting effect is unknown. Using brain-specific Gsα knockout mice, we show that Gsα imprinting within the central nervous system underlies these effects and that Gsα is imprinted in the paraventricular nucleus of the hypothalamus. Maternal Gsα mutation impaired melanocortin stimulation of energy expenditure but did not affect melanocortin's effect on food intake, suggesting that melanocortins may regulate energy balance in the central nervous system through both Gsα-dependent and -independent pathways.  相似文献   

9.
Cultures of the obligate psychrophilic diatom Fragilariopsis cylindrus (Grunow) were grown for 4 months under steady-state conditions at −1 °C and +7 °C (50 μmol photons m−2 s−1) prior to measurements in order to investigate long-term acclimation of photosynthesis to both temperatures. No differences in maximum intrinsic quantum yield of PS II (FV/FM) and relative electron transport rates could be detected at either temperature after 4 months of acclimation. Measurements of photosynthesis (relative electron transport rates) vs. irradiance (P vs. E curves) revealed similar values for relative light utilization efficiency (α = 0.57 at −1 °C, α = 0.60 at +7 °C) but higher values for irradiance levels at which photosynthesis saturates (EK) at −1 °C and, therefore, higher maximum photosynthesis (PMAX = 54 (relative units) at −1 °C, PMAX = 49 at +7 °C). Nonphotochemical quenching (NPQ) measurements at 385 μmol photons m−2 s−1 indicated higher (37%) NPQ for diatoms grown at −1 °C compared to +7 °C, which was possibly related to a 2-fold increase in the concentration of the pigment diatoxanthin and a 9-fold up-regulation of a gene encoding a fucoxanthin chlorophyll a,c-binding protein. Expression of the D1 protein encoding gene psbA was ca. 1.5-fold up-regulated at −1 °C, whereas expression levels of other genes from Photosystem II (psbC, psbU, psbO), as well as rbcL, the gene encoding the Rubisco large subunit were similar at both temperatures. However, a 2-fold up-regulation of a plastid glyceraldehyde-P dehydrogenase at −1 °C indicated enhanced Calvin cycle activity. This study revealed for the first time that a polar diatom could efficiently acclimate photosynthesis over a wide range of polar temperatures given enough time. Acclimation of photosynthesis at −1 °C was probably regulated similarly to high light acclimation.  相似文献   

10.

Background and purpose

Despite the view that only β2- as opposed to β1-adrenoceptors (βARs) couple to Gi, some data indicate that the β1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes.

Experimental approach

We used the Gs-selective (R,R)- and the Gs- and Gi-activating (R,S)-fenoterol to selectively activate β2ARs (β1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats.

Key results

PTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response.

Conclusions and implications

Together, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic Gi and Gs activity upon AC towards Gs, enhancing the effect of all cAMP-mediated inotropic agents.  相似文献   

11.
Giα1 is the inhibitory G-protein that, upon activation, reduces the activity of adenylyl cyclase. Comparison of the crystal structures of Giα1 bound to GDP•AMF or GTPγS with that of the inactive, GPD-bound protein indicates that a conformational change occurs in the activation step centered on three switch regions. The contribution of each tryptophan residue (W211 in the switch II region, W131 in the α-helical domain, and W258 in the GTPase domain) toward the intrinsic protein fluorescence was evaluated by using W211F, W131F, and W258F mutants. All three tryptophan residues contributed significantly toward the emission spectra regardless of the conformation. When activated by either GDP•AMF or GTPγS, the observed maximal-fluorescence scaled according to the solvent accessibilities of the tryptophan residues, calculated from molecular dynamics simulations. In the GDP•AMF and GTPγS, but not in the GDP, conformations, the residues W211 and R208 are in close proximity and form a π-cation interaction that results in a red shift in the emission spectra of WT, and W131F and W258F mutants, but a blue shift for the W211F mutant. The observed shifts did not show a relationship with the span of the W211-R208 bridge, but rather with changes in the total interaction energies. Trypsin digestion of the active conformations only occurred for the W211F mutant indicating that the electrostatic π-cation interaction blocks access to R208, which was consistent with the molecular dynamics simulations. We conclude that solvent accessibility and interaction energies account for the fluorescence features of Giα1.  相似文献   

12.
Cyanobacteria acclimate to environmental inorganic carbon (Ci) concentrations through re-organisations of photosynthetic function and the induction of carbon concentrating mechanisms (CCMs), which alter and constrain their subsequent acclimation to changing light. We grew cells acclimated to high Ci (4 mM) or low Ci (0.02 mM), shifted them from 50 μmol m−2 s−1 to 500 μmol m−2 s−1, and quantified their photosynthetic performance in parallel with quantitation of allocations to key indicator macromolecules. Pigments cell−1 declined, PsbA (PS II), AtpB (ATP Synthase), RbcL (Rubisco) and GlnA (Glutamine Synthetase) increased, and PsaC (PS I) remained stable through the light shift. The increase in these protein pools was slower and smaller in low Ci cells, but acted in both cell types to re-normalise the electron fluxes through the catalytic complexes back toward values before the light shift (for PsbA and GlnA) or even below the initial flux per complex (for RbcL). In contrast, an increased electron flux per PsaC was sustained for at least 6 h after the increase in light. Initially, high levels of PS II cell−1 and PS II connectivity in high Ci cells caused a more rapid net photoinactivation of PS II in high Ci cells than in low Ci cells, depressing the rate of PS II-specific electron transport (PS II ETR) to levels similar to linear ETR (net O2 evolution minus respiration). In low Ci cells, PS II ETR remained in excess of linear ETR and may have helped maintain CCM activity. The pool sizes of PsbA, AtpB and GlnA correlated with cellular growth rate, and changed at similar rates in high Ci and low Ci cells when expressed on a generational rather than chronological timescale, which has implications for differing ecology of high and low Ci cells under variable natural light.  相似文献   

13.
Gossypol has been proven to be a very effective male contraceptive. However, clinical trials showed that the major side effect of gossypol was hypokalemia. Gossypol occurs naturally as enantiomeric mixtures of (+)-gossypol and (−)-gossypol. The (−)-gossypol is found to be the active component of antifertility. 11β-Hydroxysteroid dehydrogenase 2 (11βHSD2) has been demonstrated to be a mineralocorticoid receptor (MR) protector by inactivating active glucocorticoids including corticosterone (CORT) in rats, and therefore mutation or suppression of 11βHSD2 causes hypokalemia and hypertension. In the present study, the potency of gossypol enantiomers was tested for the inhibition of 11βHSD1 and 2 in rat and human. Both (+) and (−)-gossypols showed a potent inhibition of 11βHSD2 with the half maximal inhibitory concentration (IC50) of 0.61 and 1.33 μM for (+) and (−)-gossypols, respectively in rats and 1.05 and 1.90 μM for (+) and (−)-gossypols, respectively in human. The potency of gossypol to inhibit 11βHSD1 was far less; the IC50 was ≥100 μM for racemic gossypol. The gossypol-induced hypokalemia is likely associated with its potent inhibition of kidney 11βHSD2.  相似文献   

14.
Photoautotrophic cultivation of Euglena gracilis results in cells with high α-tocopherol content but the final cell concentration is usually very low due to the difficulty of supplying light efficiently to the photobioreactor. On the other hand, Euglena grows heterotrophically to high cell concentrations, using various organic carbon sources, but the α-tocopherol contents of heterotrophically grown cells are usually very low. Sequential heterotrophic/photoautotrophic cultivation, by which cells are grown heterotrophically to high cell concentrations and then transferred to photoautotrophic culture for accumulation of α-tocopherol was therefore investigated for efficient α-tocopherol production. In batch culture, using glucose as the organic carbon source, the cellular α-tocopherol content increased from 120 μg g−1 at the end of heterotrophic phase to more than 400 μg g−1 at the end of the photoautotrophic phase. By using ethanol as the organic carbon source during the heterotrophic phase, adding corn steep liquor as a nitrogen source and optimizing light supply during the photoautotrophic phase, the α-tocopherol content of the cells at the end of the photoautotrophic phase increased to 1700 μg g−1. A system consisting of a mini-jar fermentor (for the heterotrophic phase) and an internally illuminated photobioreactor (for the photoautotrophic phase) was then constructed for continuous sequential heterotrophic/photoautotrophic cultivation. The cells were continuously cultivated heterotrophically in the mini-jar fermentor and the effluent was continuously passed through the photobioreactor for α-tocopherol accumulation. In this way, it was possible to produce 7 g l−1 cells containing about 1100 μg α-tocopherol per g-cell continuously for more than 420 h. The continuous process resulted in α-tocopherol productivity of 100 μg l−1 h−1 which is about 9.5 and 4.6 times higher than those obtained in batch photoautotrophic culture and batch heterotrophic cultures, respectively.  相似文献   

15.
The Gs and Gi pathways interact to control the levels of intracellular cAMP. Although coincident signaling through Gs and Gi-coupled receptors can attenuate Gs-stimulated cAMP levels, it is not known if prior activation of the Gi pathway can affect signaling by Gs-coupled receptors. We have found that activated Gαo/i interact with RGS20, a GTPase activating protein for members of the Gαο/i family. Interaction between Gαo/i and RGS20 results in decreased cellular levels of RGS20. This decrease was induced by activated Gαo and Gαi2 but not by Gαq, Gαi1 or Gαi3. The Gαo/i-induced decrease in RGS20 can be blocked by proteasomal inhibitors lactacystin or MG132. Activated Gαo stimulates the ubiquitination of RGS20. The serotonin-1A receptor that couples to Go/i reduces the levels of RGS20 and this effect is blocked by lactacystin, suggesting that Go/i promotes the degradation of RGS20. Expression of RGS20 attenuates the inhibition of β-adrenergic receptor-induced cAMP levels mediated by the serotonin-1A receptor. Prior activation of the serotonin-1A receptor results in loss of the RGS20-mediated attenuation, and the loss of attenuation is blocked when lactacystin is included during the prior treatment. These observations suggest that Go/i-coupled receptors, by stimulating the degradation of RGS20, can regulate how subsequent activation of the Gs and Gi pathways controls cellular cAMP levels, thus allowing for signal integration.  相似文献   

16.
Parathyroid hormone (PTH) is an important regulator of osteoblast function and is the only anabolic therapy currently approved for treatment of osteoporosis. The PTH receptor (PTH1R) is a G protein-coupled receptor that signals via multiple G proteins including Gsα. Mice expressing a constitutively active mutant PTH1R exhibited a dramatic increase in trabecular bone that was dependent upon expression of Gsα in the osteoblast lineage. Postnatal removal of Gsα in the osteoblast lineage (P-GsαOsxKO mice) yielded markedly reduced trabecular and cortical bone mass. Treatment with anabolic PTH(1–34) (80 μg/kg/day) for 4 weeks failed to increase trabecular bone volume or cortical thickness in male and female P-GsαOsxKO mice. Surprisingly, in both male and female mice, PTH administration significantly increased osteoblast numbers and bone formation rate in both control and P-GsαOsxKO mice. In mice that express a mutated PTH1R that activates adenylyl cyclase and protein kinase A (PKA) via Gsα but not phospholipase C via Gq/11 (D/D mice), PTH significantly enhanced bone formation, indicating that phospholipase C activation is not required for increased bone turnover in response to PTH. Therefore, although the anabolic effect of intermittent PTH treatment on trabecular bone volume is blunted by deletion of Gsα in osteoblasts, PTH can stimulate osteoblast differentiation and bone formation. Together these findings suggest that alternative signaling pathways beyond Gsα and Gq/11 act downstream of PTH on osteoblast differentiation.  相似文献   

17.
A model is proposed for the d-galactoside-H+(OH) transporter of Escherichia coli that accounts for essentially all the experimental observations established for this system to date. In this model, the functional unit is postulated to be a dimer (consisting of two copies of lacY-specified polypeptide) which spans the membrane with a 2-fold symmetry axis in the membrane plane (Lancaster, J.R. (1978) J. Theor. Biol. 75, 35–50). The functional dimer is assumed to possess a single pore flanked by an inner gate (gi) and an outer gate (go) and encompassing two oppositely oriented galactoside binding sites, designated m and μ. When go is open and gi is closed under non-energized conditions, binding site m adopts a configuration defined as State A (i.e., moA) exhibiting high affinity toward Class Ga galactosides (thiodigalactoside, melibiose, α-p-nitrophenylgalactoside) but low affinity for Class Gb galactosides (lactose, β-o-nitrophenylgalactoside, β-isopropylthiogalactoside), whereas binding site μ adopts State B (i.e., μoB) displaying relatively high affinity toward Class Gb galactosides but comparatively low affinity for Class Ga galactosides; further, each moA : μoB dimer contains one thiol group whose reaction with N-ethylmaleimide inactivates the transporter unless blocked by galactoside binding at site moA, while the second homologous thiol of the dimer is unreactive toward thiol reagents. Translocation of the moA : μoB dimer involves closing of go followed by opening of gi, and causes the two thiols (as well as sites m and μ) to interchange roles in a symmetrical fashion: moA : μoB ↔ miB : μiA. In the presence of a substantial (negative) transmembrane Δμ~H+, the m : μ dimer is postulated to undergo an electrogenic protein conformational change to a second form, *(m : μ), in which both sites m and μ possess low affinity toward internal Class Gb substrates; galactoside transport in both m : μ and *(m : μ) is assumed to be coupled to H+-symport (OH-antiport) with a stoichiometry of approximately 1 : 1. Finally, five characteristic predictions of the half-sites model are outlined for further tests of its validity.  相似文献   

18.
The nitrogen uptake and growth capabilities of the potentially harmful, raphidophycean flagellate Heterosigma akashiwo (Hada) Sournia were examined in unialgal batch cultures (strain CCMP 1912). Growth rates as a function of three nitrogen substrates (ammonium, nitrate and urea) were determined at saturating and sub-saturating photosynthetic photon flux densities (PPFDs). At saturating PPFD (110 μE m−2 s−1), the growth rate of H. akashiwo was slightly greater for cells grown on NH4+ (0.89 d−1) compared to cells grown on NO3 or urea, which had identical growth rates (0.82 d−1). At sub-saturating PPFD (40 μE m−2 s−1), both urea- and NH4+-grown cells grew faster than NO3-grown cells (0.61, 0.57 and 0.46 d−1, respectively). The N uptake kinetic parameters were investigated using exponentially growing batch cultures of H. akashiwo and the 15N-tracer technique. Maximum specific uptake rates (Vmax) for unialgal cultures grown at 15 °C and saturating PPFD (110 μE m−2 s−1) were 28.0, 18.0 and 2.89 × 10−3 h−1 for NH4+, NO3 and urea, respectively. The traditional measure of nutrient affinity—the half saturation constants (Ks) were similar for NH4+ and NO3 (1.44 and 1.47 μg-at N L−1), but substantially lower for urea (0.42 μg-at N L−1). Whereas the α parameter (α = Vmax/Ks), which is considered a more robust indicator for substrate affinity when substrate concentrations are low (<Ks), were 19.4, 12.2 and 6.88 × 10−3 h−1/(μg-at N L−1) for NH4+, NO3 and urea, respectively. These laboratory results demonstrate that at both saturating and sub-saturating N concentrations, N uptake preference follows the order: NH4+ > NO3 > urea, and suggests that natural blooms of H. akashiwo may be initiated or maintained by any of the three nitrogen substrates examined.  相似文献   

19.
The objective of this study was to examine the expression and activity of cytosolic phospholipase A2 (cPLA2) in relation to prostaglandin E2 (PGE2) synthesis in human amnion-derived WISH cells in response to stimulation by interleukin-1β (IL-1β). cPLA2 activity was characterized by sensitivity to heat and acid treatment, stability to dithiothreitol, and inhibition by the specific inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3). Treatment of WISH cells with IL-1β (0.01–1 ng/mL) for up to 24 h resulted in a significant increase in PGE2 release in a concentration- and time-dependent manner accompanied by increases both in total cellular cPLA2 activity and in cPLA2 protein levels detected by Western blot analysis. The parallel increase in total cellular cPLA2 activity and cPLA2 protein level indicates that IL-1β may induce the synthesis of CPLA2. Incubation of the cells with 10 μM AACOCF3 for 24 h significantly inhibited IL-1β-induced PGE2 production strongly suggesting that cPLA2 mediates IL-1β-induced PGE2 formation. In unstimulated cells, there is appreciable total cellular cPLA2 activity and protein, but these cells produce low amounts of PGE2 until stimulated by IL-1β, suggesting that cPLA2 translocation from cytosol to the membrane is necessary for its bioactivity. In contrast to IL-1β, treatment with phorbol ester (12-O-tetradecanoyl phorbol-13-acetate, TPA, 10−10−10−6 M) for 24 h significantly inhibited total cellular cPLA2 activity in a concentration-dependent manner. The amount of total cellular cPLA2 protein seen on Western blot remained unchanged following TPA treatment. These data suggest that in WISH cells, IL-1β induces both translocation to the membrane and de novo synthesis of cPLA2 protein to sustain prostaglandin (PG) synthesis. In contrast, TPA may only cause cPLA2 translocation but no increase in cPLA2 protein synthesis, resulting in limited PFG synthesis. Our results provide a mechanism for the effect of IL-1β on prostaglandin synthesis in human amnion cells and provide support for a role of cPLA2 in the mechanism initiating human parturition.  相似文献   

20.
The effects of long-term depolarization on frog skeletal muscle Cav1.1 channels were assessed. Voltage-clamp and Western-blot experiments revealed that long-term depolarization brings about a drastic reduction in the amplitude of currents flowing through Cav1.1 channels and in the levels of the α1s subunit, the main subunit of muscle L-type channels. The decline of both phenomena was prevented by the action of the protease inhibitors E64 (50 μM) and leupeptin (50 μM). In contrast, long-term depolarization had no effect on β1, the auxiliary subunit of α1s. The levels of mRNAs coding the α1s and the β1 subunits were measured by RNase protection assays. Neither the content of the α1s nor the β1 subunit mRNAs were affected by long-term depolarization, indicating that the synthesis of Cav1.1 channels remained unaffected. Taken together, our experiments suggest that the reduction in the amplitude of membrane currents and in the α1s subunit levels is caused by increased degradation of this subunit by a Ca2+-dependent protease.  相似文献   

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