scholarly journals Measuring Rubisco activity: challenges and opportunities of NADH-linked microtiter plate-based and 14C-based assays

2020 ◽  
Vol 71 (18) ◽  
pp. 5302-5312 ◽  
Author(s):  
Cristina R G Sales ◽  
Anabela Bernardes da Silva ◽  
Elizabete Carmo-Silva
Keyword(s):  
Response Curve ◽  
Crop Production ◽  
Crop Improvement ◽  
Carbon Assimilation ◽  
Microtiter Plate ◽  
Rubisco Activity ◽  
Challenges And Opportunities ◽  
Radiometric Assay ◽  
Menten Constant ◽  
Concentration Response Curve

Abstract Rubisco is central to carbon assimilation, and efforts to improve the efficiency and sustainability of crop production have spurred interest in phenotyping Rubisco activity. We tested the hypothesis that microtiter plate-based methods provide comparable results to those obtained with the radiometric assay that measures the incorporation of 14CO2 into 3-phosphoglycerate (3-PGA). Three NADH-linked assays were tested that use alternative coupling enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glycerolphosphate dehydrogenase (GlyPDH); phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH); and pyruvate kinase (PK) and lactate dehydrogenase (LDH). To date there has been no thorough evaluation of their reliability by comparison with the 14C-based method. The three NADH-linked assays were used in parallel to estimate (i) the 3-PGA concentration–response curve of NADH oxidation, (ii) the Michaelis–Menten constant for ribulose-1,5-bisphosphate, (iii) fully active and inhibited Rubisco activities, and (iv) Rubisco initial and total activities in fully illuminated and shaded leaves. All three methods correlated strongly with the 14C-based method, and the PK–LDH method showed a strong correlation and was the cheapest method. PEPC–MDH would be a suitable option for situations in which ADP/ATP might interfere with the assay. GAPDH–GlyPDH proved more laborious than the other methods. Thus, we recommend the PK–LDH method as a reliable, cheaper, and higher throughput method to phenotype Rubisco activity for crop improvement efforts.

Cholinergic stimulation modulates negative inotropic effect of cocaine on ferret ventricular myocardium

1996 ◽  
Vol 270 (2) ◽  
pp. H678-H684
Author(s):  
L. Miao ◽  
Z. Qiu ◽  
J. P. Morgan
Keyword(s):  
Response Curve ◽  
Negative Inotropic Effect ◽  
Ventricular Myocardium ◽  
Inotropic Effect ◽  
Dependent Manner ◽  
Cholinergic Stimulation ◽  
Concentration Dependent Manner ◽  
Cell Shortening ◽  
Inotropic State ◽  
Concentration Response Curve

We tested the hypothesis that the negative inotropic effect (NIE) of cocaine is mediated, at least in part, by cholinergic stimulation and can be correlated with the degree of adenosine 3',5'-cyclic monophosphate (cAMP) dependency of the inotropic state. Cardiac myocytes were isolated from left ventricles of ferrets and loaded with the fluorescent Ca2+ indicator indo 1. Cells were placed in physiological solution containing 2.0 mM Ca2+ and stimulated at 0.5 Hz and 30 degrees C. Cocaine decreased peak cell shortening and peak intracellular Ca2+ in a concentration-dependent manner (10(-8)-10(-4) M). The concentration-response curve of cocaine was shifted significantly downward compared with those of lidocaine and procaine in the same range of concentrations. Atropine (10(-6) M) shifted the concentration-response curve of cocaine, but not those of lidocaine and procaine, rightward, with a pA2 value (7.66) similar to that obtained with carbachol (7.99). With prior addition of isoproterenol (ISO, 10(-8) M) or increased Ca2+ (4.0 mM) to increase cell shortening to the same degree (approximately 60%), cocaine and carbachol decreased contractility to a significantly greater extent in ISO-stimulated myocytes. To clarify whether these treatments changed responsiveness of the contractile elements to Ca2+, the effect of 2,3-butanedione monoxime, an agent that interferes with the interaction of myosin and actin, was tested with previous addition of ISO or increased Ca2+, and no differential effect occurred. Therefore, we postulate that 1) the NIE of cocaine on myocytes is caused by decreased Ca2+ availability; 2) this effect is due to specific stimulation of cholinergic receptors in addition to other direct myocardial (probably local anesthetic) effects; and 3) the NIE correlates with the level of cAMP dependence of the inotropic state.

scholarly journals Agonist Efficiency Estimated from Concentration Response Curve

2021 ◽  
Vol 120 (3) ◽  
pp. 57a
Author(s):  
Dinesh Indurthi ◽  
Anthony Auerbach
Keyword(s):  
Response Curve ◽  
Concentration Response Curve

scholarly journals The Cellular Prion Protein Prevents Copper-Induced Inhibition of P2X4Receptors

2011 ◽  
Vol 2011 ◽  
pp. 1-6
Author(s):  
Ramón A. Lorca ◽  
Lorena Varela-Nallar ◽  
Nibaldo C. Inestrosa ◽  
J. Pablo Huidobro-Toro
Keyword(s):  
Amino Acids ◽  
Synaptic Transmission ◽  
Prion Protein ◽  
Adenosine Triphosphate ◽  
Response Curve ◽  
Physiological Function ◽  
Cellular Prion Protein ◽  
Synaptic Activity ◽  
Receptor Subtype ◽  
Concentration Response Curve

Although the physiological function of the cellular prion protein (PrPC) remains unknown, several evidences support the notion of its role in copper homeostasis. PrPCbinds Cu2+through a domain composed by four to five repeats of eight amino acids. Previously, we have shown that the perfusion of this domain prevents and reverses the inhibition by Cu2+of the adenosine triphosphate (ATP)-evoked currents in the P2X4receptor subtype, highlighting a modulatory role for PrPCin synaptic transmission through regulation of Cu2+levels. Here, we study the effect of full-length PrPCin Cu2+inhibition of P2X4receptor when both are coexpressed. PrPCexpression does not significantly change the ATP concentration-response curve in oocytes expressing P2X4receptors. However, the presence of PrPCreduces the inhibition by Cu2+of the ATP-elicited currents in these oocytes, confirming our previous observations with the Cu2+binding domain. Thus, our observations suggest a role for PrPCin modulating synaptic activity through binding of extracellular Cu2+.

gamma-Aminobutyric acid-induced response in rat dissociated paratracheal ganglion cells

1992 ◽  
Vol 67 (5) ◽  
pp. 1367-1374 ◽  
Author(s):  
S. Itabashi ◽  
K. Aibara ◽  
H. Sasaki ◽  
N. Akaike
Keyword(s):  
Response Curve ◽  
Ganglion Cells ◽  
Aminobutyric Acid ◽  
Equilibrium Potential ◽  
Induced Response ◽  
Dependent Manner ◽  
Gamma Aminobutyric Acid ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
Concentration Response Curve

1. The pharmacologic properties of gamma-aminobutyric acid (GABA)-induced Cl- current (ICl) were studied in the paratracheal ganglion cells freshly dissociated from 7- to 10-day-old rat trachea in a whole-cell recording mode by the use of a conventional patch-clamp technique. 2. GABA- and muscimol-induced currents increased sigmoidally in a concentration-dependent manner, and both currents reversed at approximately -3 mV, which was close to the Cl- equilibrium potential (ECl). 3. Strychnine (STR) at low concentration and bicuculline (BIC) inhibited GABA response competitively, whereas STR at the higher concentrations, benzylpenicillin (PCG), or picrotoxin (PTX) inhibited noncompetitively. Inhibition of GABA response by PCG but not other antagonists was voltage dependent, indicating that PCG acts as a Cl- channel blocker. 4. The concentration-response curve of pentobarbital sodium (PB)-induced ICl was bell shaped. At concentrations higher than 10(-3) M, both the peak and plateau currents decreased, and a transient "hump" current appeared immediately after washing out PB. In the presence of PB, the concentration-response curve of GABA shifted toward left without changing the maximum response. 5. Although diazepam (DZP) at concentration used did not induce a response, it potentiated the GABA response in a concentration-dependent manner between 10(-8) and 10(-6) M. DZP also caused a parallel shift toward left in the concentration-response curve of GABA. 6. PB or DZP further enhanced the GABA response in the presence of the other agent. 7. It is concluded that the properties of GABAA receptors in the paratracheal ganglion cells are essentially similar to those reported in other preparations.

scholarly journals Climate Variability and Change in Bihar, India: Challenges and Opportunities for Sustainable Crop Production

2017 ◽  
Vol 9 (11) ◽  
pp. 1998 ◽  
Author(s):  
Kindie Tesfaye ◽  
Pramod Aggarwal ◽  
Fasil Mequanint ◽  
Paresh Shirsath ◽  
Clare Stirling ◽  
...  
Keyword(s):  
Climate Variability ◽  
Crop Production ◽  
Climate Variability And Change ◽  
Sustainable Crop Production ◽  
Challenges And Opportunities

Renal microvascular effects of endothelin

1990 ◽  
Vol 259 (2) ◽  
pp. F217-F221 ◽  
Author(s):  
R. M. Edwards ◽  
W. Trizna ◽  
E. H. Ohlstein
Keyword(s):  
Response Curve ◽  
Contractile Response ◽  
Maximum Response ◽  
Lumen Diameter ◽  
Rabbit Kidney ◽  
Significant Shift ◽  
Efferent Arteriole ◽  
Endothelin 1 ◽  
Afferent Arterioles ◽  
Concentration Response Curve

The effects of endothelin 1, 2, and 3 (ET-1, -2, -3) on lumen diameter of individual afferent and efferent arterioles dissected from rabbit kidney were examined. ET-1 produced concentration-dependent and long-lasting decreases in lumen diameter in both arterioles. The 50% maximum response (EC50) values were 1.4 +/- 0.41 and 0.9 +/- 0.65 nM for afferent and efferent arterioles, respectively. In afferent arterioles, ET-2 produced decreases in lumen diameter (EC50 = 3.3 +/- 1.75 nM) that were indistinguishable from ET-1. However, ET-3 was considerably less potent (EC50 = 21.9 +/- 6.0 nM, P less than 0.05) than ET-1 or ET-2. Similar results were obtained in the efferent arteriole in which the EC50 for ET-2 (0.25 +/- 0.1 nM) was similar to ET-1, but ET-3 was significantly less potent (EC50 = 2.6 +/- 0.4 nM, P less than 0.05). Nicardipine (0.01-1 microM) produced concentration-dependent shifts in the ET-1 concentration-response curve in afferent arterioles. Verapamil (1 microM) also caused a significant shift in the ET-1 response curve. The contractile response to ET-1 was significantly more sensitive to nicardipine than was the response to norepinephrine. In contrast, the response of efferent arterioles to ET-1 and norepinephrine was unaffected by nicardipine or verapamil. The results demonstrate that ETs are potent vasoconstrictors of both the pre- and postglomerular microvasculature and may play a role in the regulation of renal hemodynamics.(ABSTRACT TRUNCATED AT 250 WORDS)

Superoxide anion impairs Ca2+mobilization in cultured human nasal epithelial cells

1999 ◽  
Vol 277 (6) ◽  
pp. L1089-L1095
Author(s):  
Tetsuya Koyama ◽  
Masahiro Oike ◽  
Sohtaro Komiyama ◽  
Yushi Ito
Keyword(s):  
Superoxide Dismutase ◽  
Epithelial Cells ◽  
Superoxide Anion ◽  
Response Curve ◽  
Krebs Solution ◽  
Nasal Epithelial Cells ◽  
Human Nasal Epithelial Cells ◽  
Bath Application ◽  
Intracellular Ca ◽  
Concentration Response Curve

We examined the effects of superoxide anion ([Formula: see text]) on the intracellular Ca2+ concentration in cultured human nasal epithelial cells. The cells were exposed to[Formula: see text] by pretreatment with xanthine (X) and xanthine oxidase (XO); control cells were treated with X alone. When Ca2+-containing Krebs solution was reperfused in the thapsigargin-treated, store-depleted cells, reapplication-induced intracellular Ca2+ concentration elevation was significantly smaller in X/XO-treated cells than in the control cells, suggesting that [Formula: see text] impairs Ca2+ release-activated Ca2+ entry (CRAC). Bath application of ATP induced a steep Ca2+ transient in both control and X/XO-treated cells. However, the concentration-response curve of the ATP-induced Ca2+ transient was shifted to a higher concentration in X/XO-treated cells. The impairments of CRAC and ATP-induced Ca2+ transient induced by X/XO were reversed by superoxide dismutase. Furthermore, all these X/XO-induced effects were also observed in cells pretreated with pyrogallol, also an [Formula: see text] donor. These results indicate that [Formula: see text] impairs at least two mechanisms involved in Ca2+ mobilization in human nasal epithelial cells, i.e., CRAC and ATP-induced Ca2+ release.

Water Use Efficiency as a Constraint and Target for Improving the Resilience and Productivity of C3and C4Crops

2019 ◽  
Vol 70 (1) ◽  
pp. 781-808 ◽  
Author(s):  
Andrew D.B. Leakey ◽  
John N. Ferguson ◽  
Charles P. Pignon ◽  
Alex Wu ◽  
Zhenong Jin ◽  
...  
Keyword(s):  
Water Use Efficiency ◽  
Water Use ◽  
Crop Production ◽  
Crop Improvement ◽  
Canopy Structure ◽  
Complex Trait ◽  
Carbon Gain ◽  
Crop Development ◽  
Component Traits ◽  
Use Efficiency

The ratio of plant carbon gain to water use, known as water use efficiency (WUE), has long been recognized as a key constraint on crop production and an important target for crop improvement. WUE is a physiologically and genetically complex trait that can be defined at a range of scales. Many component traits directly influence WUE, including photosynthesis, stomatal and mesophyll conductances, and canopy structure. Interactions of carbon and water relations with diverse aspects of the environment and crop development also modulate WUE. As a consequence, enhancing WUE by breeding or biotechnology has proven challenging but not impossible. This review aims to synthesize new knowledge of WUE arising from advances in phenotyping, modeling, physiology, genetics, and molecular biology in the context of classical theoretical principles. In addition, we discuss how rising atmospheric CO2concentration has created and will continue to create opportunities for enhancing WUE by modifying the trade-off between photosynthesis and transpiration.

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