2009 Workers' Compensation Drug Trend Report An analysis of trend and a forecast for the future Express Scripts, Inc.One Express WaySt. Louis, Missouri 63121 Published April 2010 Emily Cox, PhD, RPh Yakov Svirnovskiy Jennifer Kaburick, RN Ruth Martinez, RPh The authors would like to thank the analysts, researchers, reviewers and sponsors for the 2009 Workers' Compensation Drug Trend Report. We also recognize generous assistance from individuals throughout the Express Scripts organization, including the Clinical, Creative Management Services, Product Management and Research teams.
Doi:10.1016/j.jplph.2005.03.006Journal of Plant Physiology 162 (2005) 1087—1094 Modulation of carbonic anhydrase activity in twonitrogen fixing cyanobacteria, Nostoc calcicola andAnabaena sp.
Pranita Jaiswala,, Radha Prasannaa, Ajai Kumar Kashyapb aCentre for Conservation and Utilization of Blue Green Algae (CCUBGA), Indian Agricultural Research Institute (IARI),110012 New Delhi, IndiabDepartment of Botany, Banaras Hindu University, Varanasi-221005, India Received 28 June 2004; accepted 3 March 2005 The activity of enzyme carbonic anhydrase (CA) was investigated in two diazotrophic cyanobacteria, Anabaena sp. (ARM 629) and Nostoc calcicola, in the presence of CO2/ NaHCO3 and different inhibitors. The CA activity increased when the cells were Carbonic anhydrase; pretreated with a high concentration of CO2/NaHCO3 and then transferred to ambient level CO2. Maximum activity of CA was observed after 8 h of incubation in light on transfer of cells from high Ci to ambient level CO2, and was low when incubated indark. Addition of the photosynthetic inhibitor DCMU brought about a differentialreduction in CA activity, depending on the carbon source (NaHCO3/CO2). CA inhibitors– ethoxyzolamide (EZ) and acetazolamide (AZ) – inhibited the enzyme activity in boththe genera, but the extent of inhibition was greater in Anabaena sp. than in N.
calcicola. Such a variation in extent of inhibition/stimulation of CA activity beingdifferent in the two genera reflects differences in their inherent potential and geneticbackground. The relevance of such cyanobacterial strains as CO2 sinks is alsodiscussed.
& 2005 Elsevier GmbH. All rights reserved.
). Natural photosynthesis, and to alesser extent solution into the ocean sink, removes Carbon dioxide (CO from the atmosphere about half of the CO 2) is known to be responsible for an estimated 60% of the global warming from emitted by anthropogenic activities. In this con- the green house gases produced by human activities text, planktonic microalgae can serve as efficient Corresponding author. Tel.: +91 011 25848431; fax: +91 011 25741648.
E-mail addresses: [email protected] (P. Jaiswal), [email protected] (R. Prasanna).
0176-1617/$ - see front matter & 2005 Elsevier GmbH. All rights reserved.
doi:10.1016/j.jplph.2005.03.006 P. Jaiswal et al.
vehicles for converting atmospheric CO2 into reported that induction of CA is regulated at carbohydrates. It is now well documented that transcriptional level and is strongly induced when cyanobacteria possess a CO2 concentrating me- algae are grown in a low-CO2 environment chanism (CCM), which enables them to maintain In addition to the decrease in high rates of CO2 fixation, and also grow under low CO2 partial pressure, induction of CA is also controlled by other factors such as light and Carbonic anhydrase (CA) (EC 184.108.40.206), an im- In the present study, we report the differential portant component of CCM is a regulation of CA activity in two diazotrophic metallozyme which catalyses the inter-conversion cyanobacteria Nostoc calcicola and Anabaena sp.
and CO2, i.e. CO2+H2O2HCO3 +H+, a (ARM 629) in the presence of CO2/NaHCO3 and CA reaction which occurs spontaneously, but too slowly inhibitors and discuss their significant role as ‘CO2 to meet the physiological needs of a cell. It is an ancient enzyme widespread in the Archaea andBacteria domains However, CA Materials and methods more than compensates for this problem with aturnover rate of 106 M s1 (). CA plays Organisms and growth conditions an important role in photosynthesis in cyanobac-teria and higher plants with a C4 carbon fixing N. calcicola Breb, an ‘usar' land isolate (solentz pathway. Recent reviews have focused on several forms of plant CA (Multiple CAs have –11.0), has been maintained in Algal Research Laboratory, Department of Botany, Ba- been detected in many cyanobacterial species, but naras Hinhu University, for the last 20 years suggested that many ). Anabaena sp. (ARM 629), a cyanobacterial genomes appear to lack any identi- rice field isolate was obtained from the germplasm fiable CA genes. However, recent evidence for the of the Centre for conservation and Utilization of presence of b and g classes of metabolic diverse Blue Green Algae, IARI, New Delhi, India. Both the prokaryotic species has demonstrated that these genera were cultivated and maintained in com- enzymes may have a far more extensive and bined inorganic nitrogen-free Allen fundamental role in prokaryotic biology than previously recognized. reported a 5 trace elements and FeEDTA as the source of iron. For the purpose of novel CA (-class CA) in Prochlorococcus sp. and cultivation, the medium was diluted two-fold Synechococcus sp., which has an evolutionary except for trace elements and FeEDTA. The lineage distinct from those previously recognized cultures were incubated at 2571 in animals, plants and other prokaryotes.
nated with daylight fluorescent tubes with a photon CA is located in carboxysome ( flux density of 25 mE m2 s1 on the surface of which serves as a diffusion barrier for CO2 leakage culture vessels. The pH of the medium was while still allowing ions like HCO maintained at 7.8 by using 10 mM HEPES buffer.
enter (The ability of Differing levels of CO carboxysomes to act as a gaseous leak barrier may 2 were maintained in the gas phase of culture vessels (200 ml air-tight gas be in some way analogous to the bundle sheath of sampling vials) by water displacement. The cul- C4 plants. In addition to carboxysomal CA, the Ci tures were flushed with air+CO pump also has a CA-like function associated with 2 mixture (flow rate 0.5 L min1) two times a day for 30 min. Filter the transport step such that HCO sterilized (porosity 0.44 mm, Millipore Membrane delivered to the cell interior filter) sodium bicarbonate or potassium bicarbo- ). Any mutation leading to loss of carboxyso- nate were supplemented in the growth medium to mal CA results in cells which cannot grow at the desired level.
limiting levels of CO2 Cells devoid of carboxysomal CA accumulate HCO at a higher level than do wild type cells, pre- Chlorophyll a estimation sumably because of the inability of cells to convertHCO to CO2 for photosynthesis ( The cyanobacterial sample was centrifuged Most of the work regarding (5000g, 5 min), the pellet suspended in 80% (v/v) regulation of CA activity has been done using the acetone and incubated overnight at 4 1C in dark.
green alga Chlamydomonas reinhardtii The suspension was recentrifuged and the super- natant was analysed spectrometrically at 663 nm.
Carbonic anhydrase activity in two N2-fixing cyanobacteria The photopigments were quantified in terms of mg mg1 protein using specific absorption coeffi- Carbonic anhydrase activity CA activity was assayed electrometrically using a modified procedure of .
The sample was assayed at 3 1C by adding cells (equivalent to 200 mg Chl a) to 3 ml of HEPES buffer (pH 8.0) and sonicated, for 10 min. The reactionwas initiated by addition of 2 ml ice-cold CO CA activity (EU µg The time required for the pH to decrease from 8.0 to 7.0 was measured. The enzyme activity in thetest sample was calculated using the equation: EU ¼ 10(T0/T1), where EU is the enzyme unit, T0the time required for pH change when sample is present, and T1 the time required for pH change when sterilized distilled water was used in place of Figure 1. Carbonic anhydrase (CA) activity in Nostoccalcicola (K) and Anabaena sp. ('). Cells were grown ingraded concentrations of NaHCO Statistical analysis 3 for 5 days, centrifuged, resuspended in fresh medium and incubated at ambientatmosphere in light. CA activity was measured after 3 h All values were recorded in triplicate and of incubation. Vertical bars represent standard devia- statistical analysis (ANOVA) was performed using Sigma Stat (2).
out bicarbonate), in the case of N. calcicola, was consistently much lower in comparison with Ana-baena sp.; the maximum difference recorded was Preliminary investigations ( around 2.2-fold. However, in both the cases, CA ) were carried out to evaluate the tolerance activity increased gradually in cells grown at limits of N. calcicola and Anabaena sp in bicarbo- increasing levels of NaHCO3. Both organisms ex- nate and CO2 environments. These results indicated hibited maximum enzyme activity when grown in that both the organisms under investigation could 250 mM NaHCO3, which were 3.3- and 3.9-fold grow up to a concentration of 250 mM NaHCO3 in higher than the values recorded in control condi- the medium. However, N. calcicola had a higher tions in N. calcicola and Anabaena sp., respectively.
specific growth rate at all the concentrations of Statistical analyses revealed that there were bicarbonate employed compared to Anabaena sp.
significant differences in the CA activity of the At the maximum concentration of bicarbonate, the two cyanobacteria in cells grown in graded con- specific growth rates of N. calcicola and Anabaena centrations of bicarbonate [Fð1;4Þ ¼ 78:4; po0:001].
sp. were 0.028 and 0.02, respectively. Similar The CA activity in N. calcicola and Anabaena sp.
experiments were also conducted with graded pretreated with differing concentrations of CO2 (as concentrations of CO2 in the culture vessel atmo- for NaHCO3) is presented in It is evident that sphere, and the specific growth rate of Anabaena CO2 grown cells also showed an increase in CA sp. was higher than N. calcicola under comparable concentrations of CO2. Anabaena sp. could also Although the increment in enzyme activity was tolerate a two-fold higher concentration of CO2 as not significant till 2.5% CO2 in N. calcicola, it compared to N. calcicola.
increased sharply in cells grown in 5% and 10% CO2.
In the present investigation, cells of N. calcicola Maximum enzyme activity was recorded at 10% and Anabaena sp. were initially grown at high CO2, which was also the maximum tolerance limit concentrations of NaHCO3 for 5 days, and such cells for the cyanobacteria, and the enzyme activity at were transferred to ambient levels of CO2, and CA this concentration was 3.2-fold higher than the activity was measured (). We observed that control. Anabaena sp. also showed maximum CA the enzyme activity under control conditions (with- activity in 10% CO2 grown cells, which was 1.8 times P. Jaiswal et al.
higher than the control. Further increase in CO2 The addition of PS II inhibitor DCMU was concentration did not cause any change in enzyme examined in N. calcicola and Anabaena sp, in order activity. The enzyme activity in Anabaena sp. was to understand its effect on CA activity (We significantly higher than N. calcicola at all the observed that, irrespective of the nature of concentrations of CO2. Statistical analyses revealed inorganic carbon species in the pre-incubation that there were significant differences in the CA phase, addition of DCMU (at the time of transfer activity of the two cyanobacteria in cells grown in of cells from optimum level Ci to ambient CO2) [Fð1;4Þ ¼ 78:4; resulted in a decline in CA activity. Although this decline in CA activity after addition of DCMU wasconsistent for the two cyanobacterial strains grownin both the inorganic carbon species, the influenceof DCMU on enzyme activity was more severe in N.
calcicola than Anabaena sp. In N. calcicola areduction in enzyme activity of 73.9% and 67.5%, respectively, was recorded in NaHCO3 and CO2grown cells (when transferred to ambient level CO2 in presence of DCMU) while in Anabaena sp., these values were approximately 67% and 53%, respec- A time course induction and regulation of CA activity in N. calcicola was carried out usinginhibitors of CA activity The cells grown in an optimal concentration of bicarbonate wereharvested in log phase and DCMU and, either CA activity (EU µg acetazolamide (AZ) or ethoxyzolamide (EZ) wasadded at concentrations of 10, 50 and 25 mM, respectively. The cultures were incubated underphotoautotrophic growth conditions and CA activity was determined. At the time of incubation, the cells showed an expression of 2.6 EU mg1 Chl a CA activity. The cells incubated in light withoutinhibitors showed a gradual increase in enzyme Figure 2. Carbonic anhydrase (CA) activity in Nostoc activity with increase in period of incubation, and calcicola (K) and Anabaena sp. ('). Cells were grown in reached a level which was 4.2 times higher (at 8 h), graded concentrations of CO2for 5 days, centrifuged, than the enzyme activity recorded at 0 h. The cells resuspended in fresh medium and incubated at ambientatmosphere in light. CA activity was measured after 3 h incubated in the dark showed a gradual decrease in of incubation. Vertical bars represent standard devia- the enzyme activity which was 81% lower compared to the light incubated cells. The CA inhibitors EZ Effect of PS II inhibitor DCMU on carbonic anhydrase activity of Nostoc calcicola and Anabaena sp. at ambient CO2 level and growth promoting concentrations of NaHCO3 and CO2 Culture conditions CA activity (EU)a Anabaena sp.
Ambient CO2+DCMUb Ambient CO2+DCMUb Cells were grown in optimum growth-promoting concentrations of CO2 (1.5% and 5% for Nostoc calcicola and Anabaena sp.,respectively) for 5 days. Cells were harvested by centrifugation, washed and resuspended in fresh medium and incubated for 3 h inconditions indicated in column II. Cells grown in ambient level CO2 served as control. Data presented are mean of threeobservations7SD.
aEU ¼ Enzyme unit mg1 Chl a.
bDCMU ¼ 3(3,4-dichlorophenyl)1,1-dimethyl urea (10 mM).
Carbonic anhydrase activity in two N2-fixing cyanobacteria A similar experiment was performed with Ana- baena sp. to observe the induction in CA activityunder different conditions. Initial (0 h) enzyme activity was 4.5 EU mg1 Chl a and there wasa gradual increase in enzyme activity up to 4 h ). Upon further incubation, there was arapid increase in the enzyme activity, which reached 19.6 EU mg1 Chl a at the end of 8 h ofincubation. As compared to N. calcicola, the enzyme activity at 8 h was higher in Anabaena sp.
Incubation of cells of Anabaena sp in dark brought about a slight increase in enzyme activity. However, at the end of 8 h, it reached almost the same level as recorded at 0 h (4.5 mg1 Chl a). If the cells were exposed to DCMU or AZ or EZ in light, the enzymeactivity was of the same level as observed for dark incubated cells. Addition of azide in the cellsincubated in light was correlated with an increase CA-ACTIVITY (EU µg Figure 3. Carbonic anhydrase (CA) activity in Nostoc calcicola. Cyanobacterium was grown in NaHCO3 (75 mM)for 5 days followed by centrifugation and washing. Pellets were suspended in fresh medium and incubated (A) [in dark ('), 10 mM DCMU (m), 50 mM acetazolamide (.)25 mM ethoxyzolamide (&)] and (B) [azide in presence(') and absence (m) of light]. The cells incubated in light without any inhibitor served as control (K). Vertical CA-ACTIVITY (EU µg bars represent 7standard deviations.
and AZ also inhibited the CA activity of N. calcicolaand the activity was 3.1 and 2.9 folds lower as compared to that of control grown cells at 8 h.
Photosystem II inhibitor DCMU also inhibited en- zyme activity, and the activity was 3.6 folds loweras compared to control at 8 h of incubation.
To observe the effect of respiratory inhibitors on CA activity, azide was added to the culture at 0 h of incubation ). The induction in enzymeactivity in the cells incubated in light in the Figure 4. Carbonic anhydrase (CA) activity in Anabaenasp. Cyanobacterium was grown in 5% CO absence of any inhibitors was similar to the earlier followed by centrifugation and washing. Pellets were experiment. The enzyme activity in the cells suspended in fresh medium and incubated (A) [in dark treated with azide and incubated in dark declined ('), 10 mM DCMU (m), 50 mM acetazolamide (.) 25 mM to a level of 0.7 EU mg1 Chl a. A slight increase in ethoxyzolamide (&)] and (B) [azide in presence (') and enzyme activity in azide treated cells when absence (m) of light]. The cells incubated in light without incubated in light, indicated that light may restore any inhibitor served as control (K). Vertical bars the enzyme activity to a certain extent.
represent 7standard deviations.
P. Jaiswal et al.
in activity upto 6 h (but this effect higher in cells grown in ordinary air (low Ci cells) declined thereafter. However, the cells incubated than in cells grown in air enriched with 2–4% CO2, in dark in the presence of azide showed a gradual i.e. high Ci cells decrease in CA activity, and at 8 h it was only 50% However, when the high CO2 grown cells were level of the activity recorded at 0 h.
transferred to low CO2, CA activity was appreciablyenhanced. The present investigation indicates thatCA activity in both the genera increases graduallywhen cells grown in increasing concentration of inorganic carbon are transferred to low Ci condition(ambient CO2).
Biological fixation of CO2 through photosynthesis In order to monitor the induction of CA in the appears to be the easily manageable and most cost cyanobacterial strains used in the present study, effective way to combat the inevitable danger of the cyanobacteria were grown at high Ci conditions increasing the concentration of CO2 in the atmo- for 5 days and then transferred to low Ci in dark/ sphere. Cyanobacteria are known to grow luxur- light in the presence or absence of inhibitors. The iantly in diverse habitats and have been utilized in results clearly indicated that there was induction in reclamation of ‘usar' soil, and can therefore act as CA activity when transferred to low Ci, which was sinks for CO2. This investigation was undertaken to further enhanced in light. These observations are in evaluate the CA activity of two cyanobacterial accordance with that of strains (one of which was a ‘usar' soil isolate) as a who showed such induction of CA activity in C.
prelude to their utilization as CO2 scavengers.
reinhardtii both in light and dark, although in dark Preliminary studies () it was much lower than in light. However, according had indicated that both these organisms could to induction of CA in C.
tolerate upto 250 mM NaHCO3. Interestingly, in the reinhardtii does not take place in dark at all in present study, both the strains behaved differently response to a shift from high Ci to low Ci. It was towards the two carbon sources (CO2 and NaHCO3).
believed that CA induction is regulated at tran- It was observed that both N2-fixing cyanobacteria scriptional level have a basal level of CA activity when grown in and the role of light has not been ambient atmosphere. However, when cells grown in defined in this regard to date. Since transport and elevated level of inorganic carbon are transferred accumulation of HCO is energy dependent, light to ambient CO2, CA activity was enhanced in both could serve as the source of energy through the organisms.
It is now well known that apparent affinity of whether light and dark affect the CA activity cyanobacterial Rubisco is 20 times lower than the directly or indirectly through the supply of ATP concentration of CO2 in the medium, at equilibrium and reductant, DCMU (10 mM) was added to cyano- with air (It is this CCM bacterial cells during incubation phase. It was which enables cyanobacteria to maintain high rates observed that DCMU suppressed the enzyme activ- of CO2 fixation and growth at low CO2 ity indicating the role of NADPH2 resulting from ). CCM is induced when cell grown in high non-cyclic photophosphorylation as an energizer.
CO2 is transferred to low CO2 and induction of CCM These results are consistent with those of is correlated with induction of enzyme CA who reported that addition of DCMU to low-CO2 cells immediately after their transfer from CA enzyme appreciably speeds up the reaction, high CO2 inhibits the induction of CA in C.
i.e. conversion of HCO to CO2, with a rate of 106 M S1 (Regardless of the nature Most cyanobacteria are strict photoautotrophs i taken up, it is HCO3 which is delivered inside and the energy demand for various metabolic cytosol (There is a possibility that processes are fulfilled by the operation of PS II Ci pump may have CA-like function, which operates and PS I (followed by during CO2 transport and may facilitate its energy maintenance of intracellular ATP in the range of uptake. showed that CO2- 2.5–3 uM ATP g1 dry wt. ( dependent photosynthesis in high Ci grown cells of The treatment of cells with DCMU resulted Synechococcus PCC 7942 was inhibited by CA in inhibition of CA activity, and the residual activity inhibitor EZ. The Ci pump is the possible target could be attributed to cyclic photophosphorylation for inhibition by CA inhibitor EZ, and hence was ). However, the ATP consistent with the view that Ci pump may have CA- pool cannot be maintained for a longer period by like function. CA activity was found to be much cyclic photophosphorylation alone ( Carbonic anhydrase activity in two N2-fixing cyanobacteria and net photophophorylation is when Anabaena sp. was cultivated in CO2 atmo- required for induction of CA activity. In addition to sphere and for Nostoc sp., bicarbonate proved to be photophorylation, oxidative phosphorylation can better. The variation in CA activity observed during also contribute to short-term maintenance of the the course of experiment possibly reflects the ATP pool, but transfer of the organism from light to inherent potential and genetic background of the dark results in more than 80% reduction in the ATP organisms with respect to their original habitat, as pool within 15 min ( evident from Anabaena sp. exhibiting higher level Severe reduction in CA activity in dark may be a of CA activity than N. calcicola. Such studies on reflection of reduced level of ATP pool as in dark other cyanobacteria isolated from different ecolo- only oxidative phophorylation is operative and in gical niche may prove helpful for providing a better cyanobacteria the apparent rate of ATP synthesis in understanding of the CCMs and thereby illustrate dark is very slow as compared to that under light the importance of these organisms as CO2 sinks. In ). Since cyanobacter- the present scenario, wherein problems related to ia show a low rate of dark endogenous respiration global warming have reached social dimensions, ) azide, an inhibitor of these ubiquitous organisms may serve to absorb CO2 electron transport at cytochrome oxidase level and alleviate the related problems to a significant inhibits the oxidative phosphorylation and is also known to inhibitrespiratory ATP production in cyanobacteria (In order to ascertain whetherrespiratory electron transport contributes ATP as an energy source for CA induction, azide was added tothe cell in the incubation phase. The results Financial assistance to one of the authors (PJ) in indicated that azide suppressed the enzyme activ- the form of fellowship (GATE-JRF SRF; CSIR-RAship) ity and the effect was more pronounced when azide is gratefully acknowledged. I am also thankful to was added in the dark; it is probable that in light the Head, Department of Botany, Banaras Hindu the effect of azide is somewhat mediated by the University, for providing necessary laboratory facil- supply of ATP from photophosphorylation. To date, the role of light has been discussed at the transportlevel, affecting energization of Ci transport system.
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Int. J. Technology Management, Vol. 64, No. 1, 2014 Image-focused social media for a market analysis of tourism consumption María Pilar Latorre-Martínez* and Tatiana Iñíguez-Berrozpe Escuela de Turismo Universitaria de Zaragoza, Plaza Ecce Homo, 3, 50003, Zaragoza, Spain E-mail: [email protected] E-mail: [email protected] *Corresponding author