Evaluación del uso y de productividad del agua de la Comunidad de Regantes “Río Adaja”

El agua de riego en España se ha reducido del 80 % al 70% tras la rehabilitación de los sistemas tradicionales de riego y el incremento de riegos a presión. La política española ha favorecido la creación de nuevos regadíos con fines sociales, para asentar a la población rural en zonas con disponibilidad de recursos hídricos. Este contexto es aplicable a la Comunidad de Regantes “Rio Adaja” (CCRR), que comenzó a funcionar en 2010 por lo que se la ha elegido para evaluar el uso y productividad del agua y manejo del riego en CCRR modernizadas de la cuenca del Duero. El estudio del manejo del riego se realizó con evaluaciones de campo, el primer año de funcionamiento, en una muestra de sistemas de riego (pivotes centrales, ramales de avance frontal, cobertura total) representativa de los sistemas predominantes en la CCRR. Además, se analizó la carta de riego propuesta por el fabricante de los pivotes centrales, considerando una distribución de caudal continua a lo largo del ramal, y se propuso una nueva carta con emisores de riego que mejoraban la uniformidad de aplicación del agua. El uso del agua en la CCRR se evaluó considerando tanto los indicadores de eficiencia del riego: suministro relativo de riego (anual relative irrigation supply, ARIS), suministro relativo del agua (anual relativewater supply, ARWS), suministro relativo de precipitación (rainfall relative supply, RRS) como los de productividad: productividad del agua (water productivity, WP) productividad del agua de riego (irrigation water productivity, IWP) y productividad de la evapotranspiración (evapotranspiration water productivity, ETWP). Primero, se determinaron: las necesidades hídricas de los cultivos para mantener un contenido de humedad óptimo en su zona radical, el coeficiente dual del cultivo, el agua disponible total (ADP) y agua fácilmente aprovechable (AFA). Después, se estimaron las necesidades hídricas de los cultivos considerando tres años tipo: húmedo, normal y seco correspondientes a la probabilidad de disponibilidad de la precipitación del 20, 50 y 80%, respectivamente. Así mismo, se realizó una encuesta a los regantes de la CCRR para conocer la dosis de riego y rendimiento anual de los cultivos principales durante sus tres años de funcionamiento: 2010-2011, 2011-2012 y 2012-2013.Finalmente, se simuló el efecto del riego y su manejo en la producción de los cultivos y en la productividad del agua. Además, el modelo de simulación AQUACROP (Geerts et al., 2010) se ha utilizado para estudiar la mejora del uso del agua de los cultivos de la CCRR. Dado que este modelo requiere de calibración específica para cada cultivo y cada zona y dado que, de todos los cultivos de la CCRR, sólo el girasol cumplía el requisito, este cultivo fue elegido para estudiar si la estrategia de riego deficitario mejoraría el uso del agua. Los resultados obtenidos indican que el 90% de los sistemas de riego evaluados distribuye el agua con una uniformidad adecuada (CUC≥75%). La simulación de la distribución del agua con las cartas de riego propuestas por el fabricante en pivotes centrales resultó en coeficientes CUC< 75% y sus valores mejoraban al eliminar el aspersor distal. La uniformidad del riego mejoraría si se trabajase con la carta de riego propuesta y que se compone por emisores de riego seleccionados en este estudio. En la mayoría de los cultivos, se aplicó riegos deficitarios (ARIS < 1 en los dos primeros años de funcionamiento de la CCRR y riegos excedentarios (ARIS > 1) el tercer año siendo significativas las diferencias observadas. El manejo del riego fue bueno (0,9 ≤ ARWS ≤1,2) en la mayoría de los cultivos. Así mismo, los indicadores de productividad del agua (WP e IWP (€.m-3)) varió entre cultivos y años estudiados y, destacan los valores observados en: cebolla, patata, zanahoria y cebada. En general, la productividad del agua en los riegos deficitarios fue mayor observándose además, que los índices de productividad mayores correspondieron al año con precipitación mayor aunque, las diferencias entre sus valores medios no fueron significativas en las tres campañas de riego estudiadas. Los resultados apuntan a que la metodología del balance hídrico y las herramientas presentadas en este trabajo (uniformidad de distribución de agua, indicadores de eficiencia del uso de agua y de su productividad) son adecuadas para estudiar el manejo del agua en CCRR. En concreto, la uniformidad en la aplicación del agua de la CCRR mejoraría seleccionando emisores de riego que proporcionen una mayor uniformidad de distribución del agua, lo que conllevaría a cambiar el diámetro de la boquilla de los emisores y/o eliminar el aspersor distal. Así mismo, puede ser de interés adoptar estrategias de riego deficitario para incrementar la productividad en el uso del agua, y las rentas de los regantes, para lo cual se propone utilizar un patrón de cultivos de referencia. Finalmente, el riego deficitario puede ser una estrategia para mejorar la eficiencia y productividad en el uso del agua de la CCRR siempre que lleve asociado un manejo del riego adecuado que resulta, relativamente, más fácil cuando se dispone de sistemas de riego con una uniformidad de aplicación alta. Sin embargo su aplicación no sería aconsejable en los cultivos de remolacha azucarera, regado con sistemas de riego con un coeficiente de uniformidad de Christiansen CUC < 75%, y maíz, regado con sistemas de riego con un coeficiente de uniformidad de Christiansen CUC < 65%. ABSTRACT The irrigation scheme modernization and the increase of sprinkler irrigation area have reduced the irrigation water use from 80 to 70%. The national irrigation policy favored the creation of new irrigation schemes with the purpose to settle the rural population in areas with availability in water resources. Within this context, the irrigation district “Río Adaja” (CCRR) started in 2010 so, it has been chosen as a case study to evaluate the water use and the irrigation management in a modernized CCRR. Several field evaluations were carried out during the first operation year, in a sample of irrigation systems (center pivot, moving lateral and solid set) selected among all the systems in the CCRR. Likewise, the manufacturer irrigation chart for the center pivot systems has been considered and the pressure and discharge distribution along the pivot have been estimated, assuming a continuous flow along the pipe. Then; the sprinkler nozzles were selected order to increase the uniformity on water application. The water use in the CCRR has been assessed by considering the water use efficiency indicators: annual relative irrigation supply (ARIS), annual relative water supply (ARWS), relative rainfall supply (RRS) and also the productivity indicators: water productivity (WP), irrigation water productivity (IWP) and evapotranspiration water productivity (ETWP). On the one hand, it has been determined the crop water requirement (to maintain the optimal soil water content in the rooting zone), the dual crop coefficient, the total available water and the readily available water. The crop water requirement was estimated by considering the typical wet, normal and dry years which correspond to the probability of effective precipitation exceedance of 20, 50 and 80%, respectively. On the other hand, the irrigation depth and crop yield by irrigation campaign have been considered for the main crops in the area. This information was obtained from a farmer’s survey in 2010-2011, 2011-2012 and 2012-2013. For sunflower, the irrigation effect and its management on the crop yield and water productivity have been simulated. Also a deficit irrigation strategy, which improves the water resources, has been determined by means of AQUACROP (FAO). The results showed that 90% of the evaluated irrigation systems have adequate irrigation water application uniformity (CUC ≥ 75%). The CUC values in center pivots, which were calculated using the manufacturer irrigation chart, are below < 75% . However, these values would increase with the change of emitter nozzle to the proposed nozzles selection. The results on water use showed a deficit irrigation management (ARIS < 1), in most of crops during the first two operation years, and an excess in irrigation for the third year (ARIS > 1) although non-significant difference was observed. In most cases, the management of irrigation is adequate (0,9≤ ARWS≤ 1,2) although there are differences among crops. Likewise, the productivity indicators (WP and IWP (€.m-3)) varied among crops and with irrigation events. The highest values corresponded to onion, potato, carrot and barley. The values for deficit irrigation were the highest and the productivity indicators increased the year with the highest effective precipitation. Nevertheless, the differences between the average values of these indicators by irrigation campaign were non-significant. This study highlights that the soil water balance methodology and other tools used in the methodology are adequate to study the use and productivity of water in the irrigation district. In fact, the water use in this CCRR can be improved if the irrigation systems were designed with higher water distribution uniformity what would require the change of sprinkler nozzles and/or eliminate the end gun. Likewise, it is advisable to set up deficit irrigation strategies to increase the water productivity taking into account certain limits on water application uniformities. In this respect, a reference cropping pattern has been proposed and the limits for water uniformity have been calculated for several crops.

Functional transitions in myosin: Formation of a critical salt-bridge and transmission of effect to the sensitive tryptophan

For analyzing the mechanism of energy transduction in the “motor” protein, myosin, it is opportune both to model the structural change in the hydrolytic transition, ATP (myosin-bound) + H2O → ADP⋅Pi (myosin-bound) and to check the plausibility of the model by appropriate site-directed mutations in the functional system. Here, we made a series of mutations to investigate the role of the salt-bridge between Glu-470 and Arg-247 (of chicken smooth muscle myosin) that has been inferred from crystallography to be a central feature of the transition [Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M., & Rayment, I. (1995) Biochemistry 34, 8960–8972]. Our results suggest that whether in the normal, or in the inverted, direction an intact salt-bridge is necessary for ATP hydrolysis, but when the salt-bridge is in the inverted direction it does not support actin activation. Normally, fluorescence changes result from adding nucleotides to myosin; these signals are reported by Trp-512 (of chicken smooth muscle myosin). Our results also suggest that structural impairments in the 470–247 region interfere with the transmission of these signals to the responsive Trp.

Purification and characterization of acetone carboxylase from Xanthobacter strain Py2

Acetone metabolism in the aerobic bacterium Xanthobacter strain Py2 proceeds by a carboxylation reaction forming acetoacetate as the first detectable product. In this study, acetone carboxylase, the enzyme catalyzing this reaction, has been purified to homogeneity and characterized. Acetone carboxylase was comprised of three polypeptides with molecular weights of 85,300, 78,300, and 19,600 arranged in an α2β2γ2 quaternary structure. The carboxylation of acetone was coupled to the hydrolysis of ATP and formation of 1 mol AMP and 2 mol inorganic phosphate per mol acetoacetate formed. ADP was also formed during the course of acetone consumption, but only accumulated at low, substoichiometric levels (≈10% yield) relative to acetoacetate. Inorganic pyrophosphate could not be detected as an intermediate or product of acetone carboxylation. In the absence of CO2, acetone carboxylase catalyzed the acetone-dependent hydrolysis of ATP to form both ADP and AMP, with ADP accumulating to higher levels than AMP during the course of the assays. Acetone carboxylase did not have inorganic pyrophosphatase activity. Acetone carboxylase exhibited a Vmax for acetone carboxylation of 0.225 μmol acetoacetate formed min−1⋅mg−1 at 30°C and pH 7.6 and apparent Km values of 7.80 μM (acetone), 122 μM (ATP), and 4.17 mM (CO2 plus bicarbonate). These studies reveal molecular properties of the first bacterial acetone-metabolizing enzyme to be isolated and suggest a novel mechanism of acetone carboxylation coupled to ATP hydrolysis and AMP and inorganic phosphate formation.

Molecular cloning of a peroxisomal Ca2+-dependent member of the mitochondrial carrier superfamily

A cDNA from a novel Ca2+-dependent member of the mitochondrial solute carrier superfamily was isolated from a rabbit small intestinal cDNA library. The full-length cDNA clone was 3,298 nt long and coded for a protein of 475 amino acids, with four elongation factor-hand motifs located in the N-terminal half of the molecule. The 25-kDa N-terminal polypeptide was expressed in Escherichia coli, and it was demonstrated that it bound Ca2+, undergoing a reversible and specific conformational change as a result. The conformation of the polypeptide was sensitive to Ca2+ which was bound with high affinity (Kd ≈ 0.37 μM), the apparent Hill coefficient for Ca2+-induced changes being about 2.0. The deduced amino acid sequence of the C-terminal half of the molecule revealed 78% homology to Grave disease carrier protein and 67% homology to human ADP/ATP translocase; this sequence homology identified the protein as a new member of the mitochondrial transporter superfamily. Northern blot analysis revealed the presence of a single transcript of about 3,500 bases, and low expression of the transporter could be detected in the kidney but none in the liver. The main site of expression was the colon with smaller amounts found in the small intestine proximal to the ileum. Immunoelectron microscopy localized the transporter in the peroxisome, although a minor fraction was found in the mitochondria. The Ca2+ binding N-terminal half of the transporter faces the cytosol.

Significance of chaperonin 10-mediated inhibition of ATP hydrolysis by chaperonin 60

Chaperonins are essential for the folding of proteins in bacteria, mitochondria, and chloroplasts. We have functionally characterized the yeast mitochondrial chaperonins hsp60 and hsp10. In the presence of ADP, one molecule of hsp10 binds to hsp60 with an apparent Kd of 0.9 nM and a second molecule of hsp10 binds with a Kd of 24 nM. In the presence of ATP, the purified yeast chaperonins mediate the refolding of mitochondrial malate dehydrogenase. Hsp10 inhibits the ATPase activity of hsp60 by about 40%. Hsp10(P36H) is a point mutant of hsp10 that confers temperature-sensitive growth to yeast. Consistent with the in vivo phenotype, refolding of mitochondrial malate dehydrogenase in the presence of purified hsp10(P36H) and hsp60 is reduced at 25°C and abolished at 30°C. The affinity of hsp10(P36H) to hsp60 as well as to Escherichia coli GroEL is reduced. However, this decrease in affinity does not correlate with the functional defect, because hsp10(P36H) fully assists the GroEL-mediated refolding of malate dehydrogenase at 30°C. Refolding activity, rather, correlates with the ability of hsp10(P36H) to inhibit the ATPase of GroEL but not that of hsp60. Based on our findings, we propose that the inhibition of ATP hydrolysis is mechanistically coupled to chaperonin-mediated protein folding.

Cytokine suppression of protease activation in wild-type p53-dependent and p53-independent apoptosis

M1 myeloid leukemic cells overexpressing wild-type p53 undergo apoptosis. This apoptosis can be suppressed by some cytokines, protease inhibitors, and antioxidants. We now show that induction of apoptosis by overexpressing wild-type p53 is associated with activation of interleukin-1β-converting enzyme (ICE)-like proteases, resulting in cleavage of poly(ADP- ribose) polymerase and the proenzyme of the ICE-like protease Nedd-2. Activation of these proteases and apoptosis were suppressed by the cytokine interleukin 6 or by a combination of the cytokine interferon γ and the antioxidant butylated hydroxyanisole, and activation of poly(ADP-ribose) polymerase and apoptosis were suppressed by some protease inhibitors. In a clone of M1 cells that did not express p53, vincristine or doxorubicin induced protease activation and apoptosis that were not suppressed by protease inhibitors, but were suppressed by interleukin 6. In another myeloid leukemia (7-M12) doxorubicin also induced protease activation and apoptosis that were not suppressed by protease inhibitors, but were suppressed by granulocyte–macrophage colony-stimulating factor. The results indicate that (i) overexpression of wild-type p53 by itself or treatment with cytotoxic compounds in wild-type p53-expressing or p53-nonexpressing myeloid leukemic cells is associated with activation of ICE-like proteases; (ii) cytokines exert apoptosis-suppressing functions upstream of protease activation; (iii) the cytotoxic compounds induce additional pathways in apoptosis; and (iv) cytokines can also suppress these other components of the apoptotic machinery.

Subunit rotation in Escherichia coli FoF1–ATP synthase during oxidative phosphorylation

We report evidence for proton-driven subunit rotation in membrane-bound FoF1–ATP synthase during oxidative phosphorylation. A βD380C/γC87 crosslinked hybrid F1 having epitope-tagged βD380C subunits (βflag) exclusively in the two noncrosslinked positions was bound to Fo in F1-depleted membranes. After reduction of the β–γ crosslink, a brief exposure to conditions for ATP synthesis followed by reoxidation resulted in a significant amount of βflag appearing in the β–γ crosslinked product. Such a reorientation of γC87 relative to the three β subunits can only occur through subunit rotation. Rotation was inhibited when proton transport through Fo was blocked or when ADP and Pi were omitted. These results establish FoF1 as the second example in nature where proton transport is coupled to subunit rotation.

Activation of a caspase 3-related cysteine protease is required for glutamate-mediated apoptosis of cultured cerebellar granule neurons

Neurotoxicity induced by overstimulation of N-methyl-d-aspartate (NMDA) receptors is due, in part, to a sustained rise in intracellular Ca2+; however, little is known about the ensuing intracellular events that ultimately result in cell death. Here we show that overstimulation of NMDA receptors by relatively low concentrations of glutamate induces apoptosis of cultured cerebellar granule neurons (CGNs) and that CGNs do not require new RNA or protein synthesis. Glutamate-induced apoptosis of CGNs is, however, associated with a concentration- and time-dependent activation of the interleukin 1β-converting enzyme (ICE)/CED-3-related protease, CPP32/Yama/apopain (now designated caspase 3). Further, the time course of caspase 3 activation after glutamate exposure of CGNs parallels the development of apoptosis. Moreover, glutamate-induced apoptosis of CGNs is almost completely blocked by the selective cell permeable tetrapeptide inhibitor of caspase 3, Ac-DEVD-CHO but not by the ICE (caspase 1) inhibitor, Ac-YVAD-CHO. Western blots of cytosolic extracts from glutamate-exposed CGNs reveal both cleavage of the caspase 3 substrate, poly(ADP-ribose) polymerase, as well as proteolytic processing of pro-caspase 3 to active subunits. Our data demonstrate that glutamate-induced apoptosis of CGNs is mediated by a posttranslational activation of the ICE/CED-3-related cysteine protease caspase 3.

Nucleotide-dependent conformational change near the fulcrum region in Dictyostelium myosin II

In skeletal muscle myosin, the reactive thiols (SH1 and SH2) are close to a proposed fulcrum region that is thought to undergo a large conformational change. The reactive thiol region is thought to transmit the conformational changes induced by the actin–myosin–ATP interactions to the lever arm, which amplifies the power stroke. In skeletal muscle myosin, SH1 and SH2 can be chemically cross-linked in the presence of nucleotide, trapping the nucleotide in its pocket. Although the flexibility of the reactive thiol region has been well studied in skeletal muscle myosin, crystal structures of truncated nonmuscle myosin II from Dictyostelium in the presence of various ATP analogs do not show changes at the reactive thiol region that would be consistent with the SH1–SH2 cross-linking observed for muscle myosin. To examine the dynamics of the reactive thiol region in Dictyostelium myosin II, we have examined a modified myosin II that has cysteines at the muscle myosin SH1 and SH2 positions. This myosin is specifically cross-linked at SH1–SH2 by a chemical cross-linker in the presence of ADP, but not in its absence. Furthermore, the cross-linked species traps the nucleotide, as in the case of muscle myosin. Thus, the Dictyostelium myosin II shares the same dynamic behavior in the fulcrum region of the molecule as the skeletal muscle myosin. This result emphasizes the importance of nucleotide-dependent changes in this part of the molecule.

Kinesin’s processivity results from mechanical and chemical coordination between the ATP hydrolysis cycles of the two motor domains

Kinesin is a processive motor protein: A single molecule can walk continuously along a microtubule for several micrometers, taking hundreds of 8-nm steps without dissociating. To elucidate the biochemical and structural basis for processivity, we have engineered a heterodimeric one-headed kinesin and compared its biochemical properties to those of the wild-type two-headed molecule. Our construct retains the functionally important neck and tail domains and supports motility in high-density microtubule gliding assays, though it fails to move at the single-molecule level. We find that the ATPase rate of one-headed kinesin is 3–6 s−1 and that detachment from the microtubule occurs at a similar rate (3 s−1). This establishes that one-headed kinesin usually detaches once per ATP hydrolysis cycle. Furthermore, we identify the rate-limiting step in the one-headed hydrolysis cycle as detachment from the microtubule in the ADP⋅Pi state. Because the ATPase and detachment rates are roughly an order of magnitude lower than the corresponding rates for two-headed kinesin, the detachment of one head in the homodimer (in the ADP⋅Pi state) must be accelerated by the other head. We hypothesize that this results from internal strain generated when the second head binds. This idea accords with a hand-over-hand model for processivity in which the release of the trailing head is contingent on the binding of the forward head. These new results, together with previously published ones, allow us to propose a pathway that defines the chemical and mechanical cycle for two-headed kinesin.

Syncytiotrophoblastic giant cells in teratocarcinoma-like tumors derived from Parp-disrupted mouse embryonic stem cells

The enzyme poly(ADP-ribose) polymerase (Parp) catalyzes poly(ADP-ribosyl)ation reaction and is involved in DNA repair and cell death induction upon DNA damages. Meanwhile, poly(ADP-ribosyl)ation of chromosome-associated proteins is suggested to be implicated in the regulation of gene expression and cellular differentiation, both of which are important in tumorigenesis. To investigate directly the role of Parp deficiency in tumorigenicity and differentiation of embryonic stem (ES) cells during tumor formation, studies were conducted by using wild-type J1 (Parp+/+) ES cells and Parp+/− and Parp−/− ES clones generated by disrupting Parp exon 1. These ES cells, irrespective of the Parp genotype, produced tumors phenotypically similar to teratocarcinoma when injected s.c. into nude mice. Remarkably, all tumors derived from Parp−/− clones contained syncytiotrophoblastic giant cells (STGCs), which possess single or multiple megalo-nuclei. The STGCs were present within large areas of intratumoral hemorrhage. In contrast, neither STGC nor hemorrhage was observed in tumors of both wild-type J1 cells and Parp+/− clones. Electron microscopic examination showed that the STGCs possess microvilli on the cell surface and contained secretory granules in the cytoplasm. Furthermore, the cytoplasms of STGCs were strongly stained with antibody against mouse prolactin, which could similarly stain trophoblasts in placenta. These morphological and histochemical features indicate that the STGCs in teratocarcinoma-like tumors derived from Parp−/− clones belong to the trophoblast cell lineage. Our findings thus suggest that differentiation of ES cells into STGCs was possibly induced by the lack of Parp during the development of teratocarcinoma.

The kinetic mechanism of myosin V

Myosin V is an unconventional myosin proposed to be processive on actin filaments, analogous to kinesin on a microtubule [Mehta, A. D., et al. (1999) Nature (London) 400, 590–593]. To ascertain the unique properties of myosin V that permit processivity, we undertook a detailed kinetic analysis of the myosin V motor. We expressed a truncated, single-headed myosin V construct that bound a single light chain to study its innate kinetics, free from constraints imposed by other regions of the molecule. The data demonstrate that unlike any previously characterized myosin a single-headed myosin V spends most of its kinetic cycle (>70%) strongly bound to actin in the presence of ATP. This kinetic tuning is accomplished by increasing several of the rates preceding strong binding to actin and concomitantly prolonging the duration of the strongly bound state by slowing the rate of ADP release. The net result is a myosin unlike any previously characterized, in that ADP release is the rate-limiting step for the actin-activated ATPase cycle. Thus, because of a number of kinetic adaptations, myosin V is tuned for processive movement on actin and will be capable of transporting cargo at lower motor densities than any other characterized myosin.

ATPases and phosphate exchange activities in magnesium chelatase subunits of Rhodobacter sphaeroides

Three separate proteins, BchD, BchH, and BchI, together with ATP, insert magnesium into protoporphyrin IX. An analysis of ATP utilization by the subunits revealed the following: BchH catalyzed ATP hydrolysis at the rate of 0.9 nmol per min per mg of protein. BchI and BchD, tested individually, had no ATPase activity but, when combined, hydrolyzed ATP at the rate of 117.9 nmol/min per mg of protein. Magnesium ions were required for the ATPase activities of both BchH and BchI+D, and these activities were inhibited 50% by 2 mM o-phenanthroline. BchI additionally catalyzed a phosphate exchange reaction from ATP and ADP. We conclude that ATP hydrolysis by BchI+D is required for an activation step in the magnesium chelatase reaction, whereas ATPase activity of BchH and the phosphate exchange activity of BchI participate in subsequent reactions leading to the insertion of Mg2+ into protoporphyrin IX.

A functional homolog of a yeast tRNA splicing enzyme is conserved in higher eukaryotes and in Escherichia coli

tRNA splicing in the yeast Saccharomyces cerevisiae requires an endonuclease to excise the intron, tRNA ligase to join the tRNA half-molecules, and 2′-phosphotransferase to transfer the splice junction 2′-phosphate from ligated tRNA to NAD, producing ADP ribose 1′′–2′′ cyclic phosphate (Appr>p). We show here that functional 2′-phosphotransferases are found throughout eukaryotes, occurring in two widely divergent yeasts (Candida albicans and Schizosaccharomyces pombe), a plant (Arabidopsis thaliana), and mammals (Mus musculus); this finding is consistent with a role for the enzyme, acting in concert with ligase, to splice tRNA or other RNA molecules. Surprisingly, functional 2′-phosphotransferase is found also in the bacterium Escherichia coli, which does not have any known introns of this class, and does not appear to have a ligase that generates junctions with a 2′-phosphate. Analysis of the database shows that likely members of the 2′-phosphotransferase family are found also in one other bacterium (Pseudomonas aeruginosa) and two archaeal species (Archaeoglobus fulgidus and Pyrococcus horikoshii). Phylogenetic analysis reveals no evidence for recent horizontal transfer of the 2′-phosphotransferase into Eubacteria, suggesting that the 2′-phosphotransferase has been present there since close to the time that the three kingdoms diverged. Although 2′-phosphotransferase is not present in all Eubacteria, and a gene disruption experiment demonstrates that the protein is not essential in E. coli, the continued presence of 2′-phosphotransferase in Eubacteria over large evolutionary times argues for an important role for the protein.

Involvement of caspase-dependent activation of cytosolic phospholipase A2 in tumor necrosis factor-induced apoptosis

Tumor necrosis factor (TNF)-induced apoptosis is mediated by caspases, which are cysteine proteases related to interleukin 1β-converting enzyme. We report here that TNF-induced activation of caspases results in the cleavage and activation of cytosolic phospholipase A2 (cPLA2) and that activated cPLA2 contributes to apoptosis. Inhibition of caspases by expression of a cowpox virus-derived inhibitor, CrmA, or by a specific tetrapeptide inhibitor of CPP32/caspase-3, acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), inhibited TNF-induced activation of cPLA2 and apoptosis. TNF-induced activation of cPLA2 was accompanied by a cleavage of the 100-kDa cPLA2 to a 70-kDa proteolytic fragment. This cleavage was inhibited by Ac-DEVD-CHO in a similar manner as that of poly(ADP)ribose polymerase, a known substrate of CPP32/caspase-3. Interestingly, specific inhibition of cPLA2 enzyme activity by arachidonyl trifluoromethylketone (AACOCF3) partially inhibited TNF-induced apoptosis without inhibition of caspase activity. Thus, our results suggest a novel caspase-dependent activation pathway for cPLA2 during apoptosis and identify cPLA2 as a mediator of TNF-induced cell death acting downstream of caspases.