Phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxylase kinase isoenzymes play an important role in the filling and quality of Arabidopsis thaliana seed

Three plant-type phosphoenolpyruvate carboxylase (PPC1 to PPC3) and two phosphoenolpyruvate carboxylase kinase (PPCKs: PPCK1 and 2) genes are present in the Arabidopsis thaliana genome. In seeds, all PPC genes were found to be expressed. Examination of individual ppc mutants showed little reduction of PEPC protein and global activity, with the notable exception of PPC2 which represent the most abundant PEPC in dry seeds. Ppc mutants exhibited moderately lower seed parameters (weight, area, yield, germination kinetics) than wild type. In contrast, ppck1-had much altered (decreased) yield. At the molecular level, ppc3-was found to be significantly deficient in global seed nitrogen (nitrate, amino-acids, and soluble protein pools). Also, N-deficiency was much more marked in ppck1-, which exhibited a tremendous loss of 95% and 90% in nitrate and proteins, respectively. The line ppck2-had accumulated amino-acids but lower levels of soluble proteins. Regarding carboxylic acid pools, Krebs cycle intermediates were found to be diminished in all mutants; this was accompanied by a consistent decrease in ATP. Lipids were stable in ppc mutants, however ppck1-seeds accumulated more lipids while ppck2-seeds showed high level of polyunsaturated fatty acid oleic and linolenic (omega 3). Altogether, the results indicate that the complete PEPC and PPCK family are needed for normal C/N metabolism ratio, growth, development, yield and quality of the seed.Ministerio de Economía y Competitividad AGL2012-35708, AGL2016-75413-PJunta de Andalucía P12-FQM-48

Phytoplankton, bacterioplankton and nitrate reductase activity distribution in relation to physical structure in the northern Alborán Sea and Gulf of Cadiz (southern Iberian Peninsula)

The IctioAlborán-Cadiz 95 cruise (July 1995) covered the northwest sector of the Alborán Sea, the Straits of Gibraltar and the Gulf of Cadiz. We present and discuss the distribution of phytoand bacterioplankton abundance, nitrate concentration and nitrate reductase (NR) activity according to circulation patterns described previously in the literature. The water-column integrated values of chlorophyll-a in the Alborán Sea were higher and more variable (12 to 318 mg m⁻³) than in the Gulf of Cadiz (3 to 49 mg m⁻³). This reflects the higher dynamism of the Alborán sector, related to the existence of characteristic mesoscale physical structures. The plankton (2-80 μm equivalent spherical diameter) in the upwelling region of the Alborán Sea had a biovolume 30 times higher than in the nutrient-poorer water of the Gulf of Cadiz. Large organisms were abundant and dominated the size spectrum of one coastal station in the Alborán zone, whereas the slope of the size spectrum became more negative at the Gulf of Cadiz station, as a consequence of the dominance of small cells. Bacterial abundance maxima ( 3 X 10⁶ bact ml⁻¹) also occurred in the upwelling region. We found relatively high nitrate and chlorophyll a concentrations in Cape Trafalgar's cold surface waters, probably due to the interaction between topography and strong tidal currents. The spatial pattern of the NR activity is discussed according to chlorophyll a, nitrate and bacteria distributions. The existence of high NR activity but low chlorophyll a and bacterial abundance suggests that this could be related with dissimilatory activity in large-sized particles.La campaña IctioAlborán-Cádiz 95 (julio de 1995) cubrió el sector noroeste del mar de Alborán, estrecho de Gibraltar y golfo de Cádiz. En este trabajo se presentan y discuten las distribuciones de abundancia de fitoplancton y bacterioplancton, concentración de nitrato y actividad nitrato reductasa (NR) de acuerdo con los patrones de circulación descritos en la bibliografía. Los valores de clorofila a integrados en la columna de agua en Alborán fueron mayores y más variables (12 a 318 mg/m³) que en el golfo de Cádiz (3 a 49 mg/m³). Esto refleja el mayor dinamismo de la zona de Alborán, relacionado con la existencia de estructuras físicas características a mesoescala. El análisis de la comunidad nano-microplanctónica (2-80 μm, diametro esférico equivalente) de la región de afloramiento del mar de Alborán mostró un biovolumen 30 veces mayor que el de aguas más pobres en nutrientes del golfo de Cádiz. Por otro lado, el espectro de tamaños en una estación costera de Alborán está dominado por organismos planctónicos grandes, mientras que la pendiente del espectro de tamaño se hace más negativa en una estación del golfo de Cádiz, como consecuencia de la mayor abundancia de plancton de pequeño tamaño. En la región de afloramiento se dieron también máximos de abundancia bacteriana ( 3 X 10⁶ bact ml⁻¹). Se han encontrado valores relativamente altos de nitrato y clorofila a en las frías aguas superficiales cercanas al cabo de Trafalgar, lo que se debe, probablemente, a la interacción entre topografía y fuertes corrientes mareales. Se discute el patrón espacial de la actividad NR en relación con las distribuciones de clorofila a, nitrato y bacterias. En muestras con valores elevados de actividad NR pero bajos de clorofila y abundancia de bacterias, se sugiere que esta actividad se relaciona con actividad disimilatoria en agregados de gran tamaño.Instituto Español de Oceanografí

New insights into the post-translational modification of multiple phosphoenolpyruvate carboxylase isoenzymes by phosphorylation and monoubiquitination during sorghum seed development and germination

Phosphoenolpyruvate carboxylase (PEPC; E.C. 4.1.1.31) was characterized in developing and germinating sorghum seeds, focusing on the transcript and polypeptide abundance of multiple plant-type phosphoenolpyruvate carboxylase (PTPC) genes, and the post-translational modification of each isoenzyme by phosphorylation versus monoubiquitination during germination. We observed high levels of SbPPC4 (Sb07g014960) transcripts during early development (stage I), and extensive transcript abundance of SbPPC2 (Sb02g021090) and SbPPC3 (Sb04g008720) throughout the entire life cycle of the seed. Although tandem mass spectrometry (MS) analysis of immunopurified PTPC indicated that four different PTPC isoenzymes were expressed in the developing and germinating seeds, SbPPC3 was the most abundant isozyme of the developing seed, and of the embryo and the aleurone layer of germinating seeds. In vivo phosphorylation of the different PTPC isoenzymes at their conserved N-terminal seryl phosphorylation site during germination was also established by MS/MS analysis. Furthermore, three of the four isoenzymes were partially monoubiquitinated, with MS/MS pinpointing SbPPC2 and SbPPC3 monoubiquitination at the conserved Lys-630 and Lys-624 residues, respectively. Our results demonstrate that monoubiquitination and phosphorylation simultaneously occur in vivo with different PTPC isozymes during seed germination. In addition, we show that PTPC monoubiquitination in germinating sorghum seeds always increases at stage II (emergence of the radicle), is maintained during the aerobic period of rapid cell division and reserve mobilization, and remains relatively constant until stage IV–V when coleoptiles initiate the formation of the photosynthetic tissues.España, Minsiterio de Economía y Competitividad AGL2012-35708,España, Junta de Andalucía P06-CVI-02186 and BIO29

Diseño de intercambio vial tipo glorieta en la intersección vial entre las av Cáceres y la calle San Ramón – Piura – región Piura

En esta Tesis de investigación titulada “Diseño de intercambio vial tipo glorieta en la intersección vial entre las av Cáceres y la calle San Ramón – Piura – Región Piura”, se analiza la mencionada intersección semaforizada, en la que se identificó que no existe una alternativa de solución frente al caos vehicular que se da en ella. El objetivo principal de esta investigación es dar una solución integral a la problemática existente a través de una rotonda o glorieta, la cual reduzca el congestionamiento vehicular que muchas veces genera accidentes de tránsito. Se ha realizado el estudio de tráfico con un conteo manual de vehículos por siete (7) días, de lunes a domingo durante las 24 horas del día y se han tomado los tiempos semafóricos en la intersección, después de recopilar la información de campo se calcularon volúmenes y flujos vehiculares para intervalos de 15 minutos que sirvieron para la determinación del día de máxima demanda vehicular y las horas pico. Se determinó la capacidad vehicular, grado de saturación y nivel de servicio en la intersección semaforizada aplicando la metodología HCM 2010. Posterior a ello, se realizó el diseño geométrico de una glorieta, en base a parámetros establecidos por la normativa peruana DG-2018 y también haciendo uso de normas internacionales como la española y la estadounidense. Finalmente se realizó la evaluación de la capacidad vehicular y nivel de servicio en la intersección simulando la implementación de la rotonda diseñada mediante la utilización de metodología HCM 2010. De esta manera se determinó las mejoras que genera la implementación de una glorieta.In this Research Thesis entitled “Diseño de intercambio vial tipo glorieta en la intersección vial entre las av Cáceres y la calle San Ramón – Piura – Región Piura”, the aforementioned traffic light intersection is analyzed, in which it was identified that there is no alternative solution to the vehicular chaos that occurs in it. The main objective of this research is to provide a comprehensive solution to the existing problem through a roundabout or roundabout, which reduces vehicular congestion that often generates traffic accidents. The traffic study has been carried out with a manual vehicle count for seven (7) days, from Monday to Sunday 24 hours a day and the traffic signals at the intersection have been taken, after compiling the field information, they were calculated vehicle volumes and flows for 15-minute intervals that were used to determine the day of maximum vehicle demand and peak hours. The vehicle capacity, degree of saturation and service level at the signalized intersection were determined using the 2010 HCM methodology. Subsequently, the geometric design of a gazebo was carried out, based on parameters established by Peruvian regulations DG-2018 and also making use of international standards such as Spanish and American. Finally, the evaluation of the vehicle capacity and service level at the intersection was performed simulating the implementation of the roundabout designed using the 2010 HCM methodology. In this way, the improvements generated by the implementation of a roundabout were determined.Tesi

Red-light effects sensitized by methylene blue on nitrate reductase from spinach (Spinacia Oleracea L.) leaves

Nitrate reductase from spinach (Spinacia oleracea L.) leaves, which had been inactivated in vitro by incubation with N A D H and cyanide, was fully reactivated in minutes when irradiated in anaerobic conditions with red light in the presence o f m ethylene blue. Both the rate and the extent of reactivation increased with light intensity ( 6 to 100 W - m “ 2) and dye concentration (1 to 10 jiM). On the contrary, photoreactivation was com pletely abolished when N A D H or ethylenediaminetetra-acetic acid were present during irradiation. W e propose that methylene blue, when photo excited, exhibits a redox potential positive enough to reoxidise the CN~-reduced molybdenum complex settled in the inactive enzyme, thus causing its reactivation. On the other hand, prolonged irradiation o f nitrate reductase, under air and in the presence o f methylene blue, promoted an oxygen-dependent irreversible inactivation o f the two partial activities of the enzyme. This inactivation was markedly enhanced in 77% deuterated water and greatly prevented by azide, which indicates that singlet oxygen is the species primarily involved in the photooxidative inactivation o f the enzym

In vivo monoubiquitination of anaplerotic phosphoenolpyruvate carboxylase occurs at Lys624 in germinating sorghum seeds

Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is an important cytosolic regulatory enzyme that plays a pivotal role in numerous physiological processes in plants, including seed development and germination. Previous studies demonstrated the occurrence of immunoreactive PEPC polypeptides of ~110kDa and 107kDa (p110 and p107, respectively) on immunoblots of clarified extracts of germinating sorghum (Sorghum bicolor) seeds. In order to establish the biochemical basis for this observation, a 460kDa PEPC heterotetramer composed of an equivalent ratio of p110 and p107 subunits was purified to near homogeneity from the germinated seeds. Mass spectrometry established that p110 and p107 are both encoded by the same plant-type PEPC gene (CP21), but that p107 was in vivo monoubiquitinated at Lys624 to form p110. This residue is absolutely conserved in vascular plant PEPCs and is proximal to a PEP-binding/catalytic domain. Anti-ubiquitin IgG immunodetected p110 but not p107, whereas incubation with a deubiquitinating enzyme (USP-2 core) efficiently converted p110 into p107, while relieving the enzyme’s feedback inhibition by l-malate. Partial PEPC monoubiquitination was also detected during sorghum seed development. It is apparent that monoubiquitination at Lys624 is opposed to phosphorylation at Ser7 in terms of regulating the catalytic activity of sorghum seed PEPC. PEPC monoubiquitination is hypothesized to fine-tune anaplerotic carbon flux according to the cell’s immediate physiological requirements for tricarboxylic acid cycle intermediates needed in support of biosynthesis and carbon–nitrogen interactions.España, Ministerio de Economía y Competitividad AGL2012-35708España Junta de Andalucía P06-CVI-02186 BIO29

Proline content of sugar beet storage roots: Response to water deficit and nitrogen fertilization at field conditions

Drought stress is one of the major factors causing profit loss of the sugar beet crop. The accumulation of proline, an indicator of water stress, has been reported in response to osmotic and salt stress in sugar beet leaves, but there is little information about its levels in storage roots. Proline in storage roots is potentially useful as indicator of situations that lead to decreased yield and quality of the root, such as drought stress and excess nitrogen. This study has been focused towards proline quantification in storage roots from field trials concerning irrigation, sugar beet variety and nitrogen fertilization. Water deficit is the main factor leading to proline accumulation in sugar beet roots. Excess N supply also increases proline levels, partially by increasing leaf area index (LAI) and exacerbating drought stress. The two varieties studied (Claudia and Ramona) had different responses to water shortage and to nitrogen. Maximum proline levels were measured in Claudia roots subjected to a combination of water shortage and excess N. A positive and significant correlation was found between proline and glucose levels in sugar beet roots, pointing to a relationship between stress responses, carbohydrate catabolism, and proline and glucose accumulation. This proposal was supported by the effect of treatments with di-1-p-menthene (anti-transpirant) and with DMDP (2,5-dihydroxymethyl-3,4-dihydroxypyrrolidina, a glycosidase inhibitor), which lead to decreased level of proline in non-irrigated Claudia sugar beet roots.Subdirección General de Proyectos de Investigación Científica y Técnica de España y FEDER (Unión Europea) IFD97-0893-CO3-01Universidad de Sevilla USE-MONTE P-96Junta de Andalucía (PAI) CVI 29

Polymerized-Type I Collagen Downregulates Inflammation and Improves Clinical Outcomes in Patients with Symptomatic Knee Osteoarthritis Following Arthroscopic Lavage: A Randomized, Double-Blind, and Placebo-Controlled Clinical Trial

Objectives. Polymerized-type I collagen (polymerized collagen) is a downmodulator of inflammation and cartilage regenerator biodrug. Aim. To evaluate the effect of intraarticular injections of polymerized collagen after arthroscopic lavage on inflammation and clinical improvement in patients with knee osteoarthritis (OA). Methods. Patients (n = 19) were treated with 6 intraarticular injections of 2 mL of polymerized collagen (n = 10) or 2 mL of placebo (n = 9) during 3 months. Followup was 3 months. The primary endpoints included Lequesne index, pain on a visual analogue scale (VAS), WOMAC, analgesic usage, the number of Tregs and proinflammatory/anti-inflammatory cytokine-expressing peripheral cells. Secondary outcomes were Likert score and drug evaluation. Clinical and immunological improvement was determined if the decrease in pain exceeds 20 mm on a VAS, 20% of clinical outcomes, and inflammatory parameters from baseline. Urinary levels of C-terminal crosslinking telopeptide of collagen type II (CTXII) and erythrocyte sedimentation rate (ESR) were determined. Results. Polymerized collagen was safe and well tolerated. Patients had a statistically significant improvement (P < 0.05) from baseline versus polymerized collagen and versus placebo at 6 months on Lequesne index, VAS, ESR, Tregs IL-1β, and IL-10 peripheral-expressing cells. Urinary levels of CTXII were decreased 44% in polymerized collagen versus placebo. No differences were found on incidence of adverse events between groups. Conclusion. Polymerized collagen is safe and effective on downregulation of inflammation in patients with knee OA

Calcium and the control of C4 photosynthesis

http://www.sherpa.ac.uk/romeo/search.ph

Effects of ammonium nitrate, cesium chloride and tetraethylammonium on high-affinity potassium uptake in habanero pepper plantlets (Capsicum chinense Jacq.)

Potassium (K+) is an essential nutrient and the most abundant cation in plant cells. Plants have a wide variety of transport systems for K+ acquisition that catalyze K+ uptake across a wide spectrum of external K+ concentrations and mediate K+ movement within the plant, as well as its release into the environment. The KUP/HAK/KT transporter family plays a key role in K+ homeostasis in plant cells. The present study demonstrates that habanero pepper plantlets have a clear pattern of K+ uptake when resupplemented with K+ after K+ starvation. Habanero pepper plantlets, re-supplemented with a solution containing low concentrations of K+ after 72, 96 or 120 h of K+ starvation were able to decrease the amount of K+ in the solution at different time points. To study the effect of NH4+, we added different concentrations of NH4NO3 to the medium solution and demonstrated that NH4+ inhibited K+ uptake in a dose-dependent manner. When the plantlets were subjected to K+ starvation for 72 h and then resupplemented with 50 or 100 μM K+, exposure to K+ channel blockers (10 mM CsCl and 20 mM TEA) decreased their K+ uptake compared with the control treatment. A model demonstrating the process of K+ uptake through an NH4+-insensitive component was proposed.Key words: Potassium, high affinity transporters, channel blockers, ammonium