Pinpointing Synechococcus Rubisco large subunit sections involved in heterologous holoenzyme formation in Escherichia coli

Aims@#Heterologous holoenzyme formation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) has been a challenge due to a limited understanding of its biogenesis. Unlike bacterial Rubiscos, eukaryotic Rubiscos are incompatible with the Escherichia coli (E. coli) chaperone system to fold and assemble into the functional hexadecameric conformation (L8S8), which comprises eight large subunits (RbcL) and eight small subunits (RbcS). Our previous study reported three sections (residues 248-297, 348-397 and 398-447) within the RbcL of Synechococcus elongatus PCC6301, which may be important for the formation of L8S8 in E. coli. The present study further examined these three sections separately, dividing them into six sections of 25 residues (i.e., residues 248-272, 273-297, 348-372, 373-397, 398-422 and 423-447).@*Methodology and results@#Six chimeric Rubiscos with each section within the RbcL from Synechococcus replaced by their respective counterpart sequence from Chlamydomonas reinhardtii were constructed and checked for their effect on holoenzyme formation in E. coli. The present study shows that Section 1 (residues 248-272; section of Synechococcus RbcL replaced by corresponding Chlamydomonas sequence), Section 2 (residues 273-297), Section 3 (residues 348-372) and Section 6 (residues 423-447) chimeras failed to fold and assemble despite successful expression of both RbcL and RbcS. Only Section 4 (residues 373-397) and 5 (residues 398-422) chimeras could form L8S8 in E. coli.@*Conclusion, significance and impact of study@#GroEL chaperonin mediates the folding of bacterial RbcL in E. coli. Therefore, residues 248-297, 348-372 and 423-447 of Synechococcus RbcL may be important for interacting with the GroEL chaperonin for successful holoenzyme formation in E. coli.

Phylogenetic relationships of plant species from the flowering desert of the Atacama Region / Relaciones filogenéticas de especies de plantas que crecen en el desierto florido de la Región de Atacama

Every 3 to 7 year angiosperms species of the flowering desert appear in the Atacama Region of Chile, as a result of the climatic phenomenon "El Niño". Our objective was to evaluate the universality of matK and rbcL barcode markers of these species, and validate their taxon through phylogenetic relationships. Argemone hunnemannii, Oenothera coquimbensis, Malesherbia humilis, Leucocoryne appendiculata, Loasa elongata, Nicotiana solanifolia, Stachys grandidentata, Aristolochia chilensis, Alstroemeria kingii and Adesmia eremophila, almost all classified as endemic to Chile, were collected in Pan de Azúcar and Llanos de Challe National Park (Atacama Region, Chile) at the end of October 2017. The phylogeny of these ten angiosperm species from the flowering desert was analyzed using rbcL and matK markers with the maximum likelihood and Bayesian inference methods. The results showed that 70% of the species can be distinguished with the matK or rbcL locus, however, 100% were distinguished using both loci. The phylogenetic results showed that the species formed clades with high reliability and high support with both the matK and rbcL genes, when comparing our results with sequences obtained from GenBank. The matK and rbcL genes are efficient markers for analyzing phylogenetic relationships and validating the taxonomy of flowering species.

Las especies de angiospermas del Desierto Florido de la Región de Atacama de Chile aparecen cada 3 a 7 años, influenciado por el fenómeno climático "El Niño". Nuestro objetivo fue evaluar la universalidad de los marcadores de código de barra matK y rbcL de estas especies, y validar su taxón por medio de relaciones filogenéticas. Las especies Argemone hunnemannii, Oenothera coquimbensis, Malesherbia humilis, Leucocoryne appendiculata, Loasa elongata, Nicotiana solanifolia, Stachys grandidentata, Aristolochia chilensis, Alstroemeria kingii y Adesmia eremophila son clasificadas la mayoría endémicas de Chile. Estas especies fueron colectadas en el Parque Nacional Pan de Azúcar y Llanos de Challe, Región de Atacama, Chile. La colecta se realizó a fines de octubre de 2017. Con los marcadores rbcL y matK se analizó la filogenia con los métodos máxima verosimilitud e inferencia bayesiana en diez especies de angiosperma del Desierto Florido. Los resultados mostraron que el 70% de las especies pueden ser distinguidas con un locus matK o rbcL, sin embargo, el 100% se distinguió usando ambos locus. Los resultados filogenéticos mostraron que las especies formaron clados con alta fiabilidad y alto soporte tanto con los genes matK y rbcL, al comparar con accesos de secuencias obtenidas de GenBank. Lo genes matK y rbcL son marcadores eficientes para analizar relaciones filogenéticas y validar el taxón de las especies de flor.

Historical review of clinical vaccine studies at Oswaldo Cruz Institute and Oswaldo Cruz Foundation - technological development issues

This paper presents, from the perspective of technological development and production, the results of an investigation examining 61 clinical studies with vaccines conducted in Brazil between 1938-2013, with the participation of the Oswaldo Cruz Institute (IOC) and the Oswaldo Cruz Foundation (Fiocruz). These studies have been identified and reviewed according to criteria, such as the kind of vaccine (viral, bacterial, parasitic), their rationale, design and methodological strategies. The results indicate that IOC and Fiocruz have accumulated along this time significant knowledge and experience for the performance of studies in all clinical phases and are prepared for the development of new vaccines products and processes. We recommend national policy strategies to overcome existing regulatory and financing constraints.

Development of an activity-directed selection system enabled significant improvement of the carboxylation efficiency of Rubisco

Photosynthetic CO(2) fixation is the ultimate source of organic carbon on earth and thus is essential for crop production and carbon sequestration. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the first step of photosynthetic CO(2) fixation. However, the extreme low carboxylation efficiency of Rubisco makes it the most attractive target for improving photosynthetic efficiency. Extensive studies have focused on re-engineering a more efficient enzyme, but the effort has been impeded by the limited understanding of its structure-function relationships and the lack of an efficient selection system towards its activity. To address the unsuccessful molecular engineering of Rubisco, we developed an Escherichia coli-based activity-directed selection system which links the growth of host cell solely to the Rubisco activity therein. A Synechococcus sp. PCC7002 Rubisco mutant with E49V and D82G substitutions in the small subunit was selected from a total of 15,000 mutants by one round of evolution. This mutant showed an 85% increase in specific carboxylation activity and a 45% improvement in catalytic efficiency towards CO(2). The small-subunit E49V mutation was speculated to influence holoenzyme catalysis through interaction with the large-subunit Q225. This interaction is conserved among various Rubisco from higher plants and Chlamydomonas reinhardtii. Knowledge of these might provide clues for engineering Rubisco from higher plants, with the potential of increasing the crop yield.

Prospects and Problems for Identification of Poisonous Plants in China using DNA Barcodes / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Poisonous plants are a deadly threat to public health in China. The traditional clinical diagnosis of the toxic plants is inefficient, fallible, and dependent upon experts. In this study, we tested the performance of DNA barcodes for identification of the most threatening poisonous plants in China.</p><p><b>METHODS</b>Seventy-four accessions of 27 toxic plant species in 22 genera and 17 families were sampled and three DNA barcodes (matK, rbcL, and ITS) were amplified, sequenced and tested. Three methods, Blast, pairwise global alignment (PWG) distance, and Tree-Building were tested for discrimination power.</p><p><b>RESULTS</b>The primer universality of all the three markers was high. Except in the case of ITS for Hemerocallis minor, the three barcodes were successfully generated from all the selected species. Among the three methods applied, Blast showed the lowest discrimination rate, whereas PWG Distance and Tree-Building methods were equally effective. The ITS barcode showed highest discrimination rates using the PWG Distance and Tree-Building methods. When the barcodes were combined, discrimination rates were increased for the Blast method.</p><p><b>CONCLUSION</b>DNA barcoding technique provides us a fast tool for clinical identification of poisonous plants in China. We suggest matK, rbcL, ITS used in combination as DNA barcodes for authentication of poisonous plants.</p>

Molecular identification of raw materials from lian qiao bai du wan / 药学学报

Lian Qiao Bai Du Wan was used to study the identification of Chinese patent medicine by molecular marker technique. DNA was extracted through modified CTAB method. The psbA-trnH and rbcL sequences were gradient amplified, and PCR products were ligated with the pEASY-T5 vector and then transformed into Trans1-T1 cells, respectively. Clones were selected randomly and sequenced. All sequences were analyzed by BlastN and the neighbor-joining (NJ) phylogenetic tree was constructed by MEGA 4.0. The results showed that nine kinds of medicinal materials can be identified by psbA-trnH sequences, and six kinds of medicinal materials by rbcL sequences from Lian Qiao Bai Du Wan. Molecular marker technique can stably and accurately distinguish multi-origin medicinal materials in Chinese patent medicine.

Changes in PEP Carboxylase, Rubisco and Rubisco activase mRNA levels from maize (Zea mays) exposed to a chronic ozone stress

We quantified the ozone impact on levels of Zea mays L. cv. Chambord mRNAs encoding C4-phosphoenolpyruvate carboxylase (C4-PEPc), ribulose-l,5-bisphosphate carboxylase/oxygenase small and large subunits (Rubisco-SSU and Rubisco-LSU, respectively) and Rubisco activase (RCA) using real-time RT-PCR. Foliar pigment content, PEPc and Rubisco protein amounts were simultaneously determined. Two experiments were performed to study the ozone response of the 5th and the 10th leaf. For each experiment, three ozone concentrations were tested in open-top chambers: non-filtered air (NF, control) and non-filtered air containing 40 (+40) and 80 nL L-1 (+80) ozone. Regarding the 5th leaf, +40 atmosphere induced a loss in pigmentation, PEPc and Rubisco activase mRNAs. However, it was unable to notably depress carboxylase protein amounts and mRNAs encoding Rubisco. Except for Rubisco mRNAs, all other measured parameters from 5th leaf were depressed by +80 atmosphere. Regarding the 10th leaf, +40 atmosphere increased photosynthetic pigments and transcripts encoding Rubisco and Rubisco activase. Rubisco and PEPc protein amounts were not drastically changed, even if they tended to be increased. Level of C4-PEPc mRNA remained almost stable. In response to +80 atmosphere, pigments and transcripts encoding PEPc were notably decreased. Rubisco and PEPc protein amounts also declined to a lesser extent. Conversely, the level of transcripts encoding both Rubisco subunits and Rubisco activase that were not consistently disturbed tended to be slightly augmented. So, the present study suggests that maize leaves can respond differentially to a similar ozone stress.

Photosynthetic acclimation to elevated CO2 in relation to Rubisco gene expression in three C3 species.

Wheat (Triticum aestivum L. var. DL 1266-5), sunflower (Helianthus annuus L. var. MSFH 17) and mungbean [Vigna radiata (L.) Wilczek var. P 9072] were grown in field under atmospheric (360 +/- 10 cm3 m(-3), AC) and elevated (650 +/- 50 cm3 m(-3), EC) CO2 concentrations in open top chambers for entire period of growth and development. Photosynthetic acclimation to elevated CO2 was examined by comparing photosynthesis rate (Pn), Pn/Ci curves, leaf contents of RuBP carboxylase/oxygenase (Rubisco), change in the transcripts of Rubisco small subunit (SSU) gene and leaf carbohydrate constituents in AC and EC grown plants. The study indicated that photosynthetic acclimation to elevated CO2 concentration in wheat occurred because of down regulation of Rubisco, through limitation imposed on Rubisco SSU gene expression, as a consequence of sugar accumulation in the leaves. Leaf starch accumulators, sunflower and mungbean, showed no down regulation of Pn under EC. The Rubisco contents (%) in leaf soluble protein and rbcS transcript levels were not significantly affected in EC plants compared to AC plants of sunflower and mungbean. The study indicated that accumulation of excess assimilates in the leaves as starch was less inhibitory to Pn and would, therefore, be an important trait for sustenance of Pn not only under EC, but also under AC, where Pn inhibited by end products.

A new measure to study phylogenetic relations in the brown algal order Ectocarpales: the "codon impact parameter".

We analyse forty-seven chloroplast genes of the large subunit of RuBisCO, from the algal order Ectocarpales, sourced from GenBank. Codon-usage weighted by the nucleotide base-bias defines our score called the codon-impact-parameter. This score is used to obtain phylogenetic relations amongst the 47 Ectocarpales. We compare our classification with the ones done earlier.

Detection of HBV in Blood and Blood Products by PCR

The aim of this study was to establish a PCR for detecting of the hepatitis B virus (HBV) in blood and blood products. A primer pair set was designed to amplify a 513 bp fragment in the S-region of the HBV genome in the first PCR and a 233 bp fragment of first PCR amplicon in the second PCR with Rubisco (internal control). In order to assess the specificity of the PCR results, all the samples were tested cross-reactivity or interference in the assay. This method did not result in cross-reactivity with the non-HBV (HAV, HCV, HIV, CMV, HPV 18&6b, parvovirus B19/ or HSV 1&2) positive samples and was unaffected. In case of the HBV spiked blood products such as the immunogloubulin and coagulation factors, the lower detection limit of this method for the HBV DNA is 62.5 IU/ml. The PCR method is fully established in this study and will be a valuable method for the detection of the HBV in a variety of blood products, particularly, those derived from starting materials with a high titer of virus.

Metabolic responses of tropical trees to ozone pollution.

Plants fumigated with 40ppbv, 80ppbv and 120ppbv concentrations of O3 exhibited significant reduction in total chlorophyll content, RuBP carboxylase activity and net photosynthesis. The reduction in total chlorophyll activity ranged from 12 to 36% in Bauhinia variegata, 11 to 35% in Ficus infectoria and 3 to 26% in Pongamia pinnata on fumigation with O3, while the RuBP carboxylase activity was reduced by 10 to 32% in Bauhinia variegata, 10 to 23% in Ficus infectoria and 9 to 15% in Pongamia pinnata. The net photosynthesis was also reduced by 6 to 26% in B. variegata, 16 to 39% in F. infectoria and 7 to 31% in P. pinnata on fumigation with 03. The relative higher sensitivity of tropical trees to O3 suggests that the ambient air quality standards in tropical tree areas need to be stringent to prevent vegetation from air pollution.

RbcL sequence analysis of Belamcanda chinensis and related medicinal plants of Iris / 药学学报

<p><b>AIM</b>To identify "Shegan" [Belamcanda chinensis (L.) DC.] and relative medicinal plants of Iris including Iris tectorum Maxim., I. dichotoma Pall., I. germanica L. and I. japonica Thunb. by ribulose 1,5-bisphosphate carboxylase Large Gene (rbcL) sequence analysis.</p><p><b>METHODS</b>General DNA was isolated from the fresh leaves of Belamcanda chinensis and 4 Iris spp. by CTAB. A pair of primers was designed to amplify the rbcL gene and PCR Preps DNA kit was used to purify the PCR products. The rbcL sequences were determined by ABI (Applied Biosystems Inco.) Prism 310 Genetic Analyzer.</p><p><b>RESULTS</b>A fragment of about 750 bp of rbcL gene from Belamcanda chinensis and 4 Iris spp. were amplified and sequenced. The rbcL sequences of Iris tectorum, I. dichotoma Pall. and I. japonica were reported for the first time. The rbcL sequences of 5 species of Iridaceae were aligned and analyzed using Clustal (Version 8.0) and MEGA (Version 2.0.) programs. The nucleotide number of difference is from 1.000 to 20.000. The tranversions is from 0.000 to 9.000 and the transitions is from 0.000 to 14.000. Phylogenetic tree based on rbcL partial sequence data indicated that the eleven samples of 5 species clustered separately.</p><p><b>CONCLUSION</b>The sequence variation of rbcL can be used to identify Belamcanda chinensis and 4 species of relative medicinal plants of Iris. The molecular phylogenetic tree accords with the classical taxonomy.</p>

Changes in rubisco content in relation to saccharides accumulation in wheat leaves during day.

Two wheat varieties, T. durum (HD 4502) and T. aestivum (Kalyansona) were examined for photosynthesis rate and contents of sugars and rubisco protein in the flag leaf, at forenoon and afternoon at anthesis stage. A decrease in photosynthesis rate was observed in the afternoon compared to forenoon in both the varieties and was associated with an increase in non-reducing sugars and a decrease in rubisco content in the leaves.

Eficacia del tratamiento farmacológico de osteoporosis en la prevención de fracturas / Pharmacological treatment efficacy in osteoporosis for prevention of fractures

En el presente artículo se hace una exhaustiva revisión bibliográfica de las investigaciones y resultados clínicos de las drogas más utilizadas para el tratamiento de la osteoporosis, así como de la efectividad de cada una de ellas para prevenir una fractura. También se discuten los problemas más importantes para prevenir, diagnosticar y tratar esta enfermedad con un costo beneficio razonable. Al final se discuten algunas estrategias y sugerencias para definir si es necesaria o no una intervención farmacológica, y si ésta se considera necesaria, de qué tipo debe ser.

Photosynthesis and kinetic characteristics of rubisco in Hibiscus cannabinus L.

Photosynthetic characteristics in kenaf (Hibiscus cannabinus L.), a C3 plant, were compared with Abelmoschus esculentus (L.) Moench, another member of Malvaceae. Kenaf leaves exhibited significantly higher rate of photosynthesis (40 mg CO2 dm(-2) hr(-1)) which was 24.6 mg dm(-2) hr(-1) in A. esculentus. Rate of photo and dark respiration was similar in both the species. Kenaf leaf photosynthesis had a higher optimum temperature (32 degrees C) than that of A. esculentus (26 degrees C). Photosynthesis in kenaf leaves required higher saturation irradiance (1,600 micromole m(-2) sec(-1)). There was a significant correlation between photosynthetic rate and biomass yield in these species. The primary product of photosynthesis after 5 seconds of 14C-assimilation was 3-PGA in both the species. The kinetic properties of RuBP carboxylase/oxygenase were determined in the leaf extracts. Higher carboxylase activities were recorded with kenaf leaf extracts (245 pmole mg chl(-1) hr(-1)). Km (CO2) for kenaf leaf carboxylase was significantly lower (7.8 microM) than A. esculentus (13.5 microM) and corresponding difference in Vmax values of carboxylase was recorded between the two species. The kinetic characteristics of oxygenase were similar in both the extracts. These results indicated the variation in carboxylase activity and its kinetic characteristics reflected a significant difference in CO2 assimilation in C3 plants.

Channeling of the intermediates and catalytic facilitation to Rubisco in a multienzyme complex of Calvin cycle enzymes.

Calvin cycle multienzyme complex, consisting of phosphoriboisomerase, phosphoribulokinase and ribulose-1,5-bisphosphate carboxylase (Rubisco), shows ribose-5-phosphate + ATP dependent CO2 fixation activity with a small but discernible lag. Transient time analysis showed that the lag at pH 7 was independent of multienzyme concentration and was significantly lower than the expected transient time calculated from Km and Vmax of the individual enzymes, indicative of channeling of the intermediates in the enzyme complex. Channeling of ribulose-1,5-bisphosphate was found to offer a catalytic advantage to Rubisco. Rubisco shows a decrease in activity during catalysis in ribulose-1,5-bisphosphate dependent CO2 fixation reaction, due to the formation of the catalytic inhibitor. Such a decrease of Rubisco activity was not observed in ribose-5-phosphate + ATP dependent CO2 fixation reaction and the catalytic inhibitor was also not detected. These results suggested that the intermediates are channeled in the complex and channeling offers a catalytic facilitation to Rubisco.

Purification and characterization of ribulose-1,5-bisphosphate carboxylase from triticale.

Ribulose-1,5-bisphosphate carboxylase has been isolated from a synthetic cereal triticale and purified using a newly developed rapid procedure involving precipitation with ammonium sulphate (35-55% saturation), DEAE-cellulose (DE-52) chromatography and filtration through Sepharose CL-68. Molecular weights of the enzyme subunits are 15.5 and 52 kDa which corresponds to 540 kDa for the hexadecameric holoenzyme. Isoelectric focussing showed that the enzyme has a pI of 4.2. Various kinetic constants determined under aerobic conditions are: Km (CO2), 118 microM; Km (RuBP), 220 microM (at 20 mM NaHCO3) and Vmax, 690 nmole CO2 fixed/mg enzyme/min.

Identification and characterization of receptors for protein import into chloroplasts and mitochondria.

An anti-idiotypic antibody approach was used to identify chloroplast and mitochondrial protein component(s) which interact with the corresponding signal sequence. The proteins thus identified can be operationally defined as receptor(s) for import of proteins into chloroplasts and mitochondria. The import receptor(s) was found in "contact sites" between the outer and inner membrane of chloroplast envelope or of mitochondria.

Essential tryptophan residues of ribulose 1,5-bisphosphate carboxylase.

Ribulose 1,5-bisphosphate carboxylase [3-phospho-D-glyceratecarboxy-lyase (dimerizing), EC 4.1.1.39] is rapidly and irreversibly inactivated by micromolar concentrations of dimethyl (2-hydroxy-5-nitrobenzyl) sulphonium bromide (DMHNB), a tryptophan selective reagent, after reversible protection of the reactive sulphydryl groups. The inactivation followed pseudo-first-order reaction kinetics. Replots of the kinetic data indicated that no reversible enzyme-inhibitor complex was formed prior to irreversible modification. Kinetic analysis and the correlation of the spectral data at 410 nm with enzyme activity indicated that inactivation by DMHNB resulted from modification of on an average one tryptophan per 67 kDa combination of large and small subunits. Several competitive inhibitors and substrate RuBP offered strong protection against inhibition. The k1/2 (protection) for RuBP was 1.3 mM, indicating that the tryptophan residues may be located at or near the substrate binding site. Free and total sulphydryl groups were not affected by the reagent. The modified enzyme exhibited significantly reduced intrinsic fluorescence, indicating that the microenvironment of the tryptophans at the active site is significantly perturbed. Tryptic peptide profiles and CD spectral analyses suggested that inactivation may not be due to the extensive conformational changes in the enzyme molecule during modification.