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.

Functional Characterization of Pharmcogenetic Variants of Human Cytochrome P450 2C9 in Korean Populations

Human cytochrome P450 2C9 is a highly polymorphic enzyme that is required for drug and xenobiotic metabolism. Here, we studied eleven P450 2C9 genetic variants—including three novel variants F69S, L310V, and Q324X—that were clinically identified in Korean patients. P450 2C9 variant enzymes were expressed in Escherichia coli and their bicistronic membrane fractions were prepared The CO-binding spectra were obtained for nine enzyme variants, indicating P450 holoenzymes, but not for the M02 (L90P) variant. The M11 (Q324X) variant could not be expressed due to an early nonsense mutation. LC-MS/MS analysis was performed to measure the catalytic activities of the P450 2C9 variants, using diclofenac as a substrate. Steady-state kinetic analysis revealed that the catalytic efficiency of all nine P450 2C9 variants was lower than that of the wild type P450 2C9 enzyme. The M05 (R150L) and M06 (P279T) variants showed high k(cat) values; however, their K(m) values were also high. As the M01 (F69S), M03 (R124Q), M04 (R125H), M08 (I359L), M09 (I359T), and M10 (A477T) variants exhibited higher K(m) and lower k(cat) values than that of the wild type enzyme, their catalytic efficiency decreased by approximately 50-fold compared to the wild type enzyme. Furthermore, the novel variant M07 (L310V) showed lower k(cat) and K(m) values than the wild type enzyme, which resulted in its decreased (80%) catalytic efficiency. The X-ray crystal structure of P450 2C9 revealed the presence of mutations in the residues surrounding the substrate-binding cavity. Functional characterization of these genetic variants can help understand the pharmacogenetic outcomes.

Nutritional status of children from Cochabamba, Bolivia: a cross-sectional study / Estado nutricional de los niños de Cochabamba, Bolivia: un estudio transversal

OBJECTIVE:To assess the adequacy of energy and nutritional intakes compared to recommended daily intakes (RDIs) in schoolchildren from the Cochabamba region (Bolivia) and to determine micronutrient intake distributions across different ages and genders. METHODS: This nutritional study (n = 315) was part of a larger population-based crosssectional study (the "Bolkid" survey) that collected data on schoolchildren 5-16 years old in 2010 in the Cochabamba region. Information about food intake was gathered with a semiquan-titative, food-frequency, parent-administered questionnaire about l2 months before the study. Descriptive and bivariate analyses of energy and nutrient intakes were assessed. RESULTS: For all ages studied and both genders, the average energy and micronutrient intakes were acceptable but below the requirements. The diet included high amounts of fiber, some minerals (iron, magnesium, phosphorus, potassium, sodium), and vitamins (pantothenic acid, niacin, vitamins B2, B12, C, and E), but was low in calcium and vitamin D. However, more than half the children had insufficient energy intake, and low calcium, vitamin A, and vitamin D intakes, according to RDIs adjusted for age and gender; one-third of the children had insufficient folate and magnesium intakes; and adolescent girls had low iron intakes. CONCLUSIONS: Regardless of recommendations or demographic characteristics, the vast majority of children in Cochabamba consumed insufficient energy and too little calcium, folate, magnesium, and vitamin A and D. In addition, adolescent girls consumed insufficient iron. Higher energy intake for schoolchildren through increased food availability, frequency, and size portions in daily meals should be a priority for Bolivian public health institutions.

OBJETIVO:Evaluar la idoneidad del consumo energético y de nutrientes en escolares de la región de Cochabamba, Bolivia, por comparación con las cantidades diarias recomendadas (CDR), y determinar la distribución de la ingesta de micronutrientes en distintas edades y ambos sexos. MÉTODOS: Este estudio nutricional (n = 315) formó parte de un estudio transversal poblacional más amplio (la llamada encuesta Bolkid) en que se obtuvieron datos de escolares de 5 a 16 años de edad en la región de Cochabamba en el 2010. Se usó un cuestionario semicuantitativo, administrado por los padres, para obtener información acerca de la frecuencia del consumo de alimentos alrededor de 12 meses antes del estudio. Se evaluaron los resultados de análisis descriptivos y bivariados de la ingesta energética y de nutrientes. RESULTADOS: En todas las edades estudiadas y ambos sexos, las ingestas energética y de micronutrientes fueron aceptables pero inferiores a las cantidades necesarias. La alimentación tenía un alto contenido de fibra, de algunos minerales (hierro, magnesio, fósforo, potasio, sodio) y de vitaminas (ácido pantoténico, niacina, vitaminas B2, B12, C y E), pero poco contenido de calcio y vitamina D. No obstante, más de la mitad de los niños tenían una ingesta energética insuficiente e ingestas demasiado bajas de calcio, vitamina A y vitamina D, según las CDR ajustadas por edad y sexo; una tercera parte consumían cantidades insuficientes de folato y magnesio; y las adolescentes tenían ingestas de hierro demasiado bajas. CONCLUSIONES: Independientemente de las cantidades recomendadas o de las características demográficas, la gran mayoría de los niños en Cochabamba tenían un consumo energético insuficiente e ingestas demasiado bajas de calcio, folato, magnesio y vitaminas A y D. Además, las adolescentes consumían cantidades insuficientes de hierro. Las instituciones de salud pública bolivianas deberían dar prioridad a aumentar el consumo energético de los escolares propiciando una mayor disponibilidad de alimentos, un consumo más frecuente y porciones más grandes en las comidas diarias.

The role of protein phosphatase 2A B56β holoenzyme in the regulation of heavy metal CdCl2 induced cytotoxicity / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the role of holoenzyme containing Protein Phosphatase 2A B56β in regulating CdCl2 induced cytotoxicity.</p><p><b>METHOD</b>CdCl2-induced cytotoxicity in normal human cell line L-02, AFB1-transformed hepatic cell line L-02 RT-AFB1 and tumor cell line Bel7402 was measured by modified MTT assay. Stable cell lines L-02 SHAKT, L-02 SHB56β, L-02 RT-AFB1-B56β and Bel7402-B56β were generated by infecting L-02 cells or Bel7402 cells with retroviral vectors encoding lentiviral AKT shRNA, lentiviral B56β shRNA and B56β. The relative cell viability was measured in normal human cell line AFB1-transformed hepatic cell line and tumor cell line when treated by CdCl2 (0, 20, 40, 80, 160 µmol/L). After treated by wortmannin (2.5, 5.0 µmol/L) combined with 40 µmol/L CdCl2, Western blot was applied to measure the expression of associated protein in L-02.Western blot was applied to measure the expression of B56β, MT (metallothionein), AKT, and p-AKT in these cell lines treated by CdCl2.</p><p><b>RESULTS</b>The levels of MT were 0.12 ± 0.02, 0.06 ± 0.06 in L-02 RT-AFB1 and Bel7402, which were lower than L02 (0.92 ± 0.14) (F = 1 148.16, P < 0.001) when treated by 40 µmol/L CdCl2. When treated by 40 µmol/L CdCl2, the expression of p-AKT in L-02 SHAKT-1 and L-02 SHAKT-2 were 0.08 ± 0.02, 0.08 ± 0.05, which levels were lower than L-02 SHGFP (0.18 ± 0.15) (F = 724.70, P < 0.001); and the expression of MT were both 0.62 ± 0.16 in L-02 SHAKT-1 and L-02 SHAKT-2, which levels were higher than L-02 SHGFP (0.22 ± 0.14) (F = 94.73, P < 0.001). After treated by wortmannin (2.5, 5.0 µmol/L) combined with 40 µmol/L CdCl2, the expression of p-AKT in L-02 were 0.28 ± 0.07, 0.15 ± 0.11, which levels were lower than wortmannin untreated cells (0.52 ± 0.11) (F = 578.57, P < 0.001); and the expreesion of MT were 1.62 ± 0.80, 1.08 ± 0.15, which levels were higher than wortmannin untreated cells (0.69 ± 0.18) (F = 12.34, P < 0.001). When treated by 40 µmol/L CdCl2, the levels of p-AKT in L-02 SHB56β-1 and L-02 SHB56β-2 were 0.57 ± 0.13, 0.59 ± 0.02, which were higher than L-02 SHGFP (0.32 ± 0.02) (F = 87.16, P < 0.001); and the levels of MT were 0.35 ± 0.07, 0.20 ± 0.03 in L-02 SHB56β-1 and L-02 SHB56β-2, which were lower than L-02 SHGFP (1.51 ± 0.13) (F = 2 457.10, P < 0.001). After treated by 40 µmol/L CdCl2, the expression of p-AKT in L-02 RT-AFB1-B56β and Bel7402-B56β were 0.10 ± 0.11, 0.09 ± 0.01, which were lower than L-02 RT-AFB1 (0.36 ± 0.01) and Bel7402 (0.43 ± 0.11) (F = 877.62, P < 0.001); and the levels of MT were 0.92 ± 0.13, 0.95 ± 0.08 in L-02 RT-AFB1-B56β and Bel7402-B56β,which were higher than L-02 RT-AFB1 (0.44 ± 0.12) and Bel7402 (0.77 ± 0.06) (F = 51.97, P < 0.001).</p><p><b>CONCLUSION</b>Protein phosphatase 2A complexes containing B56β participated in the regulation of MT expression through direct dephosphorylation of AKT, finally affected the cytotoxicity responding to CdCl2. Our study revealed a key signaling pathways of PP2A involved in heavy metals induced cytotoxicity.</p>