Efficient production of recombinant PP2A at a low temperature using a baculovirus expression system.

Protein phosphatase 2A (PP2A) is an enzyme useful for detecting several natural toxins represented by okadaic acid and microcystins. We found that the production of the recombinant human PP2A catalytic subunit (rhPP2Ac) in High Five insect cells could markedly increase when the cells were cultured at 19 °C instead of 27 °C used under conventional conditions. The yield and purity of the enzyme increased four- and three-folds, respectively. The benefit of the altered culturing temperature was observed with the recombinant human protein phosphatase 2B but not 2Cα. The different responses among the enzymes suggest the involvement of an enzyme-specific mechanism that leads to the catalytic subunit overexpression. This is the first report to produce rhPP2Ac at a temperature lower than that used under conventional culture conditions (27 °C) used in the baculovirus expression system with High Five insect cells.

Different responses of primary normal human hepatocytes and human hepatoma cells toward cyanobacterial hepatotoxin microcystin-LR.

Microcystins (MCs) are potent hepatotoxins produced by cyanobacteria in aquatic environments. MC-LR, a representative MC, strongly inhibits protein phosphatases 1 and 2A, leading to cell collapse in animal hepatocytes due to hyperphosphorylation of the cytoskeleton and apoptosis due to stimulation of the relevant systems. However, the molecular mechanisms and the metabolic pathways responsible for MC-LR toxicity are poorly understood. In the present study, we compared the cytotoxic effects of MC-LR in two cell lines: normal human hepatocytes (h-Nheps) and human hepatoma cell line HepG2. We also discussed the suitability of cellular assays for evaluating the toxicity of MCs. To obtain further insight into the molecular mechanism, the uptake, excretion, and intracellular distribution of MC-LR were analyzed using an antibody and assay kit targeting the catalytic subunit of protein phosphatase 2A (the PP2A assay kit). The responses toward MC-LR were distinctly different between the two cell lines. In HepG2 cells, MC-LR did not induce morphological change, did not reveal cytotoxicity, and accumulated to a lesser extent despite a slightly elevated expression of the MC transporter protein. MC-LR also did not alter the MC-binding potency of subcellular proteins. All these results indicate that HepG2 cells are inappropriate for the evaluation of MC-LR toxicity. The PP2A assay kits were useful not only for assessing PP2A-inhibitory potency, but also for determining the concentration of MCs in biological systems.

Elective Cesarean section at 37 weeks is associated with the higher risk of neonatal complications.

Elective Cesarean section performed before 39 weeks of gestation may be associated with increased risk of neonatal complications. We retrospectively investigated differences in the neonatal complication rate between 684 newborns delivered by elective Cesarean section at 37 weeks of gestation (n = 390) and those delivered by the same procedure at 38 weeks (n = 294) between 2006 and 2012 at our hospital in order to ascertain whether adverse outcomes differ between the groups. Newborns delivered at 37 weeks had a significantly higher incidence of neonatal intensive care unit admission (p = 0.03), adverse respiratory complications (p < 0.01), low birth weight (p < 0.001), and hypoglycemia (p < 0.005) than those delivered at 38 weeks. Compared with normal weight neonates, low birth weight neonates were more likely to have hypoglycemia (p < 0.001). Multivariate logistic regression analysis revealed that an adverse respiratory outcome was independently associated with gestational age (p < 0.01; odds ratio [OR], 3.26; 95% confidence interval [CI], 1.36-7.81), while hypoglycemia was independently associated with birth weight (p < 0.01; OR, 16.34; 95% CI, 7.72-34.56). Respiratory disorders were significantly associated with gestational age even in normal birth weight newborns without any other complications such as hyperbirilubinemia, hypoglycemia or bacterial infections. In conclusion, the incidence of neonatal complications was higher in newborns delivered at 37 weeks of gestation than in those delivered at 38 weeks via elective Cesarean section. Thus, the procedure should be scheduled at 38 weeks to improve neonatal outcomes.

B-type natriuretic peptide for assessment of haemodynamically significant patent ductus arteriosus in premature infants.

AIM: Haemodynamically significant patent ductus arteriosus (hsPDA) is frequently observed in premature infants. This study was conducted to explore whether the blood BNP can be a valuable biomarker to assess the necessity of treatment for hsPDA in premature infants. METHODS: Serial measurements of the blood BNP were performed during the first 5 days of life in premature infants with hsPDA (Group I) and those without hsPDA (Group N). The definition of the hsPDA was the PDA requiring treatment, such as indomethacin administration and/or surgical ligation. RESULTS: Forty-six subjects were enrolled. Compared with Group N, Group I showed significantly higher level of blood BNP at postnatal 24-96 h and demonstrated the peak value at postnatal 24-48 h. With the ROC curve using the data at postnatal 24-48 h in Group I, we deduced the predictive value of 250 pg/mL of blood BNP for indomethacin treatment. Similarly, with the ROC curve using the maximal value of blood BNP within the first 5 days of life, the predictive value of 2000 pg/mL for surgical ligation was deduced. CONCLUSIONS: Blood BNP during early postnatal period can be a useful biomarker to assess the necessity of treatment for hsPDA in premature infants.

The effect of structural variation in 21 microcystins on their inhibition of PP2A and the effect of replacing cys269 with glycine.

Microcystins (MCs) are a group of cyclic heptapeptide hepatotoxins produced by Microcystis and several other genera of cyanobacteria. The representative MC, MC-LR, strongly inhibits protein phosphatase 2A (PP2A), while the inhibitory potencies of at least 60MC analogs characterized from bloom samples and cultured strains have not been fully elucidated. In this study, we determined the IC(50) values for 21MC analogs for inhibiting the recombinant PP2A catalytic subunit (rPP2Ac). Of the 21MC analogs, MC-LR was the strongest inhibitor of rPP2Ac. Comparison of the IC(50) values indicates that demethylation of the amino acids at positions 3 or 7 leads to a greater reduction in activity than the substitution of l-amino acids at positions 2 and 4. To obtain further insight into the MC-PP2A interaction, we substituted cysteine at position 269 in PP2Ac with glycine. The mutant PP2Ac (C269G) was comparable to the wild-type PP2Ac in the hydrolysis of p-NPP, but was more resistant to MCs as indicated by the greater IC(50) values. Our results indicate that cys269 in PP2Ac and N-methyldehydroalanine (Mdha) at position 7 in MCs play important roles in the enzyme-inhibitor interaction. We also determined the LC(50) values of the MCs for cytotoxicity assay. Our results indicate that there is a weak correlation between the cytotoxicity and PP2A inhibiting activities of the MCs. The MCs and rPP2Ac used in this study were of high purity and the IC(50) values were determined under the same experimental conditions, ensuring the quality of the data. The IC(50) values are of practical importance because they enable the precise conversion of the amounts of various MCs detected using instrumental methods to MC-LR equivalents.