Two propanediol utilization-like proteins of <Emphasis Type="Italic">Moorella thermoacetica</Emphasis> with phosphotransacetylase activity

Moorella thermoacetica is one of the model acetogenic bacteria for the resolution of the Wood–Ljungdahl (acetyl-CoA) pathway in which CO₂ is autotrophically assimilated yielding acetyl-CoA as central intermediate. Its further conversion into acetate relies on subsequent phosphotransacetylase (PTA) and acetate kinase reactions. However, the genome of M. thermoacetica contains no pta homologous gene. It has been speculated that the moth_0864 and moth_1181 gene products sharing similarities with an evolutionarily distinct phosphotransacylase involved in 1,2-propanediol utilization (PDUL) of Salmonella enterica act as PTAs in M. thermoacetica. Here, we demonstrate specific PTA activities with acetyl-CoA as substrate of 9.05 and 2.03 U/mg for the recombinant enzymes PDUL1 (Moth_1181) and PDUL2 (Moth_0864), respectively. Both showed maximal activity at 65 °C and pH 7.6. Native proteins (90 kDa) are homotetramers composed of four subunits with apparent molecular masses of about 23 kDa. Thus, one or both PDULs of M. thermoacetica might act as PTAs in vivo catalyzing the penultimate step of the Wood–Ljungdahl pathway toward the formation of acetate. In silico analysis underlined that up to now beside of M. thermoacetica, only Sporomusa ovata contains only PDUL like class^III-PTAs but no other phosphotransacetylases or phosphotransbutyrylases (PTBs).     

Biological mechanisms of aging predict age‐related disease co‐occurrence in patients

Genetic, environmental, and pharmacological interventions into the aging process can confer resistance to multiple age‐related diseases in laboratory animals, including rhesus monkeys. These findings imply that individual mechanisms of aging might contribute to the co‐occurrence of age‐related diseases in humans and could be targeted to prevent these conditions simultaneously. To address this question, we text mined 917,645 literature abstracts followed by manual curation and found strong, non‐random associations between age‐related diseases and aging mechanisms in humans, confirmed by gene set enrichment analysis of GWAS data. Integration of these associations with clinical data from 3.01 million patients showed that age‐related diseases associated with each of five aging mechanisms were more likely than chance to be present together in patients. Genetic evidence revealed that innate and adaptive immunity, the intrinsic apoptotic signaling pathway and activity of the ERK1/2 pathway were associated with multiple aging mechanisms and diverse age‐related diseases. Mechanisms of aging hence contribute both together and individually to age‐related disease co‐occurrence in humans and could potentially be targeted accordingly to prevent multimorbidity.     

The heme oxygenase(s)-phytochrome system of Pseudomonas aeruginosa

For many pathogenic bacteria like Pseudomonas aeruginosa heme is an essential source of iron. After uptake, the heme molecule is degraded by heme oxygenases to yield iron, carbon monoxide, and biliverdin. The heme oxygenase PigA is only induced under iron-limiting conditions and produces the unusual biliverdin isomers IXbeta and IXdelta. The gene for a second putative heme oxygenase in P. aeruginosa, bphO, occurs in an operon with the gene bphP encoding a bacterial phytochrome. Here we provide biochemical evidence that bphO encodes for a second heme oxygenase in P. aeruginosa. HPLC, (1)H, and (13)C NMR studies indicate that BphO is a "classic" heme oxygenase in that it produces biliverdin IXalpha. The data also suggest that the overall fold of BphO is likely to be the same as that reported for other alpha-hydroxylating heme oxygenases. Recombinant BphO was shown to prefer ferredoxins or ascorbate as a source of reducing equivalents in vitro and the rate-limiting step for the oxidation of heme to biliverdin is the release of product. In eukaryotes, the release of biliverdin is driven by biliverdin reductase, the subsequent enzyme in heme catabolism. Because P. aeruginosa lacks a biliverdin reductase homologue, data are presented indicating an involvement of the bacterial phytochrome BphP in biliverdin release from BphO and possibly from PigA.     

C-terminal fluorescent labeling impairs functionality of DNA mismatch repair proteins

The human DNA mismatch repair (MMR) process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2). Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency.     

Early Life Factors and Inter-Country Heterogeneity in BMI Growth Trajectories of European Children: The IDEFICS Study

Background

Starting from birth, this explorative study aimed to investigate between-country differences in body mass index (BMI) trajectories and whether early life factors explain these differences.

Methods

The sample included 7,644 children from seven European countries (Belgium, Cyprus, Germany, Hungary, Italy, Spain, Sweden) participating in the multi-centre IDEFICS study. Information on early life factors and in total 53,409 repeated measurements of height and weight from 0 to <12 years of age were collected during the baseline (2007/2008) and follow-up examination (2009/2010) supplemented by records of routine child health visits. Country-specific BMI growth curves were estimated using fractional polynomial mixed effects models. Several covariates focussing on early life factors were added to the models to investigate their role in the between-countries differences.

Results

Large between-country differences were observed with Italian children showing significantly higher mean BMI values at all ages ≥ 3 years compared to the other countries. For instance, at age 11 years mean BMI values in Italian boys and girls were 22.3 [21.9;22.8; 99% confidence interval] and 22.0 [21.5;22.4], respectively, compared to a range of 18.4 [18.1;18.8] to 20.3 [19.8;20.7] in boys and 18.2 [17.8;18.6] to 20.3 [19.8;20.7] in girls in the other countries. After adjustment for early life factors, differences between country-specific BMI curves became smaller. Maternal BMI was the factor being most strongly associated with BMI growth (p<0.01 in all countries) with associations increasing during childhood. Gestational weight gain (GWG) was weakly associated with BMI at birth in all countries. In some countries, positive associations between BMI growth and children not being breastfed, mothers’ smoking during pregnancy and low educational level of parents were found.

Conclusion

Early life factors seem to explain only some of the inter-country variation in growth. Maternal BMI showed the strongest association with children’s BMI growth.     

ATP binding site mutagenesis reveals different subunit stoichiometry of functional P2X2/3 and P2X2/6 receptors

The aim of the present experiments was to clarify the subunit stoichiometry of P2X2/3 and P2X2/6 receptors, where the same subunit (P2X2) forms a receptor with two different partners (P2X3 or P2X6). For this purpose, four non-functional Ala mutants of the P2X2, P2X3, and P2X6 subunits were generated by replacing single, homologous amino acids particularly important for agonist binding. Co-expression of these mutants in HEK293 cells to yield the P2X2 WT/P2X3 mutant or P2X2 mutant/P2X3 WT receptors resulted in a selective blockade of agonist responses in the former combination only. In contrast, of the P2X2 WT/P2X6 mutant and P2X2 mutant/P2X6 WT receptors, only the latter combination failed to respond to agonists. The effects of α,β-methylene-ATP and 2-methylthio-ATP were determined by measuring transmembrane currents by the patch clamp technique and intracellular Ca(2+) transients by the Ca(2+)-imaging method. Protein labeling, purification, and PAGE confirmed the assembly and surface trafficking of the investigated WT and WT/mutant combinations in Xenopus laevis oocytes. In conclusion, both electrophysiological and biochemical investigations uniformly indicate that one subunit of P2X2 and two subunits of P2X3 form P2X2/3 heteromeric receptors, whereas two subunits of P2X2 and one subunit of P2X6 constitute P2X2/6 receptors. Further, it was shown that already two binding sites of the three possible ones are sufficient to allow these receptors to react with their agonists.     

Quantitative liquid chromatographic–tandem mass spectrometric determination of orlistat in plasma with a quadrupole ion trap

This report evaluates the use of a quadrupolar ion trap for quantitation in a bioanalytical laboratory. The evaluation was accomplished with the cross-validation of an LC–MS–MS quantitative method previously validated on a triple quadrupole mass spectrometer. The method was a multi-level determination of the anti-obesity drug, orlistat, in human plasma. The method has been refined previously on a triple quadrupole instrument to provide rapid sample throughput with robust reproducibility at sub-nanogram detection limits. Optimization of the method on the ion trap required improved chromatographic separation of orlistat from interfering plasma matrix components coextracted during the initial liquid–liquid extraction of plasma samples. The ion trap produces full-scan collision-induced dissociation mass spectra containing characteristic orlistat fragment ions that are useful for quantitation. Data collection on the ion trap required a precursor ion isolation width of 3.0 Da and optimal quantitative results were obtained when three fragment ions were monitored with a 1.8 Da window for each ion. Although a direct cross-validation between the ion trap and the tandem triple quadrupole mass spectrometer was not possible, quantitative results for orlistat comparable to those obtained from the triple quadrupole instrument were achieved by the ion trap with the modified method. The limit of quantitation for orlistat in plasma on the ion trap was 0.3 ng ml⁻¹ with a linear dynamic range of 0.3 to 10 ng ml⁻¹. Precision and accuracy varied from 4 to 15% over the quantitation range. The overall results provide an example of the utility of an ion trap in bioanalytical work.     

Synthesis and characterization of photolabile derivatives of serotonin for chemical kinetic investigations of the serotonin 5-HT(3) receptor

A series of photolabile o-nitrobenzyl derivatives of serotonin (caged serotonin) were synthesized: the amine-linked serotonin derivatives N-(2-nitrobenzyl) serotonin (Bz-5HT) and N-(alpha-carboxy-2-nitrobenzyl) serotonin (N-CNB-5HT), and O-alpha-carboxy-2-nitrobenzyl) serotonin (O-CNB-5HT), which has the caging group attached to the phenolic OH group. All the derivatives released free serotonin when excited by 308-nm or 337-nm laser pulses. The time constant of serotonin release from N-CNB-5HT was 1. 2 ms, with a quantum yield of 0.08. This is too slow for rapid chemical kinetic measurements. O-CNB-5HT is suitable for transient kinetic investigations of the serotonin 5-HT(3) receptor. It released serotonin with a time constant of 16 micros and a quantum yield of 0.03. The biological properties of O-CNB-5HT were evaluated, and the applicability of the compound for kinetic studies of the 5-HT(3) receptor was demonstrated. O-CNB-5HT does not activate the 5-HT(3) receptor by itself, nor does it modulate the response of a cell when co-applied with serotonin. When irradiated with a 337-nm laser pulse, O-CNB-5HT released free serotonin that evoked 5-HT(3) receptor-mediated whole-cell currents in NIE-115 mouse neuroblastoma cells.