Search

Streptomyces davawensis synthesizes the antibiotic roseoflavin, one of the few known natural riboflavin analogs, and is roseoflavin resistant. It is thought that the endogenous flavokinase (EC 2.7.1.26)/ravin adenine dinucleotide (FAD) synthetase (EC 2.7.7.2) activities of roseoflavin-sensitive organisms are responsible for the antibiotic effect of roseoflavin, producing the inactive cofactors roseoflavin-5'-monophosphate (RoFMN) and roseoflavin adenine dinucleotide (RoFAD) from roseoflavin. To confirm this, the FAD-dependent Sus scrofa n-amino acid oxidase (EC 1.4.3.3) was tested with RoFAD as a cofactor and found to be inactive. It was hypothesized that a flavokinase/FAD synthetase (RibC) highly specific for riboflavin may be present in S. davawensis, which would not allow the formation of toxic RoFMN/RoFAD. The gene ribC from S. davawensis was cloned. RibC from S. davawensis was overproduced in Escherichia coli and purified. Analysis of the flavokinase activity of RibC revealed that the S. davawensis enzyme is not riboflavin specific (roseoflavin, [k.sub.cat]/ [K.sub.m] = 1.7 [10.sup.-2] [micro][M.sup.-l] [s.sup.-l]; riboflavin, [k.sub.cat]/[K.sub.m] = 7.5 [10.sup.-3] [M.sup.-1] [s.sup.-l]). Similar results were obtained for RibC from the roseoflavinsensitive bacterium Bacillus subtilis (roseoflavin, [k.sub.cat]/[K.sub.m] = 1.3 [10.sup.-2] [micrp][M.sup.-1] [s.sup.-l]; riboflavin, [k.sub.cat]/[K.sub.m][greater than or equal to] 1.3 [10.sup.-2] [micro][M.sup.1] [s.sup.-l]). Both RibC enzymes synthesized RoFAD and RoFMN. The functional expression of S. davawensis ribC did not confer roseoflavin resistance to a ribC-defective B. subtilis strain.

Riboflavin (vitamin [B.sub.2]) is the direct precursor of the flavin cofactors flavin mononucleotide and flavin adenine dinucleotide, essential components of cellular biochemistry. In this work we investigated the unrelated proteins YpaA from Bacillus subtilis and PnuX from Corynebacterium glutamicum for a role in riboflavin uptake. Based on the regulation of the corresponding genes by a riboswitch mechanism, both proteins have been predicted to be involved in flavin metabolism. Moreover, their primary structures suggested that these proteins integrate into the cytoplasmic membrane. We provide experimental evidence that YpaA is a plasma membrane protein with five transmembrane domains and a cytoplasmic C terminus. In B. subtilis, riboflavin uptake was increased when ypaA was overexpressed and abolished when ypaA was deleted. Riboflavin uptake activity and the abundance of the YpaA protein were also i ncreased when riboflavin auxotrophic mutants were grown in limiting amounts of riboflavin. YpaA-mediated riboflavin uptake was sensitive to protonophors and reduced in the absence of glucose, demonstrating that the protein requires metabolic energy for substrate translocation. In addition, we demonstrate that PnuX from C. glutamicum also is a riboflavin transporter. Transport by PnuX was not energy dependent and had high apparent affinity for riboflavin ([K.sub.m] II [micro]M). Roseoflavin, a toxic riboflavin analog, appears to be a substrate of PnuX and YpaA. We propose to designate the gene names ribU forypaA and ribM forpnuX to reflect that the encoded proteins function in riboflavin uptake and that the genes have different phylogenetic origins.

Abstract Preeclampsia is a leading cause of maternal and fetal/neonatal mortality and morbidity worldwide. The early identification of patients with an increased risk for preeclampsia is therefore one of the most important goals in obstetrics. The availability of highly sensitive and specific physiologic and biochemical markers would allow not only the detection of patients at risk but also permit a close surveillance, an exact diagnosis, timely intervention (e.g. lung maturation), as well as simplified recruitment for future studies looking at therapeutic medications and additional prospective markers. Today, several markers may offer the potential to be used, most likely in a combinatory analysis, as predictors or diagnostic tools. We present here the current knowledge on the biology of preeclampsia and review several biochemical markers which may be used to monitor preeclampsia in a future, that, we hope, is not to distant from today.

The Arabidopsis has encoded three more gene encoding proteins in addition to the previously identified HMGB1-HMGB5 proteins, which have the typical overall structure of the central high mobility group [HMG]-box DNA domain. It is shown that the Arabidopsis high-mobility group box (HMGB) protein discovered typified by the HMGB6 proteins emphasizes the structural variability of plant chromosomal HMGB proteins

Objectives: The selective enrichment of cell-free fetal DNA in maternal plasma by size fractionation leads to the improved detection of paternally inherited fetal point mutations when using conventional, real-time PCR, or as has more recently been shown by MALDI-TOF mass spectrometry. We have now examined the use of size fractionation in conjunction with mass spectrometry for the detection of a paternally inherited codon 39 mutation of the β-globin gene. Methods: Maternal plasma was obtained from an early second trimester pregnancy at risk for β-thalassemia, where the father carried the codon 39 mutation and the mother was a carrier for the IVSI-110 mutation of the β-globin gene. Cell-free DNA was analyzed by mutation-specific PCR and MALDI-TOF mass spectrometry for the presence of the codon 39 mutation. A comparison was made between total cell-free DNA and that which had been enriched for a size of 100–300 bp. Results: The paternally inherited codon 39 mutant allele was detectable in both cell-free DNA preparations, but the signal was much more pronounced and precise in the size-fractionated sample. Conclusions: Size fractionation of cell-free DNA may lead to the improved non-invasive detection of fetal point mutations for β-thalassemia by MALDI-TOF mass spectrometry. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Aim: The aim of the present study was to investigate whether increasing the bosentan dosing frequency from 2 mg kg -1 twice daily (b.i.d.) to 2 mg kg -1 three times daily (t.i.d.) in children with pulmonary arterial hypertension (PAH) (from ≥3 months to <12 years of age) would increase exposure., Methods: An open-label, prospective, randomized, multicentre, multiple-dose, phase III study was conducted. Patients (n = 64) were randomized 1:1 to receive oral doses of bosentan of 2 mg kg -1 b.i.d. or t.i.d. The main pharmacokinetic endpoint was the daily exposure to bosentan over 24 h corrected to the 2 mg kg -1 dose (AUC 0-24C ). The maximum plasma concentration corrected to the 2 mg kg -1 dose (C maxC ), the time to reach the maximum plasma concentration (t max ) and safety endpoints were also assessed., Results: The geometric mean [95% confidence interval (CI)] for AUC 0-24C was 8535 h.ng ml -1 (6936, 10 504) and 7275 h.ng ml -1 (5468, 9679) for 2 mg kg -1 b.i.d. and t.i.d., respectively [geometric mean ratio (95% CI) 0.85 (0.61, 1.20)]. The geometric mean (95% CI) for C maxC was 743 ng ml -1 (573, 963) and 528 ng ml -1 (386, 722) for 2 mg kg -1 b.i.d. and t.i.d., respectively [geometric mean ratio (95% CI) 0.71 (0.48, 1.05)]. The median (range) for t max was 3.0 h (0.0-7.5) and 3.0 h (1.0-8.0) for 2 mg kg -1 b.i.d. and t.i.d., respectively. The proportions of patients who experienced ≥1 adverse event were similar in the b.i.d. (66.7%) and t.i.d. (67.7%) groups., Conclusions: There appeared to be no clinically relevant difference in exposure to bosentan, or in safety, when increasing the frequency of bosentan dosing from b.i.d. to t.i.d. Therefore, the present study provides no indication that the dosing recommendation should be changed, and 2 mg kg -1 b.i.d. remains the recommended dosing regimen for bosentan in paediatric PAH patients., (© 2017 The Authors. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Objectives: The selective enrichment of cell-free fetal DNA in maternal plasma by size fractionation leads to the improved detection of paternally inherited fetal point mutations when using conventional, real-time PCR, or as has more recently been shown by MALDI-TOF mass spectrometry. We have now examined the use of size fractionation in conjunction with mass spectrometry for the detection of a paternally inherited codon 39 mutation of the beta-globin gene., Methods: Maternal plasma was obtained from an early second trimester pregnancy at risk for beta-thalassemia, where the father carried the codon 39 mutation and the mother was a carrier for the IVSI-110 mutation of the beta-globin gene. Cell-free DNA was analyzed by mutation-specific PCR and MALDI-TOF mass spectrometry for the presence of the codon 39 mutation. A comparison was made between total cell-free DNA and that which had been enriched for a size of 100-300 bp., Results: The paternally inherited codon 39 mutant allele was detectable in both cell-free DNA preparations, but the signal was much more pronounced and precise in the size-fractionated sample., Conclusions: Size fractionation of cell-free DNA may lead to the improved non-invasive detection of fetal point mutations for beta-thalassemia by MALDI-TOF mass spectrometry., (Copyright (c) 2009 S. Karger AG, Basel.)