Covalent flavinylation of L-aspartate oxidase from Escherichia coli using N6-(6-carboxyhexyl)-FAD succinimidoester

L-Aspartate oxidase is a flavoprotein catalyzing the first step in the de novo biosynthesis of pyridine nucleotides in E. coli. Binding of FAD to L-aspartate oxidase is relatively weak (K _d 6.7 × 10^–7 M), resulting in partial loss of the coenzyme under many experimental conditions. Only the three-dimensional structure of the apo-enzyme has been obtained so far. In order to probe the flavinbinding site of the enzyme, apo-L-aspartate oxidase has been reacted with N⁶-(6-carboxyhexyl)-FAD Succinimidoester. The structural characterization of the resulting N⁶-(6-carbamoylxyhexyl)-FAD-L-aspartate oxidase shows the covalent incorporation of 1 FAD-analog/ monomer. Residue Lys38 was identified as the target of the covalent modification. N⁶-(6-carbamoylxyhexyl)-FAD-L-aspartate oxidase shows only 2% catalytic activity as compared to the native enzyme. Comparison of some properties of the flavinylated and native enzymes suggests that, although the flavin is covalently bound to the former in the region predicted from molecular modeling studies, the microenvironment around the isoallossazine is different in the two forms.     

Studies on the partial characterization of extracted glycosaminoglycans from fish waste and its potentiality in modulating obesity through in-vitro and in-vivo

Glycoconjugate Journal - Glycosaminoglycans (GAGs) are bioactive polysaccharides or glycoconjugates found in the fish waste having significant health impacts. In the present study it has been...     

Presepsin (Soluble CD14 Subtype): Reference Ranges of a New Sepsis Marker in Term and Preterm Neonates

Objective

Presepsin (soluble CD14 subtype) has been shown to be beneficial as a sepsis marker in adults. Nevertheless, very few data are available in neonates. The aim of the present study was to determine reference ranges of presepsin in term and preterm neonates.

Methods

Healthy term neonates and preterm neonates without clinical signs of infection admitted to the Neonatal Unit were consecutively enrolled. Presepsin concentrations in whole blood were measured using a point-of-care assay system located in the Unit. Demographic data, antenatal and perinatal variables commonly affecting C-reactive protein and procalcitonin values were considered.

Results

Of the 684 neonates enrolled in the study, 484 (70.8%) were born at term and 200 (29.2%) were preterm (24–36 weeks’ gestation). In term infants, presepsin median value was 603.5 pg/mL (interquartile range: 466.5–791 pg/mL; 5th and 95th centiles: 315 and 1178 pg/mL respectively). In preterm infants, presepsin median value was slightly higher, equal to 620 pg/mL (interquartile range: 503–864 pg/mL; 5th and 95th centiles: 352 and 1370 pg/mL respectively). The reference ranges of presepsin we determined were much higher than those seen in healthy adults. No correlation between presepsin levels and postnatal age was observed, as well as no significant difference was demonstrated in preterm neonates at different gestational ages. None of the variables analyzed affected presepsin levels at a clinical significant extent.

Conclusion

For the first time, this study provides reference ranges of presepsin in term and preterm neonates. Having reliable reference values is crucial for obtaining an adequate diagnostic accuracy. Based on our results, most variables commonly affecting C-reactive protein and procalcitonin values do not affect presepsin levels, which suggests that presepsin could be an effective sepsis marker. Further investigations in large groups of neonates with sepsis are needed to determine the diagnostic and prognostic value of this biomarker.     

Nested Allele-Specific PCR Primers Distinguish Genetic Groups of Uncinula necator

Isolates of the obligately biotrophic fungus Uncinula necator cluster in three distinct genetic groups (groups I, II, and III). We designed PCR primers specific for these groups in order to monitor field populations of U. necator. We used the nucleotide sequences of the gene that encodes eburicol 14α-demethylase (CYP51) and of the ribosomal DNA internal transcribed spacer 1 (ITS1), ITS2, and 5.8S regions. We identified four point mutations (three in CYP51 and one in ITS1) that distinguished groups I and II from group III based on a sample of 132 single-spore isolates originating from Europe, Tunisia, Israel, India, and Australia. We developed a nested allele-specific PCR assay in which the CYP51 point mutations were used to detect and distinguish groups I and II from group III in crude mildewed samples from vineyards. In a preliminary study performed with samples from French vineyards in which isolates belonging to genetic groups I and III were present, we found that a shift from a population composed primarily of group I isolates to a population composed primarily of group III isolates occurred during the grapevine growing season.     

Chronic conventional resistance exercise reduces blood pressure in stage 1 hypertensive men

To investigate the antihypertensive effects of conventional resistance exercise (RE) on the blood pressure (BP) of hypertensive subjects, 15 middle-aged (46 ± 3 years) hypertensive volunteers, deprived of antihypertensive medication (reaching 153 ± 6/93 ± 2 mm Hg systolic/diastolic BP after a 6-week medication washout period) were submitted to a 12-week conventional RE training program (3 sets of 12 repetitions at 60% 1 repetition maximum, 3 times a week on nonconsecutive days). Blood pressure was measured in all phases of the study (washout, training, detraining). Additionally, the plasma levels of several vasodilators or vasoconstrictors that potentially could be involved with the effects of RE on BP were evaluated pre- and posttraining. Conventional RE significantly reduced systolic, diastolic, and mean BP, respectively, by an average of 16 (p < 0.001), 12 (p < 0.01), and 13 mm Hg (p < 0.01) to prehypertensive values. There were no significant changes of vasoactive factors from the kallikrein-kinin or renin-angiotensin systems. After the RE training program, the BP values remained stable during a 4-week detraining period. Taken together, this study shows for the first time that conventional moderate-intensity RE alone is able to reduce the BP of stage 1 hypertensive subjects free of antihypertensive medication. Moreover, the benefits of BP reduction achieved with RE training remained unchanged for up to 4 weeks without exercise.     

Role of permanent dicentric systems in carrot somatic embryogenesis

Summary A permanent dicentric chromosome system was studied on carrot cultures and regenerated somatic embryos at different stages of development. The large chromosomal variability of the cultures and the presence of the breakage-fusion bridge cycle did not interfere with the initial developmental process up to the seedling stage but subsequent growth proceeded only if healing of the broken ends or dicentric loss had occurred. The behaviour of the dicentric chromosome in culture and during somatic embryogenesis is discussed in relation to chromosomal variability, abnormal development and the somaclonal variation that such mechanisms may generate in regenerated plants.     

Structure-function relationship in spinach ferredoxin-NADP+ reductase as studied by limited proteolysis

Studies of limited proteolysis on purified ferredoxin-NADP+ reductase with various proteases were performed in the presence and absence of the flavoprotein ligands. Both the diaphorase and the ferredoxin-dependent activities of the enzyme were followed as well as the proteolytic pattern in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with further characterization of the polypeptides produced. These experiments revealed that only two regions of the flavoprotein are susceptible to the attack of the proteases used: (a) the N-terminal chain which can be cleaved only up to Lys35 and (b) the sequence segment 235-250. It can be inferred that these regions are on the surface of the protein molecule and presumably have a very flexible conformation adaptable to the protease active site. The deletion of the N-terminal region up to Thr36 of the native reductase (Mr 35,000) produced a truncated form (Mr about 31,000) which had full diaphorase activity but lost the capacity to catalyze the ferredoxin-dependent reaction. Proteolytic cleavage at the 235-250 segment of the sequence yielded a nicked protein (Mr about 30,000 by gel filtration; 23,000 plus 7,000 in denaturing electrophoresis) devoid of both activities. Protection by the flavoprotein ligands implies that the 23-35 region of the sequence is part of the binding site for ferredoxin and the 235-250 polypeptide segment is in the NADP(+)-binding site.     

DNA heterogeneity and genetic control of tumorigenesis in nicotiana tumorous and nontumorous genotypes

The possible relevance of changes in amounts of highly repetitive DNA sequences for plant differentiation and dedifferentiation processes has been suggested in several cases. Data are lacking however on (1) the genetic control of these phenomena and (2) cause-effect relationships between DNA amplification and specific ontogenetic patterns. The present study was carried out on a Nicotiana genetic system consisting of the tumorous amphidiploid N glauca X N langsdorffii, a nontumorous mutant of it, their F₁, and a backcross to the tumorous parent. Backcross segregation ratios were shown to be compatible with a “single gene” hypothesis, the F₁ plant being nontumorous but showing a low percentage of tumors induced by wounds, 6-azauracil or X-rays. In vitro studies of excised pith tissue grown on Linsmaier and Skoog medium for different periods of time showed the presence, confirmed by cytological analyses, of amplification of highly repetitive sequences only in the nontumorous stock, as judged by reassociation experiments in the first 24–96 hours of culture. CsCl analytical ultracentrifugation of those sequences showed the appearance in the same stock of a heavy DNA satellite (density = 1.721 gm/ml), whose presence was also confirmed by derivative melting curves. Amplification seemed to be essential for the initiation of cell division, which was completely inhibited in the nontumorous genotype and partially influenced in the F₁ by incorporation during the critical period (24–96 hours of the primary explant) of 5-bromo-2′-deoxy-uridine. The results are discussed in terms of an hypothesis of an integrated gene-controlled, hormone-mediated regulatory system of cell proliferation involving changes in target repetitive DNA sequences.