Estimation of the diversity between DNA calorimetric profiles,differential melting curves and corresponding melting temperatures

The Poland–Fixman–Freire formalism was adapted for modeling of calorimetric DNA melting profiles, and applied to plasmid pBR 322 and long random sequences. We studied the influence of the difference (H_GC?H_AT) between the helix‐coil transition enthalpies of AT and GC base pairs on the calorimetric melting profile and on normalized calorimetric melting profile. A strong alteration of DNA calorimetrical profile with H_GC?H_AT was demonstrated. In contrast, there is a relatively slight change in the normalized profiles and in corresponding ordinary (optical) normalized differential melting curves (DMCs). For fixed H_GC?H_AT, the average relative deviation (S) between DMC and normalized calorimetric profile, and the difference between their melting temperatures (T_cal?T_m) are weakly dependent on peculiarities of the multipeak fine structure of DMCs. At the same time, both the deviation S and difference (T_cal?T_m) enlarge with the temperature melting range of the helix‐coil transition. It is shown that the local deviation between DMC and normalized calorimetric profile increases in regions of narrow peaks distant from the melting temperature.     

Human MICAL1: Activation by the small GTPase Rab8 and small‐angle X‐ray scattering studies on the oligomerization state of MICAL1 and its complex with Rab8

Human MICAL1 is a member of a recently discovered family of multidomain proteins that couple a FAD‐containing monooxygenase‐like domain to typical protein interaction domains. Growing evidence implicates the NADPH oxidase reaction catalyzed by the flavoprotein domain in generation of hydrogen peroxide as a second messenger in an increasing number of cell types and as a specific modulator of actin filaments stability. Several proteins of the Rab families of small GTPases are emerging as regulators of MICAL activity by binding to its C‐terminal helical domain presumably shifting the equilibrium from the free – auto‐inhibited – conformation to the active one. We here extend the characterization of the MICAL1–Rab8 interaction and show that indeed Rab8, in the active GTP‐bound state, stabilizes the active MICAL1 conformation causing a specific four‐fold increase of k_cat of the NADPH oxidase reaction. Kinetic data and small‐angle X‐ray scattering (SAXS) measurements support the formation of a 1:1 complex between full‐length MICAL1 and Rab8 with an apparent dissociation constant of approximately 8 μM. This finding supports the hypothesis that Rab8 is a physiological regulator of MICAL1 activity and shows how the protein region preceding the C‐terminal Rab‐binding domain may mask one of the Rab‐binding sites detected with the isolated C‐terminal fragment. SAXS‐based modeling allowed us to propose the first model of the free full‐length MICAL1, which is consistent with an auto‐inhibited conformation in which the C‐terminal region prevents catalysis by interfering with the conformational changes that are predicted to occur during the catalytic cycle.     

Production of active pediocin PA-1 in Escherichia coli using a thioredoxin gene fusion expression approach: cloning, expression, purification, and characterization

Antimicrobial peptides possess cationic and amphipathic properties that allow for interactions with the membrane of living cells. Bacteriocins from lactic acid bacteria, in particular, are currently being studied for their potential use as food preservatives and for applications in health care. However, bacteriocin exploitation is often limited owing to low production yields. Gene cloning and heterologous protein or peptide production is one way to possibly achieve overexpression of bacteriocins to support biochemical studies. In this work, production of recombinant active pediocin PA-1 (PedA) was accomplished in Escherichia coli using a thioredoxin (trx) gene fusion (trx-pedA) expression approach. Trx-PedA itself did not show any biological activity, but upon cleavage by an enterokinase, biologically active pediocin PA-1 was obtained. Recombinant pediocin PA-1 characteristics (molecular mass, biological activity, physicochemical properties) were very similar to those of native pediocin PA-1. In addition, a 4- to 5-fold increase in production yield was obtained, by comparison with the PA-1 produced naturally by Pediococcus acidilactici PAC 1.0. The new production method, although not optimized, offers great potential for supporting further investigations on pediocin PA-1 and as a first-generation process for the production of pediocin PA-1 for high-value applications.     

Genomewide spatial correspondence between nonsynonymous divergence and neutral polymorphism reveals extensive adaptation in Drosophila

The effect of recurrent selective sweeps is a spatially heterogeneous reduction in neutral polymorphism throughout the genome. The pattern of reduction depends on the selective advantage and recurrence rate of the sweeps. Because many adaptive substitutions responsible for these sweeps also contribute to nonsynonymous divergence, the spatial distribution of nonsynonymous divergence also reflects the distribution of adaptive substitutions. Thus, the spatial correspondence between neutral polymorphism and nonsynonymous divergence may be especially informative about the process of adaptation. Here we study this correspondence using genomewide polymorphism data from Drosophila simulans and the divergence between D. simulans and D. melanogaster. Focusing on highly recombining portions of the autosomes, at a spatial scale appropriate to the study of selective sweeps, we find that neutral polymorphism is both lower and, as measured by a new statistic Q(S), less homogeneous where nonsynonymous divergence is higher and that the spatial structure of this correlation is best explained by the action of strong recurrent selective sweeps. We introduce a method to infer, from the spatial correspondence between polymorphism and divergence, the rate and selective strength of adaptation. Our results independently confirm a high rate of adaptive substitution (approximately 1/3000 generations) and newly suggest that many adaptations are of surprisingly great selective effect (approximately 1%), reducing the effective population size by approximately 15% even in highly recombining regions of the genome.     

Bioanalytical profile of the L-arginine/nitric oxide pathway and its evaluation by capillary electrophoresis

This review briefly summarizes recent progress in fundamental understanding and analytical profiling of the L-arginine/nitric oxide (NO) pathway. It focuses on key analytical references of NO actions and the experimental acquisition of these references in vivo, with capillary electrophoresis (CE) and high-performance capillary electrophoresis (HPCE) comprising one of the most flexible and technologically promising analytical platform for comprehensive high-resolution profiling of NO-related metabolites. Another aim of this review is to express demands and bridge efforts of experimental biologists, medical professionals and chemical analysis-oriented scientists who strive to understand evolution and physiological roles of NO and to develop analytical methods for use in biology and medicine.     

High-pressure treatment of polytene chromosomes improves structural resolution

The exceptional cytology provided by polytene chromosomes has made Drosophila melanogaster a premier model for chromosome studies, but full exploitation of polytene cytology is impeded by the difficulty in preparing high-quality chromosome spreads. Here we describe use of high pressure to produce formaldehyde-fixed chromosome spreads, which upon light-microscopy examination reveal structural detail previously observed only in electron microscopy preparations. We demonstrate applications to immunofluorescence and in situ hybridization.