Intrinsically bent DNA sites in the <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> third chromosome amplified domain

Bent DNA sites promote the curvature of DNA in both eukaryotic and prokaryotic chromosomes. Here, we investigate the localization and structure of intrinsically bent DNA sites in the extensively characterized Drosophila melanogaster third chromosome DAFC-66D segment (Drosophila amplicon in the follicle cells). This region contains the amplification control element ACE3, which is a replication enhancer that acts in cis to activate the major replication origin ori-β. Through both electrophoretic and in silico analysis, we have identified three major bent DNA sites in DAFC-66D. The bent DNA site (b1) is localized in the ACE3 element, whereas the other two bent DNA sites (b2 and b3) are localized in the ori-β region. Four additional bent DNA sites were identified in the intron of the S18 gene and near the TATA box of the S15, S19, and S16 genes. The identification of DNA bent sites in genomic regions previously characterized as functionally relevant for DNA amplification further supports a function for DNA bent sites in DNA replication in eukaryotes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Conservation of pBuM–2 satellite DNA sequences among geographically isolated Drosophila gouveai populations from Brazil

In this study, we have compared 34 repetition units of pBuM-2 satellite DNA of individuals from six isolated populations of Drosophila gouveai, a cactophilic member of Drosophila buzzatii cluster (repleta group). In contrast to the results of previous morphological and molecular data, which suggest differentiation among the D. gouveai populations, the sequences and the cluster analysis of pBuM-2 monomers showed that this repetitive element is highly conserved among the six D. gouveai populations (97.8% similarity), indicating a slow rate of evolution of pBuM-2 sequences at the population level. Probably, some homogenization mechanisms of tandem sequences, such as unequal crossing or gene conversion, have maintained the sequence similarity of pBuM-2 among D. gouveai populations. Alternatively, such a result may be associated with a functional role of pBuM-2 sequences, although it is not understood at present.     

Impact of Preservation Conditions on Fatty Acids,Xanthan Gum Production and Other Characteristics of <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">mangiferaeindicae</Emphasis> IBSBF 2103

The conditions of storage, cultivation and maintenance of microbial cultures should preserve the microbiological homogeneity, phenotypic and genotypic characteristics to ensure better reproducibility of metabolic production. To evaluate the influence of the storage condition on the composition of cell fatty acids, genetic profile and biochemical characteristics of Xanthomonas campestris pv. mangiferaeindicae IBSBF 2103, as well as, to identify its relationship with the yielding and viscosity of the xanthan gum produced, this study monitored the strain preserved in two simple and widely used conditions, ultra-freezer (?80 °C) and refrigeration (3–8 °C) during 5 months. Were identified and quantified 13 fatty acids. The cells preserved at ?80 °C showed more stable concentration of all fatty acids, producing more xanthan gum and with higher viscosity. The chromosomal analysis obtained with the enzyme XbaI revealed 17 distinct fragments with maximum size of 485 kilobases, without variations among the subcultures maintained in both storage conditions. The X. campestris pv. mangiferaeindicae subcultures preserved at ?80 °C showed less pronounced phenotypic variations, which had positive influence in the qualitative and quantitative characteristics of the xanthan gum produced.     

Fetal exposure to high-avidity TCR ligand enhances expansion of peripheral T regulatory cells

Lately, it has become clear that regulatory T cells (Tregs) play a major role in the maintenance of peripheral tolerance and control of autoimmunity. Despite these critical functions, the process underlying the development of Tregs remains largely undefined. Herein, altered peptide ligand (APL) variants derived from the proteolipid protein-1 (PLP1) epitope were expressed on immunoglobulins (Igs) and the resulting Ig-APLs were used to deliver the APLs from mother to fetus through the maternal placenta to influence thymic T cell selection. This delivery system was then adapted to the SJL/J mouse, a strain that expresses only the DM20 form of PLP, which lacks the dominant PLP1 epitope in the thymus during fetal and neonatal development. This model, which restores thymic T cell selection for PLP1, was then used to determine whether affinity plays a role in the development of Tregs. The findings show that fetal exposure to low-affinity peptide ligand was unable to drive development of Tregs while variants with higher affinity to the TCR resulted in significant seeding of the periphery with mature, naive Tregs. Thus, contrary to pathogenic T cells, Tregs require avid TCR-ligand interaction to undergo thymic development and maturation.     

Melanocortin-4 receptors expressed by cholinergic neurons regulate energy balance and glucose homeostasis

Melanocortin-4 receptor (MC4R) mutations cause dysregulation of energy balance and hyperinsulinemia. We have used mouse models to study the physiological roles of extrahypothalamic MC4Rs. Re-expression of MC4Rs in cholinergic neurons (ChAT-Cre, loxTB MC4R mice) modestly reduced body weight gain without altering food intake and was sufficient to normalize energy expenditure and attenuate hyperglycemia and hyperinsulinemia. In contrast, restoration of MC4R expression in brainstem neurons including those in the dorsal motor nucleus of the vagus (Phox2b-Cre, loxTB MC4R mice) was sufficient to attenuate hyperinsulinemia, while the hyperglycemia and energy balance were not normalized. Additionally, hepatic insulin action and insulin-mediated suppression of hepatic glucose production were improved in ChAT-Cre, loxTB MC4R mice. These findings suggest that MC4Rs expressed by cholinergic neurons regulate energy expenditure and hepatic glucose production. Our results also provide further evidence of the dissociation in pathways mediating the effects of melanocortins on energy balance and glucose homeostasis.     

Oxygen-evolving system and secondary quinonic acceptors are highly reduced in dark adapted Euglena cells: A thermoluminescence study

Characteristics of thermoluminescence glow curves were compared in three types of Euglena cells: (i) strictly autotrophic, Cramer and Myers cells; (ii) photoheterotrophic cells sampled from an exponentially growing culture containing lactate as substrate repressing the photosynthetic activity; (iii) semiautotrophic cells, sampled when the lactate being totally exhausted, the photosynthesis was enhanced.In autotrophic and semiautotrophic cells, composite curves were observed after series of two or more actinic flashes fired at –10°C, which can be deconvoluted into a large band peaking in the range 12–22°C and a smaller one near 40°C, This second band presents the characteristics of a typical B band (due to S_2/3Q_B ^- recombination), whereas the first one resembled the band, shifted by -15–20°C, which is observed in herbicide resistant plants. The amplitude of this major band, which was in all cases very low after one flash, exhibited oscillations of period four but rapidly damping, with maxima after two and six flashes. In contrast, photoheterotrophic Euglena displayed single, non-oscillating curves with maxima in the range 5–10°C.In autotrophic and semiautotrophic cells, oxidizing pretreatments by either a preillumination with one or more (up to twenty-five) flashes, or a far-red preillumination in the presence of methylviologen, followed by a short dark period, induced thermoluminescence bands almost single and shifted by +3–5°C, or +12°C, respectively. In autotrophic cells, far-red light plus methyl viologen treatment induced a band peaking at 31°C, as in isolated thylakoids from Euglena or higher plants, while it had barely any effect in photoheterotrophic cells.Due to metabolic activities in dark-adapted cells, a reduction of redox groups at the donor and acceptor sides of PS II dark-adapted cells is supposed to occur. Two different explanations can be proposed to explain such a shift in the position of the main band in dark-adapted autotrophic control. The first explanation would be that in these reducing conditions a decreasing value of the equilibrium constant for the reaction: S_nQ_A ^-Q_BS_nQ_AQ_B ^-, would determine the shift of the main TL band towards low temperatures, as observed in herbicide resistant material. The second explanation would be that the main band would correspond to peak III already observed in vivo and assigned to S_2/3Q_B ^2- recombinations.Abbreviations CM Cramer and Myers - D₁ a 32 kDa protein component of the PS II reaction center, psbA.gene product - D₂ a 34 kDa protein component of the PS II reaction center, psbD gene product - FR lar-red illumination - Lexpo and Lstat cells from lactate culture samples at exponential and stationary phase of growth - MV methylviologen - pBQ parabenzoquinone - PQ plastoquinone - PS II photosystem II - Q_A primary quinone electron acceptor - Q_B secondary quinone electron acceptor - TL thermoluminescence     

Effect of Time of Day on Performance,Hormonal and Metabolic Response during a 1000-M Cycling Time Trial

The aim of this study was to determine the effect of time of day on performance, pacing, and hormonal and metabolic responses during a 1000-m cycling time-trial. Nine male, recreational cyclists visited the laboratory four times. During the 1^st visit the participants performed an incremental test and during the 2^nd visit they performed a 1000-m cycling familiarization trial. On the 3^rd and 4^th visits, the participants performed a 1000-m TT at either 8 am or 6 pm, in randomized, repeated-measures, crossover design. The time to complete the time trial was lower in the evening than in the morning (88.2±8.7 versus 94.7±10.9 s, respectively, p<0.05), but there was no significant different in pacing. However, oxygen uptake and aerobic mechanical power output at 600 and 1000 m tended to be higher in the evening (p<0.07 and 0.09, respectively). There was also a main effect of time of day for insulin, cortisol, and total and free testosterone concentration, which were all higher in the morning (+60%, +26%, +31% and +22%, respectively, p<0.05). The growth hormone, was twofold higher in the evening (p<0.05). The plasma glucose was ∼11% lower in the morning (p<0.05). Glucagon, norepinephrine, epinephrine and lactate were similar for the morning and evening trials (p>0.05), but the norepinephrine response to the exercise was increased in the morning (+46%, p<0.05), and it was accompanied by a 5-fold increase in the response of glucose. Muscle recruitment, as measured by electromyography, was similar between morning and evening trials (p>0.05). Our findings suggest that performance was improved in the evening, and it was accompanied by an improved hormonal and metabolic milieu.