Genetic portrait of Lisboa immigrant population from Cabo Verde with mitochondrial DNA analysis

Journal of Genetics -     

Esculetin induces apoptosis and inhibits adipogenesis in 3T3-L1 cells

Objective: To determine the effects of esculetin, a plant phenolic compound with apoptotic activity in cancer cells, on 3T3‐L1 adipocyte apoptosis and adipogenesis. Research Methods and Procedures: 3T3‐L1 pre‐confluent preadipocytes and lipid‐filled adipocytes were incubated with esculetin (0 to 800 μM) for up to 48 hours. Viability was determined using the Cell Titer 96 Aqueous One Solution cell proliferation assay; apoptosis was quantified by measurement of single‐stranded DNA. Post‐confluent preadipocytes were incubated with esculetin for up to 6 days during maturation. Adipogenesis was quantified by measuring lipid content using Nile Red dye; cells were also stained with Oil Red O for visual confirmation of effects on lipid accumulation. Results: In mature adipocytes, esculetin caused a time‐ and dose‐related increase in adipocyte apoptosis and a decrease in viability. Apoptosis was increased after only 6 hours by 400 and 800 μM esculetin (p < 0.05), and after 48 hours, as little as 50 μM esculetin increased apoptosis (p < 0.05). In preadipocytes, apoptosis was detectable only after 48 hours (p < 0.05) with 200 μM esculetin and higher concentrations. However, results of the cell viability assay indicated a reduction in preadipocyte number in a time‐ and dose‐related manner, beginning as early as 6 hours with 400 and 800 μM esculetin (p < 0.05). Esculetin also inhibited adipogenesis of 3T3‐L1 preadipocytes. Esculetin‐mediated inhibition of adipocyte differentiation occurred during the early, intermediate, and late stages of the differentiation process. In addition, esculetin induced apoptosis during the late stage of differentiation. Discussion: These findings suggest that esculetin can alter fat cell number by direct effects on cell viability, adipogenesis, and apoptosis in 3T3‐L1 cells.     

Nonuse of bicycle helmets and risk of fatal head injury: a proportional mortality, case�Ccontrol study

Background:

The effectiveness of helmets at preventing cycling fatalities, a leading cause of death among young adults worldwide, is controversial, and safety regulations for cycling vary by jurisdiction. We sought to determine whether nonuse of helmets is associated with an increased risk of fatal head injury.

Methods:

We used a case–control design involving 129 fatalities using data from a coroner’s review of cycling deaths in Ontario, Canada, between 2006 and 2010. We defined cases as cyclists who died as a result of head injuries; we defined controls as cyclists who died as a result of other injuries. The exposure variable was nonuse of a bicycle helmet.

Results:

Not wearing a helmet while cycling was associated with an increased risk of dying as a result of sustaining a head injury (adjusted odds ratio [OR] 3.1, 95% confidence interval [CI] 1.3–7.3). We saw the same relationship when we excluded people younger than 18 years from the analysis (adjusted OR 3.5, 95% CI 1.4–8.5) and when we used a more stringent case definition (i.e., only a head injury with no other substantial injuries; adjusted OR 3.6, 95% CI 1.2–10.2).

Interpretation:

Not wearing a helmet while cycling is associated with an increased risk of sustaining a fatal head injury. Policy changes and educational programs that increase the use of helmets while cycling may prevent deaths.One cyclist dies in Canada each week, and cycling fatalities account for more than 2% of traffic fatalities, a leading cause of death in young adults.¹ Cycling safety regulations vary by jurisdiction, and controversy remains about the effectiveness of safety measures such as helmets. There is strong evidence that helmets prevent nonfatal head injuries,² but very limited evidence exists related to fatal head injuries. A meta-analysis of case–control studies showed a protective effect of helmets against head injuries, but it was based on just 4 case fatalities in which helmets were not worn.³ Another large study involving 1710 cycling collisions found a trend toward a protective effect of helmets, but included only 14 fatalities.⁴ The existing literature leaves open the possibility that helmets prevent nonfatal head injuries, but not fatal ones.We sought to determine whether cycling without a helmet was associated with an increased risk of sustaining a fatal head injury.     

A conformationally mobile cysteine residue (Cys-561) modulates Na+ and H+ activation of human CNT3

In humans, the SLC28 concentrative nucleoside transporter (CNT) protein family is represented by three Na(+)-coupled members; human CNT1 (hCNT1) and hCNT2 are pyrimidine and purine nucleoside-selective, respectively, whereas hCNT3 transports both purine and pyrimidine nucleosides and nucleoside drugs. Belonging to a phylogenetic CNT subfamily distinct from hCNT1/2, hCNT3 also mediates H(+)/nucleoside cotransport. Using heterologous expression in Xenopus oocytes, we have characterized a cysteineless version of hCNT3 (hCNT3C-). Processed normally to the cell surface, hCNT3C-exhibited hCNT3-like transport properties, but displayed a decrease in apparent affinity specific for Na(+) and not H(+). Site-directed mutagenesis experiments in wild-type and hCNT3C-backgrounds identified intramembranous Cys-561 as the residue responsible for this altered Na(+)-binding phenotype. Alanine at this position restored Na(+) binding affinity, whereas substitution with larger neutral amino acids (threonine, valine, and isoleucine) abolished hCNT3 H(+)-dependent nucleoside transport activity. Independent of these findings, we have established that Cys-561 is located in a mobile region of the hCNT3 translocation pore adjacent to the nucleoside binding pocket and that access of p-chloromercuribenzene sulfonate to this residue reports a specific H(+)-induced conformational state of the protein ( Slugoski, M. D., Ng, A. M. L., Yao, S. Y. M., Smith, K. M., Lin, C. C., Zhang, J., Karpinski, E., Cass, C. E., Baldwin, S. A., and Young, J. D. (2008) J. Biol. Chem. 283, 8496-8507 ). The present investigation validates hCNT3C- as a template for substituted cysteine accessibility method studies of CNTs and reveals a pivotal functional role for Cys-561 in Na(+)- as well as H(+)-coupled modes of hCNT3 nucleoside transport.     

Engineering E. coli Alkaline Phosphatase Yields Changes of Catalytic Activity, Thermal Stability and Phosphate Inhibition

To investigate the function of aspartic acid residue 101 and arginine residue 166 in the active site of Escherichia coli alkaline phosphatase (EAP), two single mutants D101S (Asp 101 →Ser) and R166K (Arg 166 →Lys) and a double mutant D101S/R166K of EAP were generated through site-directed mutagenesis based on over-lap PCR method. Their enzymatic kinetic properties, thermal stabilities and possible reaction mechanism were explored. In the presence of inorganic phosphate acceptor, 1 M diethanolamine buffer, the k cat for D101S mutant enzyme increased 10-fold compared to that of wild-type EAP. The mutant R166K has a 2-fold decrease of k cat relative to the wild-type EAP, but the double mutant D101S/R166K was in the middle of them, indicative of an additive effect of these two mutations. On the other hand, the catalytic efficiencies of mutant enzymes are all reduced because of a substantial increase of K m values. All three mutants were more resistant to phosphate inhibitor than the wild-type enzyme. The analysis of the kinetic data suggests that (1) the D101S mutant enzyme obtains a higher catalytic activity by allowing a faster release of the product; (2) the R166K mutant enzyme can reduce the binding of the substrate and phosphate competitive inhibitor; (3) the double mutant enzyme has characteristics of both quicker catalytic turnover number and decreased affinity for competitive inhibitor. Additionally, pre-steady-state kinetics of D101S and D101S/R166K mutants revealed a transient burst followed by a linear steady state phase, obviously different from that of wild-type EAP, suggesting that the rate-limiting step has partially change from the release of phosphate from non-covalent E-Pi complex to the hydrolysis of covalent E-Pi complex for these two mutants.     

Regulation of K+ current in human airway epithelial cells by exogenous and autocrine adenosine

The regulatory actions ofadenosine on ion channel function are mediated by four distinctmembrane receptors. The concentration of adenosine in the vicinity ofthese receptors is controlled, in part, by inwardly directed nucleosidetransport. The purpose of this study was to characterize the effects ofadenosine on ion channels in A549 cells and the role of nucleosidetransporters in this regulation. Ion replacement and pharmacologicalstudies showed that adenosine and an inhibitor of human equilibrative nucleoside transporter (hENT)-1, nitrobenzylthioinosine, activated K⁺ channels, most likely Ca²⁺-dependentintermediate-conductance K⁺ (I_K)channels. A₁ but not A₂ receptor antagonistsblocked the effects of adenosine. RT-PCR studies showed that A549 cellsexpressed mRNA for I_K-1 channels as well asA₁, A_2A, and A_2B but notA₃ receptors. Similarly, mRNA for equilibrative (hENT1 andhENT2) but not concentrative (hCNT1, hCNT2, and hCNT3) nucleosidetransporters was detected, a result confirmed in functional uptakestudies. These studies showed that adenosine controls the function ofK⁺ channels in A549 cells and that hENTs play a crucialrole in this process.

     

Production of phytohormones by root-associated saprophytic actinomycetes isolated from the actinorhizal plant Ochetophila trinervis

The aim of the present study was to evaluate phytohormone production by symbiotic and saprophytic actinomycetes isolated from the actinorhizal plant Ochetophila trinervis which had previously proved to stimulate nodulation by Frankia. Three saprophytic strains out of 122, isolated from the rhizosphere of this plant with multiple enzymatic activities were selected for plant growth experiments in pots: Streptomyces sp. (BCRU-MM40), Actinoplanes sp. (BCRU-ME3) and Micromonospora sp. (BCRU-MM18). For experiments, the symbiotic N₂-fixing strain Frankia (BCU110501), isolated from nodules of the same actinorhizal plant was used. Phytohormone production was evaluated in supernatant of non-inoculated and inoculated culture media in exponential growth phase. Indole 3-acetic acid (IAA) and gibberellic acid (GA₃) were analyzed by gas chromatography-mass spectrometry (GC–MS), while zeatine (Z) production was determined by gas chromatography-flame ionization detector and high performance liquid chromatography (HPLC fluorescent and UV). The levels of the three phytohormones produced by the saprophytic rhizoactinomycetes were higher than that produced by the symbiotic Frankia strain. Zeatine biosynthesis was higher (μg ml⁻¹) than IAA and GA₃ (ng ml⁻¹), and Micromonospora strain produced the highest levels of these phytohormones. Although O. trinervis has been shown to be intercellularly infected by Frankia without mediation of root hair deformation, when plants were co-inoculated with actinomycetes’ culture, some root hair deformation was observed. This is the first report on identification of IAA, GA₃ and Z in saprophytic actinomycetes and their potential role in plant–microbe interaction.     

A Thermostable phosphofructokinase from the extreme thermophile Thermus X-1.

Two relatively simple procedures are described for the purification of phosphofructokinase from the extreme thermophile, Thermus X-1. The native enzyme has a molecular weight of 1.32 × 10⁵ and contains four apparently identical polypeptide chains. One substrate, fructose-6-phosphate, induces a cooperative protein transition while the other substrate, ATP, does not. Phosphoenolpyruvate functions as an avid negative effector and ADP is a positive effector. The enzyme has an optimum temperature for catalysis of 80 °C. Persistence of the catalytic and allosteric properties over the temperature range 20–80 °C suggests that the same protein structure is retained throughout this temperature range. Thermus X-1 phosphofructokinase is more stable to inactivation by heat, urea, guanidine hydrochloride or acidification than the phosphofructokinases obtained from the mesophilic organisms Escherichia coli and Clostridium pasteurianum. Comparison of the amino acid compositions of the three enzymes indicates no substantive differences in their hydrophobicity, hydrogen bonding potential or average residue size. The markedly elevated optimum temperature for catalysis exhibited by the Thermus enzyme appears to result from stabilization of its catalytically functional conformational to a reversible thermal inactivation above 40 °C and to ligation of the substrate fructose-6-phosphate.     

Molecular Ageing of Alpha- and Beta-Synucleins: Protein Damage and Repair Mechanisms

Abnormal α-synuclein aggregates are hallmarks of a number of neurodegenerative diseases. Alpha synuclein and β-synucleins are susceptible to post-translational modification as isoaspartate protein damage, which is regulated in vivo by the action of the repair enzyme protein L-isoaspartyl O-methyltransferase (PIMT). We aged in vitro native α-synuclein, the α-synuclein familial mutants A30P and A53T that give rise to Parkinsonian phenotypes, and β-synuclein, at physiological pH and temperature for a time course of up to 20 days. Resolution of native α-synuclein and β-synuclein by two dimensional techniques showed the accumulation of a number of post-translationally modified forms of both proteins. The levels of isoaspartate formed over the 20 day time course were quantified by exogenous methylation with PIMT using S-Adenosyl-L-[³H-methyl]methionine as a methyl donor, and liquid scintillation counting of liberated ³H-methanol. All α-synuclein proteins accumulated isoaspartate at ∼1% of molecules/day, ∼20 times faster than for β-synuclein. This disparity between rates of isoaspartate was confirmed by exogenous methylation of synucleins by PIMT, protein resolution by one-dimensional denaturing gel electrophoresis, and visualisation of ³H-methyl esters by autoradiography. Protein silver staining and autoradiography also revealed that α-synucleins accumulated stable oligomers that were resistant to denaturing conditions, and which also contained isoaspartate. Co-incubation of approximately equimolar β-synuclein with α-synuclein resulted in a significant reduction of isoaspartate formed in all α-synucleins after 20 days of ageing. Co-incubated α- and β-synucleins, or α, or β synucleins alone, were resolved by non-denaturing size exclusion chromatography and all formed oligomers of ∼57.5 kDa; consistent with tetramerization. Direct association of α-synuclein with β-synuclein in column fractions or from in vitro ageing co-incubations was demonstrated by their co-immunoprecipitation. These results provide an insight into the molecular differences between α- and β-synucleins during ageing, and highlight the susceptibility of α-synuclein to protein damage, and the potential protective role of β-synuclein.