Photooxidation of pterin in aqueous solutions: biological and biomedical implications

Studies of the photochemical reactivity of pterin (= 2-aminopteridin-4(3H)-one; PT) in acidic (pH 5.0-6.0) and alkaline (pH 10.2-10.8) aqueous solutions have been performed. The photochemical reactions were followed by UV/VIS spectrophotometry, thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), and an enzymatic method for H2O2 determination. PT is not light-sensitive in the absence of molecular oxygen, but it undergoes photooxidation in the presence of O2, yielding several nonpteridinic products. The quantum yields for PT disappearance were found to be 8.2 (+/-0.6) x 10(-4) and 1.2 (+/-0.2) x 10(-3) in acidic and alkaline media, respectively. H2O2 was detected and quantified in irradiated solutions of PT; and its importance from a biomedical point of view is discussed. The rate constant of the chemical reaction between singlet oxygen ((1)O2) and PT was determined to be 2.5 (+/-0.2) x 10(5) l mol(-1) s(-1) in alkaline medium, and the role of (1)O2 in the photooxidation of pterin was evaluated.     

Towards a molecular dynamics consensus view of B-DNA flexibility

We present a systematic study of B-DNA flexibility in aqueous solution using long-scale molecular dynamics simulations with the two more recent versions of nucleic acids force fields (CHARMM27 and parmbsc0) using four long duplexes designed to contain several copies of each individual base pair step. Our study highlights some differences between pambsc0 and CHARMM27 families of simulations, but also extensive agreement in the representation of DNA flexibility. We also performed additional simulations with the older AMBER force fields parm94 and parm99, corrected for non-canonical backbone flips. Taken together, the results allow us to draw for the first time a consensus molecular dynamics picture of B-DNA flexibility.     

Extension of the Messapia x dicoccoides linkage map of Triticum turgidum (L.) Thell

A set of recombinant inbred lines (RIL) derived from a cross between the cultivar Messapia of durum wheat (Triticum turgidum var. durum) and the accession MG4343 of T. turgidum var. dicoccoides was analysed to increase the number of assigned markers and the resolution of the previously constructed genetic linkage map. An updated map of the durum wheat genome consisting of 458 loci was constructed. These loci include 261 Restriction Fragment Length Polymorphisms (RFLPs), 91 microsatellites (Simple Sequence Repeats, SSRs), 87 Amplified Fragment Length Polymorphisms (AFLPs), two ribosomal genes, and nine biochemical (seven seed storage proteins and two isozymes) and eight morphological markers. The loci were mapped on all 14 chromosomes of the A and B genomes, and covered a total distance of 3038.4 cM with an average distance of 6.7 cM between adjacent markers. The molecular markers were evenly distributed between the A and the B genomes (240 and 218 markers, respectively). An additional forty loci (8.8%) could not be assigned to a specific linkage group. A fraction (16.4%) of the markers significantly deviated from the expected Mendelian ratios; clusters of loci showing distorted segregation were found on the 1B, 2A, 2B, 3A, 4A, 7A and 7B chromosomes. The genetic lengths of the chromosomes range from 148.8 cM (chromosome 6B) to 318.0 cM (chromosome 2B) and approximately concur with their physical lengths. Chromosome 2B has the largest number of markers (47), while the chromosomes with the fewest markers are 3A and 6B (23). There are two gaps larger than 40 cM on chromosomes 2A and 3B. The durum wheat map was compared with the published maps of bread and durum wheats; the order of most common RFLP and SSR markers on the 14 chromosomes of the A and B genomes were nearly identical. A core-map can be extracted from the high-density Messapia x dicoccoides map and a subset of uniformly distributed markers can be used to detect and map quantitative trait loci.     

Occurrence of Fusaproliferin and Beauvericin in Fusarium-Contaminated Livestock Feed in Iowa

Fusarium fungal contaminants and related mycotoxins were investigated in eight maize feed samples submitted to the Iowa State University Veterinary Diagnostic Laboratory. Fusarium moniliforme, F. proliferatum, and F. subglutinans were isolated from seven, eight, and five samples, respectively. These strains belonged to mating populations A, D, and E of the teleomorph Gibberella fujikuroi. Fusaproliferin was detected at concentrations of 0.1 to 30 μg/g in four samples, and beauvericin was detected (0.1 to 3.0 μg/g) in five samples. Fumonisins were detected in all eight samples (1.1 to 14 μg/g). Ten of 11 strains of F. proliferatum and all 12 strains of F. subglutinans isolated from the samples produced fusaproliferin in culture on whole maize kernels (4 to 350 and 100 to 1,000 μg/g, respectively). Nine F. proliferatum strains also produced beauvericin in culture (85 to 350 μg/g), but none of the F. subglutinans strains produced beauvericin. Fumonisin B₁ was produced by all nine F. moniliforme strains (50 to 2,000 μg/g) and by 10 of the F. proliferatum strains (1,000 to 2,000 μg/g). This is the first report of the natural occurrence of fusaproliferin outside Italy and of the natural occurrence of beauvericin in North America.     

In vivo gene regulation in Salmonella spp. by a salicylate-dependent control circuit

Systems allowing tightly regulated expression of prokaryotic genes in vivo are important for performing functional studies of bacterial genes in host-pathogen interactions and establishing bacteria-based therapies. We integrated a regulatory control circuit activated by acetyl salicylic acid (ASA) in attenuated Salmonella enterica that carries an expression module with a gene of interest under control of the XylS2-dependent Pm promoter. This resulted in 20-150-fold induction ex vivo. The regulatory circuit was also efficiently induced by ASA when the bacteria resided in eukaryotic cells, both in vitro and in vivo. To validate the circuit, we administered Salmonella spp., carrying an expression module encoding the 5-fluorocytosine-converting enzyme cytosine deaminase in the bacterial chromosome or in a plasmid, to mice with tumors. Induction with ASA before 5-fluorocytosine administration resulted in a significant reduction of tumor growth. These results demonstrate the usefulness of the regulatory control circuit to selectively switch on gene expression during bacterial infection.     

Distribution, population parameters, and diet of Astropecten marginatus (Asteroidea: Astropectinidae) in the Venezuelan Atlantic coast

Astropecten marginatus is a sea star widely distributed in Northern and Eastern South America, found on sandy and muddy bottoms, in shallow and deep waters. To describe some of its ecological characteristics, we calculated it spatial-temporal distribution, population parameters (based on size and weight) and diet in the Orinoco Delta ecoregion (Venezuela). The ecoregion was divided in three sections: Golfo de Paria, Boca de Serpiente and Plataforma Deltana. Samples for the rainy and dry seasons came from megabenthos surveys of the "Línea Base Ambiental Plataforma Deltana (LBAPD)" and "Corocoro Fase I (CFI)" projects. The collected sea stars were measured, weighted and dissected by the oral side to extract their stomach and identify the preys consumed. A total of 570 sea stars were collected in LBAPD project and 306 in CFI one. The highest densities were found during the dry season in almost all sections. In LBAPD project the highest density was in "Plataforma Deltana" section (0.007 +/- 0.022 ind/m2 in dry season and 0.014 +/- 0.06 ind/m2 in rainy season) and in the CFI project the densities in "Golfo de Paria" section were 0.705 +/- 0.829 ind/m2 in rainy season and 1.027 +/- 1.107 ind/m2 in dry season. The most frequent size range was 3.1-4.6cm. The highest biomass was found in "Golfo de Paria" section (7.581 +/- 0.018 mg/m2 in dry season and 0.005 +/- 6.542 x 10(-06) mg/m2 in rainy season for 2004-2005 and 3.979 +/- 4.024 mg/m2 in dry season; and 3.117 +/- 3.137 mg/m2 in rainy season for 2006). A linear relationship was found between the sea star size and its weight but no relationship was observed between its size and the depth where it was collected. Mollusks are dominant in the sea star diet (47.4% in abundance). The diet in any of the sections, seasons or between projects or size class was heterogeneous, using multivariate ordinations (MDS) and SIMPER analysis and there was no difference in the prey number or food elements that a sea star can eat. Although A. marginatus has been described as a predator, in this study were also inferred scavenger and detritivorous habits.     

Moderate exercise training provides left ventricular tolerance to acute pressure overload

The present study evaluated the impact of moderate exercise training on the cardiac tolerance to acute pressure overload. Male Wistar rats were randomly submitted to exercise training or sedentary lifestyle for 14 wk. At the end of this period, the animals were anaesthetized, mechanically ventilated, and submitted to hemodynamic evaluation with biventricular tip pressure manometers. Acute pressure overload was induced by banding the descending aorta to induce a 60% increase of peak systolic left ventricular pressure during 120 min. This resulted in the following experimental groups: 1) sedentary without banding (SED + Sham), 2) sedentary with banding (SED + Band), and 3) exercise trained with banding (EX + Band). In response to aortic banding, SED + Band animals could not sustain the 60% increase of peak systolic pressure for 120 min, even with additional narrowing of the banding. This was accompanied by a reduction of dP/dt(max) and dP/dt(min) and a prolongation of the time constant tau, indicating impaired systolic and diastolic function. This impairment was not observed in EX + Band (P < 0.05 vs. SED + Band). Additionally, compared with SED + Band, EX + Band presented less myocardial damage, exhibited attenuated protein expression of active caspase-3 and NF-κB (P < 0.016), and showed less protein carbonylation and nitration (P < 0.05). These findings support our hypothesis that exercise training has a protective role in the modulation of the early cardiac response to pressure overload.     

Biochemical and pharmacological characterization of PhTX-I a new myotoxic phospholipase A2 isolated from Porthidium hyoprora snake venom

This paper reports the biochemical and pharmacological characterization of a new myotoxic PLA(2) (EC 3.1.1.4) called PhTX-I, purified from Porthidium hyoprora venom by one step analytical chromatography reverse phase HPLC. The homogeneity of the PhTX-I fraction and its molecular mass were initially evaluated by SDS-PAGE and confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 14.249Da and constituted of a single polipeptidic chain. Amino acid sequence was determined by "de novo sequencing," in tandem mass spectrometry, belonging to D49-PLA(2) enzyme class and exhibiting high identity (44-90%) with other myotoxics PLA(2) from snake venoms. The enzymatic investigation showed maximal activity at pH 8 and 35-45°C. This activity was dependent on Ca(2+), other cations (Mg(2+), Mn(2+), Cd(2+) and Zn(2+)) reduced notably the enzymatic activity, suggesting that the arrangement of the catalytic site presents an exclusive structure for Ca(2+). Ex vivo, whole venom and PhTX-I PLA(2) caused blockade of the neuromuscular transmission in young chick biventer cervicis preparations similar to other isolated snake venom toxins from the Bothrops genus. In vivo, both induced local myotoxicity and systemic interleukin-6 response upon intramuscular injection, additionally, induced moderate footpad edema. In vitro, both induced low cytotoxicity in skeletal muscle myoblasts, however PhTX-I PLA(2) was able to lyse myotubes.     

Purification of chondroitin precursor from Escherichia coli K4 fermentation broth using membrane processing

Recently the possibility of producing the capsular polysaccharide K4, a fructosylated chondroitin, in fed-batch experiments was assessed. In the present study, a novel downstream process to obtain chondroitin from Escherichia coli K4 fermentation broth was developed. The process is simple, scalable and economical. In particular, downstream procedures were optimized with a particular aim of purifying a product suitable for further chemical modifications, in an attempt to develop a biotechnological platform for chondroitin sulfate production. During process development, membrane devices (ultrafiltration/diafiltration) were exploited, selecting the right cassette cut-offs for different phases of purification. The operational conditions (cross-flow rate and transmembrane pressure) used for the process were determined on an ?KTA cross-flow instrument (GE Healthcare, USA), a lab-scale automatic tangential flow filtration system. In addition, parameters such as selectivity and throughput were calculated based on the analytical quantification of K4 and defructosylated K4, as well as the major contaminants. The complete downstream procedure yielded about 75% chondroitin with a purity higher than 90%.