Biosynthesis of poly-β-hydroxybutyrate (PHB) with a high molecular mass by a mutant strain of Azotobacter vinelandii (OPN)

The aim of this study was to characterize the influence of the aeration conditions on the production of PHB and its molecular mass in a mutant strain of Azotobacter vinelandii (OPN), which carries a mutation on ptsN, the gene encoding enzyme IIA^Ntr, previously shown to increase the accumulation of PHB. Cultures of A. vinelandii wild-type strain OP and its mutant derivative strain OPN were grown in 500-mL flasks, containing 100 and 200 mL of PY sucrose medium. PHB production and its molecular mass were analyzed at the end of the culture. The molecular mass (MM) was significantly influenced by the aeration conditions and strain used. A polymer with a higher molecular weight was produced under low aeration conditions for both strains. A maximal molecular mass of 2,026 kDa (equivalent to 3,670 kDa measured by GPC) was obtained with strain OPN cultured under low-aeration conditions, reaching a value two-fold higher than that obtained from the parental strain OP (MM?=?1,013 kDa) grown under the same conditions. Aeration conditions and the ptsN mutation influence the molecular mass of the PHB produced by A. vinelandii affecting in turn its physico-chemical properties.     

Water-Soluble Ruthenium Complexes Bearing Activity Against Protozoan Parasites

Parasitic illnesses are major causes of human disease and misery worldwide. Among them, both amebiasis and Chagas disease, caused by the protozoan parasites, Entamoeba histolytica and Trypanosoma cruzi, are responsible for thousands of annual deaths. The lack of safe and effective chemotherapy and/or the appearance of current drug resistance make the development of novel pharmacological tools for their treatment relevant. In this sense, within the framework of the medicinal inorganic chemistry, metal-based drugs appear to be a good alternative to find a pharmacological answer to parasitic diseases. In this work, novel ruthenium complexes [RuCl₂(HL)(HPTA)₂]Cl₂ with HL = bioactive 5-nitrofuryl containing thiosemicarbazones and PTA?=?1,3,5-triaza-7-phosphaadamantane have been synthesized and fully characterized. PTA was included as co-ligand in order to modulate complexes aqueous solubility. In fact, obtained complexes were water soluble. Their activity against T. cruzi and E. histolytica was evaluated in vitro. [RuCl₂(HL4)(HPTA)₂]Cl₂ complex, with HL4?=?N-phenyl-5-nitrofuryl-thiosemicarbazone, was the most active compound against both parasites. In particular, it showed an excellent activity against E. histolytica (half maximal inhibitory concentration (IC₅₀)?=?5.2 μM), even higher than that of the reference drug metronidazole. In addition, this complex turns out to be selective for E. histolytica (selectivity index (SI) >38). The potential mechanism of antiparasitic action of the obtained ruthenium complexes could involve oxidative stress for both parasites. Additionally, complexes could interact with DNA as second potential target by an intercalative-like mode. Obtained results could be considered a contribution in the search for metal compounds that could be active against multiple parasites.     

Inter- and intra-genetic variation of four wild populations of Prosopis using rapd-pcr fingerprints

The species of Prosopis (Leguminosae) are welladapted to grow in the arid regions of the world. In Mexico, nearly 70%of its territory accounts for arid lands and nine species of this genus arefound in the country which are currently utilized for multiple purposes. Giventhat a sustainable exploitation of this important natural resource is necessary,knowledge of the genetic variability of natural populations is required. Thegenetic variation among and within four wild populations of Prosopisglandulosa, P. juliflora, and two ofP. laevigata, located in the Mexican States of Chihuahua(1), Sinaloa (2), Guanajuato (3) and Hidalgo (4), respectively, was determinedusing random amplified polymorphic DNA (RAPDs). There was a genetic variation of72.48% among these four populations, and within them the estimated geneticvariation was 27.52%. Between the populations of P.laevigata the genetic variation obtained was 7.15%, and within them92.85% (AMOVA, P < 0.001). Based on the assumption ofWright's island model, the genetic variation found among populations correspondsto a gene flow (N_em) of 0.09, while the gene flow detected betweenthe populations of P. laevigata was 3.2. A total of 27 RAPDphenotypes were identified in the four populations; among them, 11 correspondedto P. laevigata. The phylogenetic analyses showed that thethree species are differentiated in three clades and that probably P.juliflora was the origin of the other two species. The protogynoustrait of their flowers and the outcrossing are the main factors that may explainthe genetic variation observed in these populations.     

Protein kinase C-associated kinase (PKK) mediates Bcl10-independent NF-kappa B activation induced by phorbol ester

Protein kinase C-associated kinase (PKK) is a recently described kinase of unknown function that was identified on the basis of its specific interaction with PKC beta. PKK contains N-terminal kinase and C-terminal ankyrin repeats domains linked to an intermediate region. Here we report that the kinase domain of PKK is highly homologous to that of two mediators of nuclear factor-kappa B (NF-kappa B) activation, RICK and RIP, but these related kinases have different C-terminal domains for binding to upstream factors. We find that expression of PKK, like RICK and RIP, induces NF-kappa B activation. Mutational analysis revealed that the kinase domain of PKK is essential for NF-kappa B activation, whereas replacement of serine residues in the putative activation loop did not affect the ability of PKK to activate NF-kappa B. A catalytic inactive PKK mutant inhibited NF-kappa B activation induced by phorbol ester and Ca(2+)-ionophore, but it did not block that mediated by tumor necrosis factor alpha, interleukin-1 beta, or Nod1. Inhibition of NF-kappa B activation by dominant negative PKK was reverted by co-expression of PKC beta I, suggesting a functional association between PKK and PKC beta I. PKK-mediated NF-kappa B activation required IKK alpha and IKK beta but not IKK gamma, the regulatory subunit of the IKK complex. Moreover, NF-kappa B activation induced by PKK was not inhibited by dominant negative Bimp1 and proceeded in the absence of Bcl10, two components of a recently described PKC signaling pathway. These results suggest that PKK is a member of the RICK/RIP family of kinases, which is involved in a PKC-activated NF-kappa B signaling pathway that is independent of Bcl10 and IKK gamma.     

Construction of an Aspergillus nidulans multicopy transformant for the xlnB gene and its use in purifying the minor X24 xylanase

 Using recombinant DNA techniques, an Aspergillus nidulans multicopy transformant for the gene xlnB coding for the minor X₂₄ xylanase has been constructed. When grown on glucose as sole carbon source this transformant secretes 114 U of xylanase (mg protein)^-1. In this culture condition, X₂₄ is the only xylanase secreted and the predominant protein in the culture filtrate. This strategy has been used to purify the X₂₄ enzyme to homogeneity. The purified xylanase showed a single band on sodium dodecyl sulphate/ polyacrylamide gel electrophoresis with a molecular mass of 24 kDa and had an isoelectric point of approximately 3.5. The enzyme was a non-debranching endo-1,4-β-xylan xylanohydrolase highly specific for xylans and showed optimal activity at pH 5.5 and 52°C. The X₂₄ xylanase had a Michaelis constant, K _m, of 12.43 mg oat spelt xylan ml^-1 and a V _max of 1639 μmol min^-1 (mg protein)^-1. Received: 17 May 1995/Received last revision: 25 September 1995/Accepted: 29 September 1995     

Production of 13C polyunsaturated fatty acids from the microalga Phaeodactylum tricornutum

An integrated process for the indoor production of ¹³C labelled PUFA from Phaeodactylum tricornutum is presented. The core of the process is a bubble column photobioreactor from which the exhaust gas from the reactor is returned to the culture by a low pressure compressor. To avoid accumulation of dissolved oxygen in the culture medium, the exhaust gas is bubbled through a sodium sulphite solution before returning it to the reactor. Carbon is removed from the medium before inoculating the alga, then labelled ¹³CO₂ is injected for pH control and carbon supply. The reactor has been operated in semicontinuous mode at a dilution rate of 0.01 h^–1, a biomass productivity of 0.1 g L^–1 d^–1 being obtained. Under this conditions both pH and dissolved oxygen were correctly controlled and the adequacy of the system for autotrophic production of labelled biomass was demonstrated. Analysis by GC-MS revealed that the fatty acids content of the biomass obtained was 10% d.wt., the content of eicosapentaenoic acid was 2.5% d.wt. All the fatty acids were labelled, more that 90% of the carbon present in these fatty acids was ¹³C. Element analysis of biomass and supernatant showed that 59.5% of injected carbon was assimilated into the biomass whereas 33% remained in the supernatant, and 7.5% remained undetected. Due to the high cost of ¹³CO₂ different strategies for the optimisation of labelled carbon use are proposed.     

Construction of novel chimeric proteins through the truncation of SEC2 and Sak from Staphylococcus aureus

It is an usual clinical phenomenon that cancer patients are prone to thrombosis. Until now, there have been no efficient methods or appropriate drugs to prevent and cure tumor thrombus. Therefore, the construction of a bifunctional chimeric protein for the treatment of cancer, complicated with thrombosis, is of great significance. Utilizing the superantigenic activity of staphylococcal enterotoxin C2 (SEC2) and the thrombolytic activity of staphylokinase (Sak), Sak-linker-SEC2 and SEC2-linker-Sak were constructed which had good anti-tumor and thrombolytic activities at the same time. Due to the intrinsic emetic activity of SEC2 and high molecular weight (MW) of chimeric proteins (44?kDa), their clinical applications will be restricted. In this study, novel chimeric proteins including ΔSEC2–ΔSak and ΔSak–ΔSEC2 were constructed through the truncation of SEC2 and Sak without 9-Ala linker and His-tag. Compared with the former, both the truncated proteins preserved nearly the same anti-tumor and thrombolytic activities. In addition, their MWs were only 29?kDa and their immunoreactivities were slightly lower than that of Sak-linker-SEC2 and SEC2-linker-Sak, respectively. Therefore, the novel chimeric proteins possessed merits and characteristics, such as low MS, low immunogenicity, and difunctionality which the former had not. It will be of great interest if the above-mentioned proteins can be used to cure Trousseau syndrome in clinic.     

New Data on Therangospodus oncalensis from the Berriasian Fuentesalvo Tracksite (Villar del Río,Soria, Spain): An Example of Gregarious Behaviour in Theropod Dinosaurs

Therangospodus oncalensis is a Berriasian theropod ichnotaxon from the Cameros Basin (Soria Province, Spain). We discovered new trackways assigned to this ichnotaxon during cleaning and conservation works at the type locality (Fuentesalvo), enabling us to describe it more precisely. Therangospodus oncalensis was made by a medium-sized theropod dinosaur and is characterized by a single tapering pad on each toe, a rounded heel impression and a narrow trackway. In addition to having similar morphometric characteristics, most of the trackways at the Fuentesalvo site show comparable size and biometric features. This indicates that, in terms of size and age, a homogeneous population of a single theropod species could have produced these tracks. Moreover, the similar orientation (lying within a range of only 15°) and estimated speed suggested by most of the trackways, lend weight to the hypothesis that the trackmaker of Therangospodus oncalensis was able, at least at times, to move in structured packs displaying gregarious behaviour. Furthermore, this is the first structured pack of theropods to be described.     

The Multiple-Minima Problem in Small Peptides Revisited. The Threshold Accepting Approach

Abstract

A recently reported optimization method, known as Threshold Accepting, was tested for the purpose of locating the structure of several peptide molecules with the lowest conformational energy. A comparison with previous results obtained with the Simulated Annealing technique was made. Our study indicate Threshold Accepting as a better technique in locating such structures.