Identification of poly(cis-1,4-Isoprene) degradation intermediates during growth of moderately thermophilic actinomycetes on rubber and cloning of a functional lcp homologue from Nocardia farcinica strain E1

The enrichment and isolation of thermophilic bacteria capable of rubber [poly(cis-1,4-isoprene)] degradation revealed eight different strains exhibiting both currently known strategies used by rubber-degrading mesophilic bacteria. Taxonomic characterization of these isolates by 16S rRNA gene sequence analysis demonstrated closest relationships to Actinomadura nitritigenes, Nocardia farcinica, and Thermomonospora curvata. While strains related to N. farcinica exhibited adhesive growth as described for mycolic acid-containing actinomycetes belonging to the genus Gordonia, strains related to A. nitritigenes and T. curvata formed translucent halos on natural rubber latex agar as described for several mycelium-forming actinomycetes. For all strains, optimum growth rates were observed at 50 degrees C. The capability of rubber degradation was confirmed by mineralization experiments and by gel permeation chromatography (GPC). Intermediates resulting from early degradation steps were purified by preparative GPC, and their analysis by infrared spectroscopy revealed the occurrence of carbonyl carbon atoms. Staining with Schiff's reagent also revealed the presence of aldehyde groups in the intermediates. Bifunctional isoprenoid species terminated with a keto and aldehyde function were found by matrix-assisted laser desorption ionization-time-of-flight and electrospray ionization mass spectrometry analyses. Evidence was obtained that biodegradation of poly(cis-1,4-isoprene) is initiated by endocleavage, rather than by exocleavage. A gene (lcp) coding for a protein with high homology to Lcp (latex-clearing protein) from Streptomyces sp. strain K30 was identified in Nocardia farcinica E1. Streptomyces lividans TK23 expressing this Lcp homologue was able to cleave synthetic poly(cis-1,4-isoprene), confirming its involvement in initial polymer cleavage.     

Intracerebroventricular serotonin reduces the degree of acute hypoxic ventilatory depression in peripherally chemodenervated rabbits

Hypoxia causes changes in the rate of synthesis or release of neurotransmitters in the brain. The accumulation of serotonin (5-HT) in the central nervous system might cause hypoxic respiratory depression. In the present study, we aimed to examine the role of central 5-HT on normoxic and acute hypoxic ventilatory depression (AHVD) in peripheral chemoreceptors denervated rabbits. All experiments were performed in peripherally chemodenervated rabbits anesthetized with intravenous injection of urethane (400 mg/kg) and alpha-chloralose (40 mg/kg). For intracerebroventricular (ICV) administration of 5-HT (20 microg/kg) and ketanserin (10 microg/kg), a cannula was placed in left lateral ventricle by stereotaxic method. Respiratory frequency (fR), tidal volume (VT), ventilation minute volume (VE) and systemic arterial bood pressure (BP) were recorded in each experimental phases and mean arterial pressure was calculated (MAP). Heart rate (HR) was also determined from the pulsation of BP. The effects of ICV serotonin and ICV ketanserin on the indicated parameters during air breathing (normoxia) and breathing of hypoxia (8% O2--92% N2) were investigated. During hypoxia, fR, VT, VE, MAP and HR decreased, and AHVD was thus obtained. ICV injection of 5-HT during normoxia caused significant increases in VT (P < 0.001) and in VE (P < 0.01). On the other hand, ICV 5-HT injection reduced the degree of AHVD in peripherally chemodenervated rabbits during hypoxia (fR; P < 0.05, VT; P < 0.05 and VE; P < 0.01). After ICV injection of ketanserin, the enhancement of 5-HT on VE was prevented during normoxia. On the breathing of hypoxic gas after ICV ketanserin, the degree of AHVD was augmented. In conclusion, our findings suggested that central 5-HT increases normoxic ventilation and reduces the degree of AHVD during hypoxia and that ICV ketanserin prevents the stimulatory effect of 5-HT on respiration and augments AHVD.     

The investigation of the antioxidative properties of the novel synthetic organoselenium compounds in some rat tissues

DMBA (7,12-dimethylbenz[a]anthracene) is a polycyclic aromatic hydrocarbon (PAH) known to cause tumors in rats. Selenium is an essential element with physiological non-enzymatic antioxidant properties. Because of the health problems induced by many environmental pollutants, many efforts have been undertaken in evaluating the relative antioxidant potential of selenium and synthetic organoselenium compounds. In this study, adult female Wistar rats were treated with DMBA and the novel organoselenium compounds (1-isopropyl-3-methylbenzimidazole-2-selenone [SeI] and 1,3-di-p-methoxybenzylpyrimidine-2-selenone [SeII]) in the determined doses. The protective effects of novel synthetic organoselenium compounds (SeI and SeII) against DMBA-induced changes in superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) activities and total glutathione (GSH) and malone-dialdehyde (MDA) levels of rat heart and brain were investigated. It was determined that SeI and SeII fully or partially restored enzyme activity. It was also found that lipid peroxidation was also decreased in SeI and SeII treated groups. Consequently, it was determined that novel synthetic organoselenium compounds (SeI and SeII) provided protection of antioxidant activity, and protection against lipid peroxidation measured as MDA in SeI and SeII treated groups was provided by novel synthesized organoselenium compounds. The ability of the organoselenium compounds to prevent oxidative damage induced by DMBA in rats was rationalized.     

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Observation des spermatozoïdes au fort grossissement (MSOME): intérêt et perspectives

Motile Sperm Organelle Morphology Examination (MSOME) constitutes a real improvement in ART management and outcome, as it allows detection of specific sperm anomalies on living cells, which cannot be detected by routine analysis. MSOME applied to the selection of sperm injected into the oocyte is called IMSI (Intracytoplasmic Morphologically Selected sperm Injection) and is associated with a considerable improvement of implantation, clinical pregnancy and delivery rates. A high-power microscope (X 2,000 to X 10,000) and video enhancement system are necessary and technical limitations are related to cryptozoospermia and/or severe teratozoospermia. Compared to routine sperm morphology assessment, MSOME allows the detection of subtle cephalic anomalies, such as vacuoles. These vacuoles seem to have a deleterious effect on fertilization and embryo developmentin vitro. These observations have led to a detailed classification of anomalies and this morphological diagnosis on living sperm demonstrates that most sperm selected for conventional ICSI at X 400 are actually abnormal on MSOME at X 6,000 or more. In addition to the very good results obtained in IMSI, this new approach opens up interesting prospects concerning the relationship between the phenotype of the injected sperm and its fertilization capacity and embryo development. In terms of diagnosis, MSOME could be used to select and study homogeneous groups of normal sperm or homogeneous groups of sperm exhibiting the same well defined anomaly. Such studies, associated with fine analysis of injected sperm and follow-up of each oocyte and each embryo, should provide more information about the relationship between sperm structure and function and should help to define the relevant indications for IMSI and the choice of spermatozoa to be injected.     

Preparation and characterization of polyacrylic acid/karaya gum and polyacrylic acid/tamarind seed gum adducts and utilization in textile printing

Polymerization of 20% neutralized acrylic acid (Na form), AA, in presence of Karaya gum, KG, or tamarind seed gum, TG, at AA/gum weight ratio of 1/1 and 2/1 results in PAA/KG1, PAA/KG2, PAA/TG1 and PAA/TG2 adducts, respectively (where the suffix 1 or 2 stands for AA/gum ratios of 1/1 or 2/1). Infra red spectra of adducts are examined. Aqueous pastes of adducts, native gums and GG are of non-Newtonian thixotropic flow within a shear rate range of 4–40 s⁻¹. Adduct pastes (7.5% w/v) are of higher apparent viscosities (η) than their native gums or GG, and pastes of TG adducts are of higher η than KG adducts. Except for PAA/TG2 adduct, the power law does not correlate well to the other pastes. Preliminary trials showed that adducts are excellent thickeners for reactive and acid printing on wool, silk and nylon 6. Prints by adducts are of higher color strength than those by native gums or GG. GG paste was completely destroyed after storing for 7 days, whereas η of pastes of adducts and native gums were noticeably decreased upon storing.     

Structure-activity relationships of wheat flavone O-methyltransferase: a homodimer of convenience

Wheat flavone O-methyltransferase catalyzes three sequential methylations of the flavone tricetin. Like other flavonoid O-methyltransferases, the protein is a homodimer. We demonstrate, using analytical ultracentrifugation, that perchlorate dissociates the dimer into monomers. The resulting monomers retain all their catalytic capacity, including the ability to catalyze the three successive methylations. We show, using isothermal titration calorimetry, that the binding constant for S-adenosyl-L-methionine does not change significantly as the protein dissociates. The second substrate, tricetin, binds to the dimers but could not be tested with the monomers. CD, UV and fluorescence spectroscopy show that there are substantial changes in the structure of the protein as it dissociates. The fact that there are differences between the monomers and dimers even as the monomers maintain activity may be the result of the very low catalytic capacity of this enzyme. Maximal turnover numbers for the dimers and monomers are only about 6-7 per minute. Even though the binding pockets for S-adenosyl-L-methionine, tricetin, selgin and tricin are intact, selection of a catalytically competent structure may be a very slow step during catalysis.     

In-silico modeling of the mitotic spindle assembly checkpoint

Background

The Mitotic Spindle Assembly Checkpoint (^MSAC) is an evolutionary conserved mechanism that ensures the correct segregation of chromosomes by restraining cell cycle progression from entering anaphase until all chromosomes have made proper bipolar attachments to the mitotic spindle. Its malfunction can lead to cancer.

Principle Findings

We have constructed and validated for the human ^MSAC mechanism an in silico dynamical model, integrating 11 proteins and complexes. The model incorporates the perspectives of three central control pathways, namely Mad1/Mad2 induced Cdc20 sequestering based on the Template Model, MCC formation, and APC inhibition. Originating from the biochemical reactions for the underlying molecular processes, non-linear ordinary differential equations for the concentrations of 11 proteins and complexes of the ^MSAC are derived. Most of the kinetic constants are taken from literature, the remaining four unknown parameters are derived by an evolutionary optimization procedure for an objective function describing the dynamics of the APC:Cdc20 complex. MCC:APC dissociation is described by two alternatives, namely the “Dissociation” and the “Convey” model variants. The attachment of the kinetochore to microtubuli is simulated by a switching parameter silencing those reactions which are stopped by the attachment. For both, the Dissociation and the Convey variants, we compare two different scenarios concerning the microtubule attachment dependent control of the dissociation reaction. Our model is validated by simulation of ten perturbation experiments.

Conclusion

Only in the controlled case, our models show ^MSAC behaviour at meta- to anaphase transition in agreement with experimental observations. Our simulations revealed that for ^MSAC activation, Cdc20 is not fully sequestered; instead APC is inhibited by MCC binding.