Influence of HOCl…O3 and HOCl…HOCl interactions on the stability of O3(HOCl)2 complexes: a theoretical study

We have analysed the role of HOCl…O₃ and HOCl…HOCl interactions on the stability of four estimated O₃(HOCl)₂ complexes by means of ab initio molecular orbital calculations. It is predicted that the O₃(HOCl) + HOCl reaction is more energetically favourable than (HOCl)₂ + O₃ one. In all complexes, HOCl…HOCl interaction is stronger than HOCl…O₃ one. The results show that the HOCl…O₃ interaction strengthens the HOCl…HOCl one. On the other hand, O…H interaction in HOCl…O₃ moiety is strengthened when it interacts with HOCl. Quantum theory of atoms in molecules predicts that the weak interactions in O₃(HOCl)₂ complexes have electrostatic characteristic. In all complexes, the charge transfer from O₃ to (HOCl)₂ is expected from natural bond orbital analysis.     

Cloning of casbene and neocembrene synthases from Euphorbiaceae plants and expression in Saccharomycescerevisiae

A large number of diterpenes have been isolated from Euphorbiaceae plants, many of which are of interest due to toxicity or potential therapeutic activity. Specific Euphorbiaceae diterpenes of medical interest include the latent HIV-1 activator prostratin (and related 12-deoxyphorbol esters), the analgesic resiniferatoxin, and the anticancer drug candidate ingenol 3-angelate. In spite of the large number of diterpenes isolated from these plants and the similarity of their core structures, there is little known about their biosynthetic pathways. Other than the enzymes involved in gibberellin biosynthesis, the only diterpene synthase isolated to date from the Euphorbiaceae has been casbene synthase, responsible for biosynthesis of a macrocyclic diterpene in the castor bean (Ricinus communis). Here, we have selected five Euphorbiaceae species in which to investigate terpene biosynthesis and report on the distribution of diterpene synthases within this family. We have discovered genes encoding putative casbene synthases in all of our selected Euphorbiaceae species and have demonstrated high-level casbene production through expression of four of these genes in a metabolically engineered strain of Saccharomyces cerevisiae. The only other diterpene synthase found among the five plants was a neocembrene synthase from R. communis (this being the first report of a neocembrene synthase gene). Based on the prevalence of casbene synthases, the lack of other candidates, and the structure of the casbene skeleton, we consider it likely that casbene is the precursor to a large number of Euphorbiaceae diterpenes. Casbene production levels of 31 mg/L were achieved in S. cerevisiae and we discuss strategies to further increase production by maximizing flux through the mevalonate pathway.     

Molecular pathogenesis of a new glycogenosis caused by a glycogenin-1 mutation

Glycogenin-1 initiates the glycogen synthesis in skeletal muscle by the autocatalytic formation of a short oligosaccharide at tyrosine 195. Glycogenin-1 catalyzes both the glucose-O-tyrosine linkage and the α1,4 glucosidic bonds linking the glucose molecules in the oligosaccharide. We recently described a patient with glycogen depletion in skeletal muscle as a result of a non-functional glycogenin-1. The patient carried a Thr83Met substitution in glycogenin-1. In this study we have investigated the importance of threonine 83 for the catalytic activity of glycogenin-1. Non-glucosylated glycogenin-1 constructs, with various amino acid substitutions in position 83 and 195, were expressed in a cell-free expression system and autoglucosylated in vitro. The autoglucosylation was analyzed by gel-shift on western blot, incorporation of radiolabeled UDP-(14)C-glucose and nano-liquid chromatography with tandem mass spectrometry (LC/MS/MS). We demonstrate that glycogenin-1 with the Thr83Met substitution is unable to form the glucose-O-tyrosine linkage at tyrosine 195 unless co-expressed with the catalytically active Tyr195Phe glycogenin-1. Our results explain the glycogen depletion in the patient expressing only Thr83Met glycogenin-1 and why heterozygous carriers without clinical symptoms show a small proportion of unglucosylated glycogenin-1.     

Comparison of Glutathione S-transferase Activity and Concentration in Aflatoxin-Producing and their Non-Toxigenic Counterpart Isolates

In this study, two techniques were used to compare the specific activity and total concentration of mycelial glutathione S-transferase (GST) in fungal strains isolated from natural sources. The fungi identified as Aspergillus parasiticus and Aspergillus flavus have been divided into two groups based on their ability to produce aflatoxins. Altogether 26 fungi were isolated, among which 12 were capable of producing varying levels of aflatoxin and 14 were proved to be non-toxigenic. GST specific activity in mycelial preparation was measured spectrophotometrically using 2,1-chloro-2,4-dinitrobenzene as the substrate. The results showed that the mean GST activity in toxigenic isolates was 25.06 +/- 9.8 mumol/mg protein/min which was 2.8-fold greater than that measured in non-toxigenic isolates (8.84 +/- 5.5 mumol/mg protein/min). Moreover, the GST concentration was compared in toxigenic and non-toxigenic isolates using an Enzyme Linked Immunosorbent Assay based on antigen (fungal preparation) and antibody (antibody produced against fungal GST in rabbit). The results of ELISA showed that the mean GST level in toxigenic and non-toxigenic fungi was 1.17 +/- 0.55 and 0.40 +/- 0.24, respectively. These results further confirm that the aflatoxin production in the fungal strains is correlated with GST expression and using ELISA, it is possible to discriminate aflatoxin-producing fungi from their non-toxigenic counterparts.     

Experimental and computational studies on the binding of diazinon to human serum albumin

In the present research, the binding properties of diazinon (DZN), as an organophosphorus herbicide, to human serum albumin (HSA) were investigated using combination of spectroscopic, electrochemistry, and molecular modeling techniques. Changes in the UV–Vis and FT-IR spectra were observed upon ligand binding along with a significant degree of tryptophan fluorescence quenching on complex formation. The obtained results from spectroscopic and electrochemistry experiments along with the computational studies suggest that DZN binds to residues located in subdomains IIA of HSA with binding constant about 1410.9 M^?1 at 300 K. From the thermodynamic parameters calculated according to the van’t Hoff equation, the enthalpy change ΔH° and entropy change ΔS° were found to be ?16.695 and 0.116 KJ/mol K, respectively. The primary binding pattern is determined by hydrophobic interaction and hydrogen binding occurring in so-called site I of HSA. DZN could slightly alter the secondary structure of HSA. All of experimental results are supported by computational techniques such as docking and molecular dynamics simulation using a HSA crystal model.     

A coupling of homology modeling with multiple molecular dynamics simulation for identifying representative conformation of GPCR structures: a case study on human bombesin receptor subtype-3

In this study, a computational pipeline was therefore devised to overcome homology modeling (HM) bottlenecks. The coupling of HM with molecular dynamics (MD) simulation is useful in that it tackles the sampling deficiency of dynamics simulations by providing good-quality initial guesses for the native structure. Indeed, HM also relaxes the severe requirement of force fields to explore the huge conformational space of protein structures. In this study, the interaction between the human bombesin receptor subtype-3 and MK-5046 was investigated integrating HM, molecular docking, and MD simulations. To improve conformational sampling in typical MD simulations of GPCRs, as in other biomolecules, multiple trajectories with different initial conditions can be employed rather than a single long trajectory. Multiple MD simulations of human bombesin receptor subtype-3 with different initial atomic velocities are applied to sample conformations in the vicinity of the structure generated by HM. The backbone atom conformational space distribution of replicates is analyzed employing principal components analysis. As a result, the averages of structural and dynamic properties over the twenty-one trajectories differ significantly from those obtained from individual trajectories.     

Hypoglycemic and Hypolipidemic Effects of Leucine,Zinc, and Chromium,Alone and in Combination,in Rats with Type 2 Diabetes

The main objective was to determine the elemental profile of the lung lining fluid of rats which are used as model animals in various experiments. Lung lining fluid elemental constitution obtained after bronchoalveolar lavage fluid (BALF) was analyzed by inductively coupled plasma mass spectrometry (ICP-MS) to determine the biological trace elements along with calcium and magnesium. BALF was collected from healthy rats using a tracheal cannula. However, cells in BALF were counted to monitor any underlying inflammatory lung condition. Cell free BALF samples were processed and analyzed for the elements including magnesium (Mg), calcium (Ca), chromium (Cr), manganese (Mn), iron (Fe), nickel (Ni), copper (Cu), zinc (Zn), selenium (Se), bromine (Br), and iodine (I). In view of this, calcium concentration was the highest (6318.08 ± 3094.3 μg/L) and copper concentration was the lowest (0.89 ± 0.21 μg/L). The detected elements, from high to low concentration, include Ca > Mg > Fe > Br > I > Cr > Ni > Zn > Mn > Se > Cu. Pearson’s correlation analysis revealed no significant correlation between cell count and concentration of any of the element detected in BALF. Correlation analysis also revealed significant positive correlation among Fe, I, Cr, Ni, and Mn. Ca was found to be correlated negatively with Cu and positively with Se. Br and Mg found to be positively correlated with each other. Zn remained the only element that was not found to be correlated with any of the elements in the rat BALF.     

Gamma irradiation as a useful tool for the isolation of astaxanthin-overproducing mutant strains of Phaffia rhodozyma

Astaxanthin is a carotenoid pigment responsible for the red color of the flesh of many marine animals. There is an increasing interest in the use of astaxanthin in aquaculture, chemical, pharmaceutical, and alimentary industries. Phaffia rhodozyma has been identified as the best biological source of astaxanthin. Mutagenesis was carried out using different doses of gamma irradiation (1.0, 2.0, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, and 7.0 kGy), and 10 mutant colonies (Gam1-Gam10) were obtained. Highly pigmented mutant strains produced astaxanthin at approximately 15?887.5?μg/L dry mass of yeast, whereas the parental strain produced it at 1061.64?μg/g dry mass of yeast. In the thin-layer chromatography analysis, P. rhodozyma JH-82 and Gam1 mutant strain produced the same retention factor (R(f)) values, but Gam1 showed a higher astaxanthin content than JH-82.     

Biodegradation of Petroleum Hydrocarbons in a Soil Polluted Sample by Oil-Based Drilling Cuttings

Microbial degradation of hydrocarbons in soils polluted by oil-based drilling mud and cuttings has been investigated by static methods such as composting or biopiling. Bioremediation of polluted soils by oil-based drilling cuttings through a slurry bioreactor has not previously been reported. The main aim of this work is to monitor hydrocarbon biodegradation in slurry of drilling cuttings and unpolluted soils and the effects of nutrients on it. Indigenous, bacterial-mixed culture isolated from a polluted soil by drilling cuttings adapted to drilling mud concentrations up to 15% (v/v) was done during a 15-month program. The total petroleum hydrocarbons’ (TPHs) removal efficiency in C/N/P 100/5/1 ratio was 90.5 and 79.85% under experimental and control conditions, respectively. The microbial count on the first day, 15 × 10⁷ CFUg^?1, reached 20 × 10⁹ CFUg^?1on the twenty-first day at experimental conditions. The TPH removal efficiency in C/N/P 100/10/2 was 92.5 and 82.25% at experiment and control, respectively. Increasing nitrogen and phosphorous amount couldn't increase microbial count in comparison with C/N/P ratio 100/5/1. The measured biomass contents and microbial counts in experiments were significantly higher than the control and confirmed hydrocarbons’ biodegradation during the time. Results showed that slurry bioreactors could accelerate the biodegradation of TPHs and reduce remediation time in soil polluted by oil-based drilling cuttings.     

Safety and possible outcome assessment of autologous Schwann cell and bone marrow mesenchymal stromal cell co-transplantation for treatment of patients with chronic spinal cord injury

Background aimsCell replacement therapy has become a promising issue that has raised much hope in the regeneration of central nervous system injury. Evidence indicates that successful functional recovery in patients with spinal cord injury will not simply emphasize a single therapeutic strategy. Therefore, many recent studies have used combination strategies for spinal cord regeneration.MethodsWe assessed the safety and feasibility of a bone marrow mesenchymal stromal cell and Schwann cell combination for the treatment of patients with chronic spinal cord injury. Eight subjects who received a complete traumatic spinal cord injury (American Spinal Injury Association [ASIA] classification A) enrolled in this study. The patients received this autologous combination of cells directly into the injury site. The mean duration of follow-up was approximately 24 months.ResultsNo magnetic resonance imaging evidence of neoplastic tissue overgrowth, syringomyelia or psuedomeningocele in any of the patients was seen during the study. There was no deterioration in sensory or motor function in any of the patients during the course of the study. Three patients had negligible improvement in ASIA sensory scale. No motor score improvement and no change in ASIA classification was seen. The patients had widely subjective changes in the course of the study such as urination and defecation sensation and more stability and trunk equilibrium in the sitting position.ConclusionsThere were no adverse findings at least 2 years after autologous transplantation of Schwann cell and mesenchymal stromal cell combination into the injured spinal cord. It appears that the use of this combination of cells is safe for clinical application to spinal cord regeneration.