High performance liquid chromatographic determination of topiramate in human serum using UV detection

Topiramate has no ultraviolet, visible or fluorescence absorption. Analysis of the drug in human serum has been reported by high performance liquid chromatography (HPLC) with either mass detector or fluorescence detection after precolumn derivatization using 9-fluorenylmethyl chloroformate as fluorescent labeling agent. This study was aimed to validate derivatization and analysis of topiramate in human serum with HPLC using UV detection. The drug was extracted from human serum by liquid-liquid extraction and subjected to derivatization with 9-fluorenylmethyl chloroformate. Analysis was performed on a phenyl column using of spectrophotometer detection operated at wavelength of 264 nm. A mixture of phosphate buffer (0.05M) containing triethylamine (1 ml/l, v/v; pH 2.3) and methanol (28:72, v/v) at a flow rate of 2.5 ml/min was used as mobile phase. No interference was found with endogenous substances. Validity of the method was studied and the method was precise and accurate with a linearity range from 40 ng/ml to 40 microg/ml. The limit of quantification was 40 ng/ml of serum. The correlation coefficient between HPLC methods using fluorescence and UV detections was studied and found to be 0.992.     

Isolation and Genotyping of Toxoplasma gondii Strains in Ovine Aborted Fetuses in Khorasan Razavi Province,Iran

Toxoplasmosis is an important zoonotic disease that can cause abortion in humans and animals. The aim of this study was isolation and subsequent genotyping of Toxoplasma gondii isolates in ovine aborted fetuses. During 2012-2013, 39 ovine aborted fetuses were collected from sheep flocks in Khorasan Razavi Province, Iran. The brain samples were screened for detection of the parasite DNA by nested PCR. The positive brain samples were bioassayed in Webster Swiss mice. The serum samples of mice were examined for T. gondii antibodies by IFAT at 6 weeks post inoculation, and T. gondii cysts were searched in brain tissue samples of seropositive mice. The positive samples were genotyped by using a PCR-RLFP method. Subsequently, GRA6 sequences of isolates were analyzed using a phylogenetic method. The results revealed that T. gondii DNA was detected in 54% (20/37, 95% CI 38.4-69.0%) brain samples of ovine aborted fetuses. In bioassay of mice, only 2 samples were virulent and the mice were killed at 30 days post inoculation, while the others were non-virulent to mice. The size of cysts ranged 7-22 µm. Complete genotyping data for GRA6 locus were observed in 5 of the 20 samples. PCR-RLFP results and phylogenetic analysis revealed that all of the isolated samples were closely related to type I. For the first time, we could genotype and report T. gondii isolates from ovine aborted fetuses in Khorasan Razavi Province, Iran. The results indicate that the T. gondii isolates are genetically related to type I, although most of them were non-virulent for mice.     

Biochemical characterization of an extracellular polyextremophilic α-amylase from the halophilic archaeon Halorubrum xinjiangense

An extracellular haloalkaliphilic thermostable α-amylase producing archaeon was isolated from the saltwater Lake Urmia and identified as Halorubrum xinjiangense on the basis of morphological, biochemical, and molecular properties. The enzyme was purified to an electrophoretically homogenous state by 80 % cold ethanol precipitation, followed by affinity chromatography. The concentrated pure amylase was eluted as a single peak on fast protein liquid chromatography. The molecular mass of the purified enzyme was about 60 kDa, with a pI value of 4.5. Maximum amylase activity was at 4 M NaCl or 4.5 M KCl, 70 °C, and pH 8.5. The K _m and V _max of the enzyme were determined as 3.8 mg ml^?1 and 12.4 U mg^?1, respectively. The pure amylase was stable in the presence of SDS, detergents, and organic solvents. In addition, the enzyme (20 U) hydrolyzed 69 % of the wheat starch after a 2-h incubation at 70 °C in an aqueous/hexadecane two-phase system.     

Molecular cloning and biologically active production of IpaD N-terminal region

Shigella is known as pathogenic intestinal bacteria in high dispersion and pathogenic bacteria due to invasive plasmid antigen (Ipa). So far, a number of Ipa proteins have been studied to introduce a new candidate vaccine. Here, for the first time, we examined whether the N-terminal region of IpaD^72–162 could be a proper candidate for Shigella vaccine. Initially, the DNA sequence coding N-terminal region was isolated by PCR from Shigella dysenteriae type I and cloned into pET-28a expression vector. Then, the heterologous protein was expressed, optimized and purified by affinity Ni–NTA column. Western blot analysis using, His-tag and IpaD^72–162 polyclonal antibodies, confirmed the purity and specificity of the recombinant protein, respectively. Subsequently, the high immunogenicity of the antigen was shown by ELISA. The results of the sereny test in Guinea pigs showed that IpaD^72–162 provides a protective system against Shigella flexneri 5a and S. dysenteriae type I.     

A novel soluble immune-type receptor (SITR) in teleost fish: carp SITR is involved in the nitric oxide-mediated response to a protozoan parasite

Background

The innate immune system relies upon a wide range of germ-line encoded receptors including a large number of immunoglobulin superfamily (IgSF) receptors. Different Ig-like immune receptor families have been reported in mammals, birds, amphibians and fish. Most innate immune receptors of the IgSF are type I transmembrane proteins containing one or more extracellular Ig-like domains and their regulation of effector functions is mediated intracellularly by distinct stimulatory or inhibitory pathways.

Methodology/Principal Findings

Carp SITR was found in a substracted cDNA repertoire from carp macrophages, enriched for genes up-regulated in response to the protozoan parasite Trypanoplasma borreli. Carp SITR is a type I protein with two extracellular Ig domains in a unique organisation of a N-proximal V/C2 (or I-) type and a C-proximal V-type Ig domain, devoid of a transmembrane domain or any intracytoplasmic signalling motif. The carp SITR C-proximal V-type Ig domain, in particular, has a close sequence similarity and conserved structural characteristics to the mammalian CD300 molecules. By generating an anti-SITR antibody we could show that SITR protein expression was restricted to cells of the myeloid lineage. Carp SITR is abundantly expressed in macrophages and is secreted upon in vitro stimulation with the protozoan parasite T. borreli. Secretion of SITR protein during in vivo T. borreli infection suggests a role for this IgSF receptor in the host response to this protozoan parasite. Overexpression of carp SITR in mouse macrophages and knock-down of SITR protein expression in carp macrophages, using morpholino antisense technology, provided evidence for the involvement of carp SITR in the parasite-induced NO production.

Conclusion/Significance

We report the structural and functional characterization of a novel soluble immune-type receptor (SITR) in a teleost fish and propose a role for carp SITR in the NO-mediated response to a protozoan parasite.     

On the parasitoid complex (Hymenoptera: Chalcidoidea) of Anophococcus abaii (Hemiptera: Eriococcidae), with description of Metaphycus anophococcusi Lotfalizadeh,n. sp.

The parasitoid community of Anophococcus abaii (Danzig, 1990) – one of the main pests of Haloxylon in Iran – was studied. Four species of Chalcidoidea, belonging to the families Aphelinidae, Encyrtidae, Pteromalidae, and Signiphoridae, were found. Metaphycus anophococcusi Lotfalizadeh, n. sp. is described in the zebratus species group and compared with closely allied species of Metaphycus.     

Agrobacterium-mediated transformation of alfalfa (Medicago sativa) using a synthetic cry3a gene to enhance resistance against alfalfa weevil

To introduce genetic resistance against alfalfa weevil (Hypera postica), leaves and petiole explants of three commercial alfalfa genotypes, including Km-27, Kk-14 and Syn-18 were transformed with Agrobacterium tumefaciens strains GV101, LBA4404 and AGL01. All the Agrobacterium strains used harbored the recombinant binary vector pBI121 containing a synthetic cry3a gene under the control of CaMV35S promoter as well as the nptII gene as selectable marker. Transformed explants were cultured on callus-induction medium, and the germinated somatic embryos were then transferred to the regeneration medium. The primary transformants were evaluated by PCR and Southern blot analysis. The results indicated successful integration of the target gene into the genomes of primary transgenic lines. Moreover, the expression of Cry3a protein in the transgenic plants was confirmed by ELISA method. Three transgenic lines, including TL6, TL8 and TL11 showed significantly higher levels of insect resistance against H. postica larvae (mortality rate of 73–90 % after infestation), in comparison with the control plants during the two-year bioassays. All transgenic plants were fertile and no irregular behavior in terms of growth and the morphological traits were observed. Transgenic plants developed during the course of this study are currently being grown in greenhouse and will be crossed with each other for seed production.     

DNA‐binding activity and cytotoxic and cell‐cycle arrest properties of some new coumarin derivatives: a multispectral and computational investigation

Coumarins are the most important class of natural compounds found widely in various plants. Many coumarin derivatives with different biological and pharmacological activities have been synthesized. In this study, the antiapoptotic and cytotoxic effects and DNA‐binding properties of some synthetic coumarin derivatives (4b, 4d, 4f, 4 g (DBP‐g), 4 h and 4j) against K562 cell lines were investigated using different techniques. MTT assay indicated that the DBP‐g compound was more active than other derivatives, with a IC₅₀ value of 55 μM, and therefore this compound was chosen for further investigation. Apoptosis induction was assessed using acridine orange/ethidium bromide double‐staining and cell‐cycle analysis. In addition, in vitro DNA‐binding studies were carried out using ultraviolet–visible light absorption and fluorescence spectroscopy, as well as viscosity measurement and molecular modelling studies. In vitro results indicated that DBP‐g interacted with DNA through a groove‐binding mode with a binding constant (K_b) of 1.17 × 10⁴ M^?1. In agreement with other experimental data, molecular docking studies showed that DBP‐g is a minor groove binder. Overall, it can be concluded that DBP‐g could be used as an effective and novel chemotherapeutic agent.     

Adsorption Properties of Tetracycline onto Graphene Oxide: Equilibrium,Kinetic and Thermodynamic Studies

Graphene oxide (GO) nanoparticle is a high potential effective absorbent. Tetracycline (TC) is a broad-spectrum antibiotic produced, indicated for use against many bacterial infections. In the present research, a systematic study of the adsorption and release process of tetracycline on GO was performed by varying pH, sorption time and temperature. The results of our studies showed that tetracycline strongly loads on the GO surface via π–π interaction and cation–π bonding. Investigation of TC adsorption kinetics showed that the equilibrium was reached within 15 min following the pseudo-second-order model with observed rate constants of k₂ = 0.2742–0.5362 g/mg min (at different temperatures). The sorption data has interpreted by the Langmuir model with the maximum adsorption of 323 mg/g (298 K). The mean energy of adsorption was determined 1.83 kJ/mol (298 K) based on the Dubinin–Radushkevich (D–R) adsorption isotherm. Moreover, the thermodynamic parameters such as ΔH°, ΔS° and ΔG° values for the adsorption were estimated which indicated the endothermic and spontaneous nature of the sorption process. The electrochemistry approved an ideal reaction for the adsorption under electrodic process. Simulation of GO and TC was done by LAMMPS. Force studies in z direction showed that tetracycline comes close to GO sheet by C₈ direction. Then it goes far and turns and again comes close from amine group to the GO sheet.