Molecular cloning, phylogenetic analysis, and expression profiling of a grape CMP-sialic acid transporter-like gene induced by phytohormone and abiotic stress

Nucleotide sugars are synthesized in the cytosol and nucleus and transported into the lumen of the endoplasmic reticulum and the Golgi apparatus via nucleotide sugar transporters (NSTs). Because NSTs exhibit high similarities with triose phosphate translocators (TPTs), they are classified into the NST/TPT superfamily. Here, we identified 38 members of the NST/TPT family by screening the grapevine genome and proteome 12× database. Vitis vinifera NST/TPT proteins can be classified into two groups on the basis of their phylogenetic relationships. From these, we isolated a full-length cDNA encoding a putative NST and named it VvCSTLP1. VvCSTLP1 comprises 15 exons and 14 introns and exhibits high similarities with OsCSTLP2. A search for cis-regulatory elements in the promoter region of VvCSTLP1 revealed that this protein is probably regulated by phytohormones and abiotic stresses. The VvCSTLP1 cDNA encodes an open reading frame of 1065 bp, and the predicted polypeptide is 354 amino acids long with a molecular mass of 39.11 kDa. Expression of VvCSTLP1 was elevated during early berry development, and dramatically decreased after the initiation of ripening. VvCSTLP1 was highly expressed in old leaves and mature leaves, and at low levels in young leaves, pollen, roots, and tendrils. Finally, VvCSTLP1 expression was induced in response to 1-naphthaleneacetic acid, salicylic acid (SA), and boric acid treatments, but was decreased by drought stress. The regulation of VvCSTLP1 expression led us to conclude that it may play a role in cell wall composition and structure and in the cross-talk between the auxin, SA, and abiotic stress signaling pathways.     

Species distribution and detection of Crimean Congo Hemorrhagic Fever Virus (CCHFV) in field-collected ticks in Ankara Province, Central Anatolia, Turkey

Ticks may act as vectors for a number of infectious diseases including Crimean Congo Hemorrhagic Fever (CCHF). The causative agent is Crimean Congo Hemorrhagic Fever Virus (CCHFV), a member of Bunyaviridae, causing extensive ecchymosis, visceral bleeding and hepatic dysfunction with a high fatality rate in the affected individuals. CCHF was initially recognized in Turkey in 2002 and the current number of reported cases exceeds 4,400. This study was conducted to confirm the presence of tick species established as potential CCHFV vectors and investigate CCHFV activity in ticks at Ankara province, Turkey’s second most-densely populated province, where CCHF cases were demonstrated. A total of 1,196 adult ticks, collected from various animals and vegetation in 12 sites located in 5 counties of Ankara during April–July 2010 were identified to species level. Twenty-two tick pools from county K2 were also evaluated for the presence of CCHFV RNA via a one-step real-time RT-PCR assay and reactive results were further confirmed by an in house nested RT-PCR assay. Nine tick species were identified: Rhipicephalus bursa (44.9%), R. sanguineus (18.9%), R. turanicus (18.1%), Haemaphysalis parva (8.3%), Hyalomma marginatum marginatum (5.4%), H. aegyptium (1.4%), H. anatolicum excavatum (1.3%), Hae. punctata (0.3%) and Dermacentor marginatus (0.2%). A total of five tick pools (22.7%) were reactive in real-time and nested RT-PCR assays. The pools included R. bursa, H. m. marginatum and Hae. parva ticks, collected from mammal hosts from two villages in one county. This is the first documentation of CCHFV activity in ticks from Ankara province, which indicates requirement for detailed surveillance to predict high risk zones in the region.     

The in vitro and in vivo inhibitory effects of some sulfonamide derivatives on rainbow trout (Oncorhynchus mykiss) erythrocyte carbonic anhydrase activity

The in vitro and in vivo inhibitory effects of 5-(3alpha, 12alpha-dihydroxy-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (1), 5-(3alpha, 7alpha, 12alpha-trihydroxy-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (2), 5-(3alpha, 7alpha, 12alpha-triacetoxy-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (3) and acetazolamide on rainbow trout (Oncorhynchus mykiss) (RT) erythrocyte carbonic anhydrase (CA) were investigated. The RT erythrocyte CA was obtained by affinity chromatography with a yield of 20.9%, a specific activity of 422.5EU/mg protein and a purification of 222.4-fold. The purity of the enzyme was confirmed by SDS-PAGE. Inhibitory effects of the sulfonamides and acetazolamide on the RT erythrocyte CA were determined using the CO2-Hydratase method in vitro and in vivo studies. From in vitro studies, it was found that all the compounds inhibited CA. The obtained I50 value for the sulfonamides (1), (2) and (3) and acetazolamide were 0.83, 0.049, 0.82 and 0.052 microM, respectively. From in vivo studies, it was observed that CA was inhibited by the sulfonamides (1), (2) and (3) and acetazolamide.     

Cutting edge: A common polymorphism impairs cell surface trafficking and functional responses of TLR1 but protects against leprosy

TLRs constitute an essential family of pattern recognition molecules that, through direct recognition of conserved microbial components, initiate inflammatory responses following infection. In this role, TLR1 enables host responses to a variety of bacteria, including pathogenic species of mycobacteria. In this study, we report that I602S, a common single nucleotide polymorphism within TLR1, is associated with aberrant trafficking of the receptor to the cell surface and diminished responses of blood monocytes to bacterial agonists. When expressed in heterologous systems, the TLR1 602S variant, but not the TLR1 602I variant, exhibits the expected deficiencies in trafficking and responsiveness. Among white Europeans, the 602S allele represents the most common single nucleotide polymorphism affecting TLR function identified to date. Surprisingly, the 602S allele is associated with a decreased incidence of leprosy, suggesting that Mycobacterium leprae subverts the TLR system as a mechanism of immune evasion.     

Chronic tinnitus and BDNF/GDNF CpG promoter methylations: a case–control study

Brain-derived neurotrophic factor (BDNF) and Glial-derived neurotrophic factor (GDNF) are neurotrophic factors that play key roles in the auditory pathway. While the relationship between serum levels and polymorphisms of BDNF/GDNF and chronic tinnitus is emphasized in the literature, there is no study showing the link between the promoter methylations of these genes and tinnitus. For this purpose, the relationship between chronic tinnitus and peripheral blood derived BDNF/GDNF promoter methylations was investigated to identify their role in the pathophysiology of tinnitus. In this case–control study, we examined the possible effects of BDNF/GDNF methylations in the blood samples of patients with tinnitus complaints for more than 3 months. Sixty tinnitus subjects between the ages of 18–55 and 50 healthy control subjects in the same age group who were free of any otorhinolaryngology and systemic disease were selected for examination. Methylation of total 12 CpG sites in BDNF and GDNF promoter regions were determined by the bisulfite-pyrosequencing method. Statistically significant differences were detected between BDNF CpG6 and GDNF CpG3-5-6 methylation ratios in the comparison of control group and the chronic tinnitus patients (P?=?0.002, 0.0005, 0.00003, and 0.0029, respectively). To our knowledge, this is the first study in the literature investigating the relationship between chronic tinnitus and peripheral blood derived BDNF/GDNF promoter methylations. It is believed that the current results might be supported by investigating the relationships between BDNF/GDNF methylations and genotypes in future research using higher sample sizes.

     

Spectrofluorimetric determination of tobramycin in human serum and pharmaceutical preparations by derivatization with fluorescamine

A simple, sensitive and selective spectrofluorimetric method has been developed for the determination of tobramycin (TOB) in human serum and pharmaceutical preparations. The method is based on the reaction between the primary amino group of TOB and fluorescamine in borate buffer, pH 8.5, to give a highly fluorescent derivative which is measured at 469 nm after excitation at 388 nm. The fluorescence intensity was directly proportional to the concentration over the range 300–1500 ng/mL, with a limit of detection of 65 ng/mL and limit of quantitation of 215 ng/mL. All variables were investigated to optimize the reaction conditions. The method was validated according to International Conference on Harmonization guidelines in terms of specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness. Good recoveries were obtained ranging from 97.4 to 100.64%, indicating that no interference was observed from concomitants usually present in pharmaceutical dosage forms. The method was successfully, applied for the analysis of the drug substance in its pharmaceutical preparations and spiked serum samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Development of sensitive spectrofluorimetric and spectrophotometric methods for the determination of duloxetine in capsule and spiked human plasma

A new, sensitive and selective spectrofluorimetric method has been developed for the determination of duloxetine (DLX) in capsule and spiked human plasma. DLX, as a secondary amine compound, reacts with 7‐chloro‐4‐nitrobenzofurazon (NBD‐Cl), a highly sensitive fluorogenic and chromogenic reagent used in many investigations. The method is based on the reaction between the drug and NBD‐Cl in borate buffer at pH 8.5 to yield a highly fluorescent derivative that is measured at 523 nm after excitation at 478 nm. The fluorescence intensity was directly proportional to the concentration over the range 50–250 ng/mL. The reaction product was also measured spectrophotometrically. The relation between the absorbance at 478 nm and the concentration is rectilinear over the range 1.0–12.0 µg/mL. The methods were successfully applied for the determination of this drug in pharmaceutical dosage form. The spectrofluorimetric method was also successfully applied to the determination of duloxetine in spiked human plasma. The suggested procedures could be used for the determination of DLX in pure form, capsules and human plasma being sensitive, simple and selective. Copyright © 2014 John Wiley & Sons, Ltd.     

A very simple high‐performance liquid chromatographic method with fluorescence detection for the determination of gemifloxacin in human breast milk

A high‐performance liquid chromatographic method with fluorescence detection was developed and validated for the determination of gemifloxacin in human breast milk. The proposed method allows the determination of gemifloxacin in breast milk samples without complex sample preparation. The samples were mixed with a mobile phase and filtered with a 0.45 µm polytetrafluoroethylene filter before analysis. Chromatographic separation was carried out on a C18 column (150 × 4.6 mm, 5 µm I.D.) using methanol:50 mM ortho‐phosphoric acid solution (40:60) as the mobile phase with a 1.0 mL/min flow rate. Quantitation was performed using fluorescence detection with an excitation wavelength at 272 nm and an emission wavelength at 395 nm. The linear range was found to be 0.1–2.5 µg/mL. The method was applied successfully for the determination of gemifloxacin in breast milk obtained from a breastfeeding mother after oral administration of a single tablet that included 320 mg gemifloxacin per gemifloxacin tablet. Copyright © 2015 John Wiley & Sons, Ltd.