Photodimerizations of hydroxy- and benzoylated 4-azachalcones and quantum chemical investigation of the reactions

Photodimerization reactions of compounds 4–6 gave four new cyclobutane-containing compounds (7–9) with full control over the stereochemistry at the four stereogenic centers. These new cyclobutane-containing compounds had β-truxinic (7a), δ-truxinic (7b and 9), and ε-truxillic (8) structures. However, o-, m-, and p-hydroxy 4-azachalcones (1–3) did not give photochemical cyclization products under any conditions (in solvent or in their solid or molten states). Experimental data suggested the possibility of frontier orbital control over stereochemical behavior, so some theoretical calculations were performed. Full geometrical optimization of compounds 1–9 was performed via DFT B3LYP/6-31⁺G**, and their electronic structures were also investigated. The geometries of the singlet and triplet states were initially optimized by density functional theory (DFT) and the configuration interaction singles (CIS) B3LYP/3-21⁺G** level. An additional calculation was performed for the triplet state using the ground-state geometry. The possible photochemical dimerization products of compounds 7–9 (a–g) and the intrinsic reaction coordinates (IRCs) of the reactions of compounds 4–6 were calculated theoretically by the DFT/3-21⁺G** method. The configurations (reactant, transition state, product, and reaction pathway) corresponding to the stationary points (minima or saddle points) were determined. The intrinsic reaction coordinates were followed to verify the energy profiles that connect each TS to the appropriate local minimum. The dimeric products expected from the calculations coincided with the dimers produced experimentally.     

Comparison of methods for detection of Blastocystis infection in routinely submitted stool samples, and also in IBS/IBD Patients in Ankara, Turkey

Background

This study compared diagnostic methods for identifying Blastocystis in stool samples, and evaluated the frequency of detection of Blastocystis in patients with irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD).

Results and Discussion

From a set of 105 stool specimens submitted for routine parasitological analysis, 30 were identified as positive for Blastocystis by the culture method. From that group of 30 positives, Lugol''s stain, trichrome staining, and an immunofluorescence assay identified 11, 15, and 26 samples as positive respectively. Using culture as a standard, the sensitivity of Lugol''s stain was 36.7%, trichrome staining was 50%, and the IFA stain was 86.7%. The specificity of Lugol''s stain was 91%, trichrome staining was 100%, and the IFA stain was 97.3%. In the group of 27 IBS and IBD patients, using all methods combined, we detected Blastocystis in 67% (18/27) of the patients. Blastocystis was detected in 33% (2/6) of IBD patients and 76% (16/21) of IBS patients. For comparison, trichrome staining alone, the method most frequently used in many countries, would have only identified Blastocystis infection in 29% (6/21) of the IBS patients. No parasitic co-infections were identified in the IBS/IBD patients. Most Blastocystis-positive IBS/IBD patients were over 36 with an average length of illness of 4.9 years.

Conclusions

Most IBS patients in this study were infected with Blastocystis. IFA staining may be a useful alternative to stool culture, especially if stool specimens have been chemically preserved.     

High-performance liquid chromatography assay for N-acetylcysteine in biological samples following derivatization with N-(1-pyrenyl)maleimide

N-Acetylcysteine is a thiol antioxidant with expanding clinical importance. A sensitive, rapid method for determining reduced N-acetylcysteine (NAC) concentration in biological samples has been developed which uses a modified reversed-phase high-performance liquid chromatography (HPLC) technique in conjunction with the derivatizing agent N-(1-pyrenyl)maleimide (NPM). The NAC-NPM adduct was analyzed by HPLC with fluorescence detection. The calibration curve for NAC was linear over the range 8–2500 nM and the coefficient of variation obtained for the within-run precision and the between-run precision for 0.5 mM NAC was 1.5% and 2.7%, respectively. Relative recovery of NAC from biological materials ranged between 86% and 96% and the limit of quantitation from biological samples was 32 nM. These results suggest practical advantages relative to other widely-accepted methods of NAC measurement.     

Population diversity and multiplicity of infection in Theileria annulata

The tick-borne apicomplexan parasite Theileria annulata is endemic in many sub-tropical countries and causes the bovine disease tropical theileriosis. Although the parasite is known to be highly diverse, detailed information is lacking on the genetic structure of natural populations and levels of multiplicity of infection in the cattle host. With the widespread deployment of live attenuated vaccines and the emergence of drug-resistant parasites in the field, it is vital to appreciate the factors which shape genetic diversity of the parasite both within individual hosts and in the wider population. This study addresses these issues and represents an extensive genetic analysis of T. annulata populations in two endemic countries utilising a high-throughput adaptation of a micro- and mini-satellite genotyping system. Parasite material was collected from infected cattle in defined regions of Turkey and Tunisia to allow a variety of analyses to be conducted. All animals (n = 305) were found to harbour multiple parasite genotypes and only two isolates shared an identical predominant multi-locus profile. A modelling approach was used to demonstrate that host age, location and vaccination status play a measurable role in determining multiplicity of infection in an individual animal. Age was shown to positively correlate with multiplicity of infection and while positive vaccination status exerted a similar effect, it was shown to be due not simply to the presence of the immunising genotype. Importantly, no direct evidence was found for the immunising genotype spreading or recombining within the local parasite community. Genetic analysis confirmed the tentative conclusion of a previous study that the parasite population appears to be, in general, panmictic. Nevertheless, evidence supporting linkage disequilibrium and a departure from panmixia was uncovered in some localities and a number of explanations for these findings are advanced.     

Hsa-miR-584-5p as a novel candidate biomarker in Turkish men with severe coronary artery disease

Molecular Biology Reports - Coronary artery disease (CAD) is still the preliminary cause of mortality and morbidity in the developed world. Identification of novel predictive and therapeutic...     

Salt stress effects on the photosynthetic electron transport chain in two chickpea lines differing in their salt stress tolerance

The main objective of this study was to evaluate the effects of salt stress on the photosynthetic electron transport chain using two chickpea lines (Cicer arietinum L.) differing in their salt stress tolerance at the germination stage (AKN 87 and AKN 290). Two weeks after sowing, seedlings were exposed to salt stress for 2 weeks and irrigated with 200 ml of 200 mM NaCl every 2 days. The polyphasic OJIP fluorescence transient and the 820-nm transmission kinetics (photosystem I) were used to evaluate the effects of salt stress on the functionality of the photosynthetic electron transport chain. It was observed that a signature for salt stress was a combination of a higher J step (V_J), a smaller IP amplitude, and little or no effect on the primary quantum yield of PSII (φ_Po). We observed for AKN 290 a shorter leaf life cycle, which may represent a mechanism to cope with salt stress. For severely salt-stressed leaves, an inhibition of electron flow between the PQ pool and P700 was found. The data also suggest that the properties of electron flow beyond PSI are affected by salt stress.     

Heterologous expression of wildtype and mutant myocilin in High Five insect cells shows comparable effects to cultivated trabecular meshwork cells

Myocilin (MYOC, TIGR) variations are associated with juvenile and primary open angle glaucoma (POAG). To investigate consequences of MYOC wildtype overexpression and selected mutations, we established a heterologous insect cell system (High Five). Wildtype, Pro370Leu, Gln368X and Lys423Glu were cloned into a modified pIB/V5-His (pEXIV) vector with and without downstream GFP in frame fusion. Mutations were introduced by in vitro mutagenesis. Heterologous expression was shown and analysed by RT-PCR, Western blotting, immunocytochemistry and fluorescence microscopy. Extended cultivation (>14 days) resulted in accumulation of MYOC protein for all variants in growing dilated cisterns of the rough endoplasmic reticulum. Finally cell death for overexpressed wildtype and mutants occurs. A direct attachment of ribosomes to these growing vesicles preceding the cell death was observed by electron microscopy. Our observations indicate that this system is suitable to trace the intracellular effects of MYOC mutants.     

Simultaneous determination of paracetamol, caffeine and propyphenazone in ternary mixtures by micellar electrokinetic capillary chromatography

A new micellar electrokinetic capillary chromatographic method has been developed to analyze the pharmaceutical preparations containing ternary combination of paracetamol (PAR), caffeine (CAF) and propyphenazone (PRO). Best results were obtained by using 20mM pH 9.0 borate buffer containing 30mM sodiumdodecylsulphate as the background electrolyte. Diflunisal (DIF) was used as internal standard (IS). The separation was performed through a fused silica capillary (50microm internal diameter, 44cm total length, 35.5cm effective length) at 25 degrees C with the application of 3s of hydrodynamic injection at 50mbar pressure and a potential of 29kV. Detection wavelength was 200nm. Under these conditions, the migration times were found to be 5.174min for PAR, 5.513min for CAF, 7.195min for DIF, and 9.366min for PRO. Linearity ranges for the method were determined as 2-200microgmL(-1) for PAR and CAF and 3-200microgmL(-1) for PRO. Limit of detections were found as 0.6microgmL(-1) for PAR and CAF and 0.8microgmL(-1) for PRO. According to the validation study, the developed method was proved to be accurate, precise, sensitive, specific, rugged and robust. Three pharmaceutical preparations, which are produced by different drug companies in Turkey, were analyzed by the developed method. One of the same preparations was also analyzed by the derivative ratio spectro zero-crossing spectrophotometric method reported in literature. No significant differences were found statistically.     

Osmolality, temperature, and membrane lipid composition modulate the activity of betaine transporter BetP in Corynebacterium glutamicum

The gram-positive soil bacterium Corynebacterium glutamicum, a major amino acid-producing microorganism in biotechnology, is equipped with several osmoregulated uptake systems for compatible solutes, which is relevant for the physiological response to osmotic stress. The most significant carrier, BetP, is instantly activated in response to an increasing cytoplasmic K(+) concentration. Importantly, it is also activated by chill stress independent of osmotic stress. We show that the activation of BetP by both osmotic stress and chill stress is altered in C. glutamicum cells grown at and adapted to low temperatures. BetP from cold-adapted cells is less sensitive to osmotic stress. In order to become susceptible for chill activation, cold-adapted cells in addition needed a certain amount of osmotic stimulation, indicating that there is cross talk of these two types of stimuli at the level of BetP activity. We further correlated the change in BetP regulation properties in cells grown at different temperatures to changes in the lipid composition of the plasma membrane. For this purpose, the glycerophospholipidome of C. glutamicum grown at different temperatures was analyzed by mass spectrometry using quantitative multiple precursor ion scanning. The molecular composition of glycerophospholipids was strongly affected by the growth temperature. The modulating influence of membrane lipid composition on BetP function was further corroborated by studying the influence of artificial modulation of membrane dynamics by local anesthetics and the lack of a possible influence of internally accumulated betaine on BetP activity.