Anion- and proton-dependent gating of ClC-4 anion/proton transporter under uncoupling conditions

ClC-4 is a secondary active transporter that exchanges Cl⁻ ions and H⁺ with a 2:1 stoichiometry. In external SCN⁻, ClC-4 becomes uncoupled and transports anions with high unitary transport rate. Upon voltage steps, the number of active transporters varies in a time-dependent manner, resembling voltage-dependent gating of ion channels. We here investigated modification of the voltage dependence of uncoupled ClC-4 by protons and anions to quantify association of substrates with the transporter. External acidification shifts voltage dependence of ClC-4 transport to more positive potentials and leads to reduced transport currents. Internal pH changes had less pronounced effects. Uncoupled ClC-4 transport is facilitated by elevated external [SCN⁻] but impaired by internal Cl⁻ and I⁻. Block by internal anions indicates the existence of an internal anion-binding site with high affinity that is not present in ClC channels. The voltage dependence of ClC-4 coupled transport is modulated by external protons and internal Cl⁻ in a manner similar to what is observed under uncoupling conditions. Our data illustrate functional differences but also similarities between ClC channels and transporters.     

Serial Non-Invasive Assessment of Antibody Induced Nephritis in Mice Using Positron Emission Tomography

Mouse models of experimental anti-glomerular basement membrane (anti-GBM) nephritis provide an analytical tool for studying spontaneous lupus nephritis. The potential of Positron Emission Tomography (PET) was evaluated using 2-deoxy-2-[¹⁸F]fluoro-d-glucose (FDG) as a probe to monitor the progression of anti-GBM induced nephritis in a mouse model. The imaging results were compared to conventional measures of renal function and pathological changes. Serum and urinary vascular cell adhesion molecule-1 (VCAM-1) levels were used as measures of endothelial cell activation and inflammation. Following a challenge with anti-glomerular antibodies, mice exhibited peak changes in serum creatinine, proteinuria, and glomerulonephritis score at 14 days post-challenge (p.c.). In contrast, VCAM levels peaked at day 7 p.c. On dynamic PET images (0–60 min) of day 7, kidneys of the anti-GBM nephritis mice demonstrated a unique pattern of FDG uptake. Compared to the time activity curve (TAC) prior to challenge, a rightward shift was observed after the challenge. By day 10 p.c., kidney FDG uptake was lower than baseline and remained so until the study ended at 21 days p.c. During this time frame measures of renal dysfunction remained high but VCAM-1 levels declined. These changes were accompanied by an increase in kidney volume as measured by Computed Tomography (CT) and intra-abdominal fluid collection. Our results suggest that FDG-PET-CT can be used as a non-invasive imaging tool to longitudinally monitor the progression of renal disease activity in antibody mediated nephritis and the magnitude of renal FDG retention correlates better with early markers of renal inflammation than renal dysfunction.     

Influence of Cobalt and Zinc Exposure on mRNA Expression Profiles of Metallothionein and Cytocrome P450 in Rainbow Trout

The present research aims to evaluate the effects of cobalt and zinc exposure of rainbow trout (Oncorhynchus mykiss) on metallothioneins and cytocrome P450. Mature rainbow trouts were exposed to 10?mg/L CoCl(2).6H(2)O and 1?mg/L ZnSO(4).7H(2)O. After 6, 12, 24, and 48?h of treatment, expressions of muscle MT-A, MT-B, and CYP P4501A1 mRNAs were measured by means of quantitative real-time polymerase chain reaction. During the exposure experiments, no mortalities occurred. We observed that expression levels of all genes increased with exposure time. Since the organism has not learned how to completely dispose of heavy metals and tends to bioaccumulate them, our results indicate that cobalt and zinc exposure may result in accumulation of the non-eliminated metals which may lead to fish death.     

The Effects of Chromium Complex and Level on Glucose Metabolism and Memory Acquisition in Rats Fed High-Fat Diet

Conditions in which glucose metabolism is impaired due to insulin resistance are associated with memory impairment. It was hypothesized that supplemental chromium (Cr) may alleviate insulin resistance in type 2 diabetes and consequently improve memory acquisition, depending upon its source and level. In a complete randomized design experiment, male Wistar rats (n=60; weighing 200-220 g) were fed either normal (8%, normal diet (ND)) or high-fat (40%, high-fat diet (HFD)) diet and supplemented with Cr as either chromium-glycinate (CrGly) or chromium-acetate (CrAc) at doses of 0, 40, or 80 μg/kg body weight (BW) via drinking water from 8 to 20 weeks of age. Feeding HFD induced type 2 diabetes, as reflected by greater glucose/insulin ratio (2.98 vs. 2.74) comparing to feeding ND. Moreover, HFD rats had greater BW (314 vs. 279 g) and less serum (53 vs. 68 μg/L) and brain (14 vs. 24 ng/g) Cr concentrations than ND rats. High-fat diet caused a 32% reduction in expressions of glucose transporters 1 and 3 (GLUTs) in brain tissue and a 27% reduction in mean percentage time spent in the target quadrant and a 38% increase in spatial memory acquisition phase (SMAP) compared with ND. Compared with supplemental Cr as CrAc, CrGly was more effective to ameliorate response variables (i.e., restoration of tissue Cr concentration, enhancement of cerebral GLUTs expressions, and reduction of the glucose/insulin ratio and SMAP) in a dose-response manner, especially in rats fed HFD. Supplemental Cr as CrGly may have therapeutic potential to enhance insulin action and alleviate memory acquisition in a dose-dependent manner, through restoring tissue Cr reserve and enhancing cerebral GLUTs expressions.     

Gliadin polymorphism in Turkish cultivated emmer wheat [Triticum turgidum L. ssp. dicoccon (Schrank) Thell.] landraces

Gliadin polymorphism in 19 landrace populations of Turkish cultivated emmer wheat [Triticum turgidum L. ssp. dicoccon (Schrank) Thell.] was assessed using the aluminum lactic acid-polyacrylamide gel electrophoresis (A-PAGE) technique. Being a source of useful genes, landraces of wheat represent one of the most important genetic resources available to breeders for present and future genetic improvement of wheat. This is the first genetic characterization of these 19 Turkish emmer wheat landrace populations collected from their main cultivation areas. Considerably high amounts of variation were detected within and among the populations. A total of 27 alleles (n _a) were identified among all analyzed populations, 10 of them being unique to populations?C, D, H, K, L, M, and N. The highest allele number (n _a?=?7) was observed in populations?A and L, whereas the lowest number of alleles (n _a?=?3) was observed in populations?F, G, and U. The mean number of effective alleles (n _ae) was 12.33, and the mean values of gene diversity, genetic differentiation, and gene flow between populations were H _e?=?0.92, F _ST?=?0.296, and N _m ?=?0.60, respectively. Certain gliadins closely linked to dough quality, such as ??-45 and ??-35, were found in 13 and 18 of the populations, respectively. According to Pearson??s correlation coefficient values, gene diversity estimates had strong positive correlation (r _P?=?0.510; p?=?0.026 at <0.05%) with latitude. The rest of the genetic data (n _a and n _ae) obtained in the present study showed no correlation with geographic (altitude, latitude, and longitude) or climatic factors (temperature and annual rainfall). Principal component analysis was performed to explain spatial genetic variation, revealing 90.044% of total genetic variation in three components. Results obtained from this study can effectively be used in developing more efficient breeding programs to improve wheat genotypes, and to direct genetic resource conservation studies.     

Five ovine mitochondrial lineages identified from sheep breeds of the near East

Archaeozoological evidence indicates that sheep were first domesticated in the Fertile Crescent. To search for DNA sequence diversity arising from previously undetected domestication events, this survey examined nine breeds of sheep from modern-day Turkey and Israel. A total of 2027 bp of mitochondrial DNA (mtDNA) sequence from 197 sheep revealed a total of 85 haplotypes and a high level of genetic diversity. Six individuals carried three haplotypes, which clustered separately from the known ovine mtDNA lineages A, B, and C. Analysis of genetic distance, mismatch distribution, and comparisons with wild sheep confirmed that these represent two additional mtDNA lineages denoted D and E. The two haplogroup E sequences were found to link the previously identified groups A and C. The single haplogroup D sequence branched with the eastern mouflon (Ovis orientalis), urial (O. vignei), and argali (O. ammon) sheep. High sequence diversity (K = 1.86%, haplogroup D and O. orientalis) indicates that the wild progenitor of this domestic lineage remains unresolved. The identification in this study of evidence for additional domestication events adds to the emerging view that sheep were recruited from wild populations multiple times in the same way as for other livestock species such as goat, cattle, and pig.     

In vitro anticholinesterase activity of various alkaloids

In the current study, a number of alkaloids including retamine, cytisine, and sparteine (quinolizidine-type), yohimbine and vincamine (indole-type), scopolamine and atropine (tropane-type), colchicine (tropolone-type), allantoin (imidazolidine-type), trigonelline (pyridine-type) as well as octopamine, synephrine, and capsaicin (exocyclic amine-type) were tested in vitro for their inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) at 1 mg/ml concentration by the Ellman method using an ELISA microplate reader. Among the alkaloids tested, only capsaicin exerted a remarkable inhibitory effect towards both AChE and BChE [(62.7 +/- 0.79)% and (75.3 +/- 0.98)%, respectively]. While the rest of the alkaloids did not show any significant inhibition against AChE, three of the alkaloids, namely retamine, sparteine, and yohimbine, exerted a noteworthy anti-BChE effect as compared to galanthamine, the reference drug.     

Synthesis and biological evaluation of new N-(2-hydroxy-4(or 5)-nitro/aminophenyl)benzamides and phenylacetamides as antimicrobial agents

A new series of N-(2-hydroxy-4(or 5)-nitro/aminophenyl)benzamide and phenylacetamide derivatives (1a-1n, 2a-2n) were synthesized and evaluated for antibacterial and antifungal activities against Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Candida albicans, and their drug-resistant isolate. Microbiological results indicated that the compounds possessed a broad spectrum of activity against the tested microorganisms at MIC values between 500 and 1.95 microg/ml. Benzamide derivative 1d exhibited the greatest activity with MIC values of 1.95, 3.9, and 7.8 microg/ml against drug-resistant B. subtilis, B. subtilis, and S. aureus, respectively.     

In vitro investigation of the apoptotic effect of heparin on lymphoblasts by using flow cytometric DNA analysis and fluorometric caspase-3 and -8 activities

The apoptotic effect of heparin on the lymphoblasts obtained from 12 newly diagnosed children with acute lymphoblastic leukemia (ALL) was investigated in vitro. The lymphoblasts were incubated with 0, 10, and 20 U/mL heparin concentrations at 0, 1, and 2 h. The percentages of the apoptotic lymphoblasts were calculated by flow cytometry (FCM), and activities of caspase-3 and -8 were simultaneously measured by fluorometric protease activity method. The apoptotic effect of heparin on the lymphoblasts was determined in 10 and 20 U/mL heparin concentrations (p < 0.005 and p < 0.001, respectively) while no apoptosis was detected in 0 U/mL heparin concentration at 0, 1, and 2 h. The apoptotic percentages of the lymphoblasts were higher at the first hour than those at 0 and 2 h in 10 and 20 U/mL heparin levels (p < 0.001). The highest apoptosis was found at first hour in 20 U/mL heparin concentration. Increased concentrations of heparin had an increasing effect on the percentages of the apoptotic lymphoblasts. Significantly higher caspase-3 and -8 activities were determined in 10 and 20 U/mL heparin concentrations than those in 0 U/mL heparin concentration at 0, 1, and 2 h (p < 0.001). There were no significant differences between the caspase-3 and -8 activities in 10 and 20 U/mL heparin concentrations at 1 and 2 h (p > 0.05), while statistically significant differences were simultaneously detected in the apoptotic rates of the lymphoblasts (p < 0.001). This may be due to that the study included the limited patients, or measurement of the caspase activities is a more sensitive method than the FCM analysis for determination of apoptosis because the activation time of the caspases takes a long time period. It was concluded that the apoptotic effect of heparin in vitro on lymphoblasts developed due to the extrinsic pathway of apoptosis via the caspase-3 and -8 activations in newly diagnosed ALL patients.