The chicken serotonin transporter discriminates between serotonin-selective reuptake inhibitors. A species-scanning mutagenesis study

The serotonin transporter (SERT) belongs to a family of sodium chloride-dependent transporters responsible for uptake of amino acids and biogenic amines from extracellular spaces. SERT represents the main pharmacological target in the treatment of several clinical conditions, including depression and anxiety. Serotonin-selective reuptake inhibitors and tricyclic antidepressants are the most predominantly prescribed drugs in the treatment of depression. In addition to antidepressants also psychostimulants, like cocaine and amphetamines, are important SERT antagonists. In the present study, we report the cloning and characterization of chicken SERT. Although the uptake kinetic was very similar to human SERT, the pharmacological profiles differed considerably for the two species. We find that chicken SERT is capable of discriminating between different serotonin-selective reuptake inhibitors; thus, the potency of S-citalopram and paroxetine is reduced more than 40-fold. A cross-species chimera strategy was undertaken and followed by species-scanning mutagenesis. Differences in pharmacological profiles were tracked to amino acid residues 169, 172, and 586 in human SERT. Structure-activity studies on structurally related compounds indicated that species divergences in drug sensitivity between human and chicken SERT were arising from differences in coordination or recognition of an important aminomethyl pharmacophoric substructure, which is shared by all high affinity antidepressants. Consequently, we suggest that Ala(169) and Ile(172) of human SERT are important residues in sensing the N-methylation state of SERT antagonists.     

Epidemiological survey of Giardia spp. and Blastocystis hominis in an Argentinian rural community

The aim of this study was to relate personal data, socio-cultural and environmental characteristics, and the presence of symptoms/signs with the frequencies of Giardia spp. and Blastocystis hominis among a rural population in Buenos Aires Province, Argentina. Of the surveyed population (350), 3.7% were infected with only Giardia spp. or 22.9% with B. hominis, and 2.3% were infected with both protozoa. The frequency of infection according to sex; 6.1% of males were infected and 1.6% of females by Giardia spp., 26.7% and 19.5% by B. hominis, and 2.4% and 2.2% by both parasites, respectively. Giardia spp. was detected in only three adults (over 14 years), but B. hominis was more frequent in adults than in children. The prevalences of these protozoa in this community are lower than those reported by other Argentinean studies, which is probably associated with the low density of the studied population (5.95 inhab/km2). Statistical analysis revealed that a male sex, flooding of the home, the use of a latrine, and an abdominal pain were correlated with the presence of these parasites, which indicate the importance of these factors in rural communities.     

Antibiotic-induced changes of mycelial growth ofBotrytis cinerea

The effect of antibiotics and metabolic inhibitors on mycelial growth ofBotrytis cinerea was followed. Inhibitors of protein synthesis, chloramphenicol, erythromycin and tetracycline inhibit growth or sporulation ofBotrytis cinerea. Ethidium bromide, 5-fluorouracil, phenylethylalcohol and K 20 cause granulation, vacuolization and undulation of hyphae. 2,4-Dinitrophenol, boromycin, macrotetrolides, monensin, scopathricin and TX₂ at subfungistatic concentrations induce intensive branching of hyphal tipsi.e. at the site of synthesis of the cell wall. In older hyphae grown in the absence of the antibiotics the branching begins after their addition, particularly in the septum region. When comparing the results referred to here with those obtained previously and on the basis of literature data it may be assumed that the changes in polarity of growth ofBotrytis cinerea might be caused primarily or secondarily by impairing membrane functions and formation of cell walls.     

Suppression of photo-induced sporulation inTrichoderma viride by inhibitors

The mycelium ofTrichoderma viride grown in the dark under submerged conditions and transferred to membrane filters sporulated only after photoinduction. The optimum photoinduction of sporulation was reached when applying daylight for 3 min and near ultraviolet radiation (366 nm) for 10 to 30 sec. After the photoinduction pronounced synthesis of DNA, RNA and protein was observed. The photoinduced sporulation was partially or fully inhibited in the presence of phenethyl alcohol, actinomycin D, 5-fluorouracil, cycloheximide and ethidium bromide. The same inhibitors blocked also the photoinduced sporulation of surface growing colonies ofTrichoderma viride. Various inhibitors of synthesis of nucleic acids and protein, inhibitors impairing the function of membranes and certain other compounds were also effective. A part of the results presented here was included in the dissertation of J.S. and defended at the Slovak Polytechnical University in Bratislava.     

Changes in the lipid composition during the photo-induced conidiation ofTrichoderma viride

The sporulating and nonsporulating mycelium ofTrichoderma viride exhibited significant differences in the levels of triacylglycerols, sterol esters, fatty acids, gangliosides, phosphatidylethanolamine and sphingomyelin. Quantitative changes in the contents of polar and nonpolar lipids were observed also during the analyses of one-, two-, and three-day-old sporulation zones in colonies subjected to repeated photo-induction. These findings are similar to the changes observed during the differentiation of other species of fungi.     

Classification of fungal antibiotics from the salting-out paper chromatogram

Пользуясь методом хроматографии с высаливанием (salting-out paper chromatogram), мы исследовали 19 антибиотиков из грибков. В 10 случаях мы установили принадлежность к 1-й группе, в 4 случаях—принадлежность к 3-й группе и в 5 случаях—принадлежность к 4-й группе.     

Vitamin D Status,Filaggrin Genotype,and Cardiovascular Risk Factors: A Mendelian Randomization Approach

Background

Vitamin D deficiency is associated with increased cardiovascular disease risk in observational studies. Whether these associations are causal is not clear. Loss-of-function mutations in the filaggrin gene result in up to 10% higher serum vitamin D concentrations, supposedly due to a decreased UV-protection of the keratinocytes. We used a Mendelian randomization approach to estimate the causal effect of vitamin D status on serum lipids, blood pressure, body mass index, waist circumference, and the metabolic syndrome.

Methods

Three population based studies were included, Monica10 (2,656 individuals aged 40–71 years), Inter99 (6,784 individuals aged 30–60 years), and Health2006 (3,471 individuals aged 18–69 years) conducted in 1993–94, 1999–2001, and 2006–2008, respectively. Participants were genotyped for the two most common filaggrin gene mutations in European descendants R501X and 2282del4, in all three studies and further for the R2447X mutation in the Inter99 and Health2006 studies. Filaggrin genotype was used as instrumental variable for vitamin D status. Baseline measurements of serum 25-hydroxyvitamin D were performed in all three studies.

Results

Instrumental variable analyses showed a 23.8% (95% confidence interval, CI 3.0, 48.6) higher HDL cholesterol level and a 30.5% (95% CI: 0.8, 51.3) lower serum level of triglycerides per doubling of vitamin D. These associations were, however, not statistically significant when applying the Bonferroni adjusted significance level. The remaining lipids showed non-significant changes in a favorable direction. Doubling of vitamin D gave a non-significantly lower odds ratio = 0.26 (95% CI: 0.06, 1.17) of the metabolic syndrome. There were no statistically significant causal effects of vitamin D status on blood pressure, body mass index, or waist circumference.

Conclusion

Our results support a causal effect of higher vitamin D status on a more favorable lipid profile, although more studies in other populations are needed to confirm our results.     

Bacillus thuringiensis Cry2Ab is active on Anopheles mosquitoes: single D block exchanges reveal critical residues involved in activity

Cry2Aa exhibits dual activity to Lepidoptera and Diptera. Cry2Ab differs in amino acid sequence from Cry2Aa by 13% and has shown significant lepidopteran activity, but no mosquitocidal activity. Previous studies implicate 23 Cry2Aa specificity-conferring residues of domain II, which differ in Cry2Ab. Nine residues are putatively involved in conferring Cry2Aa dipteran specificity. To explore Cry2Ab dipteran toxicity, site-directed mutagenesis was employed to exchange Cry2Ab residues with Cry2Aa D (dipteran) block residues. Cry2Ab wild type demonstrated high toxicity (LC(50) of 540 ng mL(-1)) to Anopheles gambiae, but not to Aedes or Culex, within a 24-h time period. Cry2Ab should be reclassified as a dual active Cry toxin. Cry2Ab mutagenesis revealed critical residues for Cry2Ab protein function, as well as enhanced activity against the malarial mosquito, An. gambiae.