Halogenated derivatives of boldine with high selectivity for alpha1A-adrenoceptors in rat cerebral cortex

The selectivity of 3-nitrosoboldine and different halogenated derivatives of boldine (3-bromoboldine, 3,8-dibromoboldine and 3-chloroboldine) for alpha1-adrenoceptor subtypes was studied by examining [3H]-prazosin competition binding in rat cerebral cortex. In the competition experiments [3H]-prazosin binding was inhibited completely by all the compounds tested. The inhibition curves displayed shallow slopes which could be subdivided into high and low affinity components. The relative order of affinity and selectivity for alpha1A-adrenoceptors was 3-bromoboldine = 3,8-dibromoboldine = 3-chloroboldine > boldine > 3-nitrosoboldine. The competition curves for 3-bromoboldine remained shallow and biphasic following chloroethylclonidine treatment. Whereas the relative contribution of the high affinity sites increased, the 3-bromoboldine affinities at its high and low affinity sites remained similar to those obtained in untreated membranes. 3-Bromoboldine, 3,8-dibromoboldine, 3-chloroboldine and 3-nitrosoboldine did not significantly displace [3H]-(+)-cis-diltiazem binding to rat cerebral cortex membranes. This activity was lower than that shown by boldine. Compared to boldine, halogen (bromine or chlorine) substitution at position 3 increases the alpha1A-adrenoceptor subtype selectivity and decreases the affinity for the benzothiazepine binding site at the calcium channel. Further halogen substitution at position 8 did not significantly improve this activity with respect to 3-bromoboldine. In contrast, the NO substitution at position 3 of boldine (3-nitrosoboldine) gives a loss of affinity and selectivity for alpha1-adrenoceptor subtypes.     

Successful laparoscopic insemination with a very low number of flow cytometrically sorted boar sperm in field conditions

The aim of this study was to develop a useful procedure for laparoscopic insemination (LI) with sex-sorted boar spermatozoa that yields adequate fertility results in farm conditions. In experiment 1, we evaluated the effects of single (oviducts) and double (oviducts and tips of the uterine horns) LI with X-sorted sperm on the reproductive performance of sows. Sows (N = 109) were inseminated once as follows: (1) single LI with 0.5 × 10⁶ unsorted sperm per oviduct; (2) single LI with 0.5 × 10⁶ sex-sorted sperm per oviduct; or (3) double LI with 0.5 × 10⁶ sex-sorted sperm per oviduct and 0.5 × 10⁶ sex-sorted sperm per uterine horn. The farrowing rates were lower (P < 0.05) in sows inseminated with sex-sorted sperm (43.2% and 61.9% for the single and double insemination groups, respectively) than in sows from the unsorted group (91.3%). Within the sex-sorted groups, the farrowing rate tended (P = 0.09) to be greater in sows inseminated using double LI. There were no differences in the litter size among groups. In experiment 2, we evaluated the effect of the number of sex-sorted sperm on the reproductive performance of sows when using double LI. Sows (N = 109) were inseminated with sex-sorted sperm once using double LI with: (1) 0.5 × 10⁶ sperm per oviduct and 1 × 10⁶ sperm per uterine horn; or (2) 1 × 10⁶ sperm per oviduct and 2 × 10⁶ sperm per uterine horn. Similarly high pregnancy (90%) and farrowing (80%) rates were achieved in both groups. The sows inseminated with the highest number of sperm tended (P = 0.09) to have more piglets (10.8 ± 0.7 vs. 9.2 ± 0.6). A high female proportion (number of female births divided by the total of all births ≥0.92) was obtained in both experiments using X-sorted sperm. Our results indicate that the double LI procedure, using between 3 and 6 × 10⁶ sex-sorted sperm per sow produces adequate fertility at the farm level, making sperm-sexing technology potentially applicable in elite breeding units.     

An expanded auxin-inducible degron toolkit for Caenorhabditis elegans

The auxin-inducible degron (AID) system has emerged as a powerful tool to conditionally deplete proteins in a range of organisms and cell types. Here, we describe a toolkit to augment the use of the AID system in Caenorhabditis elegans. We have generated a set of single-copy, tissue-specific (germline, intestine, neuron, muscle, pharynx, hypodermis, seam cell, anchor cell) and pan-somatic TIR1-expressing strains carrying a co-expressed blue fluorescent reporter to enable use of both red and green channels in experiments. These transgenes are inserted into commonly used, well-characterized genetic loci. We confirmed that our TIR1-expressing strains produce the expected depletion phenotype for several nuclear and cytoplasmic AID-tagged endogenous substrates. We have also constructed a set of plasmids for constructing repair templates to generate fluorescent protein::AID fusions through CRISPR/Cas9-mediated genome editing. These plasmids are compatible with commonly used genome editing approaches in the C. elegans community (Gibson or SapTrap assembly of plasmid repair templates or PCR-derived linear repair templates). Together these reagents will complement existing TIR1 strains and facilitate rapid and high-throughput fluorescent protein::AID tagging of genes. This battery of new TIR1-expressing strains and modular, efficient cloning vectors serves as a platform for straightforward assembly of CRISPR/Cas9 repair templates for conditional protein depletion.     

Single-nucleotide polymorphisms in the Toll-like receptor 9 gene (TLR9): frequencies,pairwise linkage disequilibrium,and haplotypes in three U.S. ethnic groups and exploratory case-control disease association studies

TLR9 is a mammalian Toll-like receptor homologue that appears to function as an innate immune pattern recognition protein for motifs that are far more common in bacterial than in mammalian DNA. The gene was sequenced in 71 subjects from three self-identified U.S. ethnic groups to identify single-nucleotide polymorphisms (SNPs). A total of 20 SNPs were found of which only 20% were in the public dbSNP database. Four SNPs were relatively common in all three ethnic samples. Using these four SNPs, seven distinct haplotypes were statistically inferred, of which four accounted for 75% or more chromosomes. These four haplotypes could be distinguished from each other by the alleles of two SNPs (-1237 and 2848). Five exploratory nested case-control disease-association studies (asthma, DVT, MI, and COPD in European Americans and asthma in African Americans) were performed by genotyping DNA collected from four ongoing cohort studies. There was evidence suggesting increased risk for asthma with a C allele at -1237 (odds ratio 1.85, 95%CI 1.05 to 3.25) among European Americans (genotypes available from 67 cases and 152 controls). No other significant disease associations were detected. Replication of this finding in other, larger samples is needed. This study suggests that there is substantial diversity in human TLR9, possibly associated with asthma in Europeans but not African Americans. No association was detected with three other diseases potentially related to innate immunity.     

Feeding low-fat milk during infancy

Despite the widespread agreement that low-fat milk should not be used during infancy, there is a sizable portion of infants in the United States who were fed a diet that included low-fat milk (less than or equal to 2% fat). In 1985, 14% of infants 8 months old, 20% of infants 10 months old, and 32% of infants 12 months old were fed low-fat milk. The reasons given most often by mothers for low-fat milk use was their consideration that low-fat milk has less fat than whole cow's milk and that low-fat milk use was recommended/suggested by their physician. Nutrient intakes of infants fed low-fat milk are compared to those of infants fed whole cow's milk and infant formula. Except for fat, nutrient intakes of infants fed low-fat milk or whole cow's milk were similar. A majority of infants fed either low-fat milk or whole cow's milk received amounts of sodium, potassium, and chloride that exceeded the recommended safe and adequate ranges and amounts of iron below the RDA. These data are considered in relation to dietary requirements during infancy.     

Manganese-Mediated Lignin Degradation by Pleurotus pulmonarius

Pleurotus pulmonarius produced the strongest degradation of lignin during solid-state fermentation of [(sup14)C]lignin wheat straw with different fungi. A manganese-oxidizing peroxidase seemed to be involved in lignin attack, since the addition of Mn(sup2+) to the culture increased lignin mineralization by ca. 125%. This enzyme was purified and characterized from both solid-state fermentation and liquid cultures.     

Flavoenzyme-catalyzed redox cycling of hydroxylamino- and amino metabolites of 2,4,6-trinitrotoluene: implications for their cytotoxicity

The toxicity of 2,4,6-trinitrotoluene (TNT), a widespread environmental contaminant, is exerted through its enzymatic redox cycling and/or covalent binding of its reduction products to proteins and DNA. In this study, we examined the possibility of another cytotoxicity mechanism of the amino- and hydroxylamino metabolites of TNT, their flavoenzyme-catalyzed redox cycling. The above compounds acted as redox-cycling substrates for single-electron transferring NADPH:cytochrome P-450 reductase (P-450R) and ferredoxin:NADP(+) reductase (FNR), as well as substrates for the two-electron transferring flavoenzymes rat liver NAD(P)H:quinone oxidoreductase (NQO1) and Enterobacter cloacae NAD(P)H:nitroreductase (NR). Their reactivity in P-450R-, FNR-, and NR-catalyzed reactions increased with an increase in their single-electron reduction potential (E(1)(7)) or the decrease in the enthalpy of free radical formation. The cytotoxicity of the amino- and hydroxylamino metabolites of TNT towards bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) was partly prevented by the antioxidant N,N'-diphenyl-p-phenylene diamine and desferrioxamine, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea, thus pointing to the involvement of oxidative stress. In general, their cytotoxicity increased with an increase in their electron accepting properties, or their reactivity towards the single-electron transferring FNR and P-450R. Thus, our data imply that the flavoenzyme-catalyzed redox cycling of amino and hydroxylamino metabolites of TNT may be an important factor in their cytotoxicity.