Inhibition of adenosine 3',5'-cyclic monophosphate phosphodiesterase by colchicine: implications for glucagon and corticosteroid secretion

In the study reported, colchicine, often regarded as a specific inhibitor of microtubular function, was shown to exert a concentration-dependent inhibition of the low Km cyclic AMP phosphodiesterases of the pancreatic islet, adrenal cortex and various other tissues of the rat. The results indicated that colchicine is only slightly less active as an inhibitor of the enzyme than theophylline on a molar basis and kinetic analysis revealed that both inhibitors acted competitively in the case of the liver enzyme. Our results show that the inhibitory effect of colchicine upon cyclic AMP phosphodiesterase is a general property of the alkaloid at concentrations of 5 x 10(-5)M and above in both endocrine and non-endocrine tissues. Thus, results obtained employing colchicine at concentrations significantly greater than those which are known to lead to microtubular disaggregation must be viewed with great caution if incorrect implication of microtubular participation in biological processes is to be avoided. For example, we propose that the previously reported paradoxical stimulatory effects of colchicine on the secretion of glucagon from the rat pancreatic islet and on steroidogenesis in the rat adrenal may be due to cyclic AMP accumulation consequent upon phosphodiesterase inhibition in these endocrine tissues and not to microtubular disaggregation as has hitherto been assumed.     

Systematic biases in DNA copy number originate from isolation procedures

Background

The ability to accurately detect DNA copy number variation in both a sensitive and quantitative manner is important in many research areas. However, genome-wide DNA copy number analyses are complicated by variations in detection signal.

Results

While GC content has been used to correct for this, here we show that coverage biases are tissue-specific and independent of the detection method as demonstrated by next-generation sequencing and array CGH. Moreover, we show that DNA isolation stringency affects the degree of equimolar coverage and that the observed biases coincide with chromatin characteristics like gene expression, genomic isochores, and replication timing.

Conclusion

These results indicate that chromatin organization is a main determinant for differential DNA retrieval. These findings are highly relevant for germline and somatic DNA copy number variation analyses.     

Ligand and pathogen specificity of the Atlantic salmon serum C-type lectin

Background

An Atlantic salmon (Salmo salar) C-type lectin (SSL) binds to mannose and related sugars as well as to the surface of Aeromonas salmonicida. To characterize this lectin as a pathogen recognition receptor in salmon, aspects of its interaction with molecules and with intact pathogens were investigated.

Methods

SSL was isolated using whole-yeast-affinity and mannan-affinity chromatography. The binding of SSL to the two major surface molecules of A. salmonicida, lipopolysaccharide (LPS) and A-layer protein was investigated by western blotting and enzyme-linked immunosorbent assays. Microbial binding specificity of SSL was examined by whole cell binding assays using a range of species. Carbohydrate ligand specificity of SSL was examined using glycan array analysis and frontal affinity chromatography.

Results

SSL showed binding to bacteria and yeast including, Pseudomonas fluorescens, A. salmonicida, A. hydrophila, Pichia pastoris, and Saccharomyces cerevisiae, but there was no detectable binding to Yersinia ruckeri. In antimicrobial assays, SSL showed no activity against Escherichia coli, Bacillus subtilis, S. cerevisiae, or A. salmonicida, but it was found to agglutinate E. coli. The major surface molecule of A. salmonicida recognized by SSL was shown to be LPS and not the A-layer protein. LPS binding was mannose-inhibitable. Glycans containing N-acetylglucosamine were shown to be predominant ligands.

Conclusion

SSL has a distinct ligand preference while allowing recognition of a wide variety of related carbohydrate structures.

General Significance

SSL is likely to function as a wide-spectrum pattern recognition protein.     

Museum specimens provide reliable SNP data for population genomic analysis of a widely distributed but threatened cockatoo species

Natural history museums harbour a plethora of biological specimens which are of potential use in population and conservation genetic studies. Although technical advancements in museum genomics have enabled genome‐wide markers to be generated from aged museum specimens, the suitability of these data for robust biological inference is not well characterized. The aim of this study was to test the utility of museum specimens in population and conservation genomics by assessing the biological and technical validity of single nucleotide polymorphism (SNP) data derived from such samples. To achieve this, we generated thousands of SNPs from 47 red‐tailed black cockatoo (Calyptorhychus banksii) traditional museum samples (i.e. samples that were not collected with the primary intent of DNA analysis) and 113 fresh tissue samples (cryopreserved liver/muscle) using a restriction site‐associated DNA marker approach (DArTseq^?). Thousands of SNPs were successfully generated from most of the traditional museum samples (with a mean age of 44 years, ranging from 5 to 123 years), although 38% did not provide useful data. These SNPs exhibited higher error rates and contained significantly more missing data compared with SNPs from fresh tissue samples, likely due to considerable DNA fragmentation. However, based on simulation results, the level of genotyping error had a negligible effect on inference of population structure in this species. We did identify a bias towards low diversity SNPs in older samples that appears to compromise temporal inferences of genetic diversity. This study demonstrates the utility of a RADseq‐based method to produce reliable genome‐wide SNP data from traditional museum specimens.     

Chemical and biological pathways in the bacterial oxidation of arsenopyrite

Abstract: A moderately thermophilic mixed culture of bacteria catalysed the oxidative solubilization of arsenopyrite to give Fe(III), S(VI) and As(V). Toxic effects were observed in a few experiments due to teh build-up of As(III). The bacterial oxidation of arsenopyrite involved direct attack of the bacteria on the mineral to give AS(III). Subsequent oxidation of AS(III) to AS(V) occurred reaction with FE(III), but only in the presence of pyrite, which provide a catalytic surface. Arsenopyrite was unable to act as a catalyst. The pyrite- catalysed oxidation of As(III) to AS(V) by FE(III) usually only went to completion in the presence of bacteria, possibly due to their role in the provision of clean catalytic surfaces. Thus, toxic concentrations of As(III) may accumulate in reactors during the bacterial oxidation of arsenopyrite due to the absence of pyrite or a clean pyrite surface or to low concentrations of the effective oxidizing agent, Fe(III).     

Age-related hematologic changes in captive-reared houbara,white-bellied,and rufous-crested bustards

Blood samples were obtained at monthly intervals between April 1994 and March 1996 from captive-bred houbara (Chlamydotis undulata macqueenii), rufous-crested (Eupodotis ruficrista gindiana), and white-bellied (Eupodotis senegalensis) bustards from 4-24 wk of age. Hematology investigations were conducted to determine age-related changes and to establish reference values for growing chicks of these species. There were significant age-related changes in hematocrit, hemoglobin, and red cell count in young birds compared with those of adults. White cell counts (lymphocytes and monocytes) were higher in juvenile birds, compared with adult values.     

Conformational features of crystal-surface cellulose from higher plants

Native cellulose in higher plants forms crystalline fibrils a few nm across, with a substantial fraction of their glucan chains at the surface. The accepted crystal structures feature a flat-ribbon 21 helical chain conformation with every glucose residue locked to the next by hydrogen bonds from O-3' to O-5 and from O-2 to O-6'. Using solid-state NMR spectroscopy we show that the surface chains have a different C-6 conformation so that O-6 is not in the correct position for the hydrogen bond from O-2. We also present evidence consistent with a model in which alternate glucosyl residues are transiently or permanently twisted away from the flat-ribbon conformation of the chain, weakening the O-3' - 0-5 hydrogen bond. Previous molecular modelling and the modelling studies reported here indicate that this 'translational' chain conformation is energetically feasible and does not preclude binding of the surface chains to the interior chains, because the surface chains share the axial repeat distance of the 21 helix. Reduced intramolecular hydrogen bonding allows the surface chains to form more hydrogen bonds to external molecules in textiles, wood, paper and the living plant.     

Genome-wide RNAi of C. elegans using the hypersensitive rrf-3 strain reveals novel gene functions

RNA-mediated interference (RNAi) is a method to inhibit gene function by introduction of double-stranded RNA (dsRNA). Recently, an RNAi library was constructed that consists of bacterial clones expressing dsRNA, corresponding to nearly 90% of the 19,427 predicted genes of C. elegans. Feeding of this RNAi library to the standard wild-type laboratory strain Bristol N2 detected phenotypes for approximately 10% of the corresponding genes. To increase the number of genes for which a loss-of-function phenotype can be detected, we undertook a genome-wide RNAi screen using the rrf-3 mutant strain, which we found to be hypersensitive to RNAi. Feeding of the RNAi library to rrf-3 mutants resulted in additional loss-of-function phenotypes for 393 genes, increasing the number of genes with a phenotype by 23%. These additional phenotypes are distributed over different phenotypic classes. We also studied interexperimental variability in RNAi results and found persistent levels of false negatives. In addition, we used the RNAi phenotypes obtained with the genome-wide screens to systematically clone seven existing genetic mutants with visible phenotypes. The genome-wide RNAi screen using rrf-3 significantly increased the functional data on the C. elegans genome. The resulting dataset will be valuable in conjunction with other functional genomics approaches, as well as in other model organisms.