Fly immunity: great expectations

Preliminary analysis of the Drosophila genome sequence reveals important similarities and differences between the functioning of mammalian and invertebrate immune systems.     

Exploring dynamics of protein structure determination and homology-based prediction to estimate the number of superfamilies and folds

Background

As tertiary structure is currently available only for a fraction of known protein families, it is important to assess what parts of sequence space have been structurally characterized. We consider protein domains whose structure can be predicted by sequence similarity to proteins with solved structure and address the following questions. Do these domains represent an unbiased random sample of all sequence families? Do targets solved by structural genomic initiatives (SGI) provide such a sample? What are approximate total numbers of structure-based superfamilies and folds among soluble globular domains?

Results

To make these assessments, we combine two approaches: (i) sequence analysis and homology-based structure prediction for proteins from complete genomes; and (ii) monitoring dynamics of the assigned structure set in time, with the accumulation of experimentally solved structures. In the Clusters of Orthologous Groups (COG) database, we map the growing population of structurally characterized domain families onto the network of sequence-based connections between domains. This mapping reveals a systematic bias suggesting that target families for structure determination tend to be located in highly populated areas of sequence space. In contrast, the subset of domains whose structure is initially inferred by SGI is similar to a random sample from the whole population. To accommodate for the observed bias, we propose a new non-parametric approach to the estimation of the total numbers of structural superfamilies and folds, which does not rely on a specific model of the sampling process. Based on dynamics of robust distribution-based parameters in the growing set of structure predictions, we estimate the total numbers of superfamilies and folds among soluble globular proteins in the COG database.

Conclusion

The set of currently solved protein structures allows for structure prediction in approximately a third of sequence-based domain families. The choice of targets for structure determination is biased towards domains with many sequence-based homologs. The growing SGI output in the future should further contribute to the reduction of this bias. The total number of structural superfamilies and folds in the COG database are estimated as ~4000 and ~1700. These numbers are respectively four and three times higher than the numbers of superfamilies and folds that can currently be assigned to COG proteins.     

Estimation of relatedness among non-pedigreed Yakutian cryo-bank bulls using molecular data: implications for conservation and breed management

Background

Yakutian cattle, the last remaining native cattle breed in Siberia, are well adapted to the extreme sub-arctic conditions. Nowadays only ca. 1200 purebred animals are left in Yakutia. The semen of six Yakutian bulls was stored in a cryo-bank without any pedigree documentation because of the traditional free herding style of the population.

Methods

To clarify the genetic relatedness between these bulls and to provide recommendations to use their semen in future conservation and breed management programs, we have analysed 30 autosomal microsatellites and mitochondrial DNA sequences in 60 individuals including the six for which semen has been stored. Four relatedness estimators were calculated. In addition, we assessed the value of the cryo-bank bulls for the preservation of genetic variation of the contemporary Yakutian cattle by calculating allelic and gene diversity estimates and mean molecular coancestries.

Results

On the basis of microsatellite variability, including the Yakutian cryo-bank bulls increases the allelic variation in the contemporary population by 3% and in the male subpopulation by 13%. In terms of the mean molecular coancestries, they are less related to the contemporary cow population than the breeding bulls and therefore could be used to reduce inbreeding in the living population. Although 30 loci are insufficient to resolve definitely their relatedness categories, the data suggest four pairs of cryo-bank bulls as possible half-sibs.

Conclusions

Our results show that even relatively limited cryo-bank storage of semen can carry allelic variation through a bottleneck. We propose a breeding scheme based on the rotation of breeding females and the division of cryo-bank bulls into three groups. Thus, if molecular data (e.g. autosomal microsatellite genotypes) for the contemporary population are available and based on relatively small-scale laboratory analyses, it is possible to avoid serious mistakes in their use for breeding applications. The approach suggested here based on the use of Yakutian cryo-bank semen can be easily extended to cryo-bank materials of other animals in future breeding programs.     

Energy Coupling Efficiency in the Type I ABC Transporter GlnPQ

Solute transport via ATP binding cassette (ABC) importers involves receptor-mediated substrate binding, which is followed by ATP-driven translocation of the substrate across the membrane. How these steps are exactly initiated and coupled, and how much ATP it takes to complete a full transport cycle, are subject of debate. Here, we reconstitute the ABC importer GlnPQ in nanodiscs and in proteoliposomes and determine substrate-(in)dependent ATP hydrolysis and transmembrane transport. We determined the conformational states of the substrate-binding domains (SBDs) by single-molecule Förster resonance energy transfer measurements. We find that the basal ATPase activity (ATP hydrolysis in the absence of substrate) is mainly caused by the docking of the closed-unliganded state of the SBDs onto the transporter domain of GlnPQ and that, unlike glutamine, arginine binds both SBDs but does not trigger their closing. Furthermore, comparison of the ATPase activity in nanodiscs with glutamine transport in proteoliposomes shows that the stoichiometry of ATP per substrate is close to two. These findings help understand the mechanism of transport and the energy coupling efficiency in ABC transporters with covalently linked SBDs, which may aid our understanding of Type I ABC importers in general.     

Tezosentan increases nitric oxide signaling via enhanced hydrogen peroxide generation in lambs with surgically induced acute increases in pulmonary blood flow

We have previously shown that acute increases in pulmonary blood flow (PBF) are limited by a compensatory increase in pulmonary vascular resistance (PVR) via an endothelin‐1 (ET‐1) dependent decrease in nitric oxide synthase (NOS) activity. The mechanisms underlying the reduction in NO signaling are unresolved. Thus, the purpose of this study was to elucidate mechanisms of this ET‐1–NO interaction. Pulmonary arterial endothelial cells were acutely exposed to shear stress in the presence or absence of tezosentan, a combined ET_A/ET_B receptor antagonist. Shear increased NO_x, eNOS phospho‐Ser1177, and H₂O₂ and decreased catalase activity; tezosentan enhanced, while ET‐1 attenuated all of these changes. In addition, ET‐1 increased eNOS phospho‐Thr495 levels. In lambs, 4 h of increased PBF decreased H₂O₂, eNOS phospho‐Ser1177, and NO_X levels, and increased eNOS phospho‐Thr495, phospho‐catalase, and catalase activity. These changes were reversed by tezosentan. PEG‐catalase reversed the positive effects of tezosentan on NO signaling. In all groups, opening the shunt resulted in a rapid increase in PBF by 30 min. In vehicle‐ and tezosentan/PEG‐catalase lambs, PBF did not change further over the 4 h study period. PVR fell by 30 min in vehicle‐ and tezosentan‐treated lambs, and by 60 min in tezosentan/PEG‐catalase‐treated lambs. In vehicle‐ and tezosentan/PEG‐catalase lambs, PVR did not change further over the 4 h study period. In tezosentan‐treated lambs, PBF continued to increase and LPVR to decrease over the 4 h study period. We conclude that acute increases in PBF are limited by an ET‐1 dependent decrease in NO production via alterations in catalase activity, H₂O₂ levels, and eNOS phosphorylation. J. Cell. Biochem. 114: 435–447, 2013. © 2012 Wiley Periodicals, Inc.     

Lignan profile of Piper cubeba, an Indonesian medicinal plant

The lignan profile of the aerial part of Piper cubeba L. (Piperaceae) was determined using GC, GC–MS and HPLC. The number of lignans found in the leaves was 15, followed by berries and the stalks with respectively 13 and five lignans. This is the first investigation of lignans from the leaves and the stalks of P. cubeba. Cubebin, hinokinin, yatein, isoyatein are common lignans in the genus Piper and appeared as major components in all parts of P. cubeba investigated.     

RNA Binding and Core Complexes Constitute the U-Insertion/Deletion Editosome

Enzymes embedded into the RNA editing core complex (RECC) catalyze the U-insertion/deletion editing cascade to generate open reading frames in trypanosomal mitochondrial mRNAs. The sequential reactions of mRNA cleavage, U-addition or removal, and ligation are directed by guide RNAs (gRNAs). We combined proteomic, genetic, and functional studies with sequencing of total and complex-bound RNAs to define a protein particle responsible for the recognition of gRNAs and pre-mRNA substrates, editing intermediates, and products. This approximately 23-polypeptide tripartite assembly, termed the RNA editing substrate binding complex (RESC), also functions as the interface between mRNA editing, polyadenylation, and translation. Furthermore, we found that gRNAs represent only a subset of small mitochondrial RNAs, and yet an inexplicably high fraction of them possess 3′ U-tails, which correlates with gRNA''s enrichment in the RESC. Although both gRNAs and mRNAs are associated with the RESC, their metabolic fates are distinct: gRNAs are degraded in an editing-dependent process, whereas edited mRNAs undergo 3′ adenylation/uridylation prior to translation. Our results demonstrate that the well-characterized editing core complex (RECC) and the RNA binding particle defined in this study (RESC) typify enzymatic and substrate binding macromolecular constituents, respectively, of the ∼40S RNA editing holoenzyme, the editosome.     

The Impact of Funding through the RF President’s Grants for Young Scientists (the field – Medicine) on Research Productivity: A Quasi-Experimental Study and a Brief Systematic Review

The impact of grants on research productivity has been investigated by a number of retrospective studies. The results of these studies vary considerably. The objective of my study was to investigate the impact of funding through the RF President’s grants for young scientists on the research productivity of awarded applicants. The study compared the number of total articles and citations for awarded and rejected applicants who in 2007 took part in competitions for young candidates of science (CoS’s) and doctors of science (DoS’s) in the scientific field of medicine. The bibliometric analysis was conducted for the period from 2003 to 2012 (five years before and after the competition). The source of bibliometric data is the eLIBRARY.RU database. The impact of grants on the research productivity of Russian young scientists was assessed using the meta-analytical approach based on data from quasi-experimental studies conducted in other countries. The competition featured 149 CoS’s and 41 DoS’s, out of which 24 (16%) and 22 (54%) applicants, respectively, obtained funding. No difference in the number of total articles and citations at baseline, as well as in 2008–2012, for awarded and rejected applicants was found. The combination of data from the Russian study and other quasi-experimental studies (6 studies, 10 competitions) revealed a small treatment effect – an increase in the total number of publications over a 4–5-year period after the competition by 1.23 (95% CI 0.48–1.97). However, the relationship between the number of total publications published by applicants before and after the competition revealed that this treatment effect is an effect of the “maturation” of scientists with a high baseline publication activity – not of grant funding.     

Leptin-induced endothelial dysfunction in obesity

Hyperleptinemia accompanying obesity affects endothelial nitric oxide (NO) and is a serious factor for vascular disorders. NO, superoxide (O(2)(-)), and peroxynitrite (ONOO(-)) nanosensors were placed near the surface (5+/-2 microm) of a single human umbilical vein endothelial cell (HUVEC) exposed to leptin or aortic endothelium of obese C57BL/6J mice, and concentrations of calcium ionophore (CaI)-stimulated NO, O(2)(-), ONOO(-) were recorded. Endothelial NO synthase (eNOS) expression and L-arginine concentrations in HUVEC and aortic endothelium were measured. Leptin did not directly stimulate NO, O(2)(-), or ONOO(-) release from HUVEC. However, a 12-h exposure of HUVEC to leptin increased eNOS expression and CaI-stimulated NO (625+/-30 vs. 500+/-24 nmol/l control) and dramatically increased cytotoxic O(2)(-) and ONOO(-) levels. The [NO]-to-[ONOO(-)] ratio ([NO]/[ONOO(-)]) decreased from 2.0+/-0.1 in normal to 1.30+/-0.1 in leptin-induced dysfunctional endothelium. In obese mice, a 2.5-fold increase in leptin concentration coincided with 100% increase in eNOS and about 30% decrease in intracellular L-arginine. The increased eNOS expression and a reduced l-arginine content led to eNOS uncoupling, a reduction in bioavailable NO (250+/-10 vs. 420+/-12 nmol/l control), and an elevated concentration of O(2)(-) (240%) and ONOO(-) (70%). L-Arginine and sepiapterin supplementation reversed eNOS uncoupling and partially restored [NO]/[ONOO(-)] balance in obese mice. In obesity, leptin increases eNOS expression and decreases intracellular l-arginine, resulting in eNOS an uncoupling and depletion of endothelial NO and an increase of cytotoxic ONOO(-). Hyperleptinemia triggers an endothelial NO/ONOO(-) imbalance characteristic of dysfunctional endothelium observed in other vascular disorders, i.e., atherosclerosis and diabetes.     

Localization of ubiquinone-8 in the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae

Na(+) is the second major coupling ion at membranes after protons, and many pathogenic bacteria use the sodium-motive force to their advantage. A prominent example is Vibrio cholerae, which relies on the Na(+)-pumping NADH:quinone oxidoreductase (Na(+)-NQR) as the first complex in its respiratory chain. The Na(+)-NQR is a multisubunit, membrane-embedded NADH dehydrogenase that oxidizes NADH and reduces quinone to quinol. Existing models describing redox-driven Na(+) translocation by the Na(+)-NQR are based on the assumption that the pump contains four flavins and one FeS cluster. Here we show that the large, peripheral NqrA subunit of the Na(+)-NQR binds one molecule of ubiquinone-8. Investigations of the dynamic interaction of NqrA with quinones by surface plasmon resonance and saturation transfer difference NMR reveal a high affinity, which is determined by the methoxy groups at the C-2 and C-3 positions of the quinone headgroup. Using photoactivatable quinone derivatives, it is demonstrated that ubiquinone-8 bound to NqrA occupies a functional site. A novel scheme of electron transfer in Na(+)-NQR is proposed that is initiated by NADH oxidation on subunit NqrF and leads to quinol formation on subunit NqrA.