A semantic analysis of the annotations of the human genome

The correct interpretation of any biological experiment depends in an essential way on the accuracy and consistency of the existing annotation databases. Such databases are ubiquitous and used by all life scientists in most experiments. However, it is well known that such databases are incomplete and many annotations may also be incorrect. In this paper we describe a technique that can be used to analyze the semantic content of such annotation databases. Our approach is able to extract implicit semantic relationships between genes and functions. This ability allows us to discover novel functions for known genes. This approach is able to identify missing and inaccurate annotations in existing annotation databases, and thus help improve their accuracy. We used our technique to analyze the current annotations of the human genome. From this body of annotations, we were able to predict 212 additional gene-function assignments. A subsequent literature search found that 138 of these gene-functions assignments are supported by existing peer-reviewed papers. An additional 23 assignments have been confirmed in the meantime by the addition of the respective annotations in later releases of the Gene Ontology database. Overall, the 161 confirmed assignments represent 75.95% of the proposed gene-function assignments. Only one of our predictions (0.4%) was contradicted by the existing literature. We could not find any relevant articles for 50 of our predictions (23.58%). The method is independent of the organism and can be used to analyze and improve the quality of the data of any public or private annotation database.     

Differentiation of mitochondrial DNA and Y chromosomes in Russian populations

The genetic composition of the Russian population was investigated by analyzing both mitochondrial DNA (mtDNA) and Y-chromosome loci polymorphisms that allow for the different components of a population gene pool to be studied, depending on the mode of DNA marker inheritance. mtDNA sequence variation was examined by using hypervariable segment I (HVSI) sequencing and restriction analysis of the haplogroup-specific sites in 325 individuals representing 5 Russian populations from the European part of Russia. The Y-chromosome variation was investigated in 338 individuals from 8 Russian populations (including 5 populations analyzed for mtDNA variation) using 12 binary markers. For both uniparental systems most of the observed haplogroups fell into major West Eurasian haplogroups (97.9% and 99.7% for mtDNA and Y-chromosome haplogroups, respectively). Multidimensional scaling analysis based on pairwise F(ST) values between mtDNA HVSI sequences in Russians compared to other European populations revealed a considerable heterogeneity of Russian populations; populations from the southern and western parts of Russia are separated from eastern and northern populations. Meanwhile, the multidimensional scaling analysis based on Y-chromosome haplogroup F(ST) values demonstrates that the Russian gene pool is close to central-eastern European populations, with a much higher similarity to the Baltic and Finno-Ugric male pools from northern European Russia. This discrepancy in the depth of penetration of mtDNA and Y-chromosome lineages characteristic for the most southwestern Russian populations into the east and north of eastern Europe appears to indicate that Russian colonization of the northeastern territories might have been accomplished mainly by males rather than by females.     

β1 Integrin NPXY Motifs Regulate Kidney Collecting-Duct Development and Maintenance by Induced-Fit Interactions with Cytosolic Proteins

Loss of β1 integrin expression inhibits renal collecting-system development. Two highly conserved NPXY motifs in the distal β1 tail regulate integrin function by associating with phosphtyrosine binding (PTB) proteins, such as talin and kindlin. Here, we define the roles of these two tyrosines in collecting-system development and delineate the structural determinants of the distal β1 tail using nuclear magnetic resonance (NMR). Mice carrying alanine mutations have moderate renal collecting-system developmental abnormalities relative to β1-null mice. Phenylalanine mutations did not affect renal collecting-system development but increased susceptibility to renal injury. NMR spectra in bicelles showed the distal β1 tail is disordered and does not interact with the model membrane surface. Alanine or phenylalanine mutations did not alter β1 structure or interactions between α and β1 subunit transmembrane/cytoplasmic domains; however, they did decrease talin and kindlin binding. Thus, these studies highlight the fact that the functional roles of the NPXY motifs are organ dependent. Moreover, the β1 cytoplasmic tail, in the context of the adjacent transmembrane domain in bicelles, is significantly different from the more ordered, membrane-associated β3 integrin tail. Finally, tyrosine mutations of β1 NPXY motifs induce phenotypes by disrupting their interactions with critical integrin binding proteins like talins and kindlins.     

The effect of DS16570511, a new inhibitor of mitochondrial calcium uniporter,on calcium homeostasis,metabolism, and functional state of cultured cortical neurons and isolated brain mitochondria

BackgroundDisorders of mitochondrial Ca²⁺ homeostasis play a key role in the glutamate excitotoxicity of brain neurons. DS16570511 (DS) is a new penetrating inhibitor of mitochondrial Ca²⁺ uniporter complex (MCUC). The paper examines the effects of DS on the cultivated cortical neurons and isolated mitochondria of the rat brain.MethodsThe functions of neurons and mitochondria were examined using fluorescence microscopy, XF24 microplate-based сell respirometry, ion-selective microelectrodes, spectrophotometry, and polarographic technique.ResultsAt the doses of 30 and 45 μM, DS reliably slowed down the onset of glutamate-induced delayed calcium deregulation of neurons and suppressed their death. 30 μM DS caused hyperpolarization of mitochondria of resting neurons, and 45 μM DS temporarily depolarized neuronal mitochondria. It was also demonstrated that 30–60 μM DS stimulated cellular respiration. DS was shown to suppress Ca²⁺ uptake by isolated brain mitochondria. In addition, DS inhibited ADP-stimulated mitochondrial respiration and ADP-induced decrease in the mitochondrial membrane potential. It was found that DS inhibited the activity of complex II of the respiratory chain. In the presence of Ca²⁺, high DS concentrations caused a collapse of the mitochondrial membrane potential.ConclusionsThe data obtained indicate that, in addition to the inhibition of MCUC, DS affects the main energy-transducing functions of mitochondria.General significanceThe using DS as a tool for studying MCUC and its functional role in neuronal cells should be done with care, bearing in mind multiple effects of DS, a proper evaluation of which would require multivariate analysis.     

Methane emissions in two drained peat agro-ecosystems with high and low agricultural intensity

Methane (CH₄) emissions were compared for an intensively and extensively managed agricultural area on peat soils in the Netherlands to evaluate the effect of reduced management on the CH₄ balance. Chamber measurements (photoacoustic methods) for CH₄ were performed for a period of three years in the contributing landscape elements in the research sites. Various factors influencing CH₄ emissions were evaluated and temperature of water and soil was found to be the main driver in both sites. For upscaling of CH₄ fluxes to landscape scale, regression models were used which were specific for each of the contributing landforms. Ditches and bordering edges were emission hotspots and emitted together between 60% and 70% of the total terrestrial CH₄ emissions. Annual terrestrial CH₄ fluxes were estimated to be 203 (±48%), 162 (±60%) and 146 (±60%) kg CH₄ ha^?1 and 157 (±63%), 180 (±54%) and 163 (±59%) kg CH₄ ha^?1 in the intensively managed site and extensively managed site, for 2006, 2007 and 2008 respectively. About 70% of the CH₄ was emitted in the summer period. Farm based emissions caused per year an additional 257 kg CH₄ ha^?1 and 172 kg CH₄ ha^?1 for the intensively managed site and extensively managed site, respectively. To further evaluate the effect of agricultural activity on the CH₄ balance, the annual CH₄ fluxes of the two managed sites were also compared to the emissions of a natural peat site with no management and high ground water levels. By comparing the terrestrial and additional farm based emissions of the three sites, we finally concluded that transformation of intensively managed agricultural land to nature development will lead to an increase in terrestrial CH₄ emission, but will not by definition lead to a significant increase in CH₄ emission when farm based emissions are included.     

A Comparison of the Effects of the GLP-1 Analogue Liraglutide and Insulin Glargine on Endothelial Function and Metabolic Parameters: A Randomized,Controlled Trial Sapporo Athero-Incretin Study 2 (SAIS2)

Objectives

GLP-1 improves hyperglycemia, and it has been reported to have favorable effects on atherosclerosis. However, it has not been fully elucidated whether GLP-1 is able to improve endothelial function in patients with type 2 diabetes. Therefore, we investigated the efficacy of the GLP-1 analogue, liraglutide on endothelial function and glycemic metabolism compared with insulin glargine therapy.

Materials and Methods

In this multicenter, prospective randomized parallel-group comparison study, 31 diabetic outpatients (aged 60.3 ± 10.3 years with HbA1c levels of 8.6 ± 0.8%) with current metformin and/or sulfonylurea treatment were enrolled and randomly assigned to receive liraglutide or glargine therapy once daily for 14 weeks. Flow mediated dilation (FMD), a comprehensive panel of hemodynamic parameters (Task Force Monitor), and serum metabolic markers were assessed before and after the treatment period.

Results

A greater reduction (worsening) in %FMD was observed in the glargine group, although this change was not statistically different from the liraglutide group (liraglutide; 5.7 to 5.4%, glargine 6.7 to 5.7%). The augmentation index, C-peptide index, derivatives of reactive oxygen metabolites and BMI were significantly improved in the liraglutide group. Central systolic blood pressure and NT-proBNP also tended to be improved in the liraglutide-treated group, while improvements in HbA1c levels were similar between groups. Cardiac index, blood pressure and most other metabolic parameters were not different.

Conclusions

Regardless of glycemic improvement, early liraglutide therapy did not affect endothelial function but may provide favorable effects on beta-cell function and cardioprotection in type 2 diabetics without advanced atherosclerosis.

Trial Registration

UMIN Clinical Trials Registry System as trial ID UMIN000005331.     

Purification and characterization of the human γ-secretase activating protein

Alzheimer's disease is a fatal neurological disorder that is a leading cause of death, with its prevalence increasing as the average life expectancy increases worldwide. There is an urgent need to develop new therapeutics for this disease. A newly described protein, the γ-secretase activating protein (GSAP), has been proposed to promote elevated levels of amyloid-β production, an activity that seems to be inhibited using the well-establish cancer drug, imatinib (Gleevec). Despite much interest in this protein, there has been little biochemical characterization of GSAP. Here we report protocols for the recombinant bacterial expression and purification of this potentially important protein. GSAP is expressed in inclusion bodies, which can be solubilized using harsh detergents or urea; however, traditional methods of refolding were not successful in generating soluble forms of the protein that contained well-ordered and homogeneous tertiary structure. However, GSAP could be solubilized in detergent micelle solutions, where it was seen to be largely α-helical but to adopt only heterogeneous tertiary structure. Under these same conditions, GSAP did not associate with either imatinib or the 99-residue transmembrane C-terminal domain of the amyloid precursor protein. These results highlight the challenges that will be faced in attempts to manipulate and characterize this protein.     

Novel boronated chlorin e6-based photosensitizers: Synthesis,binding to albumin and antitumour efficacy

Chlorins, a class of plant porphyrins, are perspective as photosensitizing agents due to light absorption in the long wavelength spectral region and deeper photodamage of tissues. Aiming at optimization of antitumour properties of chlorins, we synthesized a series of boronated derivatives of chlorin e₆ and their complexes containing Zn(II), Pd(II) or Sn(IV). The compounds were synthesized by alkylation of amino or hydroxy derivatives of chlorin e₆ with 1-trifluoromethanesulfonylmethyl-o-carborane. Chlorin e₆ 13(1)-N-{2-[N-(o-carboran-1-yl)methyl]aminoethyl}amide-15(2), 17(3)-dimethyl ester (compound 5) formed complexes with serum albumin, a major porphyrin carrier. The binding constant of these complexes was ～4 times bigger than the respective value for the complexes of albumin with boron-free aminochlorin e₆. Compound 5 potently sensitized rat fibroblasts to illumination with monochromatic red light: >98% of cells were necrotic by 24 h post-illumination with 1 μM of 5. This compound demonstrated high efficacy in photodynamic therapy of rat M-1 sarcoma. After PDT with 25 mg/kg of 5 the residual tumours were significantly smaller than in animals subjected to PDT with equal concentration of boron-free aminochlorin e₆. No signs of general toxicity were detectable after PDT with 5. Thus, boronation can enhance the potency of chlorins in PDT, in particular, due to an increased binding to albumin. Our data expand the therapeutic applicability of boronated chlorins beyond boron neutron capture therapy; these agents emerge as dual efficacy photoradiosensitizers.     

Probing the structure and function of an archaeal C/D-box methylation guide sRNA

The genome of the hyperthermophilic archaeon Sulfolobus solfataricus contains dozens of small C/D-box sRNAs that use a complementary guide sequence to target 2'-O-ribose methylation to specific locations within ribosomal and transfer RNAs. The sRNAs are approximately 50-60 nucleotides in length and contain two RNA structural kink-turn (K-turn) motifs that are required for assembly with ribosomal protein L7Ae, Nop5, and fibrillarin to form an active ribonucleoprotein (RNP) particle. The complex catalyzes guide-directed methylation to target RNAs. Earlier work in our laboratory has characterized the assembly pathway and methylation reaction using the model sR1 sRNA from Sulfolobus acidocaldarius. This sRNA contains only one antisense region situated adjacent to the D-box, and methylation is directed to position U52 in 16S rRNA. Here we have investigated through RNA mutagenesis, the relationship between the sR1 structure and methylation-guide function. We show that although full activity of the guide requires intact C/D and C'/D' K-turn motifs, each structure plays a distinct role in the methylation reaction. The C/D motif is directly implicated in the methylation function, whereas the C'/D' element appears to play an indirect structural role by facilitating the correct folding of the RNA. Our results suggest that L7Ae facilitates the folding of the K-turn motifs (chaperone function) and, in addition, is required for methylation activity in the presence of Nop5 and Fib.