Synthetic muO-conotoxin MrVIB blocks TTX-resistant sodium channel NaV1.8 and has a long-lasting analgesic activity

MuO-conotoxin MrVIB is a blocker of voltage-gated sodium channels, including TTX-sensitive and -resistant subtypes. A comprehensive characterization of this peptide has been hampered by the lack of sufficient synthetic material. Here, we describe the successful chemical synthesis and oxidative folding of MrVIB that has made an investigation of the pharmacological properties and therapeutic potential of the peptide feasible. We show for the first time that synthetic MrVIB blocks rat NaV1.8 sodium channels and has potent and long-lasting local anesthetic effects when tested in two pain assays in rats. Furthermore, MrVIB can block propagation of action potentials in A- and C-fibers in sciatic nerve as well as skeletal muscle in isolated preparations from rat. Our work provides the first example of analgesia produced by a conotoxin that blocks sodium channels. The emerging diversity of antinociceptive mechanisms targeted by different classes of conotoxins is discussed.     

Will sympatric speciation fail due to stochastic competitive exclusion?

Sympatric speciation requires coexistence of the newly formed species. If divergence proceeds by small mutational steps, the new species utilize almost the same resources initially, and full speciation may be impeded by competitive exclusion in stochastic environments. We investigate this primarily ecological problem of sympatric speciation by studying the population dynamics of a diverging asexual population in a fluctuating environment. Correlation between species responses to environmental fluctuation is assumed to decrease with distance in trait space. Rapidly declining correlation in combination with high environmental variability may delay full speciation or even render it impossible. Stochastic extinctions impeding speciation are most likely when correlation decays faster than competition, for example, when demographic stochasticity is strong or when divergence is not accompanied by niche separation, such as in speciation driven entirely by sexual selection. Our general theoretical results show an interesting connection between short-term ecological dynamics and long-term, large-scale evolution.     

Acidophilic microbial communities catalyzing sludge bioleaching monitored by fluorescent in situ hybridization

Biological autotrophic sulfur oxidation processes have been proposed to remove heavy metals from wastewater treatment sludge by bioleaching. We made a characterization of the microbial population in batch and continuous sludge bioleaching reactors using fluorescent in situ hybridization of fluorescently-labeled oligonucleotidic probes targeting rRNA in a ‚top to bottom approach’. Batch incubations of sludge with 0.2% (w/v) elemental sulfur resulted in a pH value of 5. Alpha-Proteobacteria hybridizing with probe ALF1b were dominant in this incubation. Members of the Acidophilium-group (hybridizing with probe Acdp821) of Nitrospira/Leptospirillum phylum (Ntspa712 probe) and from the archaeal domain (ARCH915) were also detected. When sludge was incubated with 1% elemental sulfur in batch or continuous reactor experiments, final pH values were always below 2. Active microbial communities consisted almost exclusively of gamma-Proteobacteria (hybridizing with probe GAM42a). However, further hybridization experiments with probe Thio820 targeting Acidithiobacillus ferroxidans and Acidithiobacillus thioxidans gave negative results. A new probe, named THIO181, encompassing all known members of the genus was designed. Hybridization perfomed with THIO181 and GAM42a showed a perfect co-localization of the hybridization signals. Further hybridization experiments with probe THIO181 and THC642, specific for the species Acidithiobacillus caldus, confirmed that this bacteria was largely responsible for the sulfur oxidation reaction in our acidophilic sludge bioleaching reactors.     

Occurrence of 1-Glyceryl-1-myo-Inosityl Phosphate in Hyperthermophiles

The accumulation of compatible solutes was studied in the hyperthermophilic bacterium Aquifex pyrophilus as a function of the temperature and the NaCl concentration of the growth medium. Nuclear magnetic resonance analysis of cell extracts revealed the presence of α- and β-glutamate, di-mannosyl-di-myo-inositol phosphate, di-myo-inositol phosphate, and an additional compound here identified as 1-glyceryl-1-myo-inosityl phosphate. All solutes accumulated by A. pyrophilus are negatively charged at physiological pH. The intracellular levels of di-myo-inositol phosphate increased in response to supraoptimal growth temperature, while α- and β-glutamate accumulated in response to osmotic stress, especially at growth temperatures below the optimum. The newly discovered compound, 1-glyceryl-1-myo-inosityl phosphate, appears to play a double role in osmo- and thermoprotection, since its intracellular pool increased primarily in response to a combination of osmotic and heat stresses. This work also uncovered the nature of the unknown compound, previously detected in Archaeoglobus fulgidus (L. O. Martins et al., Appl. Environ. Microbiol. 63:896-902, 1997). The curious structural relationship between diglycerol phosphate (found only in Archaeoglobus species), di-myo-inositol phosphate (a canonical solute of hyperthermophiles), and the newly identified solute is highlighted. This is the first report on the occurrence of 1-glyceryl-1-myo-inosityl phosphate in living systems.     

Adaptive divergence in experimental populations of Pseudomonas fluorescens. II. Role of the GGDEF regulator WspR in evolution and development of the wrinkly spreader phenotype

Wrinkly spreader (WS) genotypes evolve repeatedly in model Pseudomonas populations undergoing adaptive radiation. Previous work identified genes contributing to the evolutionary success of WS. Here we scrutinize the GGDEF response regulator protein WspR and show that it is both necessary and sufficient for WS. Activation of WspR occurs by phosphorylation and different levels of activation generate phenotypic differences among WS genotypes. Five alleles of wspR, each encoding a protein with a single amino acid substitution, were generated by mutagenesis. Two alleles are constitutively active and cause the ancestral genotype to develop a WS phenotype; the phenotypic effects are allele specific and independent of phosphorylation. Three alleles contain changes in the GGDEF domain and when overexpressed in WS cause reversion to the ancestral phenotype. Ability to mimic this effect by overexpression of a liberated N-terminal domain shows that in WS, regulatory components upstream of WspR are overactive. To connect changes at the nucleotide level with fitness, the effects of variant alleles were examined in both structured and unstructured environments: alleles had adaptive and deleterious effects with trade-offs evident across environments. Despite the proclivity of mutations within wspR to generate WS, sequence analysis of wspR from 53 independently obtained WS showed no evidence of sequence change in this gene.     

Depletion of phosphatidylcholine in yeast induces shortening and increased saturation of the lipid acyl chains: evidence for regulation of intrinsic membrane curvature in a eukaryote

To study the consequences of depleting the major membrane phospholipid phosphatidylcholine (PC), exponentially growing cells of a yeast cho2opi3 double deletion mutant were transferred from medium containing choline to choline-free medium. Cell growth did not cease until the PC level had dropped below 2% of total phospholipids after four to five generations. Increasing contents of phosphatidylethanolamine (PE) and phosphatidylinositol made up for the loss of PC. During PC depletion, the remaining PC was subject to acyl chain remodeling with monounsaturated species replacing diunsaturated species, as shown by mass spectrometry. The remodeling of PC did not require turnover by the SPO14-encoded phospholipase D. The changes in the PC species profile were found to reflect an overall shift in the cellular acyl chain composition that exhibited a 40% increase in the ratio of C16 over C18 acyl chains, and a 10% increase in the degree of saturation. The shift was stronger in the phospholipid than in the neutral lipid fraction and strongest in the species profile of PE. The shortening and increased saturation of the PE acyl chains were shown to decrease the nonbilayer propensity of PE. The results point to a regulatory mechanism in yeast that maintains intrinsic membrane curvature in an optimal range.     

Predictors of belief that genetic test information about hemochromatosis should be shared with family members

We queried 101,951 white, Hispanic, black, Asian, American Indian (i.e., American Indian or Alaska Native in the United States and North American Indian, Metis, or Inuit in Canada) and Pacific Islander (including Native Hawaiian) adults who agreed to be genotypically and phenotypically screened for hemochromatosis as part of the Hemochromatosis and Iron Overload Screening (HEIRS) study about their views on sharing genetic test information with family members. Multiple logistic regression (adjusting for study site, age group, race/ethnicity, preferred language, gender, education group, income group, SF-36 General Health and Mental Health subscales, perceived benefits and limitations of genetic testing, and belief that genetic testing is a good idea) evaluated independent predictors of responding "Strongly Agree" or "Agree" versus "Disagree" or "Strongly Disagree" to the statement "Information about a person's genetic risk should be shared with family members". Agreement that genetic risk information should be shared with family members was high (93% in the overall sample of 78,952 who answered this question), but differed among racial/ethnic groups. Hispanics were significantly less likely to agree that genetic test information should be shared with family members (i.e., 88% versus 92% or more among all other ethnicities). The relationship of perceived limitations and benefits of testing, gender, and age group to the belief that information should be shared differed among racial/ethnic groups, with Spanish-preferring Hispanics being the most different from other subgroups.     

ATM- and cell cycle-dependent regulation of ATR in response to DNA double-strand breaks

It is generally thought that the DNA-damage checkpoint kinases, ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), work independently of one another. Here, we show that ATM and the nuclease activity of meiotic recombination 11 (Mre11) are required for the processing of DNA double-strand breaks (DSBs) to generate the replication protein A (RPA)-coated ssDNA that is needed for ATR recruitment and the subsequent phosphorylation and activation of Chk1. Moreover, we show that efficient ATM-dependent ATR activation in response to DSBs is restricted to the S and G2 cell cycle phases and requires CDK kinase activity. Thus, in response to DSBs, ATR activation is regulated by ATM in a cell-cycle dependent manner.     

The wild-type Schizosaccharomyces pombe mat1 imprint consists of two ribonucleotides

The imprint at the mat1 locus of Schizosaccharomyces pombe acts to initiate the replication-coupled recombination event that underlies mating-type switching. However, the nature of the imprint has been an area of dispute. Two alternative models have been proposed: one stated that the imprint is a nick in the DNA, whereas our data suggested that it consists of one or two ribonucleotides incorporated into the otherwise intact DNA duplex. Here, we verify key predictions of the RNA model by characterization of wild-type genomic DNA purified under conditions known to hydrolyse DNA-RNA-DNA hybrid strands. First, we observe one-nucleotide gap at the hydrolysed DNA, as expected from the presence of two ribonucleotides. Second, using a novel assay based on ligation-mediated PCR, a 3'-terminal ribonucleotide is detected at the hydrolysed imprint. Our observations allow the unification of available data sets characterizing the wild-type imprint.     

Addressing the problems with life-science databases for traditional uses and systems biology

A prerequisite to systems biology is the integration of heterogeneous experimental data, which are stored in numerous life-science databases. However, a wide range of obstacles that relate to access, handling and integration impede the efficient use of the contents of these databases. Addressing these issues will not only be essential for progress in systems biology, it will also be crucial for sustaining the more traditional uses of life-science databases.