Polymorphism and the secondary structure of the mitochondrial <Emphasis Type="Italic">nad1</Emphasis> gene b/c intron in <Emphasis Type="Italic">Malus</Emphasis> species and related Rosaceae species

Nucleotide sequences polymorphism of mitochondrial nad1 gene b/c intron was studied in 41 Malus accessions and 21 related Rosaceae accessions. The b/c intron sequence in genus Malus appeared to be very conservative, while in other studied Rosaceae species 126 variable sites and indels were detected in the intron sequence that varied in length from 1124 to 1456 bp. The predicted b/c intron pre-mRNA secondary structure for Malus species was determined; IBS/EBS binding sites and the boundaries of the six functional domains were identified.     

Genetical genomic determinants of alcohol consumption in rats and humans

Background

We have used a genetical genomic approach, in conjunction with phenotypic analysis of alcohol consumption, to identify candidate genes that predispose to varying levels of alcohol intake by HXB/BXH recombinant inbred rat strains. In addition, in two populations of humans, we assessed genetic polymorphisms associated with alcohol consumption using a custom genotyping array for 1,350 single nucleotide polymorphisms (SNPs). Our goal was to ascertain whether our approach, which relies on statistical and informatics techniques, and non-human animal models of alcohol drinking behavior, could inform interpretation of genetic association studies with human populations.

Results

In the HXB/BXH recombinant inbred (RI) rats, correlation analysis of brain gene expression levels with alcohol consumption in a two-bottle choice paradigm, and filtering based on behavioral and gene expression quantitative trait locus (QTL) analyses, generated a list of candidate genes. A literature-based, functional analysis of the interactions of the products of these candidate genes defined pathways linked to presynaptic GABA release, activation of dopamine neurons, and postsynaptic GABA receptor trafficking, in brain regions including the hypothalamus, ventral tegmentum and amygdala. The analysis also implicated energy metabolism and caloric intake control as potential influences on alcohol consumption by the recombinant inbred rats. In the human populations, polymorphisms in genes associated with GABA synthesis and GABA receptors, as well as genes related to dopaminergic transmission, were associated with alcohol consumption.

Conclusion

Our results emphasize the importance of the signaling pathways identified using the non-human animal models, rather than single gene products, in identifying factors responsible for complex traits such as alcohol consumption. The results suggest cross-species similarities in pathways that influence predisposition to consume alcohol by rats and humans. The importance of a well-defined phenotype is also illustrated. Our results also suggest that different genetic factors predispose alcohol dependence versus the phenotype of alcohol consumption.     

Characteristics of enzymes forming 3-methoxy-4-hydroxyphenylethyleneglycol (MOPEG) in brain

The subcellular localization and character of the enzymes forming 3-methoxy-4-hydroxyphenylethyleneglycol (MOPEG) were determined in rat brain. The aldehyde derivative of normetanephrine was produced in situ by monoamine oxidase, and two forms of aldehyde reductase were shown to metabolize the aldehyde to MOPEG. One form of the enzyme was found to have a low affinity for NADH and a higher affinity for NADPH as a cofactor, and was shown to be inhibited by pentobarbital and by high concentrations of 5-hydroxyindoleacetic acid. This enzyme form was localized primarily in the cytosol. The second aldehyde reductase had a high affinity for both NADH and NADPH, and was not inhibited to a great extent by either pentobarbital or 5-hydroxyindoleacetic acid. This second enzyme form was localized primarily in the mitochondrial fraction. The relative contribution of the two enzyme forms to MOPEG formation in homogenates was estimated, using the various inhibitors and cofactors.     

Repairing faulty genes by aminoglycosides: Development of new derivatives of geneticin (G418) with enhanced suppression of diseases-causing nonsense mutations

New pseudo-di- and pseudo-trisaccharide derivatives of the aminoglycoside drug G418 were designed, synthesized and their ability to readthrough nonsense mutations was examined in both in vitro and ex vivo systems, along with the toxicity tests. Two novel lead structures, NB74 and NB84, exhibiting significantly reduced cell toxicity and superior readthrough efficiency than those of gentamicin, were discovered. The superiority of new leads was demonstrated in six different nonsense DNA-constructs underling the genetic diseases cystic fibrosis, Duchenne muscular dystrophy, Usher syndrome and Hurler syndrome.     

Lysosomal–mitochondrial cross-talk during cell death

Lysosomes are membrane-bound organelles, which contain an arsenal of different hydrolases, enabling them to act as the terminal degradative compartment of the endocytotic, phagocytic and autophagic pathways. During the last decade, it was convincingly shown that destabilization of lysosomal membrane and release of lysosomal content into the cytosol can initiate the lysosomal apoptotic pathway, which is dependent on mitochondria destabilization. The cleavage of BID to t-BID and degradation of anti-apoptotic BCL-2 proteins by lysosomal cysteine cathepsins were identified as links to the mitochondrial cytochrome c release, which eventually leads to caspase activation. There have also been reports about the involvement of lysosome destabilization and lysosomal proteases in the extrinsic apoptotic pathway, although the molecular mechanism is still under debate. In the present article, we discuss the cross-talk between lysosomes and mitochondria during apoptosis and its consequences for the fate of the cell.     

Global marine redox changes drove the rise and fall of the Ediacara biota

The role of O₂ in the evolution of early animals, as represented by some members of the Ediacara biota, has been heavily debated because current geochemical evidence paints a conflicting picture regarding global marine O₂ levels during key intervals of the rise and fall of the Ediacara biota. Fossil evidence indicates that the diversification the Ediacara biota occurred during or shortly after the Ediacaran Shuram negative C‐isotope Excursion (SE), which is often interpreted to reflect ocean oxygenation. However, there is conflicting evidence regarding ocean oxygen levels during the SE and the middle Ediacaran Period. To help resolve this debate, we examined U isotope variations (δ²³⁸U) in three carbonate sections from South China, Siberia, and USA that record the SE. The δ²³⁸U data from all three sections are in excellent agreement and reveal the largest positive shift in δ²³⁸U ever reported in the geologic record (from ~ ?0.74‰ to ~ ?0.26‰). Quantitative modeling of these data suggests that the global ocean switched from a largely anoxic state (26%–100% of the seafloor overlain by anoxic waters) to near‐modern levels of ocean oxygenation during the SE. This episode of ocean oxygenation is broadly coincident with the rise of the Ediacara biota. Following this initial radiation, the Ediacara biota persisted until the terminal Ediacaran period, when recently published U isotope data indicate a return to more widespread ocean anoxia. Taken together, it appears that global marine redox changes drove the rise and fall of the Ediacara biota.     

Cdk1/Erk2- and Plk1-dependent phosphorylation of a centrosome protein, Cep55, is required for its recruitment to midbody and cytokinesis

Centrosomes in mammalian cells have recently been implicated in cytokinesis; however, their role in this process is poorly defined. Here, we describe a human coiled-coil protein, Cep55 (centrosome protein 55 kDa), that localizes to the mother centriole during interphase. Despite its association with gamma-TuRC anchoring proteins CG-NAP and Kendrin, Cep55 is not required for microtubule nucleation. Upon mitotic entry, centrosome dissociation of Cep55 is triggered by Erk2/Cdk1-dependent phosphorylation at S425 and S428. Furthermore, Cep55 locates to the midbody and plays a role in cytokinesis, as its depletion by siRNA results in failure of this process. S425/428 phosphorylation is required for interaction with Plk1, enabling phosphorylation of Cep55 at S436. Cells expressing phosphorylation-deficient mutant forms of Cep55 undergo cytokinesis failure. These results highlight the centrosome as a site to organize phosphorylation of Cep55, enabling it to relocate to the midbody to function in mitotic exit and cytokinesis.     

A random process may control the number of endemic species

The richness of endemic species is often recognized as an indication of the distinctiveness of certain local faunas and is used for the definition of conservation hotspots as well. Faunas of different animal taxa were considered in sets of contiguous geographical units. Comparing the faunas of different units in one set, we found an exponential increase in the number of endemics when plotted against the number of non-endemics. A model of independent stochastic population dynamics under the control of environmental oscillations produces random fluctuations in the ranges of species. Ranges of endemic species are supposedly narrower than ranges of co-occurring non-endemic species. In such a case, the flow of a random process leads to an exponential relationship between numbers of co-occurring endemic and non-endemic species. This process also produces an apparent positive correlation between total species number and the percentage of endemics.     

Microalgae Synthesize Hydrocarbons from Long-Chain Fatty Acids via a Light-Dependent Pathway

Microalgae are considered a promising platform for the production of lipid-based biofuels. While oil accumulation pathways are intensively researched, the possible existence of a microalgal pathways converting fatty acids into alka(e)nes has received little attention. Here, we provide evidence that such a pathway occurs in several microalgal species from the green and the red lineages. In Chlamydomonas reinhardtii (Chlorophyceae), a C17 alkene, n-heptadecene, was detected in the cell pellet and the headspace of liquid cultures. The Chlamydomonas alkene was identified as 7-heptadecene, an isomer likely formed by decarboxylation of cis-vaccenic acid. Accordingly, incubation of intact Chlamydomonas cells with per-deuterated D₃₁-16:0 (palmitic) acid yielded D₃₁-18:0 (stearic) acid, D₂₉-18:1 (oleic and cis-vaccenic) acids, and D₂₉-heptadecene. These findings showed that loss of the carboxyl group of a C₁₈ monounsaturated fatty acid lead to heptadecene formation. Amount of 7-heptadecene varied with growth phase and temperature and was strictly dependent on light but was not affected by an inhibitor of photosystem II. Cell fractionation showed that approximately 80% of the alkene is localized in the chloroplast. Heptadecane, pentadecane, as well as 7- and 8-heptadecene were detected in Chlorella variabilis NC64A (Trebouxiophyceae) and several Nannochloropsis species (Eustigmatophyceae). In contrast, Ostreococcus tauri (Mamiellophyceae) and the diatom Phaeodactylum tricornutum produced C₂₁ hexaene, without detectable C₁₅-C₁₉ hydrocarbons. Interestingly, no homologs of known hydrocarbon biosynthesis genes were found in the Nannochloropsis, Chlorella, or Chlamydomonas genomes. This work thus demonstrates that microalgae have the ability to convert C₁₆ and C₁₈ fatty acids into alka(e)nes by a new, light-dependent pathway.Hydrocarbons derived from fatty acids (i.e. alkanes and alkenes) are ubiquitous in plant and insect outermost tissues where they often represent a major part of cuticular waxes and play an essential role in preventing water loss from the organisms to the dry terrestrial environment (Hadley, 1989; Kunst et al., 2005). In several insect species, select cuticular alkenes also act as sex pheromones (Wicker-Thomas and Chertemps, 2010). Occurrence of alkanes or alkenes has also been reported in various microorganisms (Ladygina et al., 2006; Wang and Lu, 2013). For example, synthesis of hydrocarbons is widespread in cyanobacteria (Coates et al., 2014), and it is thought that cyanobacterial alka(e)nes contribute significantly to the hydrocarbon cycle of the upper ocean (Lea-Smith et al., 2015).Alka(e)nes of various chain lengths are important targets for biotechnology because they are major components of gasoline (mainly C5-C9 hydrocarbons), jet fuels (C5-C16), and diesel fuels (C12-C20). The alkane biosynthetic pathway of plants has been partly elucidated (Lee and Suh, 2013; Bernard and Joubès, 2013), but the use of plant hydrocarbons as a renewable source of liquid fuels is hampered by predominance of constituents with high carbon numbers (>C25), which entails solid state at ambient temperature (Jetter and Kunst, 2008). Therefore, there is great interest in the microbial pathways of hydrocarbon synthesis producing shorter chain compounds (C15-C19). In cyanobacteria, hydrocarbons are produced by two distinct pathways. The first one comprises the sequential action of an acyl-ACP reductase transforming a C15-C19 fatty acyl-ACP into a fatty aldehyde and an aldehyde-deformylating oxygenase catalyzing the oxidative cleavage of the fatty aldehyde into alka(e)ne and formic acid (Schirmer et al., 2010; Li et al., 2012). The second pathway involves a type I polyketide synthase that elongates and decarboxylates fatty acids to form alkenes with a terminal double bond (Mendez-Perez et al., 2011). Additional pathways of alkene synthesis have been described in bacteria. In Micrococcus luteus, a three-gene cluster has been shown to control the head-to-head condensation of fatty acids to form very-long-chain alkenes with internal double bonds (Beller et al., 2010). Direct decarboxylation of a long-chain fatty acid into a terminal alkene has also been reported and is catalyzed by a cytochrome P450 in Jeotgalicoccus spp. (Rude et al., 2011) and by a nonheme iron oxidase in Pseudomonas (Rui et al., 2014).Among microbes, microalgae would be ideally suited to harness the synthesis of hydrocarbons from fatty acid precursors because they are photosynthetic organisms combining a high biomass productivity (León-Bañares et al., 2004; Beer et al., 2009; Malcata, 2011; Wijffels et al., 2013) and a strong capacity to synthesize and accumulate fatty acids (Hu et al., 2008; Harwood and Guschina, 2009; Liu and Benning 2013). However, studies on microalgal alka(e)ne synthesis are scarce. In some diatoms and other algal species, a very-long-chain alkene, a C21 hexaene, has been found (Lee et al., 1970; Lee and Loeblich, 1971). Other very-long-chain alkenes have been described in the slow-growing colonial Chlorophycea Botryococcus brauni, which excretes a variety of hydrophobic compounds including C27 n-alkadienes (Metzger and Largeau, 2005; Jin et al., 2016). A decarbonylase activity converting a fatty n-aldehyde substrate to a n-alkane has been shown in B. brauni (Dennis and Kolattukudy, 1992); however, the corresponding protein has not been identified so far. Presence of shorter chain alka(e)nes in some microalga species has been reported in the context of geochemical studies (Han et al., 1968; Gelpi et al., 1970; Tornabene et al., 1980; Afi et al., 1996) but the biology of these compounds has not been investigated further.Here, we show that alka(e)nes with C15 to C17 chains can be detected in several model microalgae and originate from fatty acid metabolism. In Chlamydomonas reinhardtii and Chlorella variabilis NC64A, 7-heptadecene is identified as the major hydrocarbon produced, and we demonstrate that its synthesis depends strictly on light and uses cis-vaccenic acid as a precursor. We also show the presence of C15 to C17 alka(e)nes in Nannochloropsis sp., a model microalga from the red lineage. Absence of homologs to known hydrocarbon synthesis genes in the genomes of Chlamydomonas, Chlorella, and Nannochloropsis indicates that a hitherto unknown type of alka(e)ne-producing pathway operates in these microalgae. The wide occurrence of microalgae in marine environments suggests that microalgal alka(e)nes contribute significantly to the ocean’s hydrocarbon cycle.