Analysis of 5-methylcytosine in DNA: II. Gas chromatography

A method for analyzing 5-methylcytosine in DNA by gas chromatography is described. The method is based on degradation of the DNA to its free bases by treatment with trifluoroacetic acid and gas chromatography of the trimethylsilyl derivatives of the free bases. Chromatography of microgram amounts of derivatized material is conducted at isothermal conditions using a 3% SE-30 or 2% OV-225 column. The peak areas corresponding to cytosine and 5-methylcytosine are used to calculate the 5-methylcytosine/cytosine molar ratio in DNA. The lower limit for detection of 5-methylcytosine in DNA by this method is a 5-methylcytosine/cytosine molar ratio of 0.001.     

Cholinesterases in development and disease

Cholinesterases (ChEs) including acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are abundant in the nervous system and other tissues. Here we describe two different aspects of ChEs and the cholinergic system. The first aspect concerns the role of cholinergic transmission in central pattern generation in the neonatal rat spinal cord and the second one describes the involvement of ChEs in the pathologies of dystrophin-deficient mutant (mdx) mice, the animal model of Duchenne muscular dystrophy. Thus, this study is divided into two distinct parts. In the first part we show that AChE is abundant in ventral horn neurons, central canal-adjacent and partition neurons in all the observed segments (L2, L5, S1, and S2). AChE was also found in the intermediolateral and sacral parasympathetic nuclei of L2 and S1, respectively. Blocking the AChE by edrophonium produced non-stationary bursting in spinal cord preparations of developing rats. Cross-wavelet/coherence analyses of the data revealed epochs of locomotor-like activity (left-right and flexor-extensor alternation) followed by other rhythmic or non-rhythmic bursting patterns. Addition of exogenous ACh stabilized the rhythm and increased the incidence of locomotor-like pattern in the preparations. Thus, the cholinergic system in the spinal cord is capable of producing and modulating functional rhythmic bursts. Moreover, bath-applied edrophonium and exogenous ACh were found as potent means of modulation of the locomotor rhythm produced by stimulation of sacrocaudal afferents (SCAs). We show that a subclass of sacral neurons with contralateral funicular projections to the thoracolumbar cord is associated with the cholinergic system. This group of neurons may play a major role in the observed enhancement of the SCA-induced motor rhythm. In the second part we show that adult mdx-muscles are malformed with distorted neuromuscular junctions (nmjs) and impaired regulation of acetylcholine receptors. Examination of circulating ChE levels revealed that in mdx-sera, while AChE activity was elevated, BuChE activity was markedly lower than in wild-type (wt) sera. Thus, BuChE to AChE ratio in mouse sera decreased from 6:1 in wt control to 3:1 in mdx. Because serum ChE levels may be modulated by gonadal steroids, it is possible that lack of dystrophin in mdx-mice may affect this regulation. Further studies are in progress to determine the potential endocrine regulation of ChEs in circulation and at the nmjs of mdx- and wt-mice. These studies will help clarify whether the hormonal regulation is impaired in the mdx mutant, and whether changes in circulating ChE reflect or influence the functional deficits observed in excitable tissues of diseased states.     

Oocyte-directed depletion of connexin43 using the Cre-LoxP system leads to subfertility in female mice

A T-DNA insertion mutant of FUSCA3 (fus3-T) in Arabidopsis thaliana exhibits several of the expected deleterious effects on seed development, but not the formation of brown seeds, a colouration which results from the accumulation of large amounts of anthocyanin. A detailed phenotypic comparison between fus3-T and a known splice point mutant (fus3-3) revealed that the seeds from both mutants do not enter dormancy and can be rescued at an immature stage. Without rescue, mature fus3-3 seeds are non-viable, whereas those of fus3-T suffer only a slight loss in their germinability. A series of comparisons between the two mutants uncovered differences with respect to conditional lethality, in histological and sub-cellular features, and in the relative amounts of various storage compounds and metabolites present, leading to a further dissection of developmental processes in seeds and a partial reinterpretation of the complex seed phenotype. FUS3 function is now known to be restricted to the acquisition of embryo-dependent seed dormancy, the determination of cotyledonary cell identity, and the synthesis and accumulation of storage compounds. Based on DNA binding studies, a model is presented which can explain the differences between the mutant alleles. The fus3-T lesion is responsible for loss of function only, while the fus3-3 mutation induces various pleiotropic effects conditioned by a truncation gene product causing severe mis-differentiation.     

Aurora-B phosphorylates the myosin II heavy chain to promote cytokinesis

Cytokinesis, the final step of mitosis, is mediated by an actomyosin contractile ring, the formation of which is temporally and spatially regulated following anaphase onset. Aurora-B is a member of the chromosomal passenger complex, which regulates various processes during mitosis; it is not understood, however, how Aurora-B is involved in cytokinesis. Here, we show that Aurora-B and myosin-IIB form a complex in vivo during telophase. Aurora-B phosphorylates the myosin-IIB rod domain at threonine 1847 (T¹⁸⁴⁷), abrogating the ability of myosin-IIB monomers to form filaments. Furthermore, phosphorylation of myosin-IIB filaments by Aurora-B also promotes filament disassembly. We show that myosin-IIB possessing a phosphomimetic mutation at T¹⁸⁴⁷ was unable to rescue cytokinesis failure caused by myosin-IIB depletion. Cells expressing a phosphoresistant mutation at T¹⁸⁴⁷ had significantly longer intercellular bridges, implying that Aurora-B-mediated phosphorylation of myosin-IIB is important for abscission. We propose that myosin-IIB is a substrate of Aurora-B and reveal a new mechanism of myosin-IIB regulation by Aurora-B in the late stages of mitosis.     

Water level affects availability of optimal feeding habitats for threatened migratory waterbirds

Extensive ephemeral wetlands at Poyang Lake, created by dramatic seasonal changes in water level, constitute the main wintering site for migratory Anatidae in China. Reductions in wetland area during the last 15 years have led to proposals to build a Poyang Dam to retain high winter water levels within the lake. Changing the natural hydrological system will affect waterbirds dependent on water level changes for food availability and accessibility. We tracked two goose species with different feeding behaviors (greater white‐fronted geese Anser albifrons [grazing species] and swan geese Anser cygnoides [tuber‐feeding species]) during two winters with contrasting water levels (continuous recession in 2015; sustained high water in 2016, similar to those predicted post‐Poyang Dam), investigating the effects of water level change on their habitat selection based on vegetation and elevation. In 2015, white‐fronted geese extensively exploited sequentially created mudflats, feeding on short nutritious graminoid swards, while swan geese excavated substrates along the water edge for tubers. This critical dynamic ecotone successively exposes subaquatic food and supports early‐stage graminoid growth during water level recession. During sustained high water levels in 2016, both species selected mudflats, but also to a greater degree of habitats with longer established seasonal graminoid swards because access to tubers and new graminoid growth was restricted under high‐water conditions. Longer established graminoid swards offer less energetically profitable forage for both species. Substantial reduction in suitable habitat and confinement to less profitable forage by higher water levels is likely to reduce the ability of geese to accumulate sufficient fat stores for migration, with potential carryover effects on subsequent survival and reproduction. Our results suggest that high water levels in Poyang Lake should be retained during summer, but permitted to gradually recede, exposing new areas throughout winter to provide access for waterbirds from all feeding guilds.     

Survey of Archaeal Diversity Reveals an Abundance of Halophilic Archaea in a Low-Salt, Sulfide- and Sulfur-Rich Spring

The archaeal community in a sulfide- and sulfur-rich spring with a stream water salinity of 0.7 to 1.0% in southwestern Oklahoma was studied by cloning and sequencing of 16S rRNA genes. Two clone libraries were constructed from sediments obtained at the hydrocarbon-exposed source of the spring and the microbial mats underlying the water flowing from the spring source. Analysis of 113 clones from the source library and 65 clones from the mat library revealed that the majority of clones belonged to the kingdom Euryarchaeota, while Crenarchaeota represented less than 10% of clones. Euryarchaeotal clones belonged to the orders Methanomicrobiales, Methanosarcinales, and Halobacteriales, as well as several previously described lineages with no pure-culture representatives. Those within the Halobacteriales represented 36% of the mat library and 4% of the source library. All cultivated members of this order are obligately aerobic halophiles. The majority of halobacterial clones encountered were not affiliated with any of the currently described genera of the family Halobacteriaceae. Measurement of the salinity at various locations at the spring, as well as along vertical gradients, revealed that soils adjacent to spring mats have a much higher salinity (NaCl concentrations as high as 32%) and a lower moisture content than the spring water, presumably due to evaporation. By use of a high-salt-plus-antibiotic medium, several halobacterial isolates were obtained from the microbial mats. Analysis of 16S rRNA genes indicated that all the isolates were members of the genus Haloferax. All isolates obtained grew at a wide range of salt concentrations, ranging from 6% to saturation, and all were able to reduce elemental sulfur to sulfide. We reason that the unexpected abundance of halophilic Archaea in such a low-salt, highly reduced environment could be explained by their relatively low salt requirement, which could be satisfied in specific locations of the shallow spring via evaporation, and their ability to grow under the prevalent anaerobic conditions in the spring, utilizing zero-valent sulfur compounds as electron acceptors. This study demonstrates that members of the Halobacteriales are not restricted to their typical high-salt habitats, and we propose a role for the Halobacteriales in sulfur reduction in natural ecosystems.     

Salinity Responses of Benthic Microbial Communities in a Solar Saltern (Eilat, Israel)

The salinity responses of cyanobacteria, anoxygenic phototrophs, sulfate reducers, and methanogens from the laminated endoevaporitic community in the solar salterns of Eilat, Israel, were studied in situ with oxygen microelectrodes and in the laboratory in slurries. The optimum salinity for the sulfate reduction rate in sediment slurries was between 100 and 120‰, and sulfate reduction was strongly inhibited at an in situ salinity of 215‰. Nevertheless, sulfate reduction was an important respiratory process in the crust, and reoxidation of formed sulfide accounted for a major part of the oxygen budget. Methanogens were well adapted to the in situ salinity but contributed little to the anaerobic mineralization in the crust. In slurries with a salinity of 180‰ or less, methanogens were inhibited by increased activity of sulfate-reducing bacteria. Unicellular and filamentous cyanobacteria metabolized at near-optimum rates at the in situ salinity, whereas the optimum salinity for anoxygenic phototrophs was between 100 and 120‰.     

AtEHDs, novel Arabidopsis EH-domain-containing proteins involved in endocytosis

SUMMARY: Endocytosis is an essential process by which the eukaryotic cell internalizes exogenous material. Studies in yeast and mammalian cells have revealed that endocytosis is a complex molecular process depending on regulated interactions between a variety of proteins and lipids through specific modules. One such module is the Eps15 homology (EH) domain, a conserved modular protein-interaction domain found in several endocytic proteins. The EH-domain-containing proteins function as regulators of endocytosis through their ability to interact with other proteins involved in this process. Here we describe the isolation and characterization of two Arabidopsis EH-domain-containing proteins (AtEHD1 and AtEHD2). We show that the two proteins are involved in endocytosis in plant systems and demonstrate that the Arabidopsis EHD proteins function similarly to mammalian EHDs. Similarly to hEHD2, over-expression of AtEHD2 has an inhibitory effect on endocytosis. While transgenic plants over-expressing AtEHD1 had no detectable phenotype, downregulation of AtEHD1 caused retardation of entry of endocytosed material into plant cells.     

Beautiful faces have variable reward value: fMRI and behavioral evidence.

The brain circuitry processing rewarding and aversive stimuli is hypothesized to be at the core of motivated behavior. In this study, discrete categories of beautiful faces are shown to have differing reward values and to differentially activate reward circuitry in human subjects. In particular, young heterosexual males rate pictures of beautiful males and females as attractive, but exert effort via a keypress procedure only to view pictures of attractive females. Functional magnetic resonance imaging at 3 T shows that passive viewing of beautiful female faces activates reward circuitry, in particular the nucleus accumbens. An extended set of subcortical and paralimbic reward regions also appear to follow aspects of the keypress rather than the rating procedures, suggesting that reward circuitry function does not include aesthetic assessment.     

LITHIUM CHLORIDE EXTRACTION OF DNA FROM THE SEAWEED PORPHYRA PERFORATA (RHODOPHYTA)1

Lithium chloride facilitates the softening of cell walls resulting in a simple, quick (2 h) method for DNA extraction from the red seaweed Porphyra perforata J. Agardh. A 5-min treatment of tissues in Lid at 55°C extracts DNA that is relatively free of the viscous polysaccharides and proteins that are usually coextracted in large amounts from cell walls and cytoplasm. This protocol does not require grinding of tissues, hydroxyapatite binding, cetyl trimethyl ammonium bromide treatments, enzymatic treatments, phenol extraction, or CsCl-gradient centrifugation. The resulting DNA is of sufficient quality to be used as a template for polymerase chain reaction amplification.