The methyl donor S-Adenosylmethionine inhibits active demethylation of DNA: a candidate novel mechanism for the pharmacological effects of S-Adenosylmethionine

S-Adenosylmethionine (AdoMet) is the methyl donor of numerous methylation reactions. The current model is that an increased concentration of AdoMet stimulates DNA methyltransferase reactions, triggering hypermethylation and protecting the genome against global hypomethylation, a hallmark of cancer. Using an assay of active demethylation in HEK 293 cells, we show that AdoMet inhibits active demethylation and expression of an ectopically methylated CMV-GFP (green fluorescent protein) plasmid in a dose-dependent manner. The inhibition of GFP expression is specific to methylated GFP; AdoMet does not inhibit an identical but unmethylated CMV-GFP plasmid. S-Adenosylhomocysteine (AdoHcy), the product of methyltransferase reactions utilizing AdoMet does not inhibit demethylation or expression of CMV-GFP. In vitro, AdoMet but not AdoHcy inhibits methylated DNA-binding protein 2/DNA demethylase as well as endogenous demethylase activity extracted from HEK 293, suggesting that AdoMet directly inhibits demethylase activity, and that the methyl residue on AdoMet is required for its interaction with demethylase. Taken together, our data support an alternative mechanism of action for AdoMet as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA.     

Mercury in the marine food chain in the Southern Ocean at Macquarie Island: an analysis of a top predator,Patagonian toothfish (<Emphasis Type="Italic">Dissostichus eleginoides</Emphasis>) and a mid-trophic species,the warty squid (<Emphasis Type="Italic">Moroteuthis ingens</Emphasis>)

The characteristics and habitat of the Patagonian toothfish (Dissostichus eleginoides) are typical of fish that accumulate high concentrations of mercury. In this study, mercury determinations were made on samples of muscle tissue from Macquarie Island toothfish and the Southern Ocean deepwater warty squid (Moroteuthis ingens). The analysis of mercury in the biological tissues was made by cold vapour-atomic absorption spectrometry following acid digestion. Performance of the analytical procedure was assessed by analysis of certified reference material (DORM-2, dogfish muscle). Mercury concentrations of 16 Macquarie Island toothfish ranged from 0.12 mg kg^–1 (550 g, 381 mm TL) to 0.59 mg kg^–1 (6,100 g, 823 mm TL), with a mean concentration of 0.33±0.12 mg kg^–1. A significant correlation was found between mercury and either toothfish weight or total length. The fish analysed were juveniles, which suggests that larger individuals would have higher mercury concentrations well exceeding food standard code limits for mercury in fish (typically 0.5 mg kg^–1). Warty squid, also from around Macquarie Island, had a low mean mercury concentration of 0.086 mg kg^–1 in mantle tissue; no significant correlation existed between mercury concentration and either squid mantle length or total weight. It is postulated that the squid have a mechanism, possibly involving the digestive gland, that prevents bioaccumulation of mercury in the mantle, and presumably other body tissues.     

Developmental expression of Magmas in murine tissues and its co-expression with the GM-CSF receptor.

Magmas is a protein that is involved in GM-CSF signaling in a myeloid cell line. Its precise role in the signal transduction process is unclear. To accurately characterize Magmas expression in a variety of cells, mouse embryos and adult murine tissues were analyzed for both mRNA and protein content. Magmas expression was detected as early as the day 6.5 embryo. The level of expression was developmentally regulated. During embryogenesis, elevated Magmas was observed in several structures, including heart, liver, notochord, choroid plexus, cervical ganglion, and nasal mucosa. Muscle, pancreas, intestinal mucosa, and testes were among the adult tissues with high Magmas expression. Most cell types, including hepatocytes and skeletal, smooth, and cardiac myocytes, also expressed the GM-CSF receptor (GMR) but the relative tissue levels of GMR were not always proportional to Magmas. The expression patterns suggest that Magmas has a role in both developing and mature tissues.     

Multiplexed genotyping with sequence-tagged molecular inversion probes

We report on the development of molecular inversion probe (MIP) genotyping, an efficient technology for large-scale single nucleotide polymorphism (SNP) analysis. This technique uses MIPs to produce inverted sequences, which undergo a unimolecular rearrangement and are then amplified by PCR using common primers and analyzed using universal sequence tag DNA microarrays, resulting in highly specific genotyping. With this technology, multiplex analysis of more than 1,000 probes in a single tube can be done using standard laboratory equipment. Genotypes are generated with a high call rate (95%) and high accuracy (>99%) as determined by independent sequencing.     

Hibernation patterns of Turkish hamsters: influence of sex and ambient temperature

Turkish hamsters (Mesocricetus brandti) are a model organism for studies of hibernation, yet a detailed account of their torpor characteristics has not been undertaken. This study employed continuous telemetric monitoring of body temperature (T _b) in hibernating male and female Turkish hamsters at ambient temperatures (T _as) of 5 and 13 °C to precisely characterize torpor bout depth, duration, and frequency, as well as rates of entry into and arousal from torpor. Hamsters generated brief intervals of short (<12 h), shallow test bouts (T _b > 20 °C), followed by deep torpor bouts lasting 4–6 days at T _a = 5 °C and 2–3 days at T _a = 13 °C. Females at T _a = 5 °C had longer bouts than males, but maintained higher torpor T _b; there were no sex differences at T _a = 13 °C. Neither body mass loss nor food intake differed between the two T _as. Hamsters entered torpor primarily during the scotophase (subjective night), but timing of arousals was highly variable. Hamsters at both T _as generated short, shallow torpor bouts between deep bouts, suggesting that this species may be capable of both hibernation and daily torpor.     

The pEAQ vector series: the easy and quick way to produce recombinant proteins in plants

Promoter regions of six sugarcane Loading Stem Gene (ScLSG) alleles were analyzed using bioinformatic and transgenic approaches. Stable transgene expression analyses, on multiple independent lines per construct, revealed differences between ScLSG promoters in absolute levels and in tissue-selectivity of luciferase reporter activity. Four promoters drove peak expression in the sucrose-loading zone and maintained substantial expression throughout mature stems. One drove a pattern of gradual increase along the stem maturation profile. In general, stem: root expression ratio increased with plant age. The ScLSG5 promoter had the fewest light-enhanced and root-expression motifs in bioinformatic analysis, and drove the highest level and specificity of transgene expression in stems. This indicates the potential to further improve the stem specificity of ScLSG promoter sequences by eliminating enhancers of expression in other tissues. An intron in the 5′UTR was important for expression strength. The ScLSG promoters will be useful for research and biotechnology in sugarcane, where the tailored expression of transgenes in stems is important for enhanced accumulation of sugar or value-added products, and for development as a bioenergy feedstock.     

The effects of stimulus location on the gating of touch by heat- and cold-induced pain

The influence of heat- and cold-induced pain on tactile sensitivity, a "touch gate", was measured under conditions in which the location of the noxious stimuli was varied with respect to the tactile stimulus applied to the thenar eminence of humans. Vibrotactile thresholds were measured in the absence of pain and during administration of a painful stimulus, with the stimulus frequencies selected to activate independently the four psychophysical channels hypothesized to exist in human glabrous skin. Heat-induced pain produced by spatially co-localizing the noxious stimuli with the tactile stimuli was found, on average, to elevate threshold amplitude by 2.2 times (6.7 dB). Co-localized, cold-induced pain raised the average thresholds by about 1.5 times (3.6 dB). Heat-induced pain presented contralaterally produced no change in vibrotactile sensitivity indicating that the effect is probably not due to attentional mechanisms. Ipsilateral heat-induced pain caused an elevation in tactile thresholds even when the noxious and non-noxious stimuli were not co-localized, and the effect may seem to require that the painful stimulus be within the somatosensory region defined possibly in terms of dermatomal organization. Thus the effect is probably related to somatotopic organization and is not peripherally mediated. A brief discussion as to the possible locus of the touch gate within the nervous system is also given.     

An NMR Study of the Conformations of N-terminal Substance P Fragments and Antagonists

Abstract

Three N-terminal fragments of the neurotransmitter Substance P as well as two antagonist heptapeptides containing D-amino-acid residues were studied using different ID and 2D NMR techniques. Total nonexchangeable ¹H-NMR assignments were carried out in D₂O and the NH protons were assigned in H₂O by means of COSY experiments. The spectral data indicates that there are no preferred conformations for the backbone. The N-terminal tetrapeptide SP_1–4-OH exists as a mixture of cis/trans isomers and this effect was studied as a function of pH.     

One- and Two-Dimensional lH NMR Study of the Substance P Fragment ARG-PRO-LYS-PRO

Abstract

The Substance P fragment Arg¹Pro²-Lys³-Pro⁴ (SP_1–4) has been extensively investigated by means of proton nuclear magnetic resonance at 400 MHz. The combined application of different 2D techniques and a comparison of SP_1–4 with its derivative SPM-amide allowed the complete and unambiguous assignment of the proton NMR spectrum. Conformational data obtained from the different NMR parameters are compared with theoretical calculations. The results suggest that SP_1–4 exists, at the chosen experimental conditions, as a stretched molecule.     

Indirect Effects of Impoundment on Migrating Fish: Temperature Gradients in Fish Ladders Slow Dam Passage by Adult Chinook Salmon and Steelhead

Thermal layering in reservoirs upstream from hydroelectric dams can create temperature gradients in fishways used by upstream migrating adults. In the Snake River, Washington, federally-protected adult salmonids (Oncorhynchus spp.) often encounter relatively cool water in dam tailraces and lower ladder sections and warmer water in the upstream portions of ladders. Using radiotelemetry, we examined relationships between fish passage behavior and the temperature difference between the top and bottom of ladders (∆T) at four dams over four years. Some spring Chinook salmon (O. tshawytscha) experienced ∆T ≥ 0.5 °C. Many summer and fall Chinook salmon and summer steelhead (O. mykiss) experienced ∆T ≥ 1.0 °C, and some individuals encountered ΔT > 4.0°C. As ΔT increased, migrants were consistently more likely to move down fish ladders and exit into dam tailraces, resulting in upstream passage delays that ranged from hours to days. Fish body temperatures equilibrated to ladder temperatures and often exceeded 20°C, indicating potential negative physiological and fitness effects. Collectively, the results suggest that gradients in fishway water temperatures present a migration obstacle to many anadromous migrants. Unfavorable temperature gradients may be common at reservoir-fed fish passage facilities, especially those with seasonal thermal layering or stratification. Understanding and managing thermal heterogeneity at such sites may be important for ensuring efficient upstream passage and minimizing stress for migratory, temperature-sensitive species.