A targeted deletion upstream of <Emphasis Type="Italic">Snrpn</Emphasis> does not result in an imprinting defect

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) result from the disturbance of imprinted gene expression within human chromosome 15q11–q13. Some cases of PWS and AS are caused by microdeletions near the SNRPN gene that disrupt a regulatory element termed the imprinting center (IC). The IC has two functional components; an element at the promoter of SNRPN involved in PWS (PWS-IC) and an element 35 kilobases (kb) upstream of SNRPN involved in AS (AS-IC). To further understand the function of the IC, we sought to create a mouse model for AS-IC mutations. We have generated two deletions at a location analogous to that of the human AS-IC. Neither deletion produced an imprinting defect as indicated by DNA methylation and gene expression analyses. These results indicate that no elements critical for AS-IC function in mouse reside within the 12.8-kb deleted region and suggest that the specific location of the AS-IC is not conserved between human and mouse. Camilynn I. Brannan was Deceased     

Development of novel microsatellite markers for the grassland species Lolium multiflorum,Lolium perenne and Festuca pratensis

Twelve microsatellite markers were isolated from Lolium multiflorum. Allelic variability and cross‐species amplification were assessed on 16 individuals of each of the three grassland species L. multiflorum, Lolium perenne and Festuca pratensis. Cross‐species amplification success was 100% for L. perenne and 83% for F. pratensis. The number of alleles detected ranged from one to 14 with an average of 3.4. While three microsatellite loci were polymorphic in all three species, one marker produced species‐specific alleles in all three species. These microsatellite markers provide a valuable tool for population genetic studies within and among species of the Festuca–Lolium complex.     

Grassland yield declined by a quarter in 5 years of free-air ozone fumigation

The sensitivity of seminatural grasslands to ozone (O₃) pollution is not well known, in spite of the important function of these common ecosystems for agriculture and nature conservation. A 5‐year field experiment was carried out at a rural, mid‐elevation site at Le Mouret (Switzerland) to investigate the effect of elevated O₃ on yield and species composition of a permanent, extensively managed, species‐rich old pasture. Using a free‐air fumigation system, circular plots of 7 m diameter were exposed to either ambient air (control plots) or to air containing O₃ of approximately 1.5 × ambient concentrations. The resulting accumulated O₃ exposure over the threshold of 40 ppb for one season ranged from 13.3 to 59.5 ppm h in the elevated O₃ plots, and from 1.0 to 20.7 ppm h in the control plots. Subplots in each ring were harvested three times each year, and harvested biomass was separated into functional groups (FGs) (grasses, legumes, forbs). Productivity in both treatments decreased over time, but the yield of O₃‐exposed plots decreased faster than that of the control plots, with the reduction being twice as large by the end of the fifth season. Compared with the ambient air control, loss in annual dry matter yield was 23% after 5 years. The change in annual biomass production because of O₃ stress was accompanied by a change in the fractions of FGs, with the legume fraction showing a strong negative response. Such long‐term effects of O₃ stress could have negative implications for the maintenance of biological diversity in rural landscapes across large areas of Europe. The results from this first long‐term experiment show that a moderately elevated O₃ level reduces the productivity of intact grasslands during a 5‐year exposure under real field conditions.     

Voltage-dependent hydration and conduction properties of the hydrophobic pore of the mechanosensitive channel of small conductance

A detailed picture of water and ion properties in small pores is important for understanding the behavior of biological ion channels. Several recent modeling studies have shown that small, hydrophobic pores exclude water and ions even if they are physically large enough to accommodate them, a mechanism called hydrophobic gating. This mechanism has been implicated in the gating of several channels, including the mechanosensitive channel of small conductance (MscS). Although the pore in the crystal structure of MscS is wide and was initially hypothesized to be open, it is lined by hydrophobic residues and may represent a nonconducting state. Molecular dynamics simulations were performed on MscS to determine whether or not the structure can conduct ions. Unlike previous simulations of hydrophobic nanopores, electric fields were applied to this system to model the transmembrane potential, which proved to be important. Although simulations without a potential resulted in a dehydrated, occluded pore, the application of a potential increased the hydration of the pore and resulted in current flow through the channel. The calculated channel conductance was in good agreement with experiment. Therefore, it is likely that the MscS crystal structure is closer to a conducting than a nonconducting state.     

Kinetics of arsenic methylation by freshly isolated B6C3F1 mouse hepatocytes

The toxic and carcinogenic effects of arsenic may be mediated by both inorganic and methylated arsenic species. The methylation of arsenic(III) is thought to take place via sequential oxidative methylation and reduction steps to form monomethylarsenic (MMA) and dimethylarsenic (DMA) species, but recent evidence indicates that glutathione complexes of arsenic(III) can be methylated without oxidation. The kinetics of arsenic methylation were determined in freshly isolated hepatocytes from male B6C3F1 mice. Hepatocytes (>90% viability) were isolated by collagenase perfusion and suspended in Williams' Medium E with various concentrations of arsenic(III) (sodium m-arsenite). Aliquots of the lysed cell suspension were analyzed for arsenic species by hydride generation-atomic absorption spectrometry. The formation of MMA(III) from sodium arsenite (1 microM) was linear with respect to time for >90 min. DMA(III) formation did not become significant until 60 min. MMA(V) and DMA(V) were not consistently observed in the incubations. These results suggest that the glutathione complex mechanism of methylation plays an important role in arsenic biotransformation in mouse hepatocytes. Metabolism of arsenic(V) was not observed in mouse hepatocytes, consistent with inhibition of arsenic(V) active cellular uptake by phosphate in the medium. The formation of MMA(III) increased with increasing arsenic(III) concentrations up to approximately 2 microM and declined thereafter. The concentration dependence is consistent with a saturable methylation reaction accompanied by uncompetitive substrate inhibition of the reaction by arsenic(III). Kinetic analysis of the data suggested an apparent K(M) of approximately 3.6 microM arsenic(III), an apparent V(max) of approximately 38.9 microg MMA(III) formed/L/h/million cells, and an apparent K(I) of approximately 1.3 microM arsenic(III). The results of this study can be used in the physiologically based pharmacokinetic model for arsenic disposition in mice to predict the concentration of MMA(III) in liver and other tissues.     

A comparative study of the binding and inhibition of four copper-containing amine oxidases by azide: implications for the role of copper during the oxidative half-reaction

Copper amine oxidases (CuAOs) catalyze the oxidative deamination of primary amines operating through a ping-pong bi-bi mechanism. In this work, azide (an exogenous monodentate ligand) was used to probe the role of copper during the oxidative half-reaction of CuAO catalysis. The effects of azide on both the reductive and oxidative half-reactions of pea seedling amine oxidase (PSAO), the recombinant human kidney diamine oxidase (rhDAO), Arthrobacter globiformis amine oxidase (AGAO), and Pichia pastoris amine oxidase (PPLO) have been examined. For the reductive half-reaction, defined as the oxidation of amine substrate to an aldehyde, azide was discovered to exhibit either noncompetitive or competitive inhibition with respect to the amine, depending on the enzyme source. With regard to the oxidative half-reaction, defined as the reoxidation of the enzyme via reduction of O(2) to H(2)O(2), azide has been determined to exhibit competitive inhibition with respect to O(2) in PSAO with a calculated K(i) value that is in excellent agreement with the experimentally determined K(d) value for the Cu(II)-N(3)(-) complex. Azide was found to exhibit mixed-type/partially competitive inhibition with respect to substrate O(2) in rhDAO, with an apparent K(i) that is similar to the K(d) value for the Cu(II)-N(3)(-) complex. The competitive inhibition for PSAO and the partially competitive inhibition for rhDAO are consistent with O(2) interacting directly with copper during enzymatic reoxidation. For the enzymes AGAO and PPLO, pure noncompetitive and mixed-type/partially competitive inhibition is observed. K(i) values for reductive and oxidative half-reactions are equivalent and are lower than measured K(d) values for the Cu(II)-N(3)(-) complexes in oxidized and substrate-reduced forms of these enzymes. Given these observations, it appears that substantial inhibition of the reductive half-reaction occurs at the concentrations of azide used for the oxidative half-reaction experiments, thereby complicating kinetic interpretation. At this time, the data do not permit us to distinguish between two possibilities: (1) inhibition by azide with respect to O(2) is intrinsically competitive in CuAOs, but this effect cannot always be deconvolved experimentally from the effects of azide on the reductive half-reaction; or (2) CuAOs differ in some steps of their reoxidation mechanisms.     

Defining essential stem cell characteristics in adipose-derived stromal cells extracted from distinct anatomical sites

The discovery of adipose-derived stromal cells (ASCs) has created many opportunities for the development of patient-specific cell-based replacement therapies. We have isolated multiple cell strains of ASCs from various anatomical sites (abdomen, arms/legs, breast, buttocks), indicating widespread distribution of ASCs throughout the body. Unfortunately, there exists a general lack of agreement in the literature as to their "stem cell" characteristics. We find that telomerase activity and expression of its catalytic subunit in ASCs are both below the levels of detection, independent of age and culturing conditions. ASCs also undergo telomere attrition and eventually senesce, while maintaining a stable karyotype without the development of spontaneous tumor-associated abnormalities. Using a set of cell surface markers that have been promoted to identify ASCs, we find that they failed to distinguish ASCs from normal fibroblasts, as both are positive for CD29, CD73 and CD105 and negative for CD14, CD31 and CD45. All of the ASC isolates are multipotent, capable of differentiating into osteocytes, chondrocytes and adipocytes, while fibroblasts show no differentiation potential. Our ASC strains also show elevated expression of genes associated with pluripotent cells, Oct-4, SOX2 and NANOG, when compared to fibroblasts and bone marrow-derived mesenchymal stem cells (BM-MSCs), although the levels were lower than induced pluripotent stem cells (iPS). Together, our data suggest that, while the cell surface profile of ASCs does not distinguish them from normal fibroblasts, their differentiation capacity and the expression of genes closely linked to pluripotency clearly define ASCs as multipotent stem cells, regardless of tissue isolation location.     

Ss-bCNGa: a unique member of the bacterial cyclic nucleotide gated (bCNG) channel family that gates in response to mechanical tension

Bacterial cyclic nucleotide gated (bCNG) channels are generally a nonmechanosensitive subset of the mechanosensitive channel of small conductance (MscS) superfamily. bCNG channels are composed of an MscS channel domain, a linking domain, and a cyclic nucleotide binding domain. Among bCNG channels, the channel domain of Ss-bCNGa, a bCNG channel from Synechocystis sp. PCC 6803, is most identical to Escherichia coli (Ec) MscS. This channel also exhibits limited mechanosensation in response to osmotic downshock assays, making it the only known full-length bCNG channel to respond to hypoosmotic stress. Here, we compare and contrast the ability of Ss-bCNGa to gate in response to mechanical tension with Se-bCNG, a nonmechanosensitive bCNG channel, and Ec-MscS, a prototypical mechanosensitive channel. Compared with Ec-MscS, Ss-bCNGa only exhibits limited mechanosensation, which is most likely a result of the inability of Ss-bCNGa to form the strong lipid contacts needed for significant function. Unlike Ec-MscS, Ss-bCNGa displays a mechanical response that increases with protein expression level, which may result from channel clustering driven by interchannel cation?C?? interactions.     

Function of defensive volatiles in pedunculate oak (Quercus robur) is tricked by the moth Tortrix viridana

The indirect defences of plants are comprised of herbivore‐induced plant volatiles (HIPVs) that among other things attract the natural enemies of insects. However, the actual extent of the benefits of HIPV emissions in complex co‐evolved plant‐herbivore systems is only poorly understood. The observation that a few Quercus robur L. trees constantly tolerated (T‐oaks) infestation by a major pest of oaks (Tortrix viridana L.), compared with heavily defoliated trees (susceptible: S‐oaks), lead us to a combined biochemical and behavioural study. We used these evidently different phenotypes to analyse whether the resistance of T‐oaks to the herbivore was dependent on the amount and scent of HIPVs and/or differences in non‐volatile polyphenolic leaf constituents (as quercetin‐, kaempferol‐ and flavonol glycosides). In addition to non‐volatile metabolic differences, typically defensive HIPV emissions differed between S‐oaks and T‐oaks. Female moths were attracted by the blend of HIPVs from S‐oaks, showing significantly higher amounts of (E)‐4,8‐dimethyl‐1,3,7‐nonatriene (DMNT) and (E)‐β‐ocimene and avoid T‐oaks with relative high fraction of the sesquiterpenes α‐farnesene and germacrene D. Hence, the strategy of T‐oaks exhibiting directly herbivore‐repellent HIPV emissions instead of high emissions of predator‐attracting HIPVs of the S‐oaks appears to be the better mechanism for avoiding defoliation.     

Novel histone-derived antimicrobial peptides use different antimicrobial mechanisms

The increase in multidrug resistant bacteria has sparked an interest in the development of novel antibiotics. Antimicrobial peptides that operate by crossing the cell membrane may also have the potential to deliver drugs to intracellular targets. Buforin 2 (BF2) is an antimicrobial peptide that shares sequence identity with a fragment of histone subunit H2A and whose bactericidal mechanism depends on membrane translocation and DNA binding. Previously, novel histone-derived antimicrobial peptides (HDAPs) were designed based on properties of BF2, and DesHDAP1 and DesHDAP3 showed significant antibacterial activity. In this study, their DNA binding, permeabilization, and translocation abilities were assessed independently and compared to antibacterial activity to determine whether they share a mechanism with BF2. To investigate the importance of proline in determining the peptides' mechanisms of action, proline to alanine mutants of the novel peptides were generated. DesHDAP1, which shows significant similarities to BF2 in terms of secondary structure, translocates effectively across lipid vesicle and bacterial membranes, while the DesHDAP1 proline mutant shows reduced translocation abilities and antimicrobial potency. In contrast, both DesHDAP3 and its proline mutant translocate poorly, though the DesHDAP3 proline mutant is more potent. Our findings suggest that a proline hinge can promote membrane translocation in some peptides, but that the extent of its effect on permeabilization depends on the peptide's amphipathic properties. Our results also highlight the different antimicrobial mechanisms exhibited by histone-derived peptides and suggest that histones may serve as a source of novel antimicrobial peptides with varied properties.