Patterns of sequence variation in the mitochondrial D-loop region of shrews

Direct sequencing of the mitochondrial displacement loop (D-loop) of shrews (genus Sorex) for the region between the tRNA(Pro) and the conserved sequence block-F revealed variable numbers of 79-bp tandem repeats. These repeats were found in all 19 individuals sequenced, representing three subspecies and one closely related species of the masked shrew group (Sorex cinereus cinereus, S. c. miscix, S. c. acadicus, and S. haydeni) and an outgroup, the pygmy shrew (S. hoyi). Each specimen also possessed an adjacent 76-bp imperfect copy of the tandem repeats. One individual was heteroplasmic for length variants consisting of five and seven copies of the 79-bp tandem repeat. The sequence of the repeats is conducive to the formation of secondary structure. A termination-associated sequence is present in each of the repeats and in a unique sequence region 5' to the tandem array as well. Mean genetic distance between the masked shrew taxa and the pygmy shrew was calculated separately for the unique sequence region, one of the tandem repeats, the imperfect repeat, and these three regions combined. The unique sequence region evolved more rapidly than the tandem repeats or the imperfect repeat. The small genetic distance between pairs of tandem repeats within an individual is consistent with a model of concerted evolution. Repeats are apparently duplicated and lost at a high rate, which tends to homogenize the tandem array. The rate of D- loop sequence divergence between the masked and pygmy shrews is estimated to be 15%-20%/Myr, the highest rate observed in D-loops of mammals. Rapid sequence evolution in shrews may be due either to their high metabolic rate and short generation time or to the presence of variable numbers of tandem repeats.      

Mitochondrial proteome: altered cytochrome c oxidase subunit levels in prostate cancer

Laser capture microdissection was combined with reverse phase protein lysate arrays to quantitatively analyze the ratios of mitochondrial encoded cytochrome c oxidase subunits to nuclear encoded cytochrome c oxidase subunits, and to correlate the ratios with malignant progression in human prostate tissue specimens. Cytochrome c oxidase subunits I-III comprise the catalytic core of the enzyme and are all synthesized from mitochondrial DNA. The remaining subunits (IV-VIII) are synthesized from cellular nuclear DNA. A significant (P < 0.001, 30/30 prostate cases) shift in the relative concentrations of nuclear encoded cytochrome c oxidase subunits IV, Vb, and VIc compared to mitochondrial encoded cytochrome c oxidase subunits I and II was noted during the progression of prostate cancer from normal epithelium through premalignant lesions to invasive carcinoma. Significantly, this shift was discovered to begin even in the premalignant stage. Reverse phase protein lysate array-based observations were corroborated with immunohistochemistry, and extended to a few human carcinomas in addition to prostate. This analysis points to a role for nuclear DNA encoded mitochondrial proteins in carcinogenesis; underscoring their potential as targets for therapy while highlighting the need for full characterization of the mitochondrial proteome.     

Predicting consumer behavior: using novel mind-reading approaches

Advances in machine learning as applied to functional magnetic resonance imaging (fMRI) data offer the possibility of pretesting and classifying marketing communications using unbiased pattern recognition algorithms. By using these algorithms to analyze brain responses to brands, products, or existing marketing communications that either failed or succeeded in the marketplace and identifying the patterns of brain activity that characterize success or failure, future planned campaigns or new products can now be pretested to determine how well the resulting brain responses match the desired (successful) pattern of brain activity without the need for verbal feedback. This major advance in signal processing is poised to revolutionize the application of these brain-imaging techniques in the marketing sector by offering greater accuracy of prediction in terms of consumer acceptance of new brands, products, and campaigns at a speed that makes them accessible as routine pretesting tools that will clearly demonstrate return on investment.     

Characterization of the interaction between the N-terminal extension of human cardiac troponin I and troponin C

The N-terminal extension of cardiac troponin I (TnI) is bisphosphorylated by protein kinase A in response to beta-adrenergic stimulation. How this signal is transmitted between TnI and troponin C (TnC), resulting in accelerated Ca(2+) release, remains unclear. We recently proposed that the unphosphorylated extension interacts with the N-terminal domain of TnC stabilizing Ca(2+) binding and that phosphorylation prevents this interaction. We now use (1)H NMR to study the interactions between several N-terminal fragments of TnI, residues 1-18 (I1-18), residues 1-29 (I1-29), and residues 1-64 (I1-64), and TnC. The shorter fragments provide unambiguous information on the N-terminal regions of TnI that interact with TnC: I1-18 does not bind to TnC whereas the C-terminal region of unphosphorylated I1-29 does bind. Bisphosphorylation greatly weakens this interaction. I1-64 contains the phosphorylatable N-terminal extension and a region that anchors I1-64 to the C-terminal domain of TnC. I1-64 binding to TnC influences NMR signals arising from both domains of TnC, providing evidence that the N-terminal extension of TnI interacts with the N-terminal domain of TnC. TnC binding to I1-64 broadens NMR signals from the side chains of residues immediately C-terminal to the phosphorylation sites. Binding of TnC to bisphosphorylated I1-64 does not broaden these NMR signals to the same extent. Circular dichroism spectra of I1-64 indicate that bisphosphorylation does not produce major secondary structure changes in I1-64. We conclude that bisphosphorylation of cardiac TnI elicits its effects by weakening the interaction between the region of TnI immediately C-terminal to the phosphorylation sites and TnC either directly, due to electrostatic repulsion, or via localized conformational changes.     

ePAD,an oocyte and early embryo-abundant peptidylarginine deiminase-like protein that localizes to egg cytoplasmic sheets

Selected for its high relative abundance, a protein spot of MW approximately 75 kDa, pI 5.5 was cored from a Coomassie-stained two-dimensional gel of proteins from 2850 zona-free metaphase II mouse eggs and analyzed by tandem mass spectrometry (TMS), and novel microsequences were identified that indicated a previously uncharacterized egg protein. A 2.4-kb cDNA was then amplified from a mouse ovarian adapter-ligated cDNA library by RACE-PCR, and a unique 2043-bp open reading frame was defined encoding a 681-amino-acid protein. Comparison of the deduced amino acid sequence with the nonredundant database demonstrated that the protein was approximately 40% identical to the calcium-dependent peptidylarginine deiminase (PAD) enzyme family. Northern blotting, RT-PCR, and in situ hybridization analyses indicated that the protein was abundantly expressed in the ovary, weakly expressed in the testis, and absent from other tissues. Based on the homology with PADs and its oocyte-abundant expression pattern, the protein was designated ePAD, for egg and embryo-abundant peptidylarginine deiminase-like protein. Anti-recombinant ePAD monospecific antibodies localized the molecule to the cytoplasm of oocytes in primordial, primary, secondary, and Graafian follicles in ovarian sections, while no other ovarian cell type was stained. ePAD was also expressed in the immature oocyte, mature egg, and through the blastocyst stage of embryonic development, where expression levels began to decrease. Immunoelectron microscopy localized ePAD to egg cytoplasmic sheets, a unique keratin-containing intermediate filament structure found only in mammalian eggs and in early embryos, and known to undergo reorganization at critical stages of development. Previous reports that PAD-mediated deimination of epithelial cell keratin results in cytoskeletal remodeling suggest a possible role for ePAD in cytoskeletal reorganization in the egg and early embryo.     

Lichens toGunnera — with emphasis onAzolla

Summary N₂-fixing cyanobacteria occur in symbiotic associations with fungi (ascomycetes) as lichens and with a few green plants. The associated cyanobacterium is always a species ofNostoc orAnabaena. Only a small number of plant genera are involved but there is a remarkable range of host diversity. Associations occur with several bryophytes (e.g.Anthoceros, Blasia, Cavicularia), a pteridophyte (Azolla), cycads (nine genera includingMacrozamia andEncephalartos) and an angiosperm (Gunnera). Except forGunnera, where the cyanobacterium penetrates the plant cells, the cyanobacteria are extracellular with specialized morphological modifications and/or structures of the host plant organs providing an environment which facilitates interaction with the prokaryote.Salient aspects of current knowledge pertaining to the establishment, perpetuation, and functioning of the individual symbioses are summarized. Where possible this includes information concerning recognition and specificity, mode(s) of infection, morphological modifications/adaptations of the host plant and a synopsis of morphological, physiological and biochemical changes common to the symbiotic cyanobacteria. The latter encompasses heterocyst frequencies, enzymes involved in ammonia assimilation, photosynthetic capability and metabolic interaction with the host.TheAzolla-Anabaena symbioses, which have potential agronomic significance as an alternative nitrogen source and maintain continuity with the endophyte through the sexual cycle, are emphasized.     

Leaching potential of heavy metals (Cd, Ni, Pb, Cu and Zn) from acidic sandy soil amended with dolomite phosphate rock (DPR) fertilizers

There is an increasing concern on heavy metal leaching from the soils amended with sewage sludge. A column study was conducted to examine the extent of leaching of five important heavy metals (Cd, Ni, Pb, Cu and Zn) from an acidic sandy soil amended with different dolomite phosphate rock (DPR) fertilizers (an application rate of 1% fertilizers) developed from DPR and N-Viro (consisting of biosolids and fly ash) at 0%, 10%, 20%, 30%, 40%, 50% and 100% DPR. Ten leaching events were carried out with each event done at an interval of 7 days and with total leaching volume of 1183mm, which is equivalent to the mean annual rainfall of this region during the period of 2001-2003. Leachate was collected after each leaching event and analyzed for heavy metals. The maximum leachate concentrations of Cd, Ni, Pb, Cu and Zn were all below drinking water quality guidance limits set by Florida Department of Environmental Protection and World Health Organization, suggesting that the application of DPR fertilizers may not pose a threat to water quality by leaching. Most of leachate concentrations of Cd, Ni and Pb were below their detection limits and there were no significant differences between the control and the treatments with different DPR fertilizers. By contrast, there were higher leachate concentrations of Cu and Zn (ranging from 0.7 to 37.1mug Cu/l and 5.1 to 205.6mug Zn/l for all treatments) due to their higher contents in both the soil and different DPR fertilizers compared with Cd, Ni and Pb. The leachate concentrations of Cu and Zn for each treatment decreased with increasing leaching events. The differences in leachate concentrations of Cu and Zn between the control and the treatments with different DPR fertilizers containing N-Viro were significant, especially in the first several leaching events and, moreover, they increased with increasing proportion of N-Viro in the DPR fertilizers. There were similar trends in total losses of Cu and Zn after ten leaching events. Greater differences in both leachate concentrations and total losses of Zn between the control and the treatments containing N-Viro were noted. Total losses of Zn for the treatments containing N-Viro were 3.0-5.1 times higher than those for the control compared with 1.4-2.2 times higher for total losses of Cu, suggesting that greater proportions of Zn losses came from the DPR fertilizers due to the greater mobility of Zn in the DPR fertilizers compared with Cu.     

Attitudes toward Post‐Trial Access to Medical Interventions: A Review of Academic Literature,Legislation, and International Guidelines

There is currently no international consensus around post‐trial obligations toward research participants, community members, and host countries. This literature review investigates arguments and attitudes toward post‐trial access. The literature review found that academic discussions focused on the rights of research participants, but offered few practical recommendations for addressing or improving current practices. Similarly, there are few regulations or legislation pertaining to post‐trial access. If regulatory changes are necessary, we need to understand the current arguments, legislation, and attitudes towards post‐trial access and participants and community members. Given that clinical trials conducted in low‐income countries will likely continue, there is an urgent need for consideration of post‐trial benefits for participants, communities, and citizens of host countries. While this issue may not be as pressing in countries where participants have access to healthcare and medicines through public schemes, it is particularly important in regions where this may not be available.     

Potential Distribution and Risk Assessment of an Invasive Plant Species: A Case Study of Hymenachne amplexicaulis in Australia

Given the limited resources available for weed management, a strategic approach is required to give the “best bang for your buck.” The current study incorporates: (1) a model ensemble approach to identify areas of uncertainty and commonality regarding a species invasive potential, (2) current distribution of the invaded species, and (3) connectivity of systems to identify target regions and focus efforts for more effective management. Uncertainty in the prediction of suitable habitat for H. amplexicaulis (study species) in Australia was addressed in an ensemble-forecasting approach to compare distributional scenarios from four models (CLIMATCH; CLIMEX; boosted regression trees [BRT]; maximum entropy [Maxent]). Models were built using subsets of occurrence and environmental data. Catchment risk was determined through incorporating habitat suitability, the current abundance and distribution of H. amplexicaulis, and catchment connectivity. Our results indicate geographic differences between predictions of different approaches. Despite these differences a number of catchments in northern, central, and southern Australia were identified as high risk of invasion or further spread by all models suggesting they should be given priority for the management of H. amplexicaulis. The study also highlighted the utility of ensemble approaches in indentifying areas of uncertainty and commonality regarding the species’ invasive potential.     

Regulation of vacuolar h-pyrophosphatase by free calcium : a reaction kinetic analysis

The H⁺-translocating inorganic pyrophosphatase (H⁺-PPase) associated with vesicles of the vacuolar membrane (tonoplast) isolated from beet (Beta vulgaris L.) is subject to direct inhibition by Ca²⁺ and a number of other divalent cations (Co²⁺, Mn²⁺, Zn²⁺). By contrast, the H⁺-translocating ATPase (H⁺-ATPase) located on the same membrane is insensitive to Ca²⁺. Here we examine the mechanism and feasibility of regulation of the vacuolar H⁺-PPase by cytosolic free Ca²⁺ under the conditions thought to prevail in vivo with respect to Mg²⁺, inorganic pyrophosphate (PPi), and pH. The minimal reaction scheme that satisfactorily describes the effects of elevated Ca²⁺ or CaPPi on the enzyme is one that invokes equilibrium binding of substrate (Mg₂PPi) at one site, inhibitory binding of Mg₂PPi to a lower-affinity second site, binding of activator (Mg²⁺) at a third site, and direct binding of Ca²⁺ or CaPPi to a fourth site. Changes in enzyme activity in response to selective manipulation of either Ca²⁺ or CaPPi are explicable only if Ca²⁺, rather than CaPPi, is the inhibitory ligand. This conclusion is supported by the finding that CaPPi fails to mimic substrate in protection of the enzyme from inhibition by N-ethylmaleimide. Furthermore, the reaction scheme quantitatively and independently predicts the observed noncompetitive effects of free Ca²⁺ on the substrate concentration dependence of H⁺-PPase activity. The results are discussed in relation to the previous proposal that CaPPi is the principal inhibitory ligand of the vacuolar H⁺-PPase (M. Maeshima [1991] Eur J Biochem 196: 11-17) and the possibility that in vivo modulation of cytosolic free Ca²⁺ might constitute a specific mechanism for selective regulation of this enzyme, and consequently for stabilization of PPi levels in the cytoplasm of plant cells.