A novel series of [3.2.1] azabicyclic biaryl ethers as α3β4 and α6/4β4 nicotinic receptor agonists

We report the synthesis of a series of [3.2.1]azabicyclic biaryl ethers as selective agonists of α3- and α6-containing nicotinic receptors. In particular, compound 17a from this series is a potent α3β4 and α6/4β4 receptor agonist in terms of both binding and functional activity. Compound 17a also shows potent in vivo activity in CNS-mediated animal models that are sensitive to antipsychotic drugs. Compound 17a may thus be a useful tool for studying the role of α3β4 and α6/4β4 nicotinic receptors in CNS pharmacology.     

Evolving paradigms for repair of tissues by adult stem/progenitor cells (MSCs)

• ? Paradigm I: the haematopoietic niche
• ? Paradigm II: engraftment/differentiation
  • ‐ Early observations on engraftment and differentiation
  • ‐ Technical challenges in testing paradigm II
  • ‐ The impetus to test the paradigm II in clinical trials
  • ‐ Tests of the paradigm II with local administrations
  • ‐ Tests of paradigm II with systemic infusion
• ? Paradigm III: transient ‘quasi‐niches’
  • ‐ Unusual features of MSCs in culture
  • ‐ Cross‐talk with injured tissues
  • ‐ Modulation of inflammation in paradigm III
  • ‐ Modulation of apoptosis in paradigm III
  • ‐ Modulation of immune reactions
  • ‐ Paradigm III and the similarities to paradigm I
• ? Conclusions/perspectives
  • ‐ Why is administration of MSCs beneficial?
  • ‐ Better assays for the potency of MSCs?
  • ‐ Are MSCs pericytes?
  • ‐ Therapies with recombinant proteins?
  • ‐ Additional questions in developing therapies with MSCs

In this review, we focus on the adult stem/progenitor cells that were initially isolated from bone marrow and first referred to as colony forming units‐fibroblastic, then as marrow stromal cells and subsequently as either mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs). The current interest in MSCs and similar cells from other tissues is reflected in over 10,000 citations in PubMed at the time of this writing with 5 to 10 new publications per day. It is also reflected in over 100 registered clinical trials with MSCs or related cells ( http//www.clinicaltrials.gov ). As a guide to the vast literature, this review will attempt to summarize many of the publications in terms of three paradigms that have directed much of the work: an initial paradigm that the primary role of the cells was to form niches for haematopoietic stem cells (paradigm I); a second paradigm that the cells repaired tissues by engraftment and differentiation to replace injured cells (paradigm II); and the more recent paradigm that MSCs engage in cross‐talk with injured tissues and thereby generate microenvironments or ‘quasi‐niches’ that enhance the repair tissues (paradigm III).     

The Methanosarcina barkeri genome: comparative analysis with Methanosarcina acetivorans and Methanosarcina mazei reveals extensive rearrangement within methanosarcinal genomes

We report here a comparative analysis of the genome sequence of Methanosarcina barkeri with those of Methanosarcina acetivorans and Methanosarcina mazei. The genome of M. barkeri is distinguished by having an organization that is well conserved with respect to the other Methanosarcina spp. in the region proximal to the origin of replication, with interspecies gene similarities as high as 95%. However, it is disordered and marked by increased transposase frequency and decreased gene synteny and gene density in the distal semigenome. Of the 3,680 open reading frames (ORFs) in M. barkeri, 746 had homologs with better than 80% identity to both M. acetivorans and M. mazei, while 128 nonhypothetical ORFs were unique (nonorthologous) among these species, including a complete formate dehydrogenase operon, genes required for N-acetylmuramic acid synthesis, a 14-gene gas vesicle cluster, and a bacterial-like P450-specific ferredoxin reductase cluster not previously observed or characterized for this genus. A cryptic 36-kbp plasmid sequence that contains an orc1 gene flanked by a presumptive origin of replication consisting of 38 tandem repeats of a 143-nucleotide motif was detected in M. barkeri. Three-way comparison of these genomes reveals differing mechanisms for the accrual of changes. Elongation of the relatively large M. acetivorans genome is the result of uniformly distributed multiple gene scale insertions and duplications, while the M. barkeri genome is characterized by localized inversions associated with the loss of gene content. In contrast, the short M. mazei genome most closely approximates the putative ancestral organizational state of these species.     

Towards an ecological understanding of biological nitrogen fixation

Biogeochemistry - N limitation to primary production and other ecosystem processes is widespread. To understand the causes and distribution of N limitation, we must understand the controls of...     

Plant growth and development influenced by transgenic insertion of bacterial chitinolytic enzymes

The role of the chitinolytic enzymes in plants is not necessarilyrestricted to plant defense. Tomato plants transformed with an endochitinaseand a chitobiosidase gene from Streptomyces albidoflavus andgrowth under greenhouse conditions showed a significant reduction in plantheight, and reduced time to flowering compared with the control(non-transformed) plants. The levels of chitobiosidase and endochitinaseactivity in the transgenic tomato plants were positively correlated with earlyflowering, and negatively correlated with plant height. We have not determinedwhether these effects are exclusively due to the expression of the transgenesof endochitinase and chitobiosidase from S. albidoflavus orthe additive effect of these 2 enzymes combined with the endogenouschitinolytic enzymes produced by the plants. However, when control plants were trimmed,early flowering was observed compared with the controls that were not trimmed, whichindicates that wound induced proteins such as chitinolytic enzymes affect thetime of flowering. In addition, the expression of the endochitinase andchitobiosidase genes significantly increased the number of flowers and fruit onthe plants, resulting in an increase in yield of fruit. One of the primarygoals of crop breeding programs is to increase the productivity of plants. These twogenes were directly associated with plant productivity, and should be studied further.     

Ecological and Biogeochemical Interactions Constrain Planktonic Nitrogen Fixation in Estuaries

Many types of ecosystems have little or no N₂ fixation even when nitrogen (N) is strongly limiting to primary production. Estuaries generally fit this pattern. In contrast to lakes, where blooms of N₂-fixing cyanobacteria are often sufficient to alleviate N deficits relative to phosphorus (P) availability, planktonic N₂ fixation is unimportant in most N-limited estuaries. Heterocystic cyanobacteria capable of N₂ fixation are seldom observed in estuaries where the salinity exceeds 8–10 ppt, and blooms have never been reported in such estuaries in North America. However, we provided conditions in estuarine mesocosms (salinity over 27 ppt) that allowed heterocystic cyanobacteria to grow and fix N₂ when zooplankton populations were kept low. Grazing by macrozooplankton at population densities encountered in estuaries strongly suppressed cyanobacterial populations and N₂ fixation. The cyanobacteria grew more slowly than observed in fresh waters, at least in part due to the inhibitory effect of sulfate (SO₄ ²⁻), and this slow rate of growth increased their vulnerability to grazing. We conclude that interactions between physiological (bottom–up) factors that slow the growth rate of cyanobacteria and ecological (top–down) factors such as grazing are likely to be important regulators excluding planktonic N₂ fixation from most Temperate Zone estuaries. Received 26 April 2002; Accepted 12 July 2002.     

H<Superscript>N</Superscript>, N,C<Superscript>α</Superscript> and C<Superscript>β</Superscript> assignments of the two periplasmic domains of <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> DsbD

DsbD is a disulfide bond reductase present in the inner membrane of many Gamma-Proteobacteria. In the human pathogen Neisseria meningitidis, DsbD is required for viability and represents a potential target for the development of antibiotics. Here we report the chemical shift assignments (H^N, N, C^α and C^β) for the reduced and oxidized forms of the two periplasmic domains of N. meningitidis DsbD, n-NmDsbD and c-NmDsbD. The backbone amide resonances in all four forms were completely assigned, and the secondary structures for the core regions of the proteins were calculated using ¹³C^αβ shifts. The reduced and oxidized forms of each domain have similar secondary shifts suggesting they retain the same fold. We anticipate that these data will provide an important basis for studying the interaction between n-NmDsbD and c-NmDsbD, which is required for electron transfer across the bacterial cytoplasmic membrane.     

Melt analysis of mismatch amplification mutation assays (Melt-MAMA): a functional study of a cost-effective SNP genotyping assay in bacterial models

Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and biologically informative markers extensively used across broad scientific disciplines. Newly identified SNP markers are publicly available at an ever-increasing rate due to advancements in sequencing technologies. Efficient, cost-effective SNP genotyping methods to screen sample populations are in great demand in well-equipped laboratories, but also in developing world situations. Dual Probe TaqMan assays are robust but can be cost-prohibitive and require specialized equipment. The Mismatch Amplification Mutation Assay, coupled with melt analysis (Melt-MAMA), is flexible, efficient and cost-effective. However, Melt-MAMA traditionally suffers from high rates of assay design failures and knowledge gaps on assay robustness and sensitivity. In this study, we identified strategies that improved the success of Melt-MAMA. We examined the performance of 185 Melt-MAMAs across eight different pathogens using various optimization parameters. We evaluated the effects of genome size and %GC content on assay development. When used collectively, specific strategies markedly improved the rate of successful assays at the first design attempt from ~50% to ~80%. We observed that Melt-MAMA accurately genotypes across a broad DNA range (~100 ng to ~0.1 pg). Genomic size and %GC content influence the rate of successful assay design in an independent manner. Finally, we demonstrated the versatility of these assays by the creation of a duplex Melt-MAMA real-time PCR (two SNPs) and conversion to a size-based genotyping system, which uses agarose gel electrophoresis. Melt-MAMA is comparable to Dual Probe TaqMan assays in terms of design success rate and accuracy. Although sensitivity is less robust than Dual Probe TaqMan assays, Melt-MAMA is superior in terms of cost-effectiveness, speed of development and versatility. We detail the parameters most important for the successful application of Melt-MAMA, which should prove useful to the wider scientific community.     

West Nile virus: pending crisis for greater sage-grouse

Scientists have feared that emerging infectious diseases could complicate efforts to conserve rare and endangered species, but quantifying impacts has proven difficult until now. We report unexpected impacts of West Nile virus (WNv) on radio‐marked greater sage‐grouse (Centrocercus urophasianus), a species that has declined 45–80% and is endangered in Canada and under current consideration for federal listing in the US. We show that WNv reduced late‐summer survival an average of 25% in four radio‐marked populations in the western US and Canada. Serum from 112 sage‐grouse collected after the outbreak show that none had antibodies, suggesting that they lack resistance. The spread of WNv represents a significant new stressor on sage‐grouse and probably other at‐risk species. While managing habitat might lessen its impact on sage‐grouse populations, WNv has left wildlife and public health officials scrambling to address surface water and vector control issues in western North America.