Discovery and optimization of 3-(4-aryl/heteroarylsulfonyl)piperazin-1-yl)-6-(piperidin-1-yl)pyridazines as novel,CNS penetrant pan-muscarinic antagonists

This letter describes the synthesis and structure activity relationship (SAR) studies of structurally novel M₄ antagonists, based on a 3-(4-aryl/heteroarylsulfonyl)piperazin-1-yl)-6-(piperidin-1-yl)pyridazine core, identified from a high-throughput screening campaign. A multi-dimensional optimization effort enhanced potency at human M₄ (hM₄ IC₅₀s < 200 nM), with only moderate species differences noted, and with enantioselective inhibition. Moreover, CNS penetration proved attractive for this series (rat brain:plasma K_p = 2.1, K_p,uu = 1.1). Despite the absence of the prototypical mAChR antagonist basic or quaternary amine moiety, this series displayed pan-muscarinic antagonist activity across M_1-5 (with 9- to 16-fold functional selectivity at best). This series further expands the chemical diversity of mAChR antagonists.     

A perivascular origin for mesenchymal stem cells in multiple human organs

Mesenchymal stem cells (MSCs), the archetypal multipotent progenitor cells derived in cultures of developed organs, are of unknown identity and native distribution. We have prospectively identified perivascular cells, principally pericytes, in multiple human organs including skeletal muscle, pancreas, adipose tissue, and placenta, on CD146, NG2, and PDGF-Rbeta expression and absence of hematopoietic, endothelial, and myogenic cell markers. Perivascular cells purified from skeletal muscle or nonmuscle tissues were myogenic in culture and in vivo. Irrespective of their tissue origin, long-term cultured perivascular cells retained myogenicity; exhibited at the clonal level osteogenic, chondrogenic, and adipogenic potentials; expressed MSC markers; and migrated in a culture model of chemotaxis. Expression of MSC markers was also detected at the surface of native, noncultured perivascular cells. Thus, blood vessel walls harbor a reserve of progenitor cells that may be integral to the origin of the elusive MSCs and other related adult stem cells.     

Detection and resolution of Cryptosporidium species and species mixtures by genus-specific nested PCR-restriction fragment length polymorphism analysis, direct sequencing, and cloning

Molecular methods incorporating nested PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene of Cryptosporidium species were validated to assess performance based on limit of detection (LoD) and for detecting and resolving mixtures of species and genotypes within a single sample. The 95% LoD was determined for seven species (Cryptosporidium hominis, C. parvum, C. felis, C. meleagridis, C. ubiquitum, C. muris, and C. andersoni) and ranged from 7 to 11 plasmid template copies with overlapping 95% confidence limits. The LoD values for genomic DNA from oocysts on microscope slides were 7 and 10 template copies for C. andersoni and C. parvum, respectively. The repetitive nested PCR-RFLP slide protocol had an LoD of 4 oocysts per slide. When templates of two species were mixed in equal ratios in the nested PCR-RFLP reaction mixture, there was no amplification bias toward one species over another. At high ratios of template mixtures (>1:10), there was a reduction or loss of detection of the less abundant species by RFLP analysis, most likely due to heteroduplex formation in the later cycles of the PCR. Replicate nested PCR was successful at resolving many mixtures of Cryptosporidium at template concentrations near or below the LoD. The cloning of nested PCR products resulted in 17% of the cloned sequences being recombinants of the two original templates. Limiting-dilution nested PCR followed by the sequencing of PCR products resulted in no sequence anomalies, suggesting that this method is an effective and accurate way to study the species diversity of Cryptosporidium, particularly for environmental water samples, in which mixtures of parasites are common.     

Explicit Mentalizing Mechanisms and Their Adaptive Role in Memory Conformity

Memory conformity occurs when an individual endorses what other individuals remember about past events. Research on memory conformity is currently dominated by a ‘forensic’ perspective, which views the phenomenon as inherently undesirable. This is because conformity not only distorts the accuracy of an individual''s memory, but also produces false corroboration between individuals, effects that act to undermine criminal justice systems. There is growing awareness, however, that memory conformity may be interpreted more generally as an adaptive social behavior regulated by explicit mentalizing mechanisms. Here, we provide novel evidence in support of this emerging alternative theoretical perspective. We carried out a memory conformity experiment which revealed that explicit belief-simulation (i.e. using one''s own beliefs to model what other people believe) systematically biases conformity towards like-minded individuals, even when there is no objective evidence that they have a more accurate memory than dissimilar individuals. We suggest that this bias is functional, i.e. adaptive, to the extent that it fosters trust, and hence cooperation, between in-group versus out-group individuals. We conclude that memory conformity is, in more fundamental terms, a highly desirable product of explicit mentalizing mechanisms that promote adaptive forms of social learning and cooperation.     

Endopeptidase-24.11, a Cell-Surface Peptidace of Central Nervous System Neurons, Is Expressed by Schwann Cells in the Pig Peripheral Nervous System

Endopeptidase-24.11 is a 90-kDa surface glycoprotein with the ability to hydrolyze a variety of biologically active peptides. Interest in this enzyme is based on the consensus that it may play a role in the termination of peptide signals in the central nervous system. In the present study, we have investigated the distribution of endopeptidase-24.11 in two nerves of the peripheral nervous system of newborn pigs: the sciatic, composed of a mixture of myelinated and nonmyelinated axons, and cervical sympathetic trunk in which greater than 99% of the axons are nonmyelinated. The endopeptidase was monitored enzymatically, as well as by immunoblotting and immunocytochemistry using mono- and polyclonal anti-endopeptidase antibodies. Endopeptidase-24.11 was detected in both the sciatic nerve and the cervical sympathetic trunk. Membrane preparations from sciatic nerve hydrolyzed 125I-insulin B-chain, and more than 50% of the activity was inhibited by phosphoramidon with an IC50 concentration of 3.2 nM. Moreover, a 90-kDa polypeptide was detected by immunoblotting of sciatic nerve membranes. The type of cells expressing the endopeptidase was determined by immunohistochemistry. In teased nerve preparations, these cells were identified morphologically as myelin- and non-myelin-forming Schwann cells. Endopeptidase-24.11 was also expressed by cultured Schwann cells from sciatic nerve and cervical sympathetic trunk maintained for 3 h in vitro. The presence of endopeptidase-24.11 on the Schwann cell surface raises the possibility of a potential role for the enzyme in nerve development and/or regeneration.     

Threonine-insensitive Homoserine Dehydrogenase from Soybean: GENOMIC ORGANIZATION, KINETIC MECHANISM, AND IN VIVO ACTIVITY*

Aspartate kinase (AK) and homoserine dehydrogenase (HSD) function as key regulatory enzymes at branch points in the aspartate amino acid pathway and are feedback-inhibited by threonine. In plants the biochemical features of AK and bifunctional AK-HSD enzymes have been characterized, but the molecular properties of the monofunctional HSD remain unexamined. To investigate the role of HSD, we have cloned the cDNA and gene encoding the monofunctional HSD (GmHSD) from soybean. Using heterologously expressed and purified GmHSD, initial velocity and product inhibition studies support an ordered bi bi kinetic mechanism in which nicotinamide cofactor binds first and leaves last in the reaction sequence. Threonine inhibition of GmHSD occurs at concentrations (K_i = 160–240 mm) more than 1000-fold above physiological levels. This is in contrast to the two AK-HSD isoforms in soybean that are sensitive to threonine inhibition (K_i∼150 μm). In addition, GmHSD is not inhibited by other aspartate-derived amino acids. The ratio of threonine-resistant to threonine-sensitive HSD activity in soybean tissues varies and likely reflects different demands for amino acid biosynthesis. This is the first cloning and detailed biochemical characterization of a monofunctional feedback-insensitive HSD from any plant. Threonine-resistant HSD offers a useful biotechnology tool for manipulating the aspartate amino acid pathway to increase threonine and methionine production in plants for improved nutritional content.     

Carry‐over Effects of Size at Metamorphosis in Red‐eyed Treefrogs: Higher Survival but Slower Growth of Larger Metamorphs

Most animals have complex life histories, composed of a series of ecologically distinct stages, and the transitions between stages are often plastic. Anurans are models for research on complex life cycles. Many species exhibit plastic timing of and size at metamorphosis, due to both environmental constraints on larval growth and development and adaptive plastic responses to environmental variation. Models predicting optimal timing of metamorphosis balance cost/benefit ratios across stages, assuming that size affects growth and mortality rates in each stage. Much research has documented such effects in the larval period, but we lack an equal understanding of juvenile growth and mortality. Here, we examine how variation in size at metamorphosis in the Neotropical red‐eyed treefrog, Agalychnis callidryas, affects post‐metamorphic growth, foraging, and behavior in the lab as well as growth and survival in the field. Surprisingly, many individuals lost mass for weeks after metamorphosis. In the lab, larger metamorphs lost more mass following metamorphosis, ate similar amounts, had lower food conversion efficiencies, and grew more slowly after mass loss ceased than did smaller ones. In field cages larger metamorphs were more likely to survive than smaller ones; just one froglet died in the lab. Our data suggest that size‐specific differences in physiology and behavior influence these trends. Comparing across species and studies, large size at metamorphosis generally confers higher survival; size effects on growth rates vary substantially among species, in both magnitude and direction, but may be stronger in the tropics.     

An undecided coiled coil: the leucine zipper of Nek2 kinase exhibits atypical conformational exchange dynamics

Leucine zippers are oligomerization domains used in a wide range of proteins. Their structure is based on a highly conserved heptad repeat sequence in which two key positions are occupied by leucines. The leucine zipper of the cell cycle-regulated Nek2 kinase is important for its dimerization and activation. However, the sequence of this leucine zipper is most unusual in that leucines occupy only one of the two hydrophobic positions. The other position, depending on the register of the heptad repeat, is occupied by either acidic or basic residues. Using NMR spectroscopy, we show that this leucine zipper exists in two conformations of almost equal population that exchange with a rate of 17 s(-1). We propose that the two conformations correspond to the two possible registers of the heptad repeat. This hypothesis is supported by a cysteine mutant that locks the protein in one of the two conformations. NMR spectra of this mutant showed the predicted 2-fold reduction of peaks in the (15)N HSQC spectrum and the complete removal of cross peaks in exchange spectra. It is possible that interconversion of these two conformations may be triggered by external signals in a manner similar to that proposed recently for the microtubule binding domain of dynein and the HAMP domain. As a result, the leucine zipper of Nek2 kinase is the first example where the frameshift of coiled-coil heptad repeats has been directly observed experimentally.