Orally active achiral N-hydroxyformamide inhibitors of ADAM-TS4 (aggrecanase-1) and ADAM-TS5 (aggrecanase-2) for the treatment of osteoarthritis

A new achiral class of N-hydroxyformamide inhibitor of both ADAM-TS4 and ADAM-TS5, 2 has been discovered through modification of the complex P1 group present in historical inhibitors 1. This structural change improved the DMPK properties and greatly simplified the synthesis whilst maintaining excellent cross-MMP selectivity profiles. Investigation of structure-activity and structure-property relationships in the P1 group resulted in both ADAM-TS4 selective and mixed ADAM-TS4/5 inhibitors. This led to the identification of a pre-clinical candidate with excellent bioavailability across three species and predicting once daily dosing kinetics.     

Polyandry and multiple paternities in the threatened Agassiz’s desert tortoise, <Emphasis Type="Italic">Gopherus agassizii</Emphasis>

We used data from 17 to 20 microsatellite markers to investigate the incidence of multiple paternities in wild Agassiz’s desert tortoises, Gopherus agassizii. Neonates were sampled from clutches of eggs laid by wild mothers in nesting enclosures at Edwards Air Force Base and at the Marine Corps Air Ground Combat Center, California. We genotyped 28 clutches from 26 females sampling an average of six neonates per clutch. The number of paternal alleles was used to determine the minimum number of sires for each clutch. Based on conservative criteria requiring evidence from at least two loci to determine multiple paternity, a minimum of 64% of females were polyandrous, while a minimum of 57% of clutches were sired by multiple males. This formed one of the highest incidences of multiple paternities recorded to date in any species of tortoise. The high number of microsatellite loci involved in the analyses allowed detection of multiple paternities in clutches where this may have been missed if fewer loci were used. Our results highlighted the potential pitfalls of quantitatively comparing paternity studies based on differing sampling strategies. Finally, we summarized the conservation implications of the high rate of multiple paternities in this threatened species.     

IL-6 induced STAT3 signalling is associated with the proliferation of human muscle satellite cells following acute muscle damage

Background

Although the satellite cell (SC) is a key regulator of muscle growth during development and muscle adaptation following exercise, the regulation of human muscle SC function remains largely unexplored. STAT3 signalling mediated via interleukin-6 (IL-6) has recently come to the forefront as a potential regulator of SC proliferation. The early response of the SC population in human muscle to muscle-lengthening contractions (MLC) as mediated by STAT3 has not been studied.

Methodology/Principal Findings

Twelve male subjects (21±2 y; 83±12 kg) performed 300 maximal MLC of the quadriceps femoris at 180°•s⁻¹ over a 55° range of motion with muscle samples (vastus lateralis) and blood samples (antecubital vein) taken prior to exercise (PRE), 1 hour (T1), 3 hours (T3) and 24 hours (T24) post-exercise. Cytoplasmic and nuclear fractions of muscle biopsies were purified and analyzed for total and phosphorylated STAT3 (p-STAT3) by western blot. p-STAT3 was detected in cytoplasmic fractions across the time course peaking at T24 (p<0.01 vs. PRE). Nuclear total and p-STAT3 were not detected at appreciable levels. However, immunohistochemical analysis revealed a progressive increase in the proportion of SCs expressing p-STAT3 with ∼60% of all SCs positive for p-STAT3 at T24 (p<0.001 vs. PRE). Additionally, cMyc, a STAT3 downstream gene, was significantly up-regulated in SCs at T24 versus PRE (p<0.05). Whole muscle mRNA analysis revealed induction of the STAT3 target genes IL-6, SOCS3, cMyc (peaking at T3, p<0.05), IL-6Rα and GP130 (peaking at T24, p<0.05). In addition, Myf5 mRNA was up-regulated at T24 (p<0.05) with no appreciable change in MRF4 mRNA.

Conclusions/Significant Findings

We demonstrate that IL-6 induction of STAT3 signaling occurred exclusively in the nuclei of SCs in response to MLC. An increase in the number of cMyc+ SCs indicated that human SCs were induced to proliferate under the control of STAT3 signaling.     

Underwater application of quantitative PCR on an ocean mooring

The Environmental Sample Processor (ESP) is a device that allows for the underwater, autonomous application of DNA and protein probe array technologies as a means to remotely identify and quantify, in situ, marine microorganisms and substances they produce. Here, we added functionality to the ESP through the development and incorporation of a module capable of solid-phase nucleic acid extraction and quantitative PCR (qPCR). Samples collected by the instrument were homogenized in a chaotropic buffer compatible with direct detection of ribosomal RNA (rRNA) and nucleic acid purification. From a single sample, both an rRNA community profile and select gene abundances were ascertained. To illustrate this functionality, we focused on bacterioplankton commonly found along the central coast of California and that are known to vary in accordance with different oceanic conditions. DNA probe arrays targeting rRNA revealed the presence of 16S rRNA indicative of marine crenarchaea, SAR11 and marine cyanobacteria; in parallel, qPCR was used to detect 16S rRNA genes from the former two groups and the large subunit RuBisCo gene (rbcL) from Synecchococcus. The PCR-enabled ESP was deployed on a coastal mooring in Monterey Bay for 28 days during the spring-summer upwelling season. The distributions of the targeted bacterioplankon groups were as expected, with the exception of an increase in abundance of marine crenarchaea in anomalous nitrate-rich, low-salinity waters. The unexpected co-occurrence demonstrated the utility of the ESP in detecting novel events relative to previously described distributions of particular bacterioplankton groups. The ESP can easily be configured to detect and enumerate genes and gene products from a wide range of organisms. This study demonstrated for the first time that gene abundances could be assessed autonomously, underwater in near real-time and referenced against prevailing chemical, physical and bulk biological conditions.     

Roles of phosphorylation-dependent and -independent mechanisms in the regulation of M1 muscarinic acetylcholine receptors by G protein-coupled receptor kinase 2 in hippocampal neurons

When co-expressed with the inositol 1,4,5-trisphosphate biosensor eGFP-PH(PLC delta), G protein-coupled receptor kinase 2 (GRK2) can suppress M1 muscarinic acetylcholine (mACh) receptor-mediated phospholipase C signaling in hippocampal neurons through a phosphorylation-independent mechanism, most likely involving the direct binding of the RGS homology domain of GRK2 to G alpha(q/11). To define the importance of this mechanism in comparison with classical, phosphorylation-dependent receptor regulation by GRKs, we have examined M1 mACh receptor signaling in hippocampal neurons following depletion of GRK2 and also in the presence of non-G alpha(q/11)-binding GRK2 mutants. Depletion of neuronal GRK2 using an antisense strategy almost completely inhibited M1 mACh receptor desensitization without enhancing acute agonist-stimulated phospholipase C activity. By stimulating neurons with a submaximal agonist concentration before (R1) and after (R2) a period of exposure to a maximal agonist concentration, an index (R2/R1) of agonist-induced desensitization of signaling could be obtained. Co-transfection of neurons with either a non-G alpha(q/11)-binding (D110A) GRK2 mutant or the catalytically inactive (D110A,K220R)GRK2 did not suppress acute M1 mACh receptor-stimulated inositol 1,4,5-trisphosphate production. However, using the desensitization (R2/R1) protocol, it could be shown that expression of (D110A)GRK2 enhanced, whereas (D110A,K220R)GRK2 inhibited, agonist-induced M1 mACh receptor desensitization. In Chinese hamster ovary cells, the loss of G alpha(q/11) binding did not affect the ability of the (D110A)GRK2 mutant to phosphorylate M1 mACh receptors, whereas expression of (D110A,K220R)GRK2 had no effect on receptor phosphorylation. These data indicate that in hippocampal neurons endogenous GRK2 is a key regulator of M1 mACh receptor signaling and that the regulatory process involves both phosphorylation-dependent and -independent mechanisms.     

Enhanced tolerance to autoimmune uveitis in CD200-deficient mice correlates with a pronounced Th2 switch in response to antigen challenge

A single exposure to inhaled Ag 10 days before immunization leads to long term, Ag-specific tolerance. Respiratory tract myeloid APCs are implicated, but how regulation is invoked, and how tolerance is sustained are unclear. This study examines the in vivo function of the myeloid regulatory molecule CD200 in the process of tolerance induction. Despite earlier onset of experimental autoimmune uveitis in sham-tolerized, CD200-deficient mice, disease incidence and subsequent severity were actually reduced compared with those in wild-type mice. Protection was more effective and long term, lasting at least 28 days. Halting disease progression and tolerance in CD200(-/-) mice correlated with a marked increase in Th2-associated cytokine production by Ag-challenged splenocytes. Reduced overall disease and enhanced tolerance in the CD200-deficient mice in this model system were unexpected and may be related to altered populations of MHC class II(low) APC in the respiratory tract compared with wild-type mice together with associated activation of STAT6 in draining lymph nodes of tolerized mice. These data indicate that in the absence of default inhibitory CD200 receptor signaling, alternative, powerful regulatory mechanisms are invoked. This may represent either permissive dominant Th2 activation or an altered hierarchy of negative signaling by other myeloid cell-expressed regulatory molecules.     

Transmembrane TNF is sufficient to initiate cell migration and granuloma formation and provide acute, but not long-term, control of Mycobacterium tuberculosis infection

TNF is critical for immunity against Mycobacterium tuberculosis infection; however, the relative contributions of the soluble and transmembrane forms of TNF in this immunity are unknown. Using memTNF mice, which express only the transmembrane form of TNF, we have addressed this question. Wild-type (WT), TNF-/-, and transmembrane TNF (memTNF) mice were infected with M. tuberculosis by aerosol. TNF-/- mice developed overwhelming infection with extensive pulmonary necrosis and died after only 33 days. memTNF mice, like WT mice, contained bacterial growth for over 16 wk, developed an Ag-specific T cell response, and initially displayed compact granulomas, comprised of both lymphocytes and macrophages. Expression of mRNA for the chemokines CXCL10, CCL3, CCL5, and CCL7 was comparable in both WT and memTNF mice. As the infection progressed, however, the pulmonary lesions in memTNF mice became larger and more diffuse, with increased neutrophil accumulation and necrosis. This was accompanied by increased influx of activated memory T cells into the lungs of memTNF mice. Eventually, these mice succumbed to infection with a mean time to death of 170 days. The expression of memTNF on T cells is functionally important because the transfer of T cells from memTNF, but not TNF-/- mice, into either RAG-/- or TNF-/- mice conferred the same survival advantage on the M. tuberculosis-infected recipient mice, as the transfer of WT T cells. Therefore, memTNF, in the absence of soluble TNF, is sufficient to control acute, but not chronic, M. tuberculosis infection, in part through its expression on T cells.     

Cysteine-scanning mutagenesis and thiol modification of the Rickettsia prowazekii ATP/ADP translocase: evidence that transmembrane regions I and II, but not III, are structural components of the aqueous translocation channel

The contribution of transmembrane regions I, II, and III of the Rickettsia prowazekii ATP/ADP translocase to the structure of the putative water-filled ATP translocation channel was evaluated from the accessibility of hydrophilic, thiol-reactive, methanethiosulfonate reagents to a library of 68 independent cysteine-substitution mutants heterologously expressed in Escherichia coli. The MTS reagents used were MTSES (negatively charged) and MTSET and MTSEA (both positively charged). Mutants F036C, Y042C, and R046C (TM I), K066C and P072C (TM II), and F101C, F105C, F108C, Y113C, and P114C (TM III) had no assayable transport activity, indicating that cysteine substitution at these positions may not be tolerated. All three MTS reagents inhibit the transport of ATP in mutants of TM I (L039C, S043C, S047C, I048C) and TM II (S061C, S063C, T067C, I069C, V070C, A074C). Further, these residues appear to cluster along a single face of the transmembrane domain. Preexposure of MTS-reactive mutants S047C (TM I) and T067C (TM II) to high levels of ATP resulted in protection from MTS-mediated inhibition. This indicated that both TM I and TM II make major contributions to the structure of an aqueous ATP translocation pathway. Finally, on the basis of the lack of accessibility of charged MTS reagents to the thiol groups in mutants of TM III, it appears that TM III is not exposed to the ATP translocation channel. Cysteine substitution of residues constituting a highly conserved "phenylalanine face" in TM III resulted in ablation of ATP transport activity. Further, substituting these phenylalanine residues for either isoleucine or tyrosine also resulted in much lower transport activity, indicating that some property of phenylalanine at these positions that is not shared by cysteine, isoleucine, or tyrosine is critical to translocase activity.