Pyrrolo[2,3-d]pyrimidines containing an extended 5-substituent as potent and selective inhibitors of lck II

Pyrrolo[2,3-d]pyrimidines containing a 5-(4-phenoxyphenyl) substituent are novel, potent and selective inhibitors of lck in vitro. Exploration of C-6 position of the pyrrolo[2,3-d]pyrimidine and the terminal phenyl group structure-activity relationship (SAR) is detailed. Compound 1 is orally active in animal models.     

A conserved lipid-binding loop in the kindlin FERM F1 domain is required for kindlin-mediated αIIbβ3 integrin coactivation

The activation of heterodimeric integrin adhesion receptors from low to high affinity states occurs in response to intracellular signals that act on the short cytoplasmic tails of integrin β subunits. Binding of the talin FERM (four-point-one, ezrin, radixin, moesin) domain to the integrin β tail provides one key activation signal, but recent data indicate that the kindlin family of FERM domain proteins also play a central role. Kindlins directly bind integrin β subunit cytoplasmic domains at a site distinct from the talin-binding site, and target to focal adhesions in adherent cells. However, the mechanisms by which kindlins impact integrin activation remain largely unknown. A notable feature of kindlins is their similarity to the integrin-binding and activating talin FERM domain. Drawing on this similarity, here we report the identification of an unstructured insert in the kindlin F1 FERM domain, and provide evidence that a highly conserved polylysine motif in this loop supports binding to negatively charged phospholipid head groups. We further show that the F1 loop and its membrane-binding motif are required for kindlin-1 targeting to focal adhesions, and for the cooperation between kindlin-1 and -2 and the talin head in αIIbβ3 integrin activation, but not for kindlin binding to integrin β tails. These studies highlight the structural and functional similarities between kindlins and the talin head and indicate that as for talin, FERM domain interactions with acidic membrane phospholipids as well β-integrin tails contribute to the ability of kindlins to activate integrins.     

Purification and SAXS Analysis of the Integrin Linked Kinase,PINCH, Parvin (IPP) Heterotrimeric Complex

The heterotrimeric protein complex containing the integrin linked kinase (ILK), parvin, and PINCH proteins, termed the IPP complex, is an essential component of focal adhesions, where it interacts with many proteins to mediate signaling from integrin adhesion receptors. Here we conduct a biochemical and structural analysis of the minimal IPP complex, comprising full-length human ILK, the LIM1 domain of PINCH1, and the CH2 domain of α-parvin. We provide a detailed purification protocol for IPP and show that the purified IPP complex is stable and monodisperse in solution. Using small-angle X-ray scattering (SAXS), we also conduct the first structural characterization of IPP, which reveals an elongated shape with dimensions 120×60×40 Å. Flexibility analysis using the ensemble optimization method (EOM) is consistent with an IPP complex structure with limited flexibility, raising the possibility that inter-domain interactions exist. However, our studies suggest that the inter-domain linker in ILK is accessible and we detect no inter-domain contacts by gel filtration analysis. This study provides a structural foundation to understand the conformational restraints that govern the IPP complex.     

ASB2α, an E3 Ubiquitin Ligase Specificity Subunit,Regulates Cell Spreading and Triggers Proteasomal Degradation of Filamins by Targeting the Filamin Calponin Homology 1 Domain

Filamins are actin-binding and cross-linking proteins that organize the actin cytoskeleton and anchor transmembrane proteins to the cytoskeleton and scaffold signaling pathways. During hematopoietic cell differentiation, transient expression of ASB2α, the specificity subunit of an E3-ubiquitin ligase complex, triggers acute proteasomal degradation of filamins. This led to the proposal that ASB2α regulates hematopoietic cell differentiation by modulating cell adhesion, spreading, and actin remodeling through targeted degradation of filamins. Here, we show that the calponin homology domain 1 (CH1), within the filamin A (FLNa) actin-binding domain, is the minimal fragment sufficient for ASB2α-mediated degradation. Combining an in-depth flow cytometry analysis with mutagenesis of lysine residues within CH1, we find that arginine substitution at each of a cluster of three lysines (Lys-42, Lys-43, and Lys-135) renders FLNa resistant to ASB2α-mediated degradation without altering ASB2α binding. These lysines lie within previously predicted actin-binding sites, and the ASB2α-resistant filamin mutant is defective in targeting to F-actin-rich structures in cells. However, by swapping CH1 with that of α-actinin1, which is resistant to ASB2α-mediated degradation, we generated an ASB2α-resistant chimeric FLNa with normal subcellular localization. Notably, this chimera fully rescues the impaired cell spreading induced by ASB2α expression. Our data therefore reveal ubiquitin acceptor sites in FLNa and establish that ASB2α-mediated effects on cell spreading are due to loss of filamins.     

Relationships between lake ecology and morphological characters in Icelandic Arctic charr,Salvelinus alpinus

The common occurrence of parallel phenotypic patterns suggests that a strong relationship exists between ecological dynamics and micro‐evolution. Comparative studies from a large number of populations under varying sets of ecological drivers could contribute to a better understanding of this relationship. We used data on morphology of arctic charr (Salvelinus alpinus) and ecological factors from 35 Icelandic lakes to test the hypothesis that morphological patterns among monomorphic charr populations from different lakes are related to interlake variation in ecological characteristics. There is extensive phenotypic diversity among populations of Icelandic charr, and populations are easily distinguished based on overall body morphology. The results obtained in the present study showed that the morphological diversity of charr was related to large‐scale diversity in lake ecology. Variation in charr morphology was related to water origin (e.g. spring fed versus run‐off), bedrock age, and fish community structure. The present study shows how various ecological factors can shape the biological diversity that we observe. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 103 , 761–771.     

Evaluation of a Typhoid/Paratyphoid Diagnostic Assay (TPTest) Detecting Anti-Salmonella IgA in Secretions of Peripheral Blood Lymphocytes in Patients in Dhaka,Bangladesh

Background

Rapid and reliable diagnostic assays for enteric (typhoid and paratyphoid) fever are urgently needed. We report the characterization of novel approach utilizing lymphocyte secretions, for diagnosing patients with enteric fever by the TPTest procedure.

Methodology

TPTest detects Salmonella-specific IgA responses in lymphocyte culture supernatant. We utilized TPTest in patients with suspected enteric fever, patients with other illnesses, and healthy controls. We also evaluated simplified modifications of TPTest for adaptation in laboratories with limited facilities and equipment.

Principal Findings

TPTest was positive in 39 (27 typhoid and 12 paratyphoid A) patients confirmed by blood culture and was negative in 74 healthy individuals. Among 32 individuals with other illnesses, 29 were negative by TPTest. Of 204 individuals with suspected enteric fever who were negative by blood culture, 44 were positive by TPTest and the patients were clinically indistinguishable from patients with confirmed bacteremia, except they were more likely to be under 5 years of age. We evaluated simplifications in TPTest, including showing that lymphocytes could be recovered using lysis buffer or buffy coat method as opposed to centrifugation, that incubation of cells at 37°C did not require supplemental CO₂, and that results were available for majority of samples within 24 hours. Positive results by TPTest are transient and revert to negative during convalescence, supporting use of the test in endemic areas. The results can also be read using immunodot blot approach as opposed to ELISA. Since no true gold standard currently exists, we used a number of definitions of true positives and negatives. TPTest had sensitivity of 100% compared to blood culture, and specificity that ranged from 78–97% (73–100, 95% CI), depending on definition of true negative.

Conclusion

The TPTest is useful for identification of patients with enteric fever in an endemic area, and additional development of simplified TPTest is warranted.     

Nine new polymorphic microsatellite loci for the amplification of archived otolith DNA of Atlantic cod,Gadus morhua L.

Nine out of 22 microsatellite primers tested were successfully amplified on three samples of cod Gadus morhua L. (two contemporary and one archived otolith samples). All loci were polymorphic (5–23 alleles/locus). The average observed heterozygosity across loci and samples was 0.625, ranging from 0.294 to 0.895 at each locus. All loci were under Hardy–Weinberg equilibrium, except PGmo56 that showed significant excess of heterozygotes in all studied samples. The isolated loci were suitable for degraded DNA and therefore useful for conducting a long‐term temporal study with DNA obtained from archived otoliths of cod.