Synthesis and structural determination of stereoisomers of muscarinic ligands of the (3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicycloalkane type

Methods for the synthesis of each of the four stereoisomers of 6-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[3.2.1]octane ( 10, 11, 12 , and 13 ) and 3-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[2.2.1]heptane ( 18, 19, 20 , and 21 ), and the two stereoisomers of 3-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[2.2.2]octane ( 27 and 28 ) were developed. The relative configuration of the compounds was determined on the basis of previously described ¹H NOE experiments, and the absolute configuration of 6-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[3.2.1]octanes ( 10, 11, 12 , and 13 ) and 3-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[2.2.2]octane ( 27 and 28 ) was determined by single crystal X-ray crystallography. Optical purity was determined by capillary electrophoresis (CE) using chiral selectors as trimethyl-β-cyclodextrin and heparin dissolved in the running buffer. All the 3-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicycles had low nanomolar affinity for muscarinic receptors as determined by displacement of radiolabelled oxotremorine-M (³H-Oxo-M) and pirenzepine (³H-Pz) from cortical rat brain homogenates. The binding assay discriminated between diastereomers, but only a minor degree of enantioselectivity was observed in the binding assays. Chirality 9:739–749, 1997. © 1997 Wiley-Liss, Inc.     

Identification of an in vivo CD4+ T cell-mediated response to polymorphic membrane proteins of Chlamydia pneumoniae during experimental infection

Chlamydia pneumoniae is an obligate intracellular bacterium that causes upper and lower respiratory tract infection in humans. C. pneumoniae harbors the polymorphic membrane protein (Pmp) family with 21 different proteins with a molecular mass around 100 kDa. The Pmps are species-specific, abundant and, together with major outer membrane protein and outer membrane protein 2, the dominant proteins in the C. pneumoniae outer membrane complex. Nevertheless, it is unknown whether Pmps are recognized by the cell-mediated immune response. To address this issue, C57BL/6J mice were infected intranasally with C. pneumoniae and the immune response to primary infection was investigated. We demonstrate, as expected, that the primary response is of the Th1 type by IgG2a- and IgG1-specific sELISA (Medac) on serum. In vivo-primed spleen lymphocytes were found to be reactive to Pmp8, Pmp20 and Pmp21 in an interferon-gamma ELISpot assay. The responses were shown to be mediated by CD4(+) T cells. To our knowledge, this is the first identification of antigens recognized by CD4(+) T cells during murine C. pneumoniae infection.     

Comparison of the differential context-dependence of DNA deamination by APOBEC enzymes: correlation with mutation spectra in vivo

To investigate the extent to which in vivo mutation spectra might reflect the intrinsic specificities of active mutators, genetic and biochemical assays were used to analyse the DNA target specificities of cytidine deaminases of the APOBEC family. The results reveal the critical importance of nucleotides immediately 5' of the targeted C for the specificity of all three enzymes studied (AID, APOBEC1 and APOBEC3G). At position -1, APOBEC1 showed a marked preference for dT, AID for dA/dG and APOBEC3G a strong preference for dC. Furthermore, AID and APOBEC3G showed distinct dependence on the nucleotide at position -2 with dA/dT being favoured by AID and dC by APOBEC3G. Most if not all activity of the recombinant deaminases on free dC could be attributed to low-level contamination by host enzymes. The target preference of APOBEC3G supports it being a major but possibly not sole contributor to HIV hypermutation without making it a dominant contribution to general HIV sequence variation. The specificity of AID as deduced from the genetic assay (which relies on inactivation of sacB of Bacillus subtilis) agrees well with that deduced by Pham et al. using an in vitro assay although we postulate that major intrinsic mutational hotspots in immunoglobulin V genes in vivo might reflect favoured sites of AID action being generated by proximal DNA targets located on opposite DNA strands. The target specificity of AID also accords with the spectrum of mutations observed in B lymphoma-associated oncogenes. The possibility of deaminase involvement in non-lymphoid human tumours is hinted at by tissue-specific differences in the spectra of dC transitions in tumour-suppressor genes. Thus, the patterns of hypermutation in antibodies and retroviruses owe much to the intrinsic sequence preferences of the AID/APOBEC family of DNA deaminases: analogous biases might also contribute to the spectra of cancer-associated mutation.     

Thermal dissociation and unfolding of insulin

The thermal stability of human insulin was studied by differential scanning microcalorimetry and near-UV circular dichroism as a function of zinc/protein ratio, to elucidate the dissociation and unfolding processes of insulin in different association states. Zinc-free insulin, which is primarily dimeric at room temperature, unfolded at approximately 70 degrees C. The two monomeric insulin mutants Asp(B28) and Asp(B9),Glu(B27) unfolded at higher temperatures, but with enthalpies of unfolding that were approximately 30% smaller. Small amounts of zinc caused a biphasic thermal denaturation pattern of insulin. The biphasic denaturation is caused by a redistribution of zinc ions during the heating process and results in two distinct transitions with T(m)'s of approximately 70 and approximately 87 degrees C corresponding to monomer/dimer and hexamer, respectively. At high zinc concentrations (>or=5 Zn(2+) ions/hexamer), only the hexamer transition is observed. The results of this study show that the thermal stability of insulin is closely linked to the association state and that the zinc hexamer remains stable at much higher temperatures than the monomer. This is in contrast to studies with chemical denaturants where it has been shown that monomer unfolding takes place at much higher denaturant concentrations than the dissociation of higher oligomers [Ahmad, A., et al. (2004) J. Biol. Chem. 279, 14999-15013].     

Development of real-time PCR for detection of <Emphasis Type="Italic">Mycoplasma hominis</Emphasis>

Background  

Mycoplasma hominis is associated with pelvic inflammatory disease, bacterial vaginosis, post partum fever, sepsis and infections of the central nervous system often leading to serious conditions. Association with development of female infertility has also been suggested, but different publications present different results. We developed a sensitive and fast diagnostic real-time PCR to test clinical samples from women undergoing laparoscopic examination before fertility treatment. To develop a test for the detection and quantification of M. hominis we selected a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (gap), as a target.     

Catalase and NO CATALASE ACTIVITY1 Promote Autophagy-Dependent Cell Death in Arabidopsis

Programmed cell death often depends on generation of reactive oxygen species, which can be detoxified by antioxidative enzymes, including catalases. We previously isolated catalase-deficient mutants (cat2) in a screen for resistance to hydroxyurea-induced cell death. Here, we identify an Arabidopsis thaliana hydroxyurea-resistant autophagy mutant, atg2, which also shows reduced sensitivity to cell death triggered by the bacterial effector avrRpm1. To test if catalase deficiency likewise affected both hydroxyurea and avrRpm1 sensitivity, we selected mutants with extremely low catalase activities and showed that they carried mutations in a gene that we named NO CATALASE ACTIVITY1 (NCA1). nca1 mutants showed severely reduced activities of all three catalase isoforms in Arabidopsis, and loss of NCA1 function led to strong suppression of RPM1-triggered cell death. Basal and starvation-induced autophagy appeared normal in the nca1 and cat2 mutants. By contrast, autophagic degradation induced by avrRpm1 challenge was compromised, indicating that catalase acted upstream of immunity-triggered autophagy. The direct interaction of catalase with reactive oxygen species could allow catalase to act as a molecular link between reactive oxygen species and the promotion of autophagy-dependent cell death.     

Influence of the Chlamydia pneumoniae AR39 bacteriophage ϕCPAR39 on chlamydial inclusion morphology

The human respiratory tract pathogen Chlamydia pneumoniae AR39 is naturally infected by the bacteriophage ?CPAR39. The phage genome encodes six ORFs, [ORF8, ORF4, ORF5, and viral protein (VP) 1, VP2 and VP3]. To study the growth of the phage, antibodies were generated to VP1 and used to investigate the ?CPAR39 infection. Using immunofluorescence laser confocal microscopy and two-dimensional gel electrophoresis, we investigated the ?CPAR39 infection of C. pneumoniae AR39. It was observed that ?CPAR39 infection differentially suppressed the C. pneumoniae protein synthesis as the polymorphic membrane protein 10 and the secreted chlamydial protein Cpn0796 was hardly expressed while the secreted chlamydial protein Cpaf was expressed, but not secreted. The inclusion membrane protein, IncA, was demonstrated to surround the phage-infected abnormal reticulate bodies (RB) as well as being located in the inclusion membrane. As IncA is secreted by the type 3 secretion (T3S) system, it is likely that the T3S is disrupted in the phage-infected chlamydiae such that it accumulates around the infected RB.