Numerical and chemical classification ofStreptosporangium and some related actinomycetes

One hundred and seventeen streptosporangia from soil were compared with marker strains of the familyStreptosporangiaceae for many phenotypic properties. The data were examined using the Jaccard, pattern and simple matching coefficients with clustering achieved using average, complete and single linkage algorithms. Particular confidence was placed in the product of the pattern, average linkage analysis given the sharp definition of aggregate groups and clusters and a combination of low test error and high cophenetic correlation values. The test strains were assigned to five aggregate groups that were equated with the generaStreptosporangium (group A),Microbispora (group B),Planobispora andPlanomonospora (Group C),Kutzneria (neéStreptosporangium viridogriseum (group D), andMicrotetraspora (group E). The streptosporangia, both isolates and marker strains, were assigned to 5 major, 7 minor and 18 single membered clusters. Representative streptosporangia examined for chemical markers were characterised by the presence ofmeso-diaminopimelic acid in whole-organism hydrolysates, complex mixtures of straight- and branched chain fatty acids, di- and tetrahydrogenated menaquinones as predominant isoprenologues, and complex polar lipid patterns containing diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides and uncharacterised components. The chemical and numerical data support the taxonomic integrity of the validly described species ofStreptosporangium and suggest that the genus is markedly underspeciated.     

S-Nitroso-N-acetylpenicillamine impregnated endotracheal tubes for prevention of ventilator-associated pneumonia

The chances of ventilator-associated pneumonia (VAP) increases 6–20 folds when an endotracheal tube (ETT) is placed in a patient. VAP is one of the most common hospital-acquired infections and comprises 86% of the nosocomial pneumonia cases. This study introduces the idea of nitric oxide-releasing ETTs (NORel-ETTs) fabricated by the incorporation of the nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP) into commercially available ETTs via solvent swelling. The impregnation of SNAP provides NO release over a 7-day period without altering the mechanical properties of the ETT. The NORel-ETTs successfully reduced the bacterial infection from a commonly found pathogen in VAP, Pseudomonas aeruginosa, by 92.72 ± 0.97% when compared with the control ETTs. Overall, this study presents the incorporation of the active release of a bactericidal agent in ETTs as an efficient strategy to prevent the risk of VAP.     

The N-terminal 1000 residues of apolipoprotein B associate with microsomal triglyceride transfer protein to create a lipid transfer pocket required for lipoprotein assembly

Apolipoprotein (apo) B, the major protein component of the atherogenic low-density lipoprotein (LDL), has a pentapartite structure, NH2-betaalpha1-beta1-alpha2-beta2-alpha3-COOH, the beta domains containing multiple amphipathic beta strands and the alpha domains containing multiple amphipathic alpha helixes. We recently reported that the first 1000 residues of human apoB-100 have sequence and amphipathic motif homologies to the lipid-pocket of lamprey lipovitellin (LV) [Segrest, J. P., Jones, M. K., and Dashti, N. (1999) J. Lipid Res. 40, 1401-1416]. The lipid-pocket of LV is a small triangular space lined by three antiparallel amphipathic beta sheets, betaA, betaB, and betaD. The betaA and betaB sheets are joined together by an antiparallel alpha helical bundle, alpha domain. We proposed [Segrest, J. P., Jones, M. K., and Dashti, N. (1999) J. Lipid Res. 40, 1401-1416] that formation of a LV-like lipid-pocket is necessary for lipid-transfer to apoB-containing lipoprotein particles and that this pocket is formed by association of the region of the betaalpha1 domain homologous to the betaA and betaB sheets of LV with a betaD-like amphipathic beta sheet from microsomal triglyceride transfer protein (MTP). To test this hypothesis, we generated four truncated cDNA constructs terminating at or near the juncture of the betaalpha1 and beta1 domains: Residues 1-800 (apoB:800), 1-931 (apoB:931), 1-1000 (apoB:1000), and 1-1200 (apoB:1200). Characterization of particles secreted by stable transformants of the McA-RH7777 cell line demonstrated that (i) ApoB:800, missing the betaB domain, was secreted as a lipid-poor aggregate. (ii) ApoB:931, containing most, but not all, of the betaB domain, was secreted as lipid-poor particles unassociated with MTP. (iii) ApoB:1000, containing the entire betaB domain, was secreted as a relatively lipid-rich particle associated hydrophobically with MTP. (iv) ApoB:1200, containing the betaalpha1 domain plus 200 residues of the beta1 domain, was secreted predominantly as a lipid-poor particle but also as a minor relatively lipid-rich, MTP-associated particle. We thus have captured an intermediate in apoB-containing particle assembly, a lipid transfer competent pocket formed by association of the complete betaalpha1 domain of apoB with MTP.     

Variability in Tuberculosis Granuloma T Cell Responses Exists,but a Balance of Pro- and Anti-inflammatory Cytokines Is Associated with Sterilization

Lung granulomas are the pathologic hallmark of tuberculosis (TB). T cells are a major cellular component of TB lung granulomas and are known to play an important role in containment of Mycobacterium tuberculosis (Mtb) infection. We used cynomolgus macaques, a non-human primate model that recapitulates human TB with clinically active disease, latent infection or early infection, to understand functional characteristics and dynamics of T cells in individual granulomas. We sought to correlate T cell cytokine response and bacterial burden of each granuloma, as well as granuloma and systemic responses in individual animals. Our results support that each granuloma within an individual host is independent with respect to total cell numbers, proportion of T cells, pattern of cytokine response, and bacterial burden. The spectrum of these components overlaps greatly amongst animals with different clinical status, indicating that a diversity of granulomas exists within an individual host. On average only about 8% of T cells from granulomas respond with cytokine production after stimulation with Mtb specific antigens, and few “multi-functional” T cells were observed. However, granulomas were found to be “multi-functional” with respect to the combinations of functional T cells that were identified among lesions from individual animals. Although the responses generally overlapped, sterile granulomas had modestly higher frequencies of T cells making IL-17, TNF and any of T-1 (IFN-γ, IL-2, or TNF) and/or T-17 (IL-17) cytokines than non-sterile granulomas. An inverse correlation was observed between bacterial burden with TNF and T-1/T-17 responses in individual granulomas, and a combinatorial analysis of pair-wise cytokine responses indicated that granulomas with T cells producing both pro- and anti-inflammatory cytokines (e.g. IL-10 and IL-17) were associated with clearance of Mtb. Preliminary evaluation suggests that systemic responses in the blood do not accurately reflect local T cell responses within granulomas.     

2,4-Diaminopyrimidine MK2 inhibitors. Part I: Observation of an unexpected inhibitor binding mode

MK2 is a Ser/Thr kinase of significant interest as an anti-inflammatory drug discovery target. Here we describe the development of in vitro tools for the identification and characterization of MK2 inhibitors, including validation of inhibitor interactions with the crystallography construct and determination of the unique binding mode of 2,4-diaminopyrimidine inhibitors in the MK2 active site. Use of these tools in the optimization of a potent and selective inhibitor lead series is described in the accompanying Letter.     

Methotrexate binding causes structural and functional changes in lung cystatin

Regulation of cysteine proteinases and their inhibitors is of utmost importance in diseases like lung cancer, chronic inflammatory conditions such as asthma, emphysema, and idiopathic pulmonary fibrosis. Protease-antiprotease imbalance accelerates disease progression. In the present study, the effect of antineoplastic and antirheumatic drug methotrexate (MTX) on lung cystatin (a cysteine protease inhibitor) was studied to explore drug induced changes in functional and structural integrity of the protein. The basic binding interaction was studied by UV-absorption, FT-IR and fluorescence spectroscopy. The quenching of protein fluorescence confirmed the binding of MTX with goat lung cystatin (GLC-I). Stern-Volmer analysis of MTX-GLC-I system at different temperatures indicates the presence of static component in the quenching mechanism. The thermodynamic parameters ΔH? and ΔS? were -3.8 kJ/mol and 94.97 J?mol?1?K?1, respectively, indicating that both hydrogen bonds and hydrophobic interactions played a major role in the binding of MTX to GLC-I. Methotrexate (7 μM) caused complete inactivation of lung cystatin after 6 hours. The results of FT-IR spectroscopy reflect perturbation of the goat lung cystatin on interaction with MTX. Methotrexate induced loss of function change in the inhibitor could provide a rationale for the off target tissue injury caused by the drug and for the design of agents against such an injury.     

Robust anti-tumor immunity and memory in Rag-1-deficient mice following adoptive transfer of cytokine-primed splenocytes and tumor CD80 expression

Successful immunotherapy of solid tumors has proven difficult to achieve. The aim of the current study was to further investigate the effects of peripheral CD80-mediated co-stimulation on the efficacy of polyclonal anti-tumor effector CTL in an adoptive transfer model. Splenocytes obtained from wild-type mice immunized with CD80-transduced EL4 tumor cells were expanded in vitro in the presence of either IL-12 or IL-15 and irradiated CD80-transduced EL4 tumor cells. Polyclonal CD8 T cells were the major subset in the effector population. Primed effector cells were adoptively transferred into immuno-deficient Rag-1-deficient mice which were then challenged with syngeneic vector-control or CD80-transduced EL4 tumor cells. Expression of CD80 enhanced the elimination of EL4 tumors and mouse survival. Both IL-12 and IL-15 cultured cells had enhanced cytotoxicity. Importantly, anti-tumor memory was maintained without tumor evasion following re-challenge with either CD80-transduced and vector-control EL4 cells. We also show, using antibody-mediated depletion, that endogenous NK cells present in Rag-1-deficent mice exert anti-EL4 tumor activity that is enhanced by CD80 expression. Collectively these data show that peripheral co-stimulation by tumor expression of CD80 results in enhanced anti-tumor efficacy of NK and polyclonal effector T cells, and suggest that TCR repertoire diversity helps protect against tumor escape and provides memory with resultant robust immunity to subsequent tumor challenge irrespective of CD80 status.     

A distal methyl substituent attenuates mitochondrial protein synthesis inhibition in oxazolidinone antibacterials

Oxazolidinone analogs bearing substituted piperidine or azetidine C-rings are described. Analogs with a methyl group at the 3-position of the azetidine ring or the 4-position of the piperidine ring exhibited reduced mitochondrial protein synthesis inhibition while retaining good antibacterial potency.