Tuberculosis is associated with expansion of a motile,permissive and immunomodulatory CD16+ monocyte population via the IL-10/STAT3 axis

The human CD14⁺ monocyte compartment is composed by two subsets based on CD16 expression. We previously reported that this compartment is perturbed in tuberculosis (TB) patients, as reflected by the expansion of CD16⁺ monocytes along with disease severity. Whether this unbalance is beneficial or detrimental to host defense remains to be elucidated. Here in the context of active TB, we demonstrate that human monocytes are predisposed to differentiate towards an anti-inflammatory (M2-like) macrophage activation program characterized by the CD16⁺CD163⁺MerTK⁺pSTAT3⁺ phenotype and functional properties such as enhanced protease-dependent motility, pathogen permissivity and immunomodulation. This process is dependent on STAT3 activation, and loss-of-function experiments point towards a detrimental role in host defense against TB. Importantly, we provide a critical correlation between the abundance of the CD16⁺CD163⁺MerTK⁺pSTAT3⁺ cells and the progression of the disease either at the local level in a non-human primate tuberculous granuloma context, or at the systemic level through the detection of the soluble form of CD163 in human sera. Collectively, this study argues for the pathogenic role of the CD16⁺CD163⁺MerTK⁺pSTAT3⁺ monocyte-to-macrophage differentiation program and its potential as a target for TB therapy, and promotes the detection of circulating CD163 as a potential biomarker for disease progression and monitoring of treatment efficacy.     

PHYTOALEXIN DEFICIENT 4 affects reactive oxygen species metabolism,cell wall and wood properties in hybrid aspen (Populus tremula L. × tremuloides)

The PHYTOALEXIN DEFICIENT 4 (PAD4) gene in Arabidopsis thaliana (AtPAD4) is involved in the regulation of plant – pathogen interactions. The role of PAD4 in woody plants is not known; therefore, we characterized its function in hybrid aspen and its role in reactive oxygen species (ROS)‐dependent signalling and wood development. Three independent transgenic lines with different suppression levels of poplar PAD expression were generated. All these lines displayed deregulated ROS metabolism, which was manifested by an increased H₂O₂ level in the leaves and shoots, and higher activities of manganese superoxide dismutase (MnSOD) and catalase (CAT) in the leaves in comparison to the wild‐type plants. However, no changes in non‐photochemical quenching (NPQ) between the transgenic lines and wild type were observed in the leaves. Moreover, changes in the ROS metabolism in the pad4 transgenic lines positively correlated with wood formation. A higher rate of cell division, decreased tracheid average size and numbers, and increased cell wall thickness were observed. The results presented here suggest that the Populus tremula × tremuloides PAD gene might be involved in the regulation of cellular ROS homeostasis and in the cell division – cell death balance that is associated with wood development.     

Isolation and restriction endonuclease analysis of mycobacterial DNA

A method for the isolation of DNA from mycobacteria propagated in vitro is described that utilizes organic solvents to extract lipoidal components from the outer membrane, and digestion with a protease (nagarse) and lysozyme to penetrate the cell wall. The mycobacterial cells were lysed by the addition of detergent and the DNA was purified by digestion with pronase, sequential phenol and chloroform extractions, and digestion with RNAase A. The isolated DNA, which was obtained in good yields, was of a relatively high Mr and could be readily digested by restriction endonucleases. By this method, the genomes of Mycobacterium avium, M. intracellulare, M. lepraemurium, 'M. lufu', M. marinum, M. phlei, M. scrofulaceum, M. smegmatis and M. tuberculosis were isolated and the restriction endonuclease digestion patterns analysed. Each species could be distinguished by the digestion patterns, indicating that this approach can be used for identifying mycobacterial species. This approach is also sufficiently sensitive to differentiate strains since we were able to distinguish two independently isolated strains of M. tuberculosis, H37 and H4. In addition, no evidence was obtained for the presence of methylcytosine residues in the sequences 5'.CCGG.3',5'.CCCGGG.3',5'.CC(A/T) GG.3' or for methyladenine at 5'.GATC.3' in the DNA of the nine mycobacterial species examined using pairs of restriction enzymes that recognize and cleave at the same nucleotide sequence but differ in their sensitivity to 5-methylcytosine or 6N-methyladenine.     

Qualitative modeling of normal blood coagulation and its pathological states using stochastic activity networks

We have developed a method for representing biological pathways and simulating their behavior based on the use of stochastic activity networks (SANs). SANs, an extension of the original Petri net, have been used traditionally to model flow systems including data-communications networks and manufacturing processes. We apply the methodology to the blood coagulation cascade, a biological flow system, and present the representation method as well as results of simulation studies based on published experimental data. In addition to describing the dynamic model, we also present the results of its utilization to perform simulations of clinical states including hemophilia's A and B as well as sensitivity analysis of individual factors and their impact on thrombin production.     

Lipid metabolism during bacterial growth, sporulation, and germination: differential synthesis of individual branched- and normal-chain fatty acids during spore germination and outgrowth of Bacillus thuringiensis.

The biosynthesis of individual branched- and normal-chain fatty acids during Bacillus thuringiensis spore germination and outgrowth was studied by comparing pulsed and continuous labeling of these fatty acids with [U-14C]acetate. The relative specific activity of each fatty acid varies with time as the cell progresses through outgrowth. However, fatty acid synthesis does occur in two distinct phases. Upon germination, acetate is incorporated only into the iso-isomers i-C13, i-C14, and i-C16; no normal or anteiso synthesis occurs. Subsequent to T30, the full complement of branched- and normal-chain homologues is formed and there is a dramatic enhancement in the overall rate of fatty acid synthesis. Significantly, this rate increase coincides with a marked shift from the synthesis of short-chain to long-chain fatty acids. These findings illustrate a dichotomy in synthesis that may result from initial fatty acid formation by preexisting spore fatty acid biosynthetic enzymes in the absence of de novo protein synthesis. Elucidation of the timing and kinetics of individual fatty acid formation provides a biochemical profile of activities directly related to membrane differentiation and cellular development.     

The development of resistance to methotrexate in a mouse melanoma cell line

PG19T3 mouse melanoma cells were selected for resistance to methotrexate. Nine sub-lines that are resistant to concentrations of methotrexate ranging from 1.27×10^–7 M, to 1×10^–4M methotrexate were selected and characterised in terms of their content of dihydrofolate reductase activity and their chromosomes. The intracellular level of dihydrofolate reductase activity increases with increasing resistance such that at the highest level of resistance PG19T3:MTX_R ^{10–4 M} cells contain approximately 1,000 fold more enzyme activity than the parental PG19T3 cells. It is shown that the enhanced activity is due to an increase in the amount of the enzyme rather than any structural change to the enzyme in resistant cellls. Comparisons of pH activity profiles, profiles under different activating conditions and titrations with methotrexate suggest that the sensitive and resistant cells contain identical dihydrofolate reductases. Analysis of the chromosomes of resistant cells shows the presence of up to 5 large marker chromosomes which contain homogeneously staining regions after G-banding. These same regions stain intensely after C-banding and fluoresce brightly after staining with Hoechst 33258. The size of homogeneously staining regions increases throughout the process of selection. For one marker chromosome this increase may have been mediated via a ring chromosome.     

The oligomerization of a family of four genetically clustered human gastrointestinal mucins

Mucins are synthesized and secreted by many epithelia. They are complex glycoproteins that offer cytoprotection. In their functional configuration, mucins form oligomers by a biosynthetic process that is poorly understood. A family of four human gastrointestinal mucin genes (MUC2, MUC5AC, MUC5B, and MUC6) is clustered to chromosome 11p15.5. To study oligomerization of these related mucins, we performed metabolic labeling experiments with [35S]amino acids in LS174T cells, and isolated mucin precursors by specific immunoprecipitations that were analyzed on SDS-PAGE. Each of the precursors of MUC2, MUC5AC, MUC5B, and MUC6 formed a single species of disulfide-linked homo-oligomer within 1 h after pulse labeling. Based on apparent molecular masses, these oligomeric precursors were most likely dimers. Inhibition of vesicular RER-to-Golgi transport, with brefeldin A and CCCP, did not affect the dimerization of MUC2 precursors, localizing dimerization to the RER. O-Glycosylation of MUC2 followed dimerization. Inhibition of N- glycosylation by tunicamycin retarded, but did not inhibit, dimerization, indicating that N-glycans play a role in efficient dimerization of MUC2 precursors. Based on sequence homology, the ability of MUC2, MUC5AC, MUC5B and MUC6 to dimerize most likely resides in their C-terminal domains. Thus, the RER-localized dimerization of secretory mucins likely proceeds by similar mechanisms, which is an essential step in the formation of the human gastrointestinal mucus- gels.