Functional chararacterization of the enzymes TabB and TabD involved in tabtoxin biosynthesis by Pseudomonas syringae.

Pseudomonas syringae pv. tabaci ATCC 11528 produces tabtoxin, a ß-lactam-containing dipeptide phytotoxin. Tabtoxinine-ß-lactam (TßL), one of tabtoxin's constituent amino acids, structurally mimics lysine, and many of the proteins encoded by the tabtoxin biosynthetic gene cluster are homologs of lysine biosynthetic enzymes, suggesting that the tabtoxin and lysine biosynthetic routes parallel one another. We cloned and expressed TabB and TabD, predicted homologs of tetrahydrodipicolinate (THDPA)-N-acyltransferase and N-acyl-THDPA aminotransferase, respectively, to determine their activities in vitro. We confirmed that TabB succinylates THDPA and that TabD is a PLP-dependent aminotransferase that utilizes glutamate as an amine donor. Surprisingly, we also found that though TabD could utilize the TabB product N-succinyl-THDPA as a substrate, THDPA itself was also recognized. These observations reveal that TabB functionally duplicates DapD, the THDPA-N-succinyltransferase involved in lysine biosynthesis, and reinforce the close relationship between the metabolic logics underpinning the respective biosynthetic pathways.

Maximal exercise increases mucosal associated invariant T cell frequency and number in healthy young men.

PURPOSE: Mucosal associated invariant T (MAIT) cells have properties of the innate and acquired immune systems. While the response to vigorous exercise has been established for most leukocytes, MAIT cells have not been investigated. Therefore, the purpose was to determine if MAIT cell lymphocytosis occurs with acute maximal aerobic exercise and if this response is influenced by exercise duration, cardiovascular fitness, or body composition. METHODS: Twenty healthy young males with moderate fitness levels performed an extended graded exercise test until volitional fatigue. Peripheral blood mononuclear cells were isolated from venous blood obtained prior and immediately after exercise and were labeled to identify specific T cell populations using flow cytometry. RESULTS: The percentage of MAIT cells relative to total T cells significantly increased from 3.0 to 3.8% and absolute MAIT cell counts increased by 2.2-fold following maximal exercise. MAIT cell subpopulation proportions were unchanged with exercise. Within cytotoxic T lymphocytes (CTL), MAIT cells consisted of 8% of these cells and this remained constant after exercise. MAIT cell counts and changes with exercise were not affected by body composition, VO2peak, or exercise duration. CONCLUSIONS: Maximal exercise doubled MAIT cell numbers and showed preferential mobilization within total T cells but the response was not influenced by fitness levels, exercise duration, or body composition. These results suggest that acute exercise could be used to offset MAIT cell deficiencies observed with certain pathologies. MAIT cells also make up a substantial proportion of CTLs, which may have implications for cytotoxicity assays using these cells.

Exercise-induced modulation of monocytes in breast cancer survivors.

BACKGROUND: Exercise training reduces inflammation in breast cancer survivors; however, the mechanism is not fully understood. OBJECTIVES: The effects of acute and chronic exercise on monocyte toll-like receptor (TLR2 and 4) expression and intracellular cytokine production were examined in sedentary breast cancer survivors. METHODS: Eleven women with stage I, II, or III breast cancer within one year of treatment completion performed an acute, intermittent aerobic exercise trial. Blood samples were obtained before, immediately, and 1 h after a 45-min acute exercise trial that was performed before and after 16 weeks of combined aerobic and resistance. LPS-stimulated intracellular IL-1ß, TNF, and IL-6 production, and TLR2 and TLR4 expression were evaluated in CD14+CD16- and CD14+CD16+ monocytes using flow cytometry. RESULTS: Exercise training decreased IL-1ß+CD14+CD16- proportion (24.6%, p=0.016), IL-1ß+CD14+CD16- mean fluorescence intensity (MFI) (-9989, p=0.014), IL-1ß+CD14+CD16+ MFI (-11101, p=0.02), and IL-6+CD14+CD16- proportion (16.9%, P=0.04). TLR2 and TLR4 expression did not change following exercise training but decreased 1 h after acute exercise in CD14+CD16- (-63, p=0.002) and CD14+CD16+ (-18, p=0.006) monocytes, respectively. Immediately after the acute exercise, both monocyte subgroup cell concentration increased, with CD14+CD16+ concentrations being decreased at 1 h post without changes in intracellular cytokine production. CONCLUSIONS: Exercise training reduced monocyte intracellular pro-inflammatory cytokine production, especially IL-1ß, although these markers did not change acutely. While acute exercise downregulated the expression of TLR2 and TLR4 on monocytes, this was not sustained over the course of training. These results suggest that the anti-inflammatory effect of combined aerobic and resistance exercise training in breast cancer survivors may be, in part, due to reducing resting monocyte pro-inflammatory cytokine production.

Exercise Increases Mucosal-associated Invariant T Cell Cytokine Expression but Not Activation or Homing Markers.

Mucosal-associated invariant T (MAIT) cells have properties of both the innate and adaptive immune systems but are an understudied population within exercise immunology. These lymphocytes aggregate at the mucous membranes, but it is unknown if submaximal exercise alters their circulating numbers or function. PURPOSE: To determine the MAIT cell response to submaximal exercise on activation and homing marker expression and stimulated cytokine production. METHODS: Twenty healthy, young, recreationally active males cycled for 40 min at 86% of VT after an overnight fast. Peripheral blood mononuclear cells were isolated and labeled to identify specific MAIT cell populations using flow cytometry. Cytokine production after stimulation was also determined. RESULTS: Mucosal-associated invariant T cells were 2.9% of T cells and increased to 3.9% after exercise and with recovery whereas cell numbers significantly increased by 91.5% after exercise before returning to resting levels. Chemokine and activation marker absolute cell number significantly increased while expression levels remained constant but the high levels of CCR5 may help direct MAIT cells to sites of inflammation. After stimulation, TNFα expression significantly increased after exercise before returning to baseline with a similar trend for IFNγ. CONCLUSIONS: The MAIT cell numbers undergo a partial biphasic response after submaximal exercise and appear to be preferentially mobilized within T cells; however, the magnitude of the submaximal response was attenuated relative to maximal exercise. Stimulated MAIT cells increase TNFα expression, indicating greater responsiveness to pathogens after acute exercise.