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1.
FIGURE 6.

FIGURE 6. From: Exposure to a Cutinase-like Serine Esterase Triggers Rapid Lysis of Multiple Mycobacterial Species.

Sensitivity of various mycobacterial species to TDMH. A, lytic activity of 8 μm TDMH against 107 cfu/ml of four other mycobacterial species and E. coli. The percentage survival reflects the ratio of the number of viable bacilli before and after the 48-h exposure. B, effect on the viability of 107 cfu/ml suspension of M. marinum upon exposure to TDMH (S124A) mutant equivalent to 8 μm of the wild-type TDMH. The error bars in A and B represent standard errors of three independent experiments.

Yong Yang, et al. J Biol Chem. 2013 January 4;288(1):382-392.
2.
FIGURE 4.

FIGURE 4. From: Exposure to a Cutinase-like Serine Esterase Triggers Rapid Lysis of Multiple Mycobacterial Species.

A, two-dimensional radio-TLC of total apolar and polar lipids equivalent to 35,000 cpm extracted from cells treated with either buffer or TDMH for 2 h. Apolar lipids were resolved in solvent (Sol.) systems A, C, and D, and polar lipids were resolved in solvent system E as described previously (39). Positions of phthiocerol dimycocerosic acid (PDIM), PIMs, phosphatidylinositol (PI), phosphatidylethanolamine (PE), and diphosphatidylglycerol (DPG) are marked based on Ref. 29. Positions of TMM, TDM, and FM are marked based on the migratory pattern of purified reference standards. B, radio-TLC showing FM release when [14C]TDM, but not [14C]TMM, is incubated with 5 μg of TDMH.

Yong Yang, et al. J Biol Chem. 2013 January 4;288(1):382-392.
3.
FIGURE 1.

FIGURE 1. From: Exposure to a Cutinase-like Serine Esterase Triggers Rapid Lysis of Multiple Mycobacterial Species.

Loss of M. tuberculosis viability upon TDMH exposure. A, viability of 106 cfu/ml bacilli after exposure to various concentrations of TDMH for 24 h in PBST. In the control (0 μm) experiment, an equivalent volume of storage buffer was used. B, exposure of various densities of M. tuberculosis (mc27000) to 8 μm TDMH over 72-h period. C, viability of bacilli after TDMH exposure in growth media for mycobacteria (Sauton's media and 7H9 with or without OADC) or for macrophage (DMEM with and without serum). PBST was used as a control. D, effects of equivalent concentration of constituent amino acid of Sauton's media (26 mm l-Asn) on the antimycobacterial activity of TDMH in PBST. E, lytic activity of TDMH against M. tuberculosis in detergent-free PBS after a 2-day exposure. F, Sauton's media agar plates containing a lawn of M. tuberculosis bacilli with either storage buffer or 200 μg of TDMH spotted in the center and incubated for 3 weeks at 37 °C. The error bars represent the standard errors of three independent experiments.

Yong Yang, et al. J Biol Chem. 2013 January 4;288(1):382-392.
4.
FIGURE 5.

FIGURE 5. From: Exposure to a Cutinase-like Serine Esterase Triggers Rapid Lysis of Multiple Mycobacterial Species.

TDM is an early target of TDMH. A, quantitative analysis of relative change in FM levels in M. tuberculosis/TDMH mixture with respect to the buffer-exposed control, as described in A. Error bar indicates standard error of the densitometric data obtained from three independent experiments. B, lytic activity of 0.8 μm TDMH against M. tuberculosis (H37Rv) in the presence of 100 μg of either purified TDM, apolar lipids (apL), polar lipids (pL), PIMs, or the mAGP complex. Percentage of viability in each condition was calculated using buffer-treated cells as reference (100%). C, total radio lipids (counts/min) extracted from 100 mg of 14C-labeled cells in either three sequential cycles of sonication in 2:1 CHCl3/MeOH (total) or petroleum ether (PE-extractable) followed by re-extraction through three cycles in 2:1 CHCl3/MeOH (residual). D, percentage of TDM in ether-extracted and residual lipids with respect to the total lipids. The values were determined by calculating the total number of TDM equivalent pixels in each of the samples obtained through densitometric analysis of radio-TLC. The error bars represent standard errors of three independent experiments.

Yong Yang, et al. J Biol Chem. 2013 January 4;288(1):382-392.
5.
FIGURE 7.

FIGURE 7. From: Exposure to a Cutinase-like Serine Esterase Triggers Rapid Lysis of Multiple Mycobacterial Species.

TDMH treatment of M. tuberculosis releases nucleic acids and facilitates sensitive detection by RT-PCR. A, NA present in the TDMH/cell mixture measured at various time intervals. Equal volume of enzyme storage buffer was added in the cell suspension as a negative control. The error bars represent standard errors of three independent experiments. B, detection of M. tuberculosis NA corresponding to 16 S rRNA in molecular beacon-based RT-PCR performed on samples with ∼10 bacilli either treated with TDMH or storage buffer control. Controls with genomic DNA of M. tuberculosis as template (template ctrl) or without any template (NTC) contain equivalent amounts of TDMH. C, summary table of threshold cycles where the signal was detected in 45 independent RT-PCRs from buffer and TDMH-treated bacilli; − denotes no detectable signal. 15 independent dilutions (D1–D15) from equivalent number of broth cultures were treated in triplicates (R1–R3) with buffer or TDMH. Statistical significance between buffer and TDMH-treated samples was calculated by random effect logistic regression.

Yong Yang, et al. J Biol Chem. 2013 January 4;288(1):382-392.
6.
FIGURE 3.

FIGURE 3. From: Exposure to a Cutinase-like Serine Esterase Triggers Rapid Lysis of Multiple Mycobacterial Species.

TDMH exposure triggers early release of FM and concomitant depletion of TMM. A, radio-TLC of the lipids in cell/TDMH mixture (marked as T) incubated for specified hours (marked below each lane). Purified [14C]FM and [14C]TDM were loaded as markers. In the parallel control experiment, the cells were mixed with the storage buffer (marked as B) and processed similarly. B, radio-TLC of lipids from the cells exposed to TDMH or storage buffer incubated for shorter time (marked below each lane). Purified [14C]TDM and [14C]FM were loaded as markers. TLCs in A and B were resolved in CHCl3/MeOH/H2O (90:10:1, v/v/v). C, methyl esters of mycolic acids extracted from the insoluble mAGP fractions of M. tuberculosis treated with either buffer or TDMH for 2 and 6 h. D, radio-TLC of the lipids from TDMH and buffer-exposed cells developed in a polar solvent (CHCl3/MeOH/NH4OH, 80:20:2, v/v/v) to resolve TMM and TDM. Purified TMM and TDM were used as markers (M). E, relative change in the levels of TMM, TDM, and FM over a 48-h exposure in buffer and TDMH-exposed M. tuberculosis. The ratio between 0 (t0) and a 48-h exposure was obtained from densitometric analysis of TLCs. The error bars denote standard error in the values from three independent experiments. F, relative change in the levels of TMM and TDM in normal and heat-killed M. tuberculosis exposed to TDMH over the first 6 h of exposure. The value at each time point, obtained from densitometric analysis of TLCs, represents the percentage of the lipid relative to t0 (zero hour). The error bars denote standard error from two and three independent experiments for normal and heat-treated cells, respectively. G, TLC of apolar (developed in CHCl3/MeOH, 96:4, v/v) and polar glycolipids (developed in CHCl3/MeOH/H2O, 75:25:4, v/v/v) of heat-killed bacilli exposed to TDMH for 2 h. The major apolar and polar glycolipids, phenolic glycolipids (PGL), and PIMs are marked. For experiments in A–G, 109 cfu/ml M. tuberculosis were exposed to 8 μm TDMH.

Yong Yang, et al. J Biol Chem. 2013 January 4;288(1):382-392.
7.
FIGURE 2.

FIGURE 2. From: Exposure to a Cutinase-like Serine Esterase Triggers Rapid Lysis of Multiple Mycobacterial Species.

TDMH exposure causes lysis of M. tuberculosis. A, clearance of M. tuberculosis (H37Rv) suspension (108 cfu/ml) after 48 h in PBST containing 8 μm TDMH. In the control, an equal volume of storage buffer was added in the cell suspension. B, luciferin-luciferase-based measurement of ATP released in an M. tuberculosis (mc27000) suspension (108 cfu/ml) exposed to 8 μm TDMH (also see Fig. 1B). As a negative control, ATP was measured in culture exposed to storage buffer. C, high pressure freezing and freeze-substitution electron microscopy of 109 cfu/ml M. tuberculosis (mc27000) before (panels i–iii) and after a 12-h exposure to 8 μm TDMH (panels iv–viii). Images in panels i–iii are independent fields showing low magnification of intact cells (panel i) and high magnification of their cross-sections before TDMH exposure. A close-up view of the envelope layers is shown in the inset of panel iii. The cell inner membrane and outer layer are marked as IM and OL. Images in panels iv–viii are independent fields showing various stages of lysing bacilli in a TDMH-exposed population. Bacteria shown are as follows: panel iv, with a lesion in the outer layer (marked with arrowhead); panel v, with a distorted cytoplasm, presumably moments before the cytoplasm ejection due to the membrane lesion; panels vi and vii, in the act of releasing cytoplasmic contents, and panel viii, completely devoid of cytoplasm. Arrows point to the lesion sites. Scale bars are 100 nm for panels ii–viii, and 1 μm for panel i. D, exposure of log-phase (7-day old with OD 0.5) and stationary phase (35-day old with OD 3.0) cultures of H37Rv to 8 μm TDMH. E, loss of in vitro hydrolytic activity against TDM in the catalytic serine mutant of TDMH (S124A). F, viability of 107 cfu/ml suspension of M. tuberculosis (H37Rv) exposed to 8 μm TDMH (S124A). The error bars represent the standard errors of three independent experiments.

Yong Yang, et al. J Biol Chem. 2013 January 4;288(1):382-392.

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