Journal:

Article Title: Acetone Formation in the Vibrio Family: a New Pathway for Bacterial Leucine Catabolism

doi:

Figure Lengend Snippet: Vibrio family isolates used in this study and their ability to produce acetone in the presence of l -leucine

Article Snippet: All strains were maintained frozen at −70°C in 7% (vol/vol) dimethyl sulfoxide. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain(s) d Origin or reference a Acetone production b in: Verified by c MT glucose medium MT leucine medium V. harveyi 7OM23 41 5 (2.8) 991 (1.7) HPLC, GC-MS V. harveyi 7OM2, 7OM15, 9OM1, 9OM6, 9OM10, 9OM12 41 5–17 307–2,130 V. pelagius 7OM5 41 27 (1.0) 795 (0.8) V. pelagius 9OM11 41 5 (2.6) 425 (0.9) HPLC V. splendidus D2 This study 3 (4.4), 17 (4.4) 168 (1.7), 1,060 (2.4) HPLC V. splendidus P5 This study 3–55 (3.9–6.6; n = 4) 453–4,320 (1.4–2.8; n = 5) HPLC, GC-MS V. splendidus 1OM9 41 20 (3.0) 577 (1.5) HPLC V. splendidus 1OM13 41 74 (0.2) 888 (0.2) V. splendidus 33125 ATCC 3 (4.5) 145 (1.5) HPLC V. tubiashii 9OM7 41 7 (2.7) 427 (1.7) HPLC V. tubiashii 9SM11 41 3 (4.2) 709 (1.6) L. anguillarum 19264 ATCC 22 (3.2) 400 (2.1) L. pelagia 33784 ATCC 39 (6.2) 3,130 (1.8) HPLC, GC-MS P. angustum 25915 ATCC 388 (4.9) 3,300 (1.4) HPLC, GC-MS P. angustum 33977 ATCC 208 (3.9) 2,410 (1.9) S. alga BrY 8 5 (4.2) 6 (7.1) S. amazonensis SB2B 8 (3.0) 23 (2.8) S. frigidimarina ACAM 591, ACAM 600 6 10–12 15–19 S. putrefaciens MR-1, MR-4 28 1 1–2 S. woodyi MS32 34 1 (6.3) 150 (4.0) HPLC Open in a separate window a ATCC, American Type Culture Collection (Manassas, Va.). b Media and the method for acetone determination are described in Materials and Methods.

Techniques:

Journal: The Journal of Immunology Author Choice

Article Title: Differential Roles of CXCL2 and CXCL3 and Their Receptors in Regulating Normal and Asthmatic Airway Smooth Muscle Cell Migration

doi: 10.4049/jimmunol.1203421

Figure Lengend Snippet: CXCR1 and CXCR2 differentially regulate CXCL2- and CXCL3-induced ASMC migration. To inhibit the functionality of CXCR1 and CXCR2, serum-starved ASMCs were either 1) incubated with Abs against CXCR1 or CXCR2 for 1 h prior to migration experiment, or 2) transfected with siRNA to knock down CXCR1 or CXCR2 expression. ASMC migration toward (A) CXCL2 was completely abolished following treatment with CXCR2, but not CXCR1, neutralizing Ab. Similar results were shown after using (C) CXCR2, but not CXCR1, siRNA knockdown. In contrast, (B) CXCL3-induced ASMC migration was inhibited after neutralization of either CXCR1 or CXCR2. The results were similar with (D) CXCR1 and CXCR2 siRNA knockdown. Studies included three to four independent experiments using three to four subjects. *p < 0.05, **p < 0.01, ***p < 0.001 compared with nonstimulated controls; $$p < 0.01, $$$p < 0.001 compared with isotype or scrambled siRNA controls. Results are expressed as means ± SEM.

Article Snippet: Cells then were incubated with 1;100 of mouse allophycocyanin-conjugated anti-CXCR1 (R&D Systems), mouse monoclonal anti-CXCR2 (Invitrogen), or nonimmune isotype control (IgG) for 1 h at 4°C.

Techniques: Migration, Incubation, Transfection, Expressing, Neutralization

Journal: The Journal of Immunology Author Choice

Article Title: Differential Roles of CXCL2 and CXCL3 and Their Receptors in Regulating Normal and Asthmatic Airway Smooth Muscle Cell Migration

doi: 10.4049/jimmunol.1203421

Figure Lengend Snippet: CXCR1 and CXCR2 play different roles in CXCL2- and CXCL3-induced p38 and ERK1/2 MAPK activation. ASMCs were transfected with siRNA to knock down CXCR1 or CXCR2 expression, after which they were serum-starved and stimulated with 4 ng/ml (0.5 nM) CXCL2 or CXCL3 for 0, 5, 15, 30, 45, and 60 min. Whole-cell lysates were then collected and used in Western blot for detection of p38 and ERK1/2 MAPK pathways. CXCR2, but not CXCR1, siRNA knockdown efficiently inhibited CXCL2- induced (A) p38 and (B) ERK1/2 MAPK pathway activation. In contrast, CXCL3-induced (C) p38 and (D) ERK1/2 MAPK pathway activation significantly decreased after siRNA knockdown of either CXCR1 or CXCR2. Studies included four independent experiments using four subjects. *p < 0.05, **p < 0.01, ***p < 0.001 compared with nonstimulated controls; $p < 0.05, $$p < 0.01, $$$p < 0.001 compared with scrambled siRNA. Results are expressed as means + SEM.

Article Snippet: Cells then were incubated with 1;100 of mouse allophycocyanin-conjugated anti-CXCR1 (R&D Systems), mouse monoclonal anti-CXCR2 (Invitrogen), or nonimmune isotype control (IgG) for 1 h at 4°C.

Techniques: Activation Assay, Transfection, Expressing, Western Blot

Journal: The Journal of Immunology Author Choice

Article Title: Differential Roles of CXCL2 and CXCL3 and Their Receptors in Regulating Normal and Asthmatic Airway Smooth Muscle Cell Migration

doi: 10.4049/jimmunol.1203421

Figure Lengend Snippet: CXCL2- and CXCL3-induced asthmatic ASMC migration is mainly CXCR1- and PI3K pathway–dependent. To assess the involvement of receptors and signaling pathways in asthmatic ASMC migration, serum-starved ASMCs were incubated with Abs against CXCR1 or CXCR2 for 1 h prior to migration experiments. ASMC migration toward (A) 4 ng/ml CXCL2 was completely abolished following treatment with CXCR2, but not CXCR1, neutralizing Ab. In contrast, neutralizing CXCR1, but not CXCR2, was effective in reducing ASMC migration toward higher concentrations (B) 8 ng/ml CXCL2. ASMC migration toward either (C) 4 ng/ml or (D) 0.125 ng/ml CXCL3 was significantly inhibited following blockade of CXCR1, but not CXCR2, in asthmatic ASMCs. ASMC migration induced by 4 and 8 ng/ml of CXCL2 [(E) and (F), respectively] and 0.125 and 4 ng/ml of CXCL3 [(G) and (H), respectively] was significantly reduced after inhibition of the PI3K pathway using PI103 (PI), but not after inhibition of p38 or ERK1/2 MAPK pathways (BIRB0796 [BIRB] and PD184352 [PD], respectively). Studies included three independent experiments using three subjects. *p < 0.05, **p < 0.01, ***p < 0.001 compared with nonstimulated controls; $p < 0.05, $$p < 0.01, $$$p < 0.001 compared with isotype or DMSO control. Results are expressed as means ± SEM.

Article Snippet: Cells then were incubated with 1;100 of mouse allophycocyanin-conjugated anti-CXCR1 (R&D Systems), mouse monoclonal anti-CXCR2 (Invitrogen), or nonimmune isotype control (IgG) for 1 h at 4°C.

Techniques: Migration, Incubation, Inhibition

Journal: The Journal of Immunology Author Choice

Article Title: Differential Roles of CXCL2 and CXCL3 and Their Receptors in Regulating Normal and Asthmatic Airway Smooth Muscle Cell Migration

doi: 10.4049/jimmunol.1203421

Figure Lengend Snippet: CXCL2- and CXCL3-induced PI3K signaling pathway activation in asthmatic ASMCs is mainly regulated by CXCR1. Whole-cell lysates from asthmatic ASMCs stimulated with CXCL2 (4 and 8 ng/ml) or CXCL3 (0.125 and 4 ng/ml) for 0, 5, 15, 30, 45, and 60 min were used in Western blot. Significant activation of the PI3K pathway was detected 5 min after stimulation with CXCL2 at (A) 4 ng/ml and (B) 8 ng/ml or with CXCL3 at (C) 0.125 ng/ml and (D) 4 ng/ml. To evaluate the involvement of receptors in the PI3K activation in asthmatic ASMCs, serum-starved ASMCs were incubated with Abs against CXCR1 or CXCR2 for 1 h prior to collecting cell lysates after stimulation with CXCL2 and CXCL3. Blockade of CXCR2 completely abolished PI3K activation after stimulation with (E) 4 ng/ml CXCL2, whereas blockade of CXCR1 was effective in inhibiting PI3K activation following treatment (F) with 8 ng/ml CXCL2. Similar to the latter effect, neutralizing CXCR1, but not CXCR2, was effective in reducing PI3K activation after stimulating asthmatic ASMCs with (G) 0.125 ng/ml and (H) 4 ng/ml CXCL3. Studies included three independent experiments using three subjects. *p < 0.05, **p < 0.01 compared with nonstimulated controls; $p < 0.05, $$p < 0.01 compared with isotype control. Results are expressed as means ± SEM.

Article Snippet: Cells then were incubated with 1;100 of mouse allophycocyanin-conjugated anti-CXCR1 (R&D Systems), mouse monoclonal anti-CXCR2 (Invitrogen), or nonimmune isotype control (IgG) for 1 h at 4°C.

Techniques: Activation Assay, Western Blot, Incubation