Journal: BMC Microbiology

Article Title: Role of Mycobacterium tuberculosis pknD in the Pathogenesis of central nervous system tuberculosis

doi: 10.1186/1471-2180-12-7

Figure Lengend Snippet: Invasion and survival of M. tuberculosis pknD mutant in host-derived cells . A . BALB/c mice were infected with M. tuberculosis CDC1551 or pknD mutant, and sacrificed at days 1 and 49 after infection. The mutant for M. tuberculosis pknD was significantly attenuated (P = 0.004) in mouse brain, but not lung tissue, 49 days after infection. No defect was observed in the lungs at either time point. Bacterial burden is represented as log 10 CFU/organ for all animal experiments. B . Invasion of host-cell monolayers by wild-type CDC1551, wild-type intergenic transposon control, pknD transposon mutant (pknD:Tn), and pknD genetic complement (pknD:Comp) was examined and normalized to the wild-type control. Invasion assays were performed in brain microvascular endothelial cells (HBMEC), epithelial A549 cells, and umbilical vein endothelia (HUVEC). No difference in invasion was observed in A549 cells (P = 0.31) or HUVEC (P = 0.41). A significant reduction in invasive capacity, however, was observed in the CNS-derived HBMEC (P = 0.02). This defect was restored by genetic complementation with the native pknD/pstS2 operon. N.S. = not significantly different. C . Intracellular survival of each of the above M. tuberculosis strains was examined in HBMEC at days 1, 3, 5, and 7 after infection. The pknD:Tn mutant demonstrated an invasion and intracellular survival defect in HBMEC relative to wild-type over the course of the seven day infection. D . Survival was also examined by infection of activated J774 macrophages. No corresponding survival defect for the pknD:Tn mutant was observed in these cells during the seven day infection. A mutant for the gene Rv0442c , known to be attenuated in the macrophage model, is included as a control. All CFU counts are represented as mean ± standard deviation.

Article Snippet: Primary human brain microvascular endothelial cells and HUVEC were kind gifts from Dr. Kwang Sik Kim, Department of Pediatrics, Johns Hopkins University School of Medicine.

Techniques: Mutagenesis, Derivative Assay, Infection, Control, Standard Deviation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Lysophosphatidic acid improves corneal endothelial density in tissue culture by stimulating stromal secretion of interleukin‐1β

doi: 10.1111/jcmm.15307

Figure Lengend Snippet: Corneal stromal cell‐derived IL‐1β and CCL20 are involved in LPA‐induced proliferation of corneal endothelial cells (CECs). A, Mitomycin C‐pretreated human corneal stromal cells (HCSCs) were maintained in TC medium with or without 20 μmol/L LPA for 2 d before CM collection. ELISA showed that the concentrations of secreted IL‐1β and CCL20 were both significantly greater in the LPA‐treated group. B, The dose‐dependence of IL‐1β‐ and CCL20‐induced B4G12 cell proliferation was determined by cell counts. The number of cells was significantly higher after treatment of recombinant IL‐1β alone for 2 d. In contrast, no difference was observed in the CCL20‐treated group. C, The involvement of IL‐1β in LPA‐induced proliferation of CECs was investigated via neutralization assay of the CM. Immortalized HCECs (B4G12) co‐cultured with mitomycin C‐pretreated HCSCs in TC medium for 5 d were greater in cell number in the LPA‐treated group (20 μmol/L). Subsequent IL‐1β neutralization with 2 ng/mL IL‐1β‐specific antibody (IL‐1β Ab, clone AS10) significantly attenuated the LPA‐induced proliferation in co‐cultures. D, The proliferation‐promoting effect of IL‐1β on RCECs was also examined 2 d later. The fraction of RCECs in the co‐culture increased significantly with increasing concentrations of IL‐1β (20, 200 or 1000 pg/mL). E, The morphology and ECD of the 200 pg/mL IL‐1β‐treated tissue‐cultured rabbit corneal endothelium was examined via immunostaining for ZO‐1 on Day 2. The corneal ECD was significantly higher in the LPA‐treated cells than that in controls and had the normal hexagonal phenotype typical of RCECs. (n = 3; * P < .5, ** P < .05)

Article Snippet: Immortalized HCECs (B4G12) were purchased from Creative Bioarray (NY) and cultured in B4G12 medium (HESFM supplemented with 2% FBS and 10 ng/mL b‐FGF.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Neutralization, Cell Culture, Co-Culture Assay, Immunostaining

Journal: PPAR Research

Article Title: Stimulation of Alpha 1 -Adrenergic Receptor Ameliorates Cellular Functions of Multiorgans beyond Vasomotion through PPAR δ

doi: 10.1155/2020/3785137

Figure Lengend Snippet: The effects of α 1 -AR stimulation on the expression of mitochondrial energetic molecules, oxidative phosphorylation, and biological functions in skeletal and cardiac muscle cells and liver cells. (a, b) The expression of p-AMPK and PPAR δ in C2C12, HL1, and HepG2 cells was stimulated with 1–30 μ M midodrine for the indicated times. (c) Cytosolic calcium mobilization after midodrine treatment in C2C12 and HL1 cells. Each cell type was pretreated with the calcium reactive dye Fluo-3 AM for 45 min and then stimulated with 30 μ M midodrine for the indicated times. Green fluorescence emitted by Fluo-3 AM was detected using confocal microscopy. (d) The phosphorylation of AMPK α at Thr172 and expression of PPAR δ in C2C12 and HL1 cells after pretreatment with the calcium/calmodulin-dependent protein kinase kinase antagonist STO-609 for 25 min and treatment with midodrine. (e) Fluorescence after using the CytoPainter mitochondrial staining kit in midodrine-treated and control C2C12 cells. Original magnification was 200x. (f) The measured activity of succinate dehydrogenase (SDH) in C2C12 cells. (G) Oxygen consumption rate (OCR) in C2C12 cells treated with midodrine (30 μ M), as measured by a Seahorse XFp analyzer. (h) ATP content in C2C12 cells treated with midodrine (30 μ M) cultured with low-glucose (5.56 mM) medium. (i) Glucose transporter (GLUT) 4 protein expression in C2C12 cells treated with high glucose (HG) and midodrine (HG+Mido), HG and insulin (HG+Insulin), and the control treatment (Ctrl). (j) The uptake of 2-deoxyglucose in C2C12 skeletal muscle cells treated with midodrine. (k) OCR (measured by the Seahorse XFp analyzer) in H9C2 cells treated with midodrine (30 μ M) and cultured with low-glucose (5.56 mM) medium. (l) ATP content in H9C2 cells treated with midodrine (30 μ M). Data are expressed as the mean ± standard deviation of triplicate experiments. AMPK: adenosine monophosphate-activated protein kinase; p-AMPK: phosphorylated AMPK; PPAR δ : peroxisome proliferator-activated receptor delta; PGC-1 α : peroxisome proliferator-activated receptor gamma coactivator 1-alpha; mGLUT4: GLUT4 expression of the cell membrane; tGLUT4: total cellular expression of GLUT4; Ctrl: an untreated control group; Mido: midodrine-treated group.

Article Snippet: L6 rat skeletal muscle, C2C12 mouse skeletal muscle, HL1 and H9C2 cardiac muscle, HUVEC human umbilical vein endothelial cell line, RAW 264.7 macrophages, and 3T3-L1 mouse preadipocyte cells were purchased from a Korean cell line bank (Seoul, Korea).

Techniques: Expressing, Phospho-proteomics, Fluorescence, Confocal Microscopy, Staining, Control, Activity Assay, Cell Culture, Standard Deviation, Membrane

Journal: PPAR Research

Article Title: Stimulation of Alpha 1 -Adrenergic Receptor Ameliorates Cellular Functions of Multiorgans beyond Vasomotion through PPAR δ

doi: 10.1155/2020/3785137

Figure Lengend Snippet: The effect of midodrine on the endothelial expression of p-AMPK and p-eNOS in HUVECs; OCR analyses in H9C2 cells; intracellular fat and the expression of PPAR δ , p-AMPK, and PGC-1 α in differentiated 3T3-L1 cells; and the effects of midodrine on mRNA levels of PPAR δ , AMPK α 1 , and mannose receptor and protein levels of mannose receptor and hexokinase II in RAW 264.7 macrophage cells treated with different concentrations of midodrine. (a) The expression of phosphorylated AMPK (p-AMPK) and phosphorylated endothelial nitric oxide synthase (p-eNOS) proteins in human umbilical vein endothelial cells (HUVECs) treated with cholesterol and palmitate, and the effects from the addition of GSK0660, a PPAR δ antagonist. Ctrl: the control group; CP: the cholesterol- and palmitate-treated group; CPM: the cholesterol-, palmitate-, and midodrine-treated group. (b) The maximal oxygen consumption rate (OCR) analysis as estimated using a Seahorse XFp analyzer and ATP content measured by ELISA in H9C2 cells. (c) The effect of compound C (1 μ M) on p-AMPK expression and PPAR δ expression. (d) The effect of midodrine on intracellular lipid deposits (Oil Red O staining result) and the protein levels of PPAR δ , AMPK, and PGC-1 α in differentiated 3T3-L1 cells treated with midodrine and GSK0660. (e) The effects of midodrine on mRNA levels of PPAR δ , AMPK α 1 , and mannose receptor and protein levels of mannose receptor and hexokinase II in RAW 264.7 macrophage cells treated with different concentrations of midodrine. Ctrl: untreated control group; Mido: midodrine-treated group; Mido+GSK0660: midodrine- and GSK0660-treated group.

Article Snippet: L6 rat skeletal muscle, C2C12 mouse skeletal muscle, HL1 and H9C2 cardiac muscle, HUVEC human umbilical vein endothelial cell line, RAW 264.7 macrophages, and 3T3-L1 mouse preadipocyte cells were purchased from a Korean cell line bank (Seoul, Korea).

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Staining