Piperine augments transcription inhibitory activity of rifampicin by several fold in Mycobacterium smegmatis

A 24 : 1 (w/w) mixture of antibiotic rifampicin and a plant product piperine show remarkable growth inhibitory effect on Mycobacterium smegmatis, and this inhibition is higher than that of rifampicin alone. Interestingly, piperine alone, even at higher concentration, does not inhibit the growth of mycobacteria. As RNA polymerase is the site of action of rifampicin, the enzyme was purified from M. smegmatis and the mixture of rifampicin and piperine was found to abrogate non-specific transcription catalysed by M. smegmatis RNA polymerase. Here too the effect is higher than rifampicin alone and piperine shows no effect independently. When RNA polymerase was purified from a rifampicin-resistant strain of M. smegmatis, the enzymatic activity, otherwise resistant to rifampicin, significantly decreases in the presence of piperine along with rifampicin. Modelling studies have been carried out to explain this 'bioenhancing' behaviour of piperine

Genome-Wide Analyses of Nkx2-1 Binding to Transcriptional Target Genes Uncover Novel Regulatory Patterns Conserved in Lung Development and Tumors

The homeodomain transcription factor Nkx2-1 is essential for normal lung development and homeostasis. In lung tumors, it is considered a lineage survival oncogene and prognostic factor depending on its expression levels. The target genes directly bound by Nkx2-1, that could be the primary effectors of its functions in the different cellular contexts where it is expressed, are mostly unknown. In embryonic day 11.5 (E11.5) mouse lung, epithelial cells expressing Nkx2-1 are predominantly expanding, and in E19.5 prenatal lungs, Nkx2-1-expressing cells are predominantly differentiating in preparation for birth. To evaluate Nkx2-1 regulated networks in these two cell contexts, we analyzed genome-wide binding of Nkx2-1 to DNA regulatory regions by chromatin immunoprecipitation followed by tiling array analysis, and intersected these data to expression data sets. We further determined expression patterns of Nkx2-1 developmental target genes in human lung tumors and correlated their expression levels to that of endogenous NKX2-1. In these studies we uncovered differential Nkx2-1 regulated networks in early and late lung development, and a direct function of Nkx2-1 in regulation of the cell cycle by controlling the expression of proliferation-related genes. New targets, validated in Nkx2-1 shRNA transduced cell lines, include E2f3, Cyclin B1, Cyclin B2, and c-Met. Expression levels of Nkx2-1 direct target genes identified in mouse development significantly correlate or anti-correlate to the levels of endogenous NKX2-1 in a dosage-dependent manner in multiple human lung tumor expression data sets, supporting alternative roles for Nkx2-1 as a transcriptional activator or repressor, and direct regulator of cell cycle progression in development and tumors

Piperine augments transcription inhibitory activity of rifampicin by severalfold in Mycobacterium smegmatis

A 24:1 (w/w) mixture of antibiotic rifampicin and a plant product piperine show remarkable growth-inhibitory effect on Mycobacterium smegmatis, and this inhibition is higher than that of rifampicin alone. Interestingly, piperine alone, even at higher concentration, does not inhibit the growth of mycobacteria, As RNA polymerase is the site of action of rifampicin, the enzyme was purified from M. smegmatis and the mixture of rifampicin and piperine was found to abrogate non- specific transcription catalysed by M. smegmatis RNA polymerase, Here too the effect is higher than rifampicin alone and piperine shows no effect independently. When RNA polymerase was purified from a rifampicin-resistant strain of M. smegmatis, the enzymatic activity, otherwise resistant to rifampicin, significantly decreases in the presence of piperine along with rifampicin, Modelling studies have been carried out to explain this bioenhancing behaviour of piperine

Synthesis of an unusual polar glycopeptidolipid in glucose-limited culture of Mycobacterium smegmatis

There has been a general understanding that Mycobacterium smegmatis produces only apolar glycopeptidolipid (GPL), similar in structure to serovar non-specific GPL of Mycobacterium avium. In this study, synthesis of polar GPL in carbon-starved M. smegmatis is reported. Mass spectrometric analysis suggests the polar GPL to be a hyperglycosylated species. The earlier structural studies of polar GPLs from M. avium have invariably shown the presence of an oligosaccharide appendage to D-allo-Thr. However, a further chemical analysis using ß-elimination of the newly found polar GPL in M. smegmatis shows that the molecule still contains a monosaccharide at the D-allo-Thr, thus suggesting a new form of polar GPL

Grainyhead-like 2 (GRHL2) distribution reveals novel pathophysiological differences between human idiopathic pulmonary fibrosis and mouse models of pulmonary fibrosis

Chronic injury of alveolar lung epithelium leads to epithelial disintegrity in idiopathic pulmonary fibrosis (IPF). We had reported earlier that Grhl2, a transcriptional factor maintains alveolar epithelial cell integrity by directly regulating components of adherens and tight junctions and thus hypothesized an important role of GRHL2 in pathogenesis of IPF. Comparison of GRHL2 distribution at different stages of human lung development showed its abundance in developing lung epithelium and in adult lung epithelium. However, GRHL2 is detected in normal human lung mesenchyme only at early fetal stage (week 9). Similar Mesenchymal re-expression of GRHL2 was also observed in IPF. Immunofluorescence analysis in serial sections from three IPF patients revealed at least two subsets of alveolar epithelial cells (AEC), based on differential GRHL2 expression and the converse fluorescence intensities for epithelial versus mesenchymal markers. Grhl2 was not detected in mesenchyme in IP bleomycin-induced injury as well as in spontaneously occurring fibrosis in HPS1/2 mutant mice, while in contrast in a radiation-induced fibrosis model, with forced Forkhead box M1 (Foxm1) expression, an overlap of Grhl2 with a mesenchymal marker was observed in fibrotic regions. Grhl2's role in alveolar epithelial cell-plasticity was confirmed by altered Grhl2 gene expression analysis in IPF and further by in vitro manipulation of its expression in alveolar epithelial cell lines. Our findings reveal important patho-physiological differences between human IPF and specific mouse models of fibrosis and support a crucial role of GRHL2 in epithelial activation in lung fibrosis and perhaps also in epithelial plasticity

Spatial and temporal pattern of Nkx2-1 protein expression in developing mouse lung.

<p>Immunohistochemistry analyses of Nkx2-1 protein expression using the Nkx2-1 antibody (ab76013) in (<i>A</i>) E11.5 lung (bar = 100 µm)) and in (<i>B</i>) E19.5 lung (bar = 100 µm). Nkx2-1 is expressed in all lung epithelial cells at E11.5, but is restricted to bronchiolar and type II alveolar cells at E19.5. Confocal immunofluorescence co-localization analysis of Nkx2-1 (green) and Ki67 (red) proteins in (<i>C</i>) E11.5 (bar = 20 µm) and (<i>D</i>) E19.5 embryonic lung (bar = 20 µm) using the Nkx2-1 antibody (ab76013) and in (<i>E</i>) E11.5 (bar = 20 µm) and (<i>F</i>) E19.5 embryonic lung (bar = 20 µm) using the Nkx2-1 antibody (07-601). Nkx2-1 and Ki67 co-localized in most epithelial nuclei at E11.5 (<i>C,E</i> inserts) but are detected in different cells at E19.5 (<i>D,F</i> inserts). (<i>G</i>) Western blot analysis of Nkx2-1 protein expression using Nkx2-1 rabbit polyclonal antibody (07-601) in developing mouse lung at the indicated time points, and adult lung, thyroid and liver. MLE15 mouse epithelial cells and H441 human lung carcinoma cells were also analyzed. Actin was used as control. Two main bands between 40 and 50 kD are detected with a differential developmental pattern of expression (n = 3) (black arrows). Other minor bands of unknown identity are also detected (*) Lb (lung buds), V (blood vessels), Br (bronchioles), E (epithelium), M (mesenchyme), A (alveolus), green arrow.</p

Grainyheadlike 2 distribution reveals novel pathophysiological differences between human idiopathic pulmonary fibrosis and mouse models of pulmonary fibrosis

Chronic injury of alveolar lung epithelium leads to epithelial disintegrity in idiopathic pulmonary fibrosis (IPF). We had reported earlier that Grhl2, a transcriptional factor maintains alveolar epithelial cell integrity by directly regulating components of adherens and tight junctions and thus hypothesized an important role of GRHL2 in pathogenesis of IPF. Comparison of GRHL2 distribution at different stages of human lung development showed its abundance in developing lung epithelium and in adult lung epithelium. However, GRHL2 is detected in normal human lung mesenchyme only at early fetal stage (week 9). Similar Mesenchymal re-expression of GRHL2 was also observed in IPF. Immunofluorescence analysis in serial sections from three IPF patients revealed at least two subsets of alveolar epithelial cells (AEC), based on differential GRHL2 expression and the converse fluorescence intensities for epithelial versus mesenchymal markers. Grhl2 was not detected in mesenchyme in IP bleomycin-induced injury as well as in spontaneously occurring fibrosis in HPS1/2 mutant mice, while in contrast in a radiation-induced fibrosis model, with forced Forkhead box M1 (Foxm1) expression, an overlap of Grhl2 with a mesenchymal marker was observed in fibrotic regions. Grhl2's role in alveolar epithelial cell-plasticity was confirmed by altered Grhl2 gene expression analysis in IPF and further by in vitro manipulation of its expression in alveolar epithelial cell lines. Our findings reveal important patho-physiological differences between human IPF and specific mouse models of fibrosis and support a crucial role of GRHL2 in epithelial activation in lung fibrosis and perhaps also in epithelial plasticity

NKX2-1 levels in human lung tumors significantly correlate with expression of developmental Nkx2-1 target genes.

<p>Heatmaps of human lung tumor genes identified in GSE12667 database showing gene expression levels of the human homologues of Nkx2-1 target genes identified in mouse lung development at E11.5 (upper panel) and E19.5 (lower panel); genes are organized according to their Pearson correlation value (y axis) to relative NKX2-1 expression level (x axis).</p

Downregulation of Nkx2-1 affects expression of proliferation-related genes in mouse lung epithelial cells.

<p>(<i>A</i>) Western blot analysis of Nkx2-1 downregulation by shRNA in MLE15 cells using the Nkx2-1 antibody 07-601 (Millipore-Upstate). Two bands between 40–50 kD are detected in mouse MLE15 cells and E17.5 lung. Densitometry analyses show a significant downregulation of Nkx2-1 major bands in shRNA transduced cells. Nkx2-1 downregulation by shRNA also reduces the level of minor bands of higher molecular weight (*, asterisks) (<i>B</i>) Representative flow cytometry analyses of DNA content and cell cycle progression in the same cells; n = 3. (<i>C</i>) Relative number of cells in each stage of the cell cycle. (D) Cell count analysis of MLE15 cells transduced with empty vector or Nkx2-1shRNA. 5000 cells were plated and grown in standard conditions for up to 4 days. Attached cells were trypzinized and counted; n = 6 (E) Real time RT-PCR analyses of Nkx2-1 mRNA expression levels in Nkx2-1 shRNA or empty vector transduced cells; n = 3; similar analyses were performed for selected targets; n = 6. Error bars represent s.e.m.; (*) p≤0.05.</p