MEHP upregulates expression of CRH and COX-2 in primary cultures of human cytotrophoblast.

<p>Primary cultures of term cytotrophoblast were exposed to different concentrations of MEHP for 24 hours with DMSO as the vehicle control. (<b>A</b>) Trypan blue exclusion test of cell viability for primary cytototrophoblast exposed to MEHP at concentrations as indicated (N = 3 independent experiments). (<b>B</b>) Representative gel patterns and analysis in whole cell lysates from three independent term placentas by Western blot assay with use of antibody as indicated. (<b>C</b>) Total RNAs were extracted and RT-qPCR was performed for assessment of CRH and COX-2 mRNA levels with normalization to GAPDH mRNA level. Bars represent the average of relative mRNA levels and error bars represent standard deviation from experiments performed in three independent term placentas. * p < 0.05; ** p < 0.01.</p

The effects of MEHP exposure on interaction of RelB/p52 with the <i>CRH/COX-2</i> gene promoters.

<p>Term cytotrophoblast were exposed to MEHP for 24 hrs at concentrations as indicated. ChIP assays were performed to determine occupancy of RelB/p52 at <i>CRH</i> (<b>A</b>) and <i>COX-2</i> (<b>B</b>), and also occupancy of RelA at the <i>CRH</i> (<b>C</b>) and <i>COX-2</i> (<b>D</b>) gene promoter. Fold enrichment was derived with normalization to a human DNA a satellite, a non-coding DNA sequence to which no transcriptional factors should bind. Rabbit IgG was used as a non-specific control. The bars indicate the average of fold enrichment with error bars representing the standard deviation from three independent experiments. * p < 0.01.</p

NIK knockdown attenuates MEHP-induced upregulation of <i>CRH</i> and <i>COX-2</i> in the human placenta.

<p>Primary term cytotrophoblast were incubated with siRNAs targeting NIK (siNIK) for 24 hrs, and then treated with MEHP for an additional 24 hrs. Scramble siRNA (Scr-siRNA) was used as the non-targeting control. Total RNAs were extracted and RT-qPCR was performed to determine mRNA levels of CRH (<b>A</b>), COX-2 (<b>B</b>), or NIK (<b>C</b>) (N = 3 independent experiments). * p < 0.05 (compared to DMSO). ** p< 0.01 (compared to DMSO). ## p < 0.01 (compared to Scr-siRNA). (<b>D</b>) Representative gel pattern of Western blot analysis on whole cell lysates from three independent experiments. (<b>E</b>) IF staining was performed to directly assess intracellular distribution of RelB. These images represent data obtained from three independent placentas. Original magnification, 200Χ.</p

<i>Bacillus anthracis</i> Inosine 5′-Monophosphate Dehydrogenase in Action: The First Bacterial Series of Structures of Phosphate Ion‑, Substrate‑, and Product-Bound Complexes

Inosine 5′-monophosphate dehydrogenase (IMPDH) catalyzes the first unique step of the GMP branch of the purine nucleotide biosynthetic pathway. This enzyme is found in organisms of all three kingdoms. IMPDH inhibitors have broad clinical applications in cancer treatment, as antiviral drugs and as immunosuppressants, and have also displayed antibiotic activity. We have determined three crystal structures of <i>Bacillus anthracis</i> IMPDH, in a phosphate ion-bound (termed “apo”) form and in complex with its substrate, inosine 5′-monophosphate (IMP), and product, xanthosine 5′-monophosphate (XMP). This is the first example of a bacterial IMPDH in more than one state from the same organism. Furthermore, for the first time for a prokaryotic enzyme, the entire active site flap, containing the conserved Arg-Tyr dyad, is clearly visible in the structure of the apoenzyme. Kinetic parameters for the enzymatic reaction were also determined, and the inhibitory effect of XMP and mycophenolic acid (MPA) has been studied. In addition, the inhibitory potential of two known <i>Cryptosporidium parvum</i> IMPDH inhibitors was examined for the <i>B. anthracis</i> enzyme and compared with those of three bacterial IMPDHs from <i>Campylobacter jejuni</i>, <i>Clostridium perfringens</i>, and <i>Vibrio cholerae</i>. The structures contribute to the characterization of the active site and design of inhibitors that specifically target <i>B. anthracis</i> and other microbial IMPDH enzymes