This study was undertaken to delineate a possible role for tissue transglutaminase (tTG), an enzyme that catalyzes protein cross-linking, in hepatic fibrogenesis. Rats were treated with CCl4 solution and then killed at different stages of liver injury and fibrogenesis. Liver tTG mRNA levels were markedly increased as early as 6 h after the first injection, peaked at 4 days and 1 wk, and remained increased for 8 wk. The enzymatic activity of tTG was increased in livers of rats treated with CCl4, in a fashion that paralleled the Northern blot results. Cell isolation experiments indicated that all hepatic cell types synthesize tTG mRNA. Increased binding to the nuclear factor-kappaB (NF-kappaB) motif of the tTG promoter was found in the nuclear extracts prepared from CCl4-treated samples. These data demonstrate an increase in tTG gene expression during hepatic injury and fibrosis, suggesting a possible role for this enzyme in stabilizing the fibrotic bands during hepatic fibrogenesis. Moreover, increased NF-kappaB binding to the tTG promoter may represent one of the mechanisms by which cell injury induces tTG transcription and thus potentiates the process of fibrogenesis.

Small-angle X-ray scattering and size-exclusion chromatography have been combined within a unified experimental set-up to obtain molecular size information. Besides providing simultaneous corroborative data bearing on the same question from two distinct experimental techniques, passing the samples over a gel filtration column immediately prior to illumination by X-rays provides both a more homogeneous sample and a continuous set of data as the concentration is extrapolated to zero. This greatly facilitates analysis of data from oligomerizing or aggregating proteins and increases the reliability of the results.

Wide-angle X-ray scattering patterns from proteins in solution contain information relevant to the determination of protein fold. At relevant scattering angles, however, these data are weak, and the degree to which they might be used to categorize the fold of a protein is unknown. Preliminary work has been performed at the BioCAT insertion-device beamline at the Advanced Photon Source which demonstrates that one can collect X-ray scattering data from proteins in solution to spacings of at least 2.2 A (q = 2.8 A(-1)). These data are sensitive to protein conformational states, and are in good agreement with the scattering predicted by the program CRYSOL using the known three-dimensional atomic coordinates of the protein. An important issue in the exploitation of this technique as a tool for structural genomics is the extent to which the high intensity of X-rays available at third-generation synchrotron sources chemically or structurally damage proteins. Various data-collection protocols have been investigated demonstrating conditions under which structural degradation of even sensitive proteins can be minimized, making this technique a viable tool for protein fold categorization, the study of protein folding, unfolding, protein-ligand interactions and domain movement.

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In the present study, pumpkin seed extract was used to synthesize copper oxide nanoparticles (CuO NPs) along with evaluating its anticancer activity using different molecular biology tools in the human colorectal cancer cell line (HCT-116). Morphological and structural properties of the biogenically synthesized CuO NPs were characterized by UV-visible spectrophotometry (UV-vis), energy-dispersive X-ray spectroscopy (EDS), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). For estimating the anticancer efficacy, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity, morphological alteration, reactive oxygen species (ROS) formation, and alterations in the mitochondrial membrane potential (MMP) were determined. SEM and TEM data revealed the formation of spherical nanoparticles possessing an average size of 20 nm. The CuO NPs showed 50% inhibitory concentration (IC50) at 25 µg/mL against the HCT-116 cell line. The treatment with IC50 concentration of CuO NPs showed significant shrinking, detachment, membrane blebbing, and shape distortion of cancer cells. Similarly, the IC50 dose of CuO NPs showed significantly early apoptosis in cancer cells compared to late apoptosis. The cancer cell line also showed a dose-dependent increase and decrease in ROS formation and MMP, respectively. The results obtained through various assays indicated significant anticancer efficacy of biogenically synthesized CuO NPs. Thus, further studies are recommended to validate our results using ex vivo and in vivo models.