Evaluation of the Chinese Medicinal Herb, Graptopetalum paraguayense, as a Therapeutic Treatment for Liver Damage in Rat Models.

The incidence of cirrhosis is rising due to the widespread occurrence of chronic hepatitis, as well as the evident lack of an established therapy for hepatic fibrosis. In the search for hepatoprotective therapeutic agents, Graptopetalum paraguayense (GP) showed greater cytotoxicity toward hepatic stellate cells than other tested herbal medicines. Histopathological and biochemical analyses suggest that GP treatment significantly prevented DMN-induced hepatic inflammation and fibrosis in rats. Microarray profiling indicated that expression of most of metabolism- and cell growth and/or maintenance-related genes recovered to near normal levels following GP treatment as classified by gene ontology and LSM analysis, was observed. ANOVA showed that expression of 64% of 256 liver damage-related genes recovered significantly after GP treatment. By examining rat liver samples with Q-RT-PCR, five liver damage-related genes were identified. Among them, Egr1 and Nrg1 may serve as necroinflammatory markers, and Btg2 may serve as a fibrosis marker. Oldr1 and Hmgcs1 were up- and down-regulated markers, respectively. A publicly accessible website has been established to provide access to these data Identification of 44 necroinflammation-related and 62 fibrosis-related genes provides useful insight into the molecular mechanisms underlying liver damage and provides potential targets for the rational development of therapeutic drugs such as GP.

Protein profilings in mouse liver regeneration after partial hepatectomy using iTRAQ technology.

Liver is unique in its capability to regenerate after an injury. Liver regeneration after a 2/3 partial hepatectomy served as a classical model and is adopted frequently to study the mechanism of liver regeneration. In the present study, semiquantitative analysis of protein expression in mouse liver regeneration following partial hepatectomy was performed using an iTRAQ technique. Proteins from pre-PHx control livers and livers regenerating for 24, 48 and 72 h were extracted and inspected using 4-plex isotope labeling, followed by liquid chromatography fractionation, mass spectrometry and statistical differential analysis. A total of 827 proteins were identified in this study. There were 270 proteins for which quantitative information was available at all the time points in both biologically duplicate experiments. Among the 270 proteins, Car3, Mif, Adh1, Lactb2, Fabp5, Es31, Acaa1b and LOC100044783 were consistently down-regulated, and Mat1a, Dnpep, Pabpc1, Apoa4, Oat, Hpx, Hp and Mt1 were up-regulated by a factor of at least 1.5 from that of the controls at one time point or more. The regulation of each differential protein was also demonstrated by monitoring its time-dependent expression changes during the regenerating process. We believe this is the first report to profile the protein changes in liver regeneration utilizing the iTRAQ proteomic technique.

Refolding of a small all beta-sheet protein proceeds with accumulation of kinetic intermediates.

The refolding kinetics of Cobrotoxin (CBTX), a small all beta-sheet protein is investigated using a variety of biophysical techniques including quenched-flow hydrogen-deuterium (H/D) exchange in conjunction with two-dimensional NMR spectroscopy. Urea-induced equilibrium unfolding of CBTX follows a two-state mechanism with no distinct intermediates. The protein is observed to fold very rapidly within 250 ms. Both the refolding and the unfolding limbs of the chevron plot of CBTX show a prominent curvature suggesting the accumulation of kinetic intermediates. Quenched-flow H/D exchange data suggest the presence of a broad continuum of kinetic intermediates between the unfolded and native states of the protein. Comparison of the native state hydrogen exchange data and the results of the quenched-flow H/D exchange experiments, reveals that the residues constituting the folding core of CBTX are not a subset of the slow exchange core. To our knowledge, this is the first report wherein the refolding of a small all beta-sheet protein is shown to be a multi-step process involving the accumulation of kinetic intermediates.

Equilibrium unfolding of an oligomeric protein involves formation of a multimeric intermediate state(s).

Superoxide dismutases (SODs) are important metalloenzymes which protect cells against oxidative stress by scavenging reactive superoxides. Missense mutations in SODs are known to lead to some familial cases of amyotrophic lateral sclerosis and several forms of cancers. In the present study, we investigate the guanidinium hydrochloride (GdnHCl)-induced equilibrium unfolding of apo-manganese superoxide dismutase (apo-MnSOD) isolated from Vibrio alginolyticus using a variety of biophysical techniques. GdnHCl-induced equilibrium unfolding of apo-MnSOD is non-cooperative and involves the accumulation of stable intermediate state(s). Results of 1-anilino-8-naphthalene sulfonate binding experiments suggest that the equilibrium intermediate state(s) accumulates maximally in 1.5M GdnHCl. The intermediate state(s) appears to be obligatory and occurs both in the unfolding and refolding pathways. Size-exclusion chromatography and sedimentation velocity data reveal that the equilibrium intermediate state(s) is multimeric. To our knowledge, this is the first report of the identification of a multimeric intermediate in the unfolding pathway(s) of oligomeric proteins. The formation and dissociation of the multimeric intermediate state(s) appears to dictate the fate of the protein either to refold to its native conformation or misfold and form aggregates as observed in amyotrophic lateral sclerosis.

Cloning, overexpression, and characterization of cobrotoxin.

Cobrotoxin (CBTX) is a highly toxic short neurotoxin, isolated from the Taiwan cobra (Naja naja atra) venom. In the present study for the first time we report the cloning and expression of CBTX in high yields (12mg/L) in Escherichia coli. CBTX fused to the IgG-binding domain of protein A (IgG-CBTX) was expressed in the soluble form. The misfolded CBTX portion (of the overexpressed fusion protein) was refolded under optimal redox conditions. The fusion protein (IgG-CBTX) was observed to undergo auto-catalytic cleavage to yield CBTX with additional 5 amino acids upstream of its N-terminal end. The far UV and near UV circular dichroism spectra of the recombinant CBTX were identical to those of the toxin isolated from the crude venom source. Recombinant CBTX was isotope labeled (15N and 13C) and all the resonances ('H, 13C, and 15N) in the protein have been unambiguously assigned. ' H '5N HSQC spectrum of recombinant CBTX revealed that the protein is in a biologically active conformation. 1H-15Nchemical shift perturbation data showed that recombinant CBTX binds to a peptide derived from the alpha7 subunit of the Torpedo acetylcholine receptor (AchR) with high affinity. The AchR peptide is found to bind to residues located at the tip of Loop-2 in CBTX. The results of the present study provide an avenue to understand the structural basis for the high toxicity exhibited by CBTX. In addition, complete resonance assignments in CBTX (reported in this study) are expected to trigger intensive research towards the design of new pharmacological agents against certain neural disorders.