Herbogenomics: from traditional Chinese medicine to novel therapeutics

Traditional Chinese medicine (TCM) has a long history of development and application and has demonstrated on evidence basis its efficacy in the treatment of many diseases affecting multiple organ systems. In particular, TCM is effective in the prevention and treatment of chronic diseases and metabolic syndromes. However, the value of TCM has not been fully recognized worldwide due to the lack of definitive information of active ingredients in almost any TCM preparation. Novel functional genomics and proteomics approaches provide alternate perspectives on the mechanism of action of TCM. The target molecules on which TCM either activates or inactivates can be identified by functional genomics and proteomics, thus the affected critical signaling pathway cascades leading to effective recovery of chronic diseases can be studied. Several TCM preparations have been available for the treatment of liver fibrosis and cirrhosis, even advanced liver cirrhosis that has been shown to be irreversible and has no US-FDA approved therapy. In the TCM-treated livers with fibrosis and cirrhosis, some critical molecules that are significantly involved in the recovery can be identified through functional genomics and proteomics studies. These molecules become novel targets for drug discovery and development and candidates for the development of gene therapy. Gene therapy developed based on this strategy for the treatment of advanced liver fibrosis and cirrhosis in animal models has obtained promising results. This process thus establishes a herbogenomics approach to understand mechanisms of action of TCM and to identify effective molecular targets for the discovery and development of novel therapeutics.     

Tenascin-R distinct domains modulate migration of neural stem/progenitor cells in vitro

Extracellular matrix (ECM) molecules constitute a "niche" that modulates the migration, proliferation, and differentiation of neural stem/progenitor cells (NSPCs). The glycoprotein Tenascin-R (TN-R) is an ECM molecule, comprising multiple domains. Either the whole TN-R molecule or its distinct domains has been demonstrated to play a very important role in the developing central nervous system. However, little is known about the effect of the TN-R domain on NSPCs, especially NSPC migration. In the present study, we first show that both TN-R domains epidermal growth factor-like repeat (EGFL) and fibronectin type III (FN)6-8 can inhibit the NSPCs migration from neurospheres in vitro. Furthermore, both the EGFL and FN6-8 domains affect the distribution of neurons generated from neurospheres, indicating that EGFL and FN6-8 domains inhibit the motility of neurons generated from neurospheres. These results suggest that TN-R has an inhibitory effect on NSPCs migration.     

Detection of gene mutations related with drug resistance in Mycobacterium leprae from leprosy patients using Touch-Down (TD) PCR

The lack of methods to identify Mycobacterium leprae with the resistance against multi-drugs quickly and specifically has hindered effective chemotherapy against M. leprae infection. To screen M. leprae with resistance against multi-drugs, the Touch-Down (TD)-PCR has been used in this study. Sequences of the folP, rpoA, B, and gyrA, B genes were analyzed for isolates of M. leprae from leprosy patients in Korea. We amplified designated region of several genes in M. leprae involved in drug resistance and could obtain the PCR products of each gene. The mutations in the particular region of folP, rpoB, and gyrB gene were certified by TD-PCR single-stranded conformational polymorphism and DNA sequencing, respectively.     

Combined analysis of the association between p73 G4C14-to-A4T14 polymorphisms and cancer risk

P73 is a structural and functional homologue of p53, and plays an important role in regulating cell cycle and apoptosis. A potentially functional polymorphism (designated as p73 G4C14-to-A4T14) has been identified in a region in exon 2 of the p73 gene, which may theoretically form a stem-loop structure and thereby affect p73 expression. Several investigations have reported the correlation between p73 G4C14-to-A4T14 polymorphism and cancer risk. However, the results are inconclusive. To further assess the association between p73 polymorphism and cancer risk, we performed meta-analysis of the data sets obtained from 26 individual studies involving 8,148 cancer patients and 8,150 controls. The association between p73 G4C14-to-A4T14 polymorphism and cancer risk was determined by crude odd ratios (OR) with 95% CI (confidential interval). AT-allele carriers were found to have a significantly increased risk of cervical cancer (AT/GC vs. GC/GC, OR = 1.63, 95% CI = 1.14–2.33; AT/AT + AT/GC vs. GC/GC, OR = 1.49, 95% CI = 1.05–2.10), colorectal cancer (AT/AT vs. AT/GC + GC/GC, OR = 1.98, 95% CI = 1.25–3.12), head and neck cancer (AT/AT + AT/GC vs. GC/GC, OR = 1.44, 95% CI = 1.06–1.96) and other cancers (AT/AT vs. GC/GC, OR = 1.78, 95% CI = 1.24–2.57; AT/AT vs. AT/GC + GC/GC, OR = 1.80, 95% CI = 1.26–2.56). In the stratified analysis of ethnicity, a significantly elevated cancer risk was found in Caucasians (AT/AT + AT/GC vs. GC/GC, OR = 1.18, 95% CI = 1.08–1.30; allele AT vs. allele GC, OR = 1.15, 95% CI = 1.06–1.24). No significant association of p73 polymorphism with the cancer risk of smoking was detected by stratified analysis by smoking status. Together, our data suggest that the p73 G4C14-to-A4T14 may be a risk factor of cancer especially in Caucasians.     

Enhanced immunodetection of cell-free synthesized erythropoietin under denaturing conditions

A monoclonal antibody produced by hydridoma cell line, ATCC HB8209, was used to detect and purify erythropoietin synthesized in a cell-free system. The antibody was raised against the N-terminal 20 residues of erythropoietin. It retained anti-erythropoietin activity in 6 M urea in which most of the cell-free synthesized erythropoietin became soluble and gave an enhanced activity of the antibody.     

Body mass and behavior in Swiss mice subjected to continuous or discontinuous food restriction and refeeding

Food restriction (FR) is hypothesized to decrease body fat content of an animal and thus prevent obesity. However, the response of energy budget to a continuous (CFR) or discontinuous FR (DFR) remains inconsistent. In the present study, effects of CFR or DFR and refeeding on energy budget and behavior were examined in male Swiss mice. CFR significantly decreased the energy expenditure associated with basal metabolic rate (BMR) and activity behavior, but not sufficiently to compensate for energy deficit and thus resulted in lower body mass and fat content. DFR mice had a significantly higher food intake on ad libitum days and showed increases in BMR and activity after 4 weeks’ DFR, which might resulted in lower body mass and less body fat than controls. After being refed ad libitum, both CFR and DFR mice had similar body mass, BMR, and behavioral patterns to controls but had 95% and 75% higher fat content. This suggested that not only CFR but also DFR would be a significant factor in the process of obesity for animals that were refed ad libitum. It also indicated that food restriction interrupted many times by periods of ad libitum feeding had the same long-term effects like continuous underfeeding.     

Comparative study of two thioredoxin peroxidases from disk abalone (Haliotis discus discus): cloning, recombinant protein purification, characterization of antioxidant activities and expression analysis

Thioredoxin peroxidase (TPx), also named peroxiredoxin (Prx), is an important peroxidase, which can protect organisms against various oxidative stresses. Two TPxs were isolated from a disk abalone (Haliotis discus discus) cDNA library, named as AbTPx1 and AbTPx2, respectively. AbTPx1 and AbTPx2 consist of 1315 and 1045 bp full-length cDNA with 753 and 597 bp open reading frames encoding 251 and 199 amino acids, respectively. The TPx signature motif 1 (FYPLDFTFVCPTEI) and motif 2 (GEVCPA) were conserved in both AbTPx1 and AbTPx2 amino acid sequences. Purified recombinant abalone TPx fusion proteins catalyzed the reduction of H2O2 and butyl hydroperoxide in peroxidase assays. Furthermore, both AbTPx fusion proteins were shown to protect super-coiled DNA from damage by metal-catalyzed oxidation (MCO) in vitro. Escherichia coli cells transformed with AbTPx1 and AbTPx2 coding sequences in pMAL-c2x showed resistance to H2O2 at 0.8 mM concentration by in vivo H2O2 tolerance assay. AbTPx1 and AbTPx2 mRNA were constitutively expressed in gill, mantle, abductor muscle and digestive tract in a tissue specific manner. Additionally, both TPxs mRNA were up-regulated in gill and digestive tract tissues against H2O2 at 3h post injection. The results indicate that AbTPx1 and AbTPx2 gene expressions are induced by oxidative stress and their respective proteins function in the detoxification of different ROS molecules to maintain efficient antioxidant defense in disk abalone.     

Magnesium ion is an effective inhibitor of the binding of Escherichia coli to heparin

We investigated the effects of ions and temperature on the binding of E. coli to heparin using a chemiluminescence electrophoretic mobility shift assay. We found that magnesium ion is an effective inhibitor of the binding. The method can be readily applied to discover agents that can block the binding.