A Study of the Effect of Shiunko, a Traditional Chinese Herbal Medicine, on Fibroblasts and Its Implication on Wound Healing Processes.

SIGNIFICANCE: In China, traditional Chinese medicine (TCM) has been used for thousands of years for various acute and chronic wound care. Thus, there is a growing need to explore the possible benefits of TCM on wound healing. RECENT ADVANCES: Nowadays, in China and some Asian countries including Korea, Japan, and Singapore, Chinese herbal therapy is used as an alternative treatment in wound care. Therefore, exploration of the possible benefits of TCM on wound healing is necessary. CRITICAL ISSUES: Development of TCM is based on the concept of Yin (negative phenomenon of nature) and Yang (positive phenomenon of nature). These opposing and complementary natural phenomena of the universe restore the normal physiological functions, consequently curing diseases and restoring health of a patient. FUTURE DIRECTIONS: Due to lack of evidence-based research, TCM treatments are not widely accepted in the western world. Using state-of-the-art technology such as proteomics, bioinformatics, and biomolecular techniques, research studies may lead to more effective remedies for wound care in the future.

A novel endogenous induction of ColE7 expression in a csrA mutant of Escherichia coli.

Carbon storage regulator A (CsrA) is an important regulator that controls central metabolic pathways and a variety of physiological functions. We found that disruption of csrA in cells containing the ColE7 operon caused a 12-fold increase in colicin E7 production. Moreover, real-time RT-PCR demonstrated a decrease of around 50 % in the lexA mRNA of the csrA mutant. However, the cellular level of RecA protein and its mRNA were not significantly different from the wild type strain. Our results suggest that a novel induction mechanism might exist in E. coli that allows the expression of ColE7 operon in response to a metabolic shift. Proteomic analysis suggested that csrA deficient mutant may adapt PEP-glyoxylate cycle for energy production. Thus, the physiological changes in the csrA mutant may be similar to carbon source limitation for initiating the expression of ColE7 operon in response to stringent environmental conditions.

The molecular basis of wound healing processes induced by lithospermi radix: a proteomics and biochemical analysis.

Lithospermi Radix (LR) is an effective traditional Chinese herb in various types of wound healing; however, its mechanism of action remains unknown. A biochemical and proteomic platform was generated to explore the biological phenomena associated with LR and its active component shikonin. We found that both LR ethanol extracts and shikonin are able to promote cell proliferation by up to 25%. The results of proteomic analysis revealed that twenty-two differentially expressed proteins could be identified when fibroblast cells were treated with LR or shikonin. The functions of those proteins are associated with antioxidant activity, antiapoptosis activity, the regulation of cell mobility, the secretion of collagen, the removal of abnormal proteins, and the promotion of cell proliferation, indicating that the efficacy of LR in wound healing may be derived from a synergistic effect on a number of factors induced by the herbal medicine. Furthermore, an animal model confirmed that LR is able to accelerate wound healing on the flank back of the SD rats. Together these findings help to pinpoint the molecular basis of wound healing process induced by LR.

A Study of the Wound Healing Mechanism of a Traditional Chinese Medicine, Angelica sinensis, Using a Proteomic Approach.

Angelica sinensis (AS) is a traditional Chinese herbal medicine that has been formulated clinically to treat various form of skin trauma and to help wound healing. However, the mechanism by which it works remains a mystery. In this study we have established a new platform to evaluate the pharmacological effects of total AS herbal extracts as well as its major active component, ferulic acid (FA), using proteomic and biochemical analysis. Cytotoxic and proliferation-promoting concentrations of AS ethanol extracts (AS extract) and FA were tested, and then the cell extracts were subject to 2D PAGE analysis. We found 51 differentially expressed protein spots, and these were identified by mass spectrometry. Furthermore, biomolecular assays, involving collagen secretion, migration, and ROS measurements, gave results that are consistent with the proteomic analysis. In this work, we have demonstrated a whole range of pharmacological effects associated with Angelica sinensis that might be beneficial when developing a wound healing pharmaceutical formulation for the herbal medicine.

Delineation of the translocation of colicin E7 across the inner membrane of Escherichia coli.

The lysis protein of the colicinogenic operon is essential for colicin release and its main function is to activate the outer membrane phospholipase A (OMPLA) for the traverse of colicin across the cell envelope. However, little is known about the involvement of the lysis protein in the translocation of colicin across the inner membrane into the periplasm. The introduction of specific point mutations into the lipobox or sorting signal sequence of the lysE7 gene resulted in the production of various forms of lysis proteins. Our experimental results indicated that cells with wild-type mature LysE7 protein exhibited higher efficiency of colicin E7 translocation across the inner membrane into the periplasm than those with premature LysE7 protein. Moreover, the degree of permeability of the inner membrane induced by the mature LysE7 protein was significantly increased as compared to the unmodified LysE7 precursor. These results suggest that the efficiency of colicin movement into the periplasm is correlated with the increase in inner membrane permeability induced by the LysE7 protein. Thus, we propose that mature LysE7 protein has two critical roles: firstly mediating the translocation of colicin E7 across the inner membrane into the periplasm, and secondly activating the OMPLA to allow colicin release.

Repression of btuB gene transcription in Escherichia coli by the GadX protein.

BACKGROUND: BtuB (B twelve uptake) is an outer membrane protein of Escherichia coli. It serves as a receptor for cobalamines uptake or bactericidal toxin entry. A decrease in the production of the BtuB protein would cause E. coli to become resistant to colicins. The production of BtuB has been shown to be regulated at the post-transcriptional level. The secondary structure of 5' untranslated region of btuB mRNA and the intracellular concentration of adenosylcobalamin (Ado-Cbl) would affect the translational efficiency and RNA stability of btuB gene. The transcriptional regulation of btuB expression is still unclear. RESULTS: To determine whether the btuB gene is also transcriptionally controlled by trans-acting factors, a genomic library was screened for clones that enable E. coli to grow in the presence of colicin E7, and a plasmid carrying gadX and gadY genes was isolated. The lacZ reporter gene assay revealed that these two genes decreased the btuB promoter activity by approximately 50%, and the production of the BtuB protein was reduced by approximately 90% in the presence of a plasmid carrying both gadX and gadY genes in E. coli as determined by Western blotting. Results of electrophoretic mobility assay and DNase I footprinting indicated that the GadX protein binds to the 5' untranslated region of the btuB gene. Since gadX and gadY genes are more highly expressed under acidic conditions, the transcriptional level of btuB in cells cultured in pH 7.4 or pH 5.5 medium was examined by quantitative real-time PCR to investigate the effect of GadX. The results showed the transcription of gadX with 1.4-fold increase but the level of btuB was reduced to 57%. CONCLUSIONS: Through biological and biochemical analysis, we have demonstrated the GadX can directly interact with btuB promoter and affect the expression of btuB. In conclusion, this study provides the first evidence that the expression of btuB gene is transcriptionally repressed by the acid responsive genes gadX and gadY.

Interaction of colicin E7 with the major coat protein (g8p) may confer limited protection on colicinogenic Escherichia coli against M13 bacteriophage infection.

Colicin release provides producer strains with a competitive advantage under certain circumstances. We found that propagation of M13 bacteriophage in cells producing colicin E7 is impaired, without alteration in the efficiency of bacteriophage adsorption, as compared with non-producing cells. In contrast to the protective effect of the colicin against M13 bacteriophage infection, the endogenously expressed colicin does not confer limited protection against transfection with M13 bacteriophage DNA. Furthermore, it was found that the translocation-receptor-binding domain and toxicity domain of the colicin are able to interact with the M13 major coat protein, g8p, during bacteriophage infection. Based on these observations, we propose that interaction between colicin E7 and g8p during infection interferes with g8p depolymerizing into the cytoplasmic membrane during bacteriophage DNA penetration, thus resulting in the limited protection against M13 bacteriophage infection.

Posttranscriptional repression of the cel gene of the ColE7 operon by the RNA-binding protein CsrA of Escherichia coli.

Carbon storage regulator (CsrA) is a eubacterial RNA-binding protein that acts as a global regulator of many functionally diverse chromosomal genes. Here, we reveal that CsrA represses expression from an extrachromosomal element of Escherichia coli, the lysis gene (cel) of the ColE7 operon (cea-cei-cel). This operon and colicin expression are activated upon SOS response. Disruption of csrA caused approximately 5-fold increase of the lysis protein. Gel mobility shift assays established that both the single-stranded loop of the T1 stem-loop distal to cei, and the putative CsrA binding site overlapping the Shine-Dalgarno sequence (SD) of the cel gene are important for CsrA binding. Substitution mutations at SD relieved CsrA-dependent repression of the cel gene in vivo. Steady-state levels and half-life of the cel mRNA were not affected by CsrA, implying that regulation is mediated at the translational level. Levels of CsrB and CsrC sRNAs, which bind to and antagonize CsrA, were drastically reduced upon induction of the SOS response, while the CsrA protein itself remained unaffected. Thus, CsrA is a trans-acting modulator that downregulates the expression of lysis protein, which may confer a survival advantage on colicinogenic E. coli under environment stress conditions.

PAL31 may play an important role as inflammatory modulator in the repair process of the spinal cord injury rat.

Functional regeneration in a complete T8 transection model Cheng et al. (1996) and most recently, acidic fibroblast growth factor (aFGF; also known as FGF-1) involved in the repair process of the spinal cord injury (SCI) rat Tsai et al. (2008) have been reported. To further reveal the mechanism of the repair process of SCI, in additionally, we have identified a 30 kDa specific protein kinase A substrate induced at 6 days after SCI. However, the induction of the transducing signal was reduced in samples treated with aFGF. The 30 kDa protein was purified and identified by mass spectrometry as a novel protein, PAL31. The results of immunohistochemical study showed that PAL31 is abundantly expressed in the epicenter of the injured spinal cord and colocalizes with ED1-positive cells (macrophages) and CD8 T lymphocytes. Over-expression of PAL31 in RAW 264.7 cells resulted in the down-regulation of macrophage chemoattractant protein 1, inducible nitric oxide synthase, and signal transducer and activator of transcription-1. However, knockdown of PAL31 by small interfering RNA seems to lead to apoptosis when the cells were treated with inflammatory inducers. These experimental results suggest that PAL31 may involve in the modulation of the inflammatory response and, at the same time, prevent apoptosis process of macrophage after SCI.

Crystal structure of Escherichia coli PNPase: central channel residues are involved in processive RNA degradation.

Bacterial polynucleotide phosphorylase (PNPase) plays a major role in mRNA turnover by the degradation of RNA from the 3'- to 5'-ends. Here, we determined the crystal structures of the wild-type and a C-terminal KH/S1 domain-truncated mutant (DeltaKH/S1) of Escherichia coli PNPase at resolutions of 2.6 A and 2.8 A, respectively. The six RNase PH domains of the trimeric PNPase assemble into a ring-like structure containing a central channel. The truncated mutant DeltaKH/S1 bound and cleaved RNA less efficiently with an eightfold reduced binding affinity. Thermal melting and acid-induced trimer dissociation studies, analyzed by circular dichroism and dynamic light scattering, further showed that DeltaKH/S1 formed a less stable trimer than the full-length PNPase. The crystal structure of DeltaKH/S1 is more expanded, containing a slightly wider central channel than that of the wild-type PNPase, suggesting that the KH/S1 domain helps PNPase to assemble into a more compact trimer, and it regulates the channel size allosterically. Moreover, site-directed mutagenesis of several arginine residues in the channel neck regions produced defective PNPases that either bound and cleaved RNA less efficiently or generated longer cleaved oligonucleotide products, indicating that these arginines were involved in RNA binding and processive degradation. Taking these results together, we conclude that the constricted central channel and the basic-charged residues in the channel necks of PNPase play crucial roles in trapping RNA for processive exonucleolytic degradation.

Involvement of acidic fibroblast growth factor in spinal cord injury repair processes revealed by a proteomics approach.

Acidic fibroblast growth factor (aFGF; also known as FGF-1) is a potent neurotrophic factor that affects neuronal survival in the injured spinal cord. However, the pathological changes that occur with spinal cord injury (SCI) and the attribution to aFGF of a neuroprotective effect during SCI are still elusive. In this study, we demonstrated that rat SCI, when treated with aFGF, showed significant functional recovery as indicated by the Basso, Beattie, and Bresnahan locomotor rating scale and the combined behavior score (p < 0.01-0.001). Furthermore proteomics and bioinformatics approaches were adapted to investigate changes in the global protein profile of the damaged spinal cord tissue when experimental rats were treated either with or without aFGF at 24 h after injury. We found that 51 protein spots, resolvable by two-dimensional PAGE, had significant differential expression. Using hierarchical clustering analysis, these proteins were categorized into five major expression patterns. Noticeably proteins involved in the process of secondary injury, such as astrocyte activation (glial fibrillary acidic protein), inflammation (S100B), and scar formation (keratan sulfate proteoglycan lumican), which lead to the blocking of injured spinal cord regeneration, were down-regulated in the contusive spinal cord after treatment with aFGF. We propose that aFGF might initiate a series of biological processes to prevent or attenuate secondary injury and that this, in turn, leads to an improvement in functional recovery. Moreover the quantitative expression level of these proteins was verified by quantitative real time PCR. Furthermore we identified various potential neuroprotective protein factors that are induced by aFGF and may be involved in the spinal cord repair processes of SCI rats. Thus, our results could have a remarkable impact on clinical developments in the area of spinal cord injury therapy.

A pilot study for screening blood donors in Taiwan by nucleic acid amplification technology: detecting occult hepatitis B virus infections and closing the serologic window period for hepatitis C virus.

BACKGROUND: Blood donors in Taiwan currently are screened for hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) infection by immunoassay. The risk of enzyme immunoassay (EIA)-negative, nucleic acid amplification technology (NAT)-reactive donations is not well understood. This study aimed to screen for such donors in Taiwan by a multiplex test (cobas TaqScreen, Roche) on a commercially available NAT system (cobas s 201 system, Roche). STUDY DESIGN AND METHODS: NAT was performed on donors without prescreening in pools of six and NAT-reactive pools were then resolved to the single donation. Individual-donor NAT-reactive samples were discriminated by a commercially available polymerase chain reaction (PCR)-based diagnostic assay (COBAS AmpliScreen, Roche). Samples with EIA- and NAT-discordant results were investigated with supplemental serologic and confirmatory tests. Each sample taken from follow-up of HBV NAT yield cases was tested for HBV serologic profile, NAT, and viral load. The sensitivity and performance efficacy were also evaluated. RESULTS: The 95 percent limit of detection (LOD) for HBV, HCV, and HIV were 5.09, 11.83, and 62.53 IU per mL, respectively. Among 10,727 seronegative donations, 12 HBV NAT yield cases (0.11%) and 1 HCV NAT yield case (0.01%) were detected. Follow-up results for 1 to 8 months showed that the HCV yield case was a window case and all HBV NAT yield cases were occult carriers. CONCLUSION: The use of NAT detected occult HBV and reduced HCV window period. The yield rate, especially occult HBV, was 10- to 100-fold higher than that in developed, HBV nonendemic countries. Therefore, NAT implementation for routine donor screening in a more cost-effective manner should contribute to safer blood transfusion in Taiwan.

Three missense mutations, including a novel 860C>T transition, and allelic enhancement phenomenon associated with ABO blood subgroups A in Taiwan.

BACKGROUND: It was estimated that approximately 25 percent of Taiwanese residents were ABO blood group A. Many subgroups of A, however, revealed ambiguous serologic typing results. This study aimed to delineate the molecular basis of the A3, Ax, and weak A phenotypes. STUDY DESIGN AND METHODS: Serologic analyses including adsorption and elution assay, serum transferases activity assay, and saliva test were performed to determine the unique phenotypes of these samples. DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism were performed to further investigate the relationships between the genetic characteristics and phenotypic features of these samples. RESULTS: Three single-nucleotide transitions (745C>T, 820G>A, and a novel 860C>T) were found in nine A3/A3B cases. In addition, the Ax and A3B subjects shared the same 860C>T mutation. This A(x) allele with 860C>T transition expressed A3B phenotype in A(x)/B101 heterozygote but Ax phenotype in A(x)/O01 heterozygote. This allelic enhancement was also observed in the weak A family with Aw05 allele, which was previously not found in Taiwan. CONCLUSION: This allelic enhancement phenomenon was prone to cause serologic discrepancy between parents and children. Genotyping could help us to resolve this problem. Thus, a novel mutation is reported among Taiwanese blood donors.

The combination of peripheral nerve grafts and acidic fibroblast growth factor enhances arginase I and polyamine spermine expression in transected rat spinal cords.

Treatment with a combination of peripheral nerve grafts and acidic fibroblast growth factor improves hind limb locomotor function after spinal cord transection. This study examined the effect of treatment on expression of arginase I (Arg I) and polyamines. Arg I expression was low in the spinal cords of normal rats but increased following spinal injury. Only fully repaired spinal cords expressed higher Arg I levels 6-14 days following repair. In 10-day repaired spinal cords, high Arg I immunoreactivity was detected in motoneurons and alternatively activated macrophages in the graft area and graft-stump edges, and high levels of the polyamine spermine were expressed by macrophages within the intercostal nerve graft. Thus, in addition to enhancing the expression of Arg I and spermine in repaired spinal cords, our treatment may recruit activated macrophages and create a more favorable environment for axonal regrowth.

A sequence-specific RNase activity derived from the interface of the dimeric immunity protein of the ColE7 operon.

Recently, two sequence-specific cleavage sites were found in the ceiE7 gene of the cea-cei-cel polycistronic transcript from the ColE7 operon. The crystal structure of the ColE7 immunity protein (ImE7) suggested that a novel ribonuclease active site is created at the interface of the dimeric structure of the protein. Frame shift mutation of the ceiE7 gene and mutation of histidine residues at the putative active site of the dimeric ImE7 protein respectively abolished and significantly reduced the observed ribonucleolytic cleavage indicating that the dimeric ImE7 protein is indeed involved in this sequence-specific cleavage at the ceiE7 mRNA. It is noteworthy that E. coli S-30 cell extracts must be added to the in vitro reactions in order to detect this ribonucleolytic cleavage. In addition, mutation of the T1 stem-loop structure located between the ceiE7 and the celE7 genes completely turned off the ribonuclease activity in vivo, implying that the T1 stem-loop structure might participate in mediating the formation of a degradosome-like complex required for this specific ribonucleolytic activity.

An E. coli lon mutant conferring partial resistance to colicin may reveal a novel role in regulating proteins involved in the translocation of colicin.

Initially, we found that a lon mutant confers partial resistance against colicin. The results of Western blotting detected a decrease in the protein expression levels of BtuB and OmpF involved in colicin translocation in the lon mutant. Moreover, 2-D gel analysis revealed that the expression level of some scavenger proteins marks the lon mutant as being in a situation similar to oxidative stress. OxyRS and SoxRS are the two major response regulators for oxidative stress. Our RT-PCR analysis revealed an elevation of expression of the oxyS gene in the lon mutant. An immunoblot assay further confirmed that overexpression of oxyS RNA can negatively control on the expression of BtuB protein. Probably the BtuB is negatively regulated by a global regulator, oxyS, induced during oxidative stress.

The critical roles of polyamines in regulating ColE7 production and restricting ColE7 uptake of the colicin-producing Escherichia coli.

The ColE7 operon is an SOS response regulon, which encodes bacteriocin ColE7 to kill susceptible Escherichia coli and its related enterobacteria under conditions of stress. We have observed for the first time that polyamines confer limited resistance against ColE7 on E. coli cells. Thus, this study aims to investigate the role of polyamines in modulating the protective effect of the E. coli cells against colicin. In the experiments, we surprisingly found that endogenous polyamines are also essential for ColE7 production, and the rate of polyamine synthesis is directly related to the SOS response. Our experimental results further indicated that exogenous polyamines suppress the expression of TolA, BtuB, OmpF, and OmpC proteins that are responsible for ColE7 uptake. Moreover, two-dimensional gel electrophoresis revealed that the production of two periplasmic proteins, PotD and OppA, is increased in E. coli cells under ColE7 exposure. Based on these observations, we propose that endogenous polyamines may play a dual role in the ColE7 system. Polyamines may participate in initiating the expression of the SOS response of the ColE7 operon and simultaneously down-regulate proteins that are essential for colicin uptake, thus conferring a survival advantage on colicin-producing E. coli under stress conditions in the natural environment.

High-resolution crystal structure of a truncated ColE7 translocation domain: implications for colicin transport across membranes.

ColE7 is a nuclease-type colicin released from Escherichia coli to kill sensitive bacterial cells by degrading the nucleic acid molecules in their cytoplasm. ColE7 is classified as one of the group A colicins, since the N-terminal translocation domain (T-domain) of the nuclease-type colicins interact with specific membrane-bound or periplasmic Tol proteins during protein import. Here, we show that if the N-terminal tail of ColE7 is deleted, ColE7 (residues 63-576) loses its bactericidal activity against E.coli. Moreover, TolB protein interacts directly with the T-domain of ColE7 (residues 1-316), but not with the N-terminal deleted T-domain (residues 60-316), as detected by co-immunoprecipitation experiments, confirming that the N-terminal tail is required for ColE7 interactions with TolB. The crystal structure of the N-terminal tail deleted ColE7 T-domain was determined by the multi-wavelength anomalous dispersion method at a resolution of 1.7 angstroms. The structure of the ColE7 T-domain superimposes well with the T-domain of ColE3 and TR-domain of ColB, a group A Tol-dependent colicin and a group B TonB-dependent colicin, respectively. The structural resemblance of group A and B colicins implies that the two groups of colicins may share a mechanistic connection during cellular import.

Identification of an essential cleavage site in ColE7 required for import and killing of cells.

Colicin E7 (ColE7), a nuclease toxin released from Escherichia coli, kills susceptible bacteria under environmental stress. Nuclease colicins are processed during translocation with only the cytotoxic nuclease domains traversing the inner membrane to cleave tRNA, rRNA, or DNA in the cytoplasm of target cells. In this study, we show that the E. coli periplasmic extract cleaves ColE7 between Lys(446) and Arg(447) in the presence or absence of its inhibitor Im7 protein. Several residues near cleavage sites were mutated, but only mutants of Arg(447) completely lost in vivo cell-killing activity. Both the full-length and the nuclease domain of Arg(447) mutants retained their nuclease activities, indicating that failure to kill cells was not a consequence of damage to the endonuclease activity of the enzyme. Moreover, the R447E ColE7 mutant was not cleaved at its 447 site by periplasmic extracts or transported into the cytoplasm of target cells. Collectively, these results suggest that ColE7 is cleaved at Arg(447) during translocation and that cleavage is an essential step for ColE7 import into the cytoplasm of target cells and its cell-killing activity. Conserved basic residues aligned with Arg(447) have also been found in other nuclease colicins, implying that the processing at this position may be common to other colicins during translocation.

Involvement of colicin in the limited protection of the colicin producing cells against bacteriophage.

The restriction/modification system is considered to be the most common machinery of microorganisms for protection against bacteriophage infection. However, we found that mitomycin C induced Escherichia coli containing ColE7-K317 can confer limited protection against bacteriophage M13K07 and lambda infection. Our study showed that degree of protection is correlated with the expression level of the ColE7 operon, indicating that colicin E7 alone or the colicin E7-immunity protein complex is directly involved in this protection mechanism. It was also noted that the degree of protection is greater against the single-strand DNA bacteriophage M13K07 than the double-strand bacteriophage(lambda). Coincidently, the K(A) value of ColE7-Im either interacting with single-strand DNA (2.94x10(5)M(-1)) or double-strand DNA (1.75x10(5)M(-1)) reveals that the binding affinity of ColE7-Im with ssDNA is 1.68-fold stronger than that of the protein complex interacting with dsDNA. Interaction between colicin and the DNA may play a central role in this limited protection of the colicin-producing cell against bacteriophages. Based on these observations, we suggest that the colicin exporting pathway may interact to some extent with the bacteriophage infection pathway leading to a limited selective advantage for and limited protection of colicin-producing cells against different bacteriophages.