Faculty Bibliography

A chimeric gene mHG (669 bp) was constructed by substitution of Clostridium thermocellum ZJL4 lichenase (CG) N-terminal fragment (except its signal sequence) for the counterpart of Bacillus sp. A3 lichenase (BG). To acquire high-level secretion of the chimeric lichenase (mHG) in Bacillus subtilis, a series of site-mutated signal peptides were designed. The activity of mHG, which was directed by an artificial hydrophobic signal peptide H1 (MMARKIAGMATSLLVIFSSSAVA) from cytoplasm into growth medium, reached 80.56 U/ml after 22-h incubation, indicating that signal peptide hydrophobicity appears to be critical for early stages in mHG export. By purification of the mHG (approximately 25.3 kDa) from cultures of B. subtilis (pBSG-H1), enzymatic property assays showed that the common optima for mHG were 70 degrees C and pH 5.0. The residual activity of mHG at 90 degrees C for 10 min was 83.45% of its maximum activity, which was almost similar to that of CG (90 degrees C, 10 min, 84.33%). This constructed shuttle expression vector with a novel signal peptide exhibited its applicability for high-level production of heterologous proteins from B. subtilis. Moreover, the high-level secreted mHG with relatively high thermostability could be a potential candidate for feed industrial applications

Gammaherpesviruses, including the human pathogens Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, are causative agents of lymphomas and other malignancies. The structural characterization of these viruses has been limited due to difficulties in obtaining adequate amount of virion particles. Here we report the first three-dimensional structural characterization of a whole gammaherpesvirus virion by an emerging integrated approach of cryo-electron tomography combined with single-particle cryo-electron microscopy, using murine gammaherpesvirus-68 (MHV-68) as a model system. We found that the MHV-68 virion consists of distinctive envelope and tegument compartments, and a highly conserved nucleocapsid. Two layers of tegument are identified: an inner tegument layer tethered to the underlying capsid and an outer, flexible tegument layer conforming to the overlying, pleomorphic envelope, consistent with the sequential viral tegumentation process inside host cells. Surprisingly, comparison of the MHV-68 virion and capsid reconstructions shows that the interactions between the capsid and inner tegument proteins are completely different from those observed in alpha and betaherpesviruses. These observations support the notion that the inner layer tegument across different subfamilies of herpesviruses has evolved significantly to confer specific characteristics related to viral-host interactions, in contrast to a highly conserved capsid for genome encapsidation and protection

PURPOSE: To explore and evaluate the protective effect of erythropoietin (EPO) on retinal cells of chemically induced diabetic rats after EPO was injected intravitreally at the onset of diabetes. METHODS: Diabetes was induced in Sprague-Dawley rats by intraperitoneal injection of streptozotocin (STZ). At the onset of diabetes, a single intravitreal injection of EPO (0.05-200 ng/eye) was performed. In the following 6 weeks, the blood retinal barrier (BRB) was evaluated by Evans blue permeation (EBP). Retinal cell death in different layers was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The retinal thickness and cell counts were examined at the light microscopic level. Electron microscopy (EM) was used to scrutinize retinal vascular and neuronal injury. Neurosensory retinas of normal and diabetic rats were used as the sources of reverse transcription-polymerase chain reaction (RT-PCR) and Western blot for the detection of EPO, EPO receptor (EpoR), and products of the extracellular signal-regulated kinase (ERK) and the signal transducers and activators of transcription 5 (STAT5) pathways. The distribution of EpoR in retinal layers was demonstrated by immunohistochemistry (IHC). RESULTS: In the diabetic rats, BRB breakdown was detected soon after the onset of diabetes, peaked at 2 weeks, and reached a plateau at 2 to 4 weeks. The number of TUNEL-positive cells increased in the neurosensory retina, especially, the outer nuclear layer (ONL) at 1 week after diabetes onset and reached a peak at 4 to 6 weeks. The retinal thickness and the number of cells in the ONL were reduced significantly. EM observations demonstrated vascular and photoreceptor cell death starting soon after the onset of diabetes. All these changes were largely prevented by EPO treatment. Upregulation of EpoR in the neurosensory retina was detected at both the transcriptional and protein levels 4 to 8 weeks after the onset of diabetes, whereas, the endogenous EPO levels of neurosensory retinas were essentially unchanged during the same period observed. In EPO-treated diabetic groups, EpoR expression remained at upregulated levels. Within 2 weeks of the onset of diabetes, activation of the ERK but not the STAT5 pathway was detected in the diabetic retina treated with EPO. CONCLUSIONS: These data demonstrate that apoptosis is an major contributor to neuronal cell death in the early course of diabetic retinopathy (DR). The upregulation of EpoR may be a compensatory response of retinal cells and tissue to diabetic stresses. The EPO/EpoR system as a maintenance-survival mechanism of retinal neurons responds to the insults of early diabetes other than ischemia. The protective function of EPO/EpoR at the least acts through the EpoR-mediated ERK pathway. Exogenous EPO administration by intravitreal injection in early diabetes may prevent retinal cell death and protect the BRB function. Therefore, this is a novel approach for treatment of early DR

Polo-like kinase 3 (Plk3) activation occurs after exposure to environmental or genotoxic stresses. Plk3 regulates cell fate through regulating cell cycle progression. UV irradiation is one of the major environmental stresses that affect corneal epithelial wound healing. In the present study, we report that UV irradiation activated Plk3 and that Plk3 interacts with AP-1 and c-Jun, which appears to be important to mediate corneal epithelial cell apoptosis after UV irradiation. Recombinant Plk3, as well as Plk3 immunoprecipitated from UV-irradiated cells, phosphorylated c-Jun in vitro. The phosphorylation of c-Jun by Plk3 immunoprecipitates was not altered by the pre-removal of JNK from the cell lysates. In addition, the effect of UV irradiation-induced phosphorylation of c-Jun and apoptosis were not significantly affected by knockdown of JNK mRNA. Co-immunoprecipitation reveals that Plk3 and c-Jun directly interacted with each other. Consistently, Plk3 co-localized with c-Jun to the nucleus after UV irradiation. Further, modulating Plk3 activities by overexpressing Plk3 or its mutants significantly affected UV irradiation-induced c-Jun activity and subsequent apoptosis. Our results thus provide for the first time that Plk3 mediates UV irradiation-induced c-Jun activation by phosphorylating c-Jun, suggesting that Plk3 plays an important role in mediating programmed cell death of corneal epithelial cells after UV irradiation

The GABAC receptor/channel rho1 subunit plays an important role in the inhibitory pathway and sensory processing in the retina and spinal cord. Although it was suggested that the rho1 subunit plays a role in olfactory sensations, the precise role of the rho1 subunit in olfactory sensory function is still not clear. In the present study, we report that olfactory function was significantly altered in rho1 subunit knockout (rho1-/-) mice compared to its wildtype counterpart. The rho1 subunit mRNA, detected by reverse transcription (RT)-PCR experiments, was expressed in the olfactory bulb of wild-type mice. Expression of rho1 subunit proteins in the olfactory bulb was detected by immunohistochemistry in mitral cells in the mitral cell layer. Neither mRNA nor proteins of the rho1 subunit were found in olfactory bulb neurons in rho1-/- mice. Alterations of olfactory function in rho1-/- mutant mice compared to their wildtype littermates were examined by olfactory behavioral test. We found that sensitivity to the smell of citral odorant in rho1-/- mice was significantly greater compared to that of wildtype mice. Our results indicate that the GABAC rho1 subunit acts in olfactory bulb neurons as an inhibitory modulator that affects the process of olfactory signaling transmission

RIG-I (retinoid acid-inducible gene-I), a putative RNA helicase with a cytoplasmic caspase-recruitment domain (CARD), was identified as a pattern-recognition receptor (PRR) that mediates antiviral immunity by inducing type I interferon production. To further study the biological function of RIG-I, we generated Rig-I(-/-) mice through homologous recombination, taking a different strategy to the previously reported strategy. Our Rig-I(-/-) mice are viable and fertile. Histological analysis shows that Rig-I(-/-) mice develop a colitis-like phenotype and increased susceptibility to dextran sulfate sodium-induced colitis. Accordingly, the size and number of Peyer's patches dramatically decreased in mutant mice. The peripheral T-cell subsets in mutant mice are characterized by an increase in effector T cells and a decrease in naive T cells, indicating an important role for Rig-I in the regulation of T-cell activation. It was further found that Rig-I deficiency leads to the downregulation of G protein alpha i2 subunit (G alpha i2) in various tissues, including T and B lymphocytes. By contrast, upregulation of Rig-I in NB4 cells that are treated with ATRA is accompanied by elevated G alpha i2 expression. Moreover, G alpha i2 promoter activity is increased in co-transfected NIH3T3 cells in a Rig-I dose-dependent manner. All these findings suggest that Rig-I has crucial roles in the regulation of G alpha i2 expression and T-cell activation. The development of colitis may be, at least in part, associated with downregulation of G alpha i2 and disturbed T-cell homeostasis

This review covers the topic of cytometric assessment of activation of Ataxia telangiectasia mutated (ATM) protein kinase and histone H2AX phosphorylation on Ser139 in response to DNA damage, particularly the damage that involves formation of DNA double-strand breaks. Briefly described are molecular mechanisms associated with activation of ATM and the downstream events that lead to recruitment of DNA repair machinery, engagement of cell cycle checkpoints, and activation of apoptotic pathway. Examples of multiparameter analysis of ATM activation and H2AX phosphorylation vis-a-vis cell cycle phase position and induction of apoptosis that employ flow- and laser scanning-cytometry are provided. They include cells treated with a variety of exogenous genotoxic agents, such as ionizing and UV radiation, DNA topoisomerase I (topotecan) and II (mitoxantrone, etoposide) inhibitors, nitric oxide-releasing aspirin, DNA replication inhibitors (aphidicolin, hydroxyurea, thymidine), and complex environmental carcinogens such as present in tobacco smoke. Also presented is an approach to identify DNA replicating (BrdU incorporating) cells based on selective photolysis of DNA that triggers H2AX phosphorylation. Listed are strategies to distinguish ATM activation and H2AX phosphorylation induced by primary DNA damage by genotoxic agents from those effects triggered by DNA fragmentation that takes place during apoptosis. While we review most published data, recent new findings also are included. Examples of multivariate analysis of ATM activation and H2AX phosphorylation presented in this review illustrate the advantages of cytometric flow- and image-analysis of these events in terms of offering a sensitive and valuable tool in studies of factors that induce DNA damage and/or affect DNA repair and allow one to explore the linkage between DNA damage, cell cycle checkpoints and initiation of apoptosis

Disruption of microtubules activates the spindle checkpoint, of which BubR1 is a major component. Our early studies show that BubR1 haplo-insufficiency results in enhanced mitotic slippage in vitro and tumorigenesis in vivo. OBJECTIVE: Given that both MAPKs/ERKs and MEK play an important role during mitosis, we investigated whether there existed regulatory relationship between the MAPK signalling pathway and BubR1. METHOD AND RESULTS: Here, we have demonstrated that BubR1 deficiency is correlated with enhanced activation of MEK and ERKs after disruption of microtubule dynamics. Specifically, treatment with nocodazole and paclitaxel resulted in hyper-activation of ERKs and MEK in BubR1(+/-) murine embryonic fibroblasts (MEF) compared to that of wild-type MEFs. This enhanced activation of ERKs and MEK was at least partly responsible for more successful proliferation completion when cells were treated with nocodazole. BubR1 knockdown via RNAi resulted in enhanced activation of ERKs and MEK in HeLa cells, correlating with inhibition of PP1, a negative regulator of MEK. Moreover, when BubR1 was partially inactivated due to premature missegregation of chromosomes after Sgo1 depletion, phosphorylation of ERKs and MEK was enhanced in mitotic cells; in contrast, little, if any activated ERKs and MEK were detected in mitotic cells induced by nocodazole. Furthermore, BubR1, activated ERKs and activated MEK all localized to spindle poles during mitosis, and also, the proteins physically interacted with each other. CONCLUSION: Our studies suggest that there exists a cross-talk between spindle checkpoint components and ERKs and MEK and that BubR1 may play an important role in mediating the cross-talk

OBJECTIVE: The exposure of mammalian cells to extracellular stress induces the expression of immediate early genes such as c-fos and c-jun and activates transcription factor activator protein-1 (AP-1). The purpose of the current study was to investigate the role of c-Fos and JunD in stress-induced cell death. MATERIALS AND METHODS: We exposed cultured primary mouse embryonic fibroblasts (MEF) to ultraviolet light (UV-C) or hydrogen peroxide (H(2)O(2)). Induction of c-Fos and JunD and activation of MAPK/ERK1/2 signalling in the presence or absence of a MAPK inhibitor were analyzed by western blotting. Activation of AP-1 transcription factors was detected by the electrophoretic mobility shift assay and immunoprecipitation. Cell death was measured by changes in caspase 3 activities and nuclear morphology. Effects of c-Fos and JunD expression on cell death were investigated by transfection. RESULTS: We found that the exposure of cultured primary MEF cells to UV or H(2)O(2) caused a significant increase in c-Fos and JunD protein levels. In addition, these two proteins formed complexes with each other and contributed to activation of AP-1 transcription complexes. More importantly, under both stress conditions, overexpression of JunD alone or overexpression of both c-Fos and JunD reduced caspase 3 activity and cell death. At the same time, UV irradiation activated the MAPK/ERK1/2 signalling pathway. The suppression of MEK1/ERK1/2 activation inhibited UV-induced expression of c-Fos and JunD and increased caspase 3 activity and cell death. CONCLUSION: Our results suggest that both UV and H(2)O(2 )induce the activation of c-Fos/JunD AP-1 complexes resulting in the prevention of cell death. Moreover, UV irradiation-induced increases in c-Fos/JunD expression in primary MEF cells are mediated through the activation of the MAPK/ERK1/2 signalling pathway

Polo-like kinases play crucial roles throughout mitosis. We previously reported that wortmannin potently inhibits Polo-like kinase 1 (Plk1). In this study, we show that wortmannin also strongly inhibits Polo-like kinase 3 (Plk3). To further characterize this inhibition, we identified the sites of labeling on Plk1 and Plk3 targeted by AX7503, a tetramethylrhodamine-wortmannin conjugate. AX7503 labeling on Plk1 and Plk3 was found to occur on a conserved ATP binding site residue. In addition, we show that wortmannin inhibits Plk3 activity in live cells at concentrations commonly used to inhibit the more well known targets of wortmannin, the phosphoinositide 3-kinases. Importantly, we found that inhibition of Plk3 by wortmannin lead to a decrease in phosphorylation of p53 on serine 20 induced by DNA damage, demonstrating the effect of wortmannin on a downstream Plk3 target. Taken together, our results suggest that wortmannin can affect multiple functions of Plk3 in cell cycle progression and at the DNA damage check point. The identification of the labeling sites of Plk1 and Plk3 by AX7503 may be useful in designing more effective compounds to target Polo-like kinases for cancer treatment and also may be useful for the structural study of Plk domains