Faculty Bibliography

Post-translational modifications of histones play a critical role in regulating genome structures and integrity. We have focused on the regulatory relationship between covalent modifications of histone H3 lysine 9 (H3K9) and H3S10 during the cell cycle. Immunofluorescence microscopy revealed that H3S10 phosphorylation in HeLa, A549, and HCT116 cells was high during prophase, prometaphase, and metaphase, whereas H3K9 monomethylation (H3K9me1) and dimethylation (H3K9me2), but not H3K9 trimethylation (H3K9me3), were significantly suppressed. When H3S10 phosphorylation started to diminish during anaphase, H3K9me1 and H3K9me2 signals reemerged. Western blot analyses confirmed that mitotic histones, extracted in an SDS-containing buffer, had little H3K9me1 and H3K9me2 signals but abundant H3K9me3 signals. However, when mitotic histones were extracted in the same buffer without SDS, the difference in H3K9me1 and H3K9me2 signals between interphase and mitotic cells disappeared. Removal of H3S10 phosphorylation by pretreatment with lambda-phosphatase unmasked mitotic H3K9me1 and H3K9me2 signals detected by both fluorescence microscopy and Western blotting. Further, H3S10 phosphorylation completely blocked methylation of H3K9 but not demethylation of the same residue in vitro. Given that several conserved motifs consisting of a Lys residue immediately followed by a Ser residue are present in histone tails, our studies reveal a potential new mechanism by which phosphorylation not only regulates selective access of methylated lysines by cellular factors but also serves to preserve methylation patterns and epigenetic programs during cell division

Polo-like kinase 3 (Plk3) is an important mediator of the cellular responses to genotoxic stresses. In this study, we examined the physiologic function of Plk3 by generating Plk3-deficient mice. Plk3(-/-) mice displayed an increase in weight and developed tumors in various organs at advanced age. Many tumors in Plk3(-/-) mice were large in size, exhibiting enhanced angiogenesis. Plk3(-/-) mouse embryonic fibroblasts were hypersensitive to the induction of hypoxia-inducible factor-1 alpha (HIF-1 alpha) under hypoxic conditions or by nickel and cobalt ion treatments. Ectopic expression of the Plk3-kinase domain (Plk3-KD), but not its Polo-box domain or a Plk3-KD mutant, suppressed the nuclear accumulation of HIF-1 alpha induced by nickel or cobalt ions. Moreover, hypoxia-induced HIF-1 alpha expression was tightly associated with a significant down-regulation of Plk3 expression in HeLa cells. Given the importance of HIF-1 alpha in mediating the activation of the 'survival machinery' in cancer cells, these studies strongly suggest that enhanced tumorigenesis in Plk3-null mice is at least partially mediated by a deregulated HIF-1 pathway

OBJECTIVE: To observe the presence of bacterial biofilms in mucosal specimens in patients operated for chronic rhinosinusitis. METHODS: A total of 12 subjects undergoing endoscopic sinus surgery were included. The control group was 6 patients with obstructive sleep apnea-hypopnea syndrome. Six patients with chronic rhinosinusitis were enrolled in the study group. Mucosa of uncinate process, ethmoid bulla or maxillary sinus was obtained during endoscopic sinus surgery. All the samples were prepared using standard methods for scanning electron microscopy. Patients' information such as age, gender, symptoms, sinus CT, endoscopic examination, skin prick test were recorded in detail. RESULTS: Standard-preparation scanning electron microscopy displaed denuded epithelium and disarrayed cilia in chronic rhinosinusitis patient's mucosa. In the study group, bacterial biofilms of different morphology were seen in five samples; One sample showed filamentous structure like fungi. Using strict scanning electron microscopy morphologic criteria, 83.3% samples in the study group were found to have micrographic evidence of biofilms. No bacterial biofilms were detected in the control group. CONCLUSIONS: In the patients undergoing surgery for chronic rhinosinusitis, different degree of mucosal injury could be found. Bacteria biofilms of different life stages were demonstrated to be present. No bacterial biofilms were detected in the control group

To obtain single chain variable fragment (scFv) and bivalent single chain variable fragment (bsFv) against transferrin receptor, up-stream and down-stream primers were designed according to the complementary sequences of FR1 region of variable heavy (VH) and FR4 of variable light (VL), respectively, which contained inter-linker G4S and the restriction endonuclease SfiI, AscI and NotI. Two pieces of scFv fragments were first amplified through PCR and then inserted into plasmid pAB1, which could express scFv protein once induced by IPTG in the host bacteria. To express scFv and bsFv, E. coli TG1 was cultured in LB broth and was induced by IPTG. The restriction enzyme digestion map and DNA sequencing demonstrated that scFv and bsFv genes were successfully inserted into the expression plasmid. SDS-PAGE and Western blotting revealed the protein band at 35kD and 60kD, which were consistent with the molecular weight of scFv and bsFv respectively. Flow cytometry showed that scFv and bsFv harbored the specific binding activity with TfR expressed in various tumor cells, and the avidity of bsFv was higher than that of the parent scFv

AIM: To study the pharmacokinetics and toxicity of intravitreal erythropoietin (EPO) for potential clinical use. METHODS: For toxicity study, 4 groups (60 rabbits) with intravitreal injection (IVit) of EPO were studied (10 U, 100 U, or 1,000 U) per eye for single injection and 0.6 U/eye (the designed therapeutic level in rabbits) for monthly injections (6X). Eye examination, flash electroretinogram (ERG), and fluorescein angiography (FA) were carried out before and after injection. The rabbits were killed for histological study at different intervals. For the pharmacokinetic study, after IVit of 5 U EPO into left eyes, 44 rabbits were killed at different intervals, and the EPO levels in vitreous, aqueous, retina and serum were analyzed by enzyme-linked immunosorbent assay. RESULTS: At all of the time points examined, the eyes were within normal limits. No significant ERG or FA change was observed. The histology of retina remained unchanged. The pharmacokinetic profile of EPO in ocular compartments was summarized as follows. The half-life times of EPO in vitreous, aqueous and serum were 2.84, 3.24 and 2.12 d, respectively; and Cmax were 4615.75, 294.31 and 1.60 U/L, respectively. EPO concentrations in the retina of the injected eye peaked at 1.36 U/g protein at 6 h following injection, with the half-life observed to be 3.42 d. CONCLUSIONS: IVit of EPO in a wide range is well tolerated and safe for rabbit eyes. At doses up to 10-fold higher than therapeutic levels, EPO has a pharmacokinetic profile with faster clearance, which is favorable for episodic IVit

BACKGROUND: Hypermethylation of promoter CpG islands is strongly correlated to transcriptional gene silencing and epigenetic maintenance of the silenced state. As well as its role in tumor development, CpG island methylation contributes to the acquisition of resistance to chemotherapy. Differential Methylation Hybridisation (DMH) is one technique used for genome-wide DNA methylation analysis. The study of such microarray data sets should ideally account for the specific biological features of DNA methylation and the non-symmetrical distribution of the ratios of unmethylated and methylated sequences hybridised on the array. We have therefore developed a novel algorithm tailored to this type of data, Methylation Linear Discriminant Analysis (MLDA). RESULTS: MLDA was programmed in R (version 2.7.0) and the package is available at CRAN 1. This approach utilizes linear regression models of non-normalised hybridisation data to define methylation status. Log-transformed signal intensities of unmethylated controls on the microarray are used as a reference. The signal intensities of DNA samples digested with methylation sensitive restriction enzymes and mock digested are then transformed to the likelihood of a locus being methylated using this reference. We tested the ability of MLDA to identify loci differentially methylated as analysed by DMH between cisplatin sensitive and resistant ovarian cancer cell lines. MLDA identified 115 differentially methylated loci and 23 out of 26 of these loci have been independently validated by Methylation Specific PCR and/or bisulphite pyrosequencing. CONCLUSION: MLDA has advantages for analyzing methylation data from CpG island microarrays, since there is a clear rational for the definition of methylation status, it uses DMH data without between-group normalisation and is less influenced by cross-hybridisation of loci. The MLDA algorithm successfully identified differentially methylated loci between two classes of samples analysed by DMH using CpG island microarrays

Hypoxia/reoxygenation stress induces the activation of specific signaling proteins and activator protein 1 (AP-1) to regulate cell cycle regression and apoptosis. In the present study, we report that hypoxia/reoxygenation stress activates AP-1 by increasing c-Jun phosphorylation and DNA binding activity through activation of Polo-like-kinase 3 (Plk3) resulting in apoptosis. The specific effect of hypoxia/reoxygenation stress on Plk3 activation resulting in c-Jun phosphorylation was the opposite of UV irradiation-induced responses that are meanly independent on activation of the stress-induced JNK signaling pathway in human corneal epithelial (HCE) cells. The effect of hypoxia/reoxygenation stress-induced Plk3 activation on increased c-Jun phosphorylation and apoptosis was also mimicked by exposure of cells to CoCl(2). Hypoxia/reoxygenation activated Plk3 in HCE cells to directly phosphorylate c-Jun proteins at phosphorylation sites Ser-63 and Ser-73, and to increase DNA binding activity of c-Jun, detected by EMSA. Further evidence demonstrated that Plk3 and phospho-c-Jun were immunocolocalized in the nuclear compartment of hypoxia/reoxygenation stress-induced cells. Increased Plk3 activity by overexpression of wild-type and dominantly positive Plk3 enhanced the effect of hypoxia/reoxygenation on c-Jun phosphorylation and cell death. In contrast, knocking-down Plk3 mRNA suppressed hypoxia-induced c-Jun phosphorylation. Our results provide a new mechanism indicating that hypoxia/reoxygenation induces Plk3 activation instead of the JNK effect to directly phosphorylate and activate c-Jun, subsequently contributing to apoptosis in HCE cells

A discovery that catalytic reactivity is intensified on reducing the size of reactor toward several nanometers scale was applied for the separation of dilute species, for example, the thiophenic compounds in transportation fuels, in the present study. A characteristic reaction of the dilute species was selected as the separation mechanism, and a nanoreactor was formed with preloading reactant/catalyst in volumes of mesopore dimension. Probability for the dilute species to contact the nanoreactor radically enlarged due to the integration of such volumes in a porous material. Because all reagents and the reaction product stayed inside the nanoreactor, separation of fuel from sulfuric compounds and the surplus chemicals used in reaction becomes simple. It was experimentally shown that the nanoreaction exhibited first-order kinetics, and all thiophenes and benzothiophenes contained in different types of model fuels were completely removed at moderate conditions

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

Identifying the sources of volatile organic compounds (VOCs) is key to reducing ground-level ozone and secondary organic aerosols (SOAs). Several receptor models have been developed to apportion sources, but an intercomparison of these models had not been performed for VOCs in China. In the present study, we compared VOC sources based on chemical mass balance (CMB), UNMIX, and positive matrix factorization (PMF) models. Gasoline-related sources, petrochemical production, and liquefied petroleum gas (LPG) were identified by all three models as the major contributors, with UNMIX and PMF producing quite similar results. The contributions of gasoline-related sources and LPG estimated by the CMB model were higher, and petrochemical emissions were lower than in the UNMIX and PMF results, possibly because the VOC profiles used in the CMB model were for fresh emissions and the profiles extracted from ambient measurements by the two-factor analysis models were 'aged'