http://dspace.cityu.edu.hk:80/handle/2031/703 Wed, 01 May 2013 03:06:35 GMT 2013-05-01T03:06:35Z http://dspace.cityu.edu.hk:80/handle/2031/6587 Title: Effects of temperature, salinity and food availability on two subtidal nassariid gastropods, Nassarius siquijorensis and Nassarius conoidalis Authors: Zhao, Qian ( 趙倩) Abstract: Owing to degradation of seabed communities by environmental stresses and human perturbations, species which have specialized diets in the local marine benthic community are gradually replaced by opportunistic species of which a number of them are scavengers. This study investigated how three major environmental factors, temperature, salinity and food availability, affected the survival, development, behaviour and physiology of the embryos, larvae and adults of two dominant subtidal scavenging gastropods, Nassarius siquijorensis and Nassarius conoidalis. Mortality and developmental rate of the embryos of N. siquijorensis and N. conoidalis were investigated at nine combinations of temperature (20, 25, and 30°C) and salinity (20, 25, 30‰). The median hatching time (HD50) was calculated using the standard logistic regression. Temperature, salinity, as well as the interaction between temperature and salinity had significant effects on both survival and HD50 for both N. siquijorensis and N. conoidalis with highest survival and fastest development at 30°C and 30‰. Larval mortality at six temperatures (10, 15, 20, 25, 30 and 35°C) and seven salinities (0, 5, 10, 15, 20, 25 and 30‰) were observed after 24 hrs and 48 hrs. Both N. siquijorensis and N. conoidalis larvae showed higher mortalities at low temperatures and salinities, especially at 10°C or at 5‰, with more than half of them died after 48 hrs. The larval mortality of N. siquijorensis under low temperatures and salinities was significantly lower than that of N. conoidalis. Respiration rate, swimming velocity, and clearance rate of the larvae of N. siquijorensis and N. conoidalis were measured at nine combinations of temperature (20, 25, and 30°C) and salinity (20, 25, 30‰). Temperature, salinity and the interaction between temperature and salinity had significant effects on oxygen consumption, clearance rate and swimming behaviour of both species. Significantly lower clearance rates and oxygen consumption rates were observed at lower salinities and temperatures, and the dispersal distance VSL was significantly higher at lower salinities. The effects of starvation on respiration, ammonia excretion, shell length, body weight, scope for growth (SFG) and body chemical composition of N. siquijorensis and N. conoidalis were studied for 32 days. During starvation, significant decreases in respiration rate and ammonia excretion rate were observed for both N. siquijorensis and N. conoidalis. Starvation, time, and the interaction between starvation and time were found to be significant in affecting SFG for both N. siquijorensis and N. conoidalis. At the end of the experiment, the unfed individuals had significantly lower percentages of protein and fat but higher percentages of carbohydrate as compared with the control for both N. siquijorensis and N. conoidalis. Significant increases in wet weight and shell length were obtained from the control for both N. siquijorensis and N. conoidalis but the differences between species were insignificant. In another experiment in which the individuals were starved for 96 days, a significantly higher mortality was obtained for N. siquijorensis as compared with N. conoidalis. The effects of starvation and distance from carrion on chemoreception were compared between N. siquijorensis and N. conoidalis. The time to reach carrion was species specific and was significantly affected by both starvation and the distance from the carrion. Under well-fed condition, mean time to detect carrion at 15 cm and 20 cm away from carrion were significantly shorter in N. siquijorensis than N. conoidalis. For the starved group, the mean time to detect carrion at 2.5 cm and 10 cm away from carrion, however, was significantly shorter in N. conoidalis than N. siquijorensis. For N. conoidalis, starved individuals were more sensitive to carrion than well-fed ones with higher percentages of individuals able to locate the carrion at 10 cm, 15 cm and 20 cm away from the carrion. In summary, embryos and larvae of N. siquijorensis are more tolerant than N. conoidalis to temperature and salinity changes. This may help explain why N. siquijorensis has a wider distribution in Hong Kong waters as compared with N. conoidalis which is restricted to southern waters where salinity is relatively constant throughout the year. Both N. conoidalis and N. siquijorensis are generalist feeders adaptive to unpredictable food supply by reducing energy expenditure through decreasing respiration and ammonia excretion when starved. In recent years, they are gradually replacing species which have specialized diets in Hong Kong waters where the benthic environment suffers from various human disturbances such as eutrophication, trawling and hypoxia which directly or indirectly affects food availability. Nevertheless, N. siquijorensis grow faster than N. conoidalis when food availability is high while N. conoidalis perfom better under starvation. Their differential responses to food availability may help explain the dominance of N. siquijorensis in Hong Kong waters where the benthic environment is highly disturbed. Notes: CityU Call Number: QL430.5.N3 Z45 2011; xviii, 195 leaves : ill. (some col.) 30 cm.; Thesis (M.Phil.)--City University of Hong Kong, 2011.; Includes bibliographical references (leaves 160-193) Sat, 01 Jan 2011 00:00:00 GMT http://dspace.cityu.edu.hk:80/handle/2031/6587 2011-01-01T00:00:00Z http://dspace.cityu.edu.hk:80/handle/2031/6586 Title: Effects of andrographolide and taxifolin on cell proliferation, cell cycle progression and apoptosis of prostate carcinoma DU145 cells Authors: Zhang, Zhongrong ( 張鐘融) Abstract: Natural products have been playing a domain role in cancer chemotherapy and prevention in the past 30 years. Traditional Chinese medicine (TCM) with thousands years of history is a huge source for discovery and investigation of effective natural herbal products. A lot of compounds isolated or derived from TCM have been suggested to possess the property of inducing cell cycle arrest, cell differentiation and apoptosis in various kinds of cancer cells. Andrographolide (Andro) which is the main bioactive component from Andrographis paniculata (穿心蓮) has been reported to have many biological effects including anti-proliferative effect on several tumor cell lines. However, there is no detailed study about the biological effects of Andro on androgen refractory prostate cancer cells. Taxifolin (Taxi), a dihydroflavonol belongs to flavonoids group, together with its glycosides are commonly found in many species of medical herbs. In recent years, experimental studies have provided growing evidences for the protective effect of flavonoids against cancer because of their beneficial actions on multiple cancer-related biological pathways (e.g. carcinogen bioactivation, cellsignaling, cell cycle regulation, angiogenesis, oxidative stress, inflammation). Although the reports on flavonoids and cancer are still limited and conflicting, some protective associations have suggested flavonoid-rich food for cancer protection. Results in the present study showed that Andro inhibited cell proliferation of androgen independent prostate cancer cell line DU145 in a time and dosedependent manner, with the IC50 value (48 h) of 13.70 μM. On the other hand, Andro exhibited little growth inhibitory effect on noncancerous human fibroblast Hs27 (IC50˃500 μM, 48h). Cell cycle analysis demonstrated that, at low concentration (˂=40 μM), Andro-treated DU145 cells accumulated at G2/M phase dose-dependently. Immunoblot of Phospho-Histone H3 (Ser10) antibody (mitotic marker) further revealed that the G2/M accumulation of DU145 cell was caused by cell cycle arrest at mitotic phase. Additionally, microtubule network was visualized by immunostaining of tubulin, which suggested that Andro treatment leaded to the formation of abnormal spindle-chromosome structure resulting in cell arrest at prometaphase. However, in vitro microtubule assembly assay indicated that Andro did not interact directly with microtubule. Double staining of AnnexinV-FITC / PI showed that Andro also induced apoptosis dose-dependently, with the highest apoptotic rate after 48-hour treatment. High concentration (80 μM) of Andro treatment directly induced cell death without a marked alteration of cell cycle distribution within the first 24 hour. Western blotting analysis revealed that Andro exposure triggered several cell cycle regulation pathways, including up-regulation of cyclin B1 and cyclin-dependent kinase (CDK) inhibitor p21(Waf1/Cip1), dephosphorylation on Tyr15 of Cdc2 and phosphorylation of Wee1, Myt1 and Cdc25C, which involved in the process of cyclin B/Cdc2 complex activation and led to cell accumulation in mitosis. Andro-induced apoptosis was associated with activation and cleavage of poly (ADP-ribose) polymerase (PARP), caspase-7, caspase-9 and caspase-3 related to mitochondria apoptotic pathway. Taxi exhibited low anti-proliferative effect on DU145 cell line (IC50˃500 μM, 48 h). On the other hand, combination of 100 μM Taxi with Andro significantly enhanced the growth inhibitory effect of the latter on DU145 cells. It was found that combinated treatments of 100 μM Taxi and Andro (10-40 μM) markedly increased G2/M accumulation in DU145 cells compared to treatments with Andro-alone, through rising of mitotic index (approximately twice with 20 μM Andro treatment). Quantification of apoptosis with flow cytometry revealed that Andro-induced apoptosis and cell death was also promoted potently by synchronous treatment of Taxi, whereas exposure to Taxi alone did not exhibit marked alteration on cell morphology, cell cycle distribution or cell viability. Western blotting analysis revealed that the mitotic rate increased by combination treatment was related to upregulation of cyclin B/Cdc2 mitotic complex and CDK inhibitor p21; the enhanced apoptosis was associated with increases of PRAP, caspase-7, and caspase-9 activation. The increase of twisted and elongated mitotic spindle after the combined treatment suggested that anti-microtubule activity of the two combined compounds was involved as a remarkable enhanced microtubule polymerization was observed. This study investigated the biological effect of Andro and Taxi on cell morphology proliferation, cell cycle, apoptosis in androgen-independent prostate cancer cell DU145, and the synergetic/additive anticancer effect with combination treatment. Hence, Andro might be effective on prostate cancer treatment and low cytotoxic Taxi might be a promising additive in combined drug treatment of prostate cancer. This study also provided experimental evidences for the potential treatment of cancer by dietary-flavonoid in combination with anti-cancer compounds. Notes: CityU Call Number: RC280.P7 Z45 2011; xviii, 198 leaves : ill. (some col.) 30 cm.; Thesis (M.Phil.)--City University of Hong Kong, 2011.; Includes bibliographical references (leaves 175-198) Sat, 01 Jan 2011 00:00:00 GMT http://dspace.cityu.edu.hk:80/handle/2031/6586 2011-01-01T00:00:00Z http://dspace.cityu.edu.hk:80/handle/2031/6585 Title: The biological activities and molecular mechanism of a herbal formula extract of Rehmanniae Radix and Astragali Radix in wound healing Authors: Zhang, Qi ( 張琦) Abstract: Wound healing is an intricate process that involves a variety of cell types, including fibroblast, platelet, neutrophil, macrophage, endothelial cell and keratinocyte, as well as a set of biochemical events. The classical model of wound healing is divided into four sequential but overlapping phases: hemostasis, inflammation, proliferation and remodeling. In pathological conditions, such as diabetes, an organized and orderly wound healing process is interrupted and results in chronic wounds. Fibroblast is the connective tissue cell. During the proliferation phase of wound healing, fibroblasts are stimulated by various growth factors to proliferate and migrate to the wound site, and produce extracellular matrix (ECM) including collagen and fibronectin. Fibroblast is one of the components making up granulation tissue in wound healing. It is also able to secrete growth factors associated with angiogenesis and epthelialization, such as vascular endothelial cell growth factor (VEGF), platelet-derived growth factor (PDGF) and transforming growth factor beta (TGFβ). A Chinese herbal formula, consisting of Rehmanniae Radix (RR) and Astragali Radix (AR), has been clinically demonstrated to improve the healing of diabetic foot ulcer in diabetic patients with insulin resistance. In vivo study has shown that RR extract reduced the wound area and promoted scar formation in a diabetic foot ulcer rat model. In the present study, the biological activities and molecular mechanism of the herbal formula were investigated for wound healing using the human foreskin fibroblast cell model. The formula was prepared based on the weight ratio of AR to RR at 2:1. Fibroblasts were exposed to water extract of the herbal formula (Formula AR-RR) in a serum-free medium. The results showed that Formula AR-RR at the concentration of 4mg/ml significantly increased cell viability and proliferation. Flow cytometry and Western blot analysis demonstrated that Formula AR-RR was able to affect fibroblast cell cycle, and stimulate cells to enter S phase with the expression of cyclin D1 protein. Fibroblast cell migration, adhesion and contraction were subsequently studied to further understand the effect of Formula AR-RR on wound healing. Formula AR-RR was found to stimulate fibroblast migration and to improve the adhesion of fibroblasts to the ECM protein, fibrinogen. Wound contraction occurs at a late stage of wound healing. The evaluation of fibroblast contraction revealed that Formula AR-RR did not significantly enhance cell contraction. Differentiation of fibroblasts into myofibroblasts is responsible for providing the force for wound contraction. Western blot demonstrated that the expression level of alpha-smooth muscle actin (α-SMA) protein, a cytoskeletal protein biomarker indicating the differentiation of fibroblast to myofibroblast, was not changed, suggesting that Formula AR-RR had no effect on the differentiation of fibroblast to myofibroblast and the promotion of cell contraction. Wound repair is dependent on the expression and deposition of ECM proteins, mostly collagen and fibronectin, in fibroblasts. Formula AR-RR was also found to elevate the expression of ECM proteins, including collagen I, III and fibronectin in fibroblasts. Because the deposition of ECM proteins is associated with collagenases responsible for the breakdown of the matrix protein, the cytoplasmic matrix metalloproteinase protein (MMP) and the gene expression of tissue matrix metalloproteinase inhibitor (TIMP) were measured in cells treated with Formula AR-RR. The results showed that the stimulation of fibroblasts by Formula AR-RR increased TIMP1 gene expression and decreased the synthesis of MMP3 protein. Many diverse biological events during wound healing, including cell growth, cell migration, angiogenesis, fibrogenesis, and inflammatory response, are driven by cytokines. In the present study, Formula AR-RR was found to induce fibroblasts to increase the synthesis of angiogenic and fibrogenic factors in the cytoplasm, including vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), transforming growth factor beta 1 (TGFβ1) and bone morphogenic protein 6 (BMP6). The induced production of VEGF, PDGF, TGFβ1 and BMP6 suggested that Formula AR-RR was able to affect blood vessel formation and maturation, help form granulation tissue, and promote acute wound repair. In addition, Formual RA-RR decreased the production of inflammatory factors, including tumor necrosis factor alpha (TNFα), chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand 3 (CXCL3), interleukin-6 (IL-6) and interleukin-8 (IL-8), which are responsible for recruiting neutrophils leading to a chronic wound. The gene expression profile of fibroblasts stimulated by Formula AR-RR was studied by using a cDNA microarray containing 10,000 human genes. One hundred and twenty-six genes with significant change of expression level were identified, with 102 genes up-regulated and 24 genes down-regulated. Further analysis identified two pathways involved in wound healing - a Wnt signaling pathway and an angiogenesis signaling pathway. Eighteen genes with significant differential regulation were classified in the Wnt signaling pathway. Proteins encoded by these genes were distributed in 11 components of this pathway, including frizzled (FZD1, 2.3-fold), casein kinase 1 (CSNK1E, 0.5-fold), cadherin (PCDH1, 4.2-fold; FAT, 3.7-fold; PCDHGC3, 2.0-fold; CDH16, 2.5-fold; CELSR3, 2.9-fold and PCDH8, 2.2-fold), protein kinase C (PRKCH, 3.0-fold and PRKCZ, 2.6-fold), protein phosphatase 2A (PPP2R5A, 2.4-fold), calcineurin (PPP3CB, 2.4-fold), B-cell lymphoma 9 (Bcl9, 8.6-fold), switched/sucrose non fermentation (SMARCC1, 4.7-fold and SMARCA5, 2.2-fold), transforming growth factor beta activated kinase 1 (ACVR1B, 2.4-fold), CREB binding protein (CREBBP, 13.4-fold), and Wnt target genes (CCND1, 2.3-fold). Proteins encoded by ten genes were identified to involve in eight components of the angiogenesis signaling pathway, including protein kinase C (PRKCH, 3.0-fold; PRKCZ, 2.6-fold), sphingosin kinase (SPHK1, 2.6-fold), mitogen activated protein kinase 4 (MAPK4, 2.6-fold), platelet-derived growth factor (PDGFA, 3.7-fold), growth factor receptor-bound protein 7 (GRB7, 2.3-fold), growth factor receptor-bound protein 14 (GRB14, 20.7-fold), frizzled (FZD1, 2.3-fold), and delta/serrate (DLL1, 6.0-fold and DLL4, 3.6-fold). These two pathways are directly related to cell proliferation, angiogenesis, and ECM formation in the process of wound healing, and provide the molecular basis for the effects of Formula AR-RR. In summary, the study showed that Formula AR-RR has obvious biological effects on wound healing through promoting fibroblast proliferation, increasing cell migration and adhesion, stimulating the synthesis of cytokines involved in angiogenesis and fibrogenesis, and controlling inflammation. Recently, some chemical components, such as formononetin and genistine, were identified from Formula AR-RR. Formononetin was reported to promote angiogenesis, and genistine was found to substantially impair inflammatory response. Identification of additional bioactive chemical components from Formula AR-RR, and analysis of their functions in the future will further help understand the mechanisms of the herbal formula and develop new therapeutics for wound healing. Notes: CityU Call Number: RM666.H33 Z36 2010; xvi, 215 leaves : ill. (some col.) 30 cm.; Thesis (Ph.D.)--City University of Hong Kong, 2010.; Includes bibliographical references (leaves 197-215) Fri, 01 Jan 2010 00:00:00 GMT http://dspace.cityu.edu.hk:80/handle/2031/6585 2010-01-01T00:00:00Z http://dspace.cityu.edu.hk:80/handle/2031/6584 Title: An investigation on the bioactive properties of the chemical components and endophytic fungi of Cephalotaxus oliveri Mast. Authors: Zeng, Lingbin ( 曾令斌) Abstract: It is widely accepted that endophytic fungi are a rich source of active compounds. Many natural products with diverse bioactivities have been obtained from these fungi, including ones, such as taxol and podophyllotoxin, which were originally thought to be isolated from plants. These results imply that endophytic fungi are a potentially large source of untapped bioactive compounds awaiting exploration. Cephalotaxus oliveri Mast. is a relic, dioecious plant species and endemic to some subtropical forests in China. Its richness in harringtonine (HT) and homoharringtonine (HHT), two phytochemical drugs used to treat leukemia and other cancers is well established. To date, little information is available on the bioactivities of the chemical components and the endophytic fungi of C. oliveri. Hence, this plant seems to be a good candidate to explore the bioactivities of its chemical components and endophytic fungi, to analyze the chemical profile and their antioxidant activities, to isolate and identify the endophytic fungi, and evaluate their bioactivity potential, and to search for HT- and/or HHT- producing strains amongst these fungi, and finally to isolate the pure active compounds from the endophytic strain with the most potent bioactivities. Antioxidant activities of C. oliveri as indicated by the DPPH (2,2-diphenyl -1-picryl hydrazyl), ABTS [2,2'-azinobis (3-ethyl- benzothiazoline-6- sulfonic acid)], superoxide, reducing power coupled with the total phenolic content assays suggest the plant has potent antioxidant potentials. The methanol extract had the most potent activities in DPPH, ABTS and reducing power assays with the highest level of phenolic content among the test extracts. LC-MS/MS and GC-MS analyses resulted in the detection of 22 compounds in the methanol extract, among which are 15 phenolic compounds. The considerable antioxidant potentials and high phenolic content suggest C. oliveri is a rich source of natural phenolic antioxidants. A total of 101 endophytic fungal strains were isolated from C. oliveri using successive surface-sterilization, fragment plating and hyphal tip methods. Nineteen representative strains, identified as either Guignardia (anamorph Phyllosticta) or Colletotrichum species by 18S rRNA gene sequencing, show evident antioxidant, anticancer and antimicrobial activities. The phenolic compounds produced by the endophytes contributed to their antioxidant activities as assessed in DPPH test. Among the endophytic strains, Guignardia sp. L79 was the most antioxidative and cytotoxic. LC-MS/MS and GC-MS analyses indicate the presence of four phenolic compounds including protocatechuic acid, gentisic acid, tyrosol and syringol as secondary metabolites in Guignardia sp. L79. High yields suggest a promising alternative source of the four phenolic compounds can be produced by controlled fungal fermentation. Tyrosol and syringol were isolated from the ethyl acetate extract of Guignardia sp. L79 culture liquid by bioactivity guide fractionation. Syringol displayed more potent antioxidant activities than the commonly used synthetic antioxidant BHT in DPPH, ABTS and reducing power assays, whereas tyrosol exhibited only weak activities in the three assays. Syringol had overt cytotoxic activities to all the three test cancer cell lines, while tyrosol exerted significant inhibitory action to KB and HeLa cells. This is the first report on tyrosol from Guignardia (anamorph Phyllosticta) species and syringol from endophytes as secondary metabolites. However, no trace of HT and HHT was detected in the culture filtrates of all the 101 endophytic strains from C. oliveri using HPLC-DAD analysis. Notes: CityU Call Number: QK494.5.C4 Z46 2011; xi, 191 leaves : ill. 30 cm.; Thesis (Ph.D.)--City University of Hong Kong, 2011.; Includes bibliographical references (leaves 144-176) Sat, 01 Jan 2011 00:00:00 GMT http://dspace.cityu.edu.hk:80/handle/2031/6584 2011-01-01T00:00:00Z