DEPT OF ZOOLOGY
| Researcher : Chan SM |
| Project Title: | From hepatopancreas to ovary: The study of vitellogenin processing and vitellogenin receptor in the shrimp |
| Investigator(s): | Chan SM |
| Department: | Zoology |
| Source(s) of Funding: | General Research Fund (GRF) |
| Start Date: | 09/2006 |
| Abstract: |
| (1) Study of the processing of vitellogenin in the hepatopancreas (2) Cloning and molecular characterization of the shrimp vitellogenin receptor (3) Development of a ligand binding procedure to study the mechanism of Vg uptake by receptor mediated endocytosis |
| Project Title: | The shrimp MeNP: A newly discovered neuroparsin potentially important in the regulation of female reproduction |
| Investigator(s): | Chan SM |
| Department: | Zoology |
| Source(s) of Funding: | General Research Fund (GRF) |
| Start Date: | 07/2007 |
| Abstract: |
| (1) The use recombinant protein for MeNP in vitro and in vivo biological assay. (2) To develop a RNA interference approach to study the function of MeNP. (3) Genome wide search for other MeNP or neuroparsin related genes in shrimp. |
| Researcher : Cheung WT |
| List of Research Outputs |
| Cheung W.T., Leung P.C. and Wong A.S.T., Cadherin switching and activation of p120 catenin signaling are mediators of gonadotropin-releasing hormone to promote tumor cell migration and invasion in ovarian cancer, Oncogene. 2010, 29: 2427-2440. |
| Cheung W.T., Leung P.C. and Wong A.S.T., GnRH promotes peritoneal adhesion and dissemination of ovarian cancer cells, 101th Annual Meeting of the American Association for Cancer Research, Washington, DC, (Abstract No. 5190). 2010. |
| Researcher : Chow BKC |
| Project Title: | A negative feedback loop involving bile acids and Small Heterodimer Partner in controlling secretin gene expression is a key to modulate bile release |
| Investigator(s): | Chow BKC |
| Department: | Zoology |
| Source(s) of Funding: | General Research Fund (GRF) |
| Start Date: | 01/2007 |
| Completion Date: | 12/2009 |
| Abstract: |
| (1) To substantiate the working model for regulating secretin gene expression by bile acids in mouse; (2) use secretin receptor WT and Ko mice to investigate 2a) in vivo functions of bile acids to negatively feeedback to control secretin gene expression: 2b) in vivo functions and cellular mechanisms of secretin in the liver to activate bile flow; (3) to investigate the potential of secretin to protect or prevent bile acids-induced liver damage. |
| Project Title: | Discovery of novel growth hormone-releasing hormones in vertebrates: from functions to evolution |
| Investigator(s): | Chow BKC |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | France/Hong Kong Joint Research Scheme - Travel Grants |
| Start Date: | 01/2008 |
| Completion Date: | 12/2009 |
| Abstract: |
| 1) To confirm the function of the newly discovered GHRH as GH-regulator in goldfish; 2) to consolidate the proposed evolutionary scheme for three important intercellular communicators, GHRH, VIP and PACAP. |
| Project Title: | Discovery of novel growth hormone-releasing hormone: from functions to evolution |
| Investigator(s): | Chow BKC |
| Department: | Zoology |
| Source(s) of Funding: | General Research Fund (GRF) |
| Start Date: | 01/2008 |
| Abstract: |
| To confirm the function of the newly discovered GHRH as GH-regulator in goldfish; to find putative function(s) of PRP in non-mammalian vertebrates; to consolidate the proposed evolutionary scheme for three important intercellular communicators, GHRH, PACAP and VIP, based on three rounds of genome duplication early on in vertebrate evolution. |
| Project Title: | Neuron-restrictive silencer factor (NRSF) regulates cell-specific expression of the human secretin receptor gene |
| Investigator(s): | Chow BKC, Lee TO |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Seed Funding Programme for Basic Research |
| Start Date: | 04/2008 |
| Completion Date: | 03/2010 |
| Abstract: |
| 1. To identify the location and function of neuron-restrictive silencer element (NRSE) within the hSR promoter 2. To confirm the in vitro/in vivo binding of NRSF to NRSE 3. To study the role of NRSF, by interacting with Sp-proteins, in regulating hSR expression. |
| Project Title: | A unified on-line learning portal for Majors and Minors offered by the newly formed School of Biological Sciences |
| Investigator(s): | Chow BKC, Pointing SB, Wong AST, Wan JMF, Lo CSC, Hau CH, Gu JD |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Run Run Shaw Research and Teaching Endowment Fund - Teaching Grants |
| Start Date: | 09/2008 |
| Abstract: |
| Purpose of the investigation To provide on-line learning support to all biological science courses that feed all the Major/Minor programmes offered by the School. This will be unique in that it’s design will create a constantly evolving resource with a simple interface that requires no programming knowledge to operate: Individual staff will have direct access to edit the web-based noticeboards, download centres, tutorial material etc. This will not only alleviate the burden of web support on technical staff but vitally it will have valuable benefits in academic staff development within the School as all can become more engaged in the development on-line learning. The project will also reinforce development of a School culture, which is vital in the early days of a merged set of departments such as these. Students will gain from a centralized resource that will enrich and add real value to their learning. Modern biological sciences rely heavily on graphical explanations and multimedia tools to convey concepts and techniques. This is particularly relevant to areas where safety and ethical issues in biology preclude traditional practical teaching. Added value will accrue as students will be able to identify a ‘brand’ associated with the new School, and this will help to foster collegiality among students and staff who have recently come together form different departments. |
| Project Title: | The 9th International Symposium on VIP, PACAP and Related Peptides Central Administration of Secretin Suppresses Food Intake in Mice |
| Investigator(s): | Chow BKC |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | URC/CRCG - Conference Grants for Teaching Staff |
| Start Date: | 10/2009 |
| Completion Date: | 10/2009 |
| Abstract: |
| N/A |
| Project Title: | THE POTENTIAL ROLE OF SECRETIN, A POSTPRANDIALLY RELEASED GUT HORMONE, IN APPETITE CONTROL |
| Investigator(s): | Chow BKC, Chan YS, Ng SM |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | General Research Fund (GRF) |
| Start Date: | 01/2010 |
| Abstract: |
| 1) To demonstrate the role of central and peripheral secretin in appetite control; 2) To investigate mechanisms through which central and peripheral SCT modulates appetite; 3) To study pathways that secretin employs to stimulate/inhibit expression of anorexigenic/orexigenic peptides. |
| Project Title: | Modulation of secretin and secretin receptor gene expressions by angiotensin II in mouse hypothalamic cells. |
| Investigator(s): | Chow BKC |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Seed Funding Programme for Basic Research |
| Start Date: | 04/2010 |
| Abstract: |
| Angiotensin II (Ang II) has diverse physiological actions leading, for example, to increases in extracellular volume, peripheral vascular resistance and blood pressure, and has also been implicated in the regulation of cell growth and differentiation. In brain, the intrinsic renin-angiotensin system (RAS) is important in regulating blood pressure and volume homeostasis. Immunohistochemical study suggested that a large number of Ang II immunoreactive fibers and Ang II receptors (AT1 receptor) are expressed in the hypothalamic paraventricular nucleus (PVN). Actually, magnocellular neurons of the hypothalamo- neurohypophysial system (HNS) play a fundamental role in the maintenance of body water homeostasis by regulating vasopressin (Vp) expression and its secretion from the posterior pituitary in response to systemic osmotic perturbations. Recent studies by our group, however, have found that secretin, in additional to Vp, could also be released from these neurosecretory cells, where transcription of the secretin gene, as well as its receptor (SCTR) gene, are activated by hyperosmolality (1). These data indicate that the increase of secretin expression results in thirst and hence polydipsia. Interestingly, the effects of secretin in brain under water deprivation are similar to those of Ang II. It is therefore possible that secretin may act as a mediating factor in the renin-angiotensin axis. In wild-type mice, centrally administration of Ang II significantly increased secretin and SCTR transcript levels in brain. More important, in metabolic cage study, Ang II-stimulated water intake was abolished in SCTR knock-out (SCTR-/-) mice. Therefore, we proposed that secretin is involved as a component within the RAS. In our data using N42 cells, the expression levels of both secretin and SCTR were increased by treatment of Ang II. Although it has clearly been established that Sp1/Sp3 ratio and the methylation status of the promoter contribute to the expressions of both genes (2,3), the precise molecular mechanism underlying the induction of these two genes in response to Ang II has not been clarified. The present study therefore aims to identify the osmotic response elements in the secretin and SCTR promoters. To investigate the mechanism that activates secretin and secretin receptor genes after Ang II treatment, three objectives are proposed: (1) Mapping of the cis-acting elements of secretin and SCTR promoters (2) Identification of the transcription factors that mediate the osmotic response (3) Functional analysis of proposed pathways by specific inhibitors and gene silencing technique |
| Researcher : Fang JW |
| List of Research Outputs |
| Lee D.C.W., Lin S.S., Law H.Y., Fang J.W., Chua D.T.T. and Lau A.S.Y., Epstein-Barr virus latent membrane protein-1 induces cytokine expression through protein kinase PKR., Hong Kong Society for Immunology 2010 Annual General Meeting and Scientific Meeting, LKS Faculty of Medicine, The University of Hong Kong, 17 April. 2010. |
| Researcher : Ko RCC |
| Project Title: | Modulation of cell-mediated immune and inflammatory responses by Trichinella pseudospiralis during the acute muscle phase of infection- the roles of adhesion molecules and chemokines |
| Investigator(s): | Ko RCC, Mak C |
| Department: | Zoology |
| Source(s) of Funding: | Small Project Funding |
| Start Date: | 11/2002 |
| Abstract: |
| To further characterise the cell-mediated immune (systemic) response during the early phase of worm infection; to determine the roles of adhesion molecules and chemokines in suppression of inflammatory/immune response after the newborn larvae have invaded the striated muscles; to test the hypothesis that this species of nematode produce effector molecules to evade host's response. |
| Project Title: | Study on the structure of TCTP gene of Trichinella pseudospiralis (Nematoda) |
| Investigator(s): | Ko RCC, Mak CH |
| Department: | Zoology |
| Source(s) of Funding: | Small Project Funding |
| Start Date: | 07/2005 |
| Abstract: |
| The present study represents a follow-up work on our previous efforts to clone and characterize the heat-induced translationally controlled tumour protein (TCTP) of a tissue dwelling nematode, Trichinella pseudopsiralis. We had succeeded to clone both the full-length and N-terminal cDNA sequences of TCTP. The full-length gene comprises 534 bp, with an open reading frame encoding 177 amino acid residues and conserved regions. The recombinant protein( 23 kDA) was also expressed. Antibodies were produced against the protein. However, more studies are required to complete the work on gene characterization and evaluation of the recombinant protein before a paper can be published. TCTP was originally found in a human tumour in 1980's. Later, it has been found in many organisms including some parasitic protozoans and helminths. It is known to play an important role in cell-cycle progression and malignant transformation of cells. The protein can also act as a histamine-release factor and has an anti-apoptotic activity. However, the extent of its functions is not known. In parasitic organisms the protein is suspected to play a key role in host-parasite interactions and in survival. The results of the present project may provide the basis for further studies on the functions of this protein. The objectives are: (1) To complete the determination of the gene structure of TCTP.(2) To elucide its genomic organization. (3). To determine its 5' translational site. (4) To confirm the size of the recombinant protein. |
| Researcher : Lee TO |
| Project Title: | Transcriptional regulation of a nasopharyngeal carcinoma tumor suppressor: RASSF1A |
| Investigator(s): | Lee TO, Chow BKC |
| Department: | Zoology |
| Source(s) of Funding: | Small Project Funding |
| Start Date: | 11/2004 |
| Abstract: |
| To elucidate the basal transcription regulation mechanisms of RASSFIA in normal - RASSF1A-expressing and RASSF1A-non-expressing NPC cells; to fine map the core promoter region of RASSF1 gene; identify transcription factor(s) that regulates RASSF1A expression. |
| Project Title: | Molecular evolution of PACAP and VIP receptors in agnathan: Searching for the ancestral PACAP/VIP receptor gene of vertebrates. |
| Investigator(s): | Lee TO, Chow BKC |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Seed Funding Programme for Basic Research |
| Start Date: | 04/2008 |
| Completion Date: | 04/2010 |
| Abstract: |
| To identify PACAP/VIP rece Background: Evolution of G protein-coupled receptors GPCRs Recently, the availability of genomic sequences from various genome projects has provided us with the opportunity to locate conserved sequence motifs that are responsible for specific functions via comparative approaches. This idea is applicable to investigating GPCRs, the biggest gene family in the animal kingdom. Using these data, the evolutionary origin of existing GPCRs, signaling pathway, ligand-receptor interactions, and other functionally relevant elements can be studied. GPCRs are crucial to a diversity of physiological functions by mediating extracellular signals and leading to cellular responses. The superfamily can be classified according to the GRAFS system, including: Glutamate, Rhodopsin, Adhesion, Frizzled and Secretin receptors [1]. The occurrence of GPCRs, as well as G-protein signaling pathways, could date back to ~1.2 billion years, from a common ancestor before plants, fungi and animals emerged. To-date, the phylogenetically “oldest” GPCRs that have been studied are fungal pheromone, cAMP-receptor-like and glutamate-receptor-like receptors [2]. Among these GPCRs, the rhodposin receptor family is the most studied. It was proposed that the first rhodopsin-like receptors appeared around 580-800 million years ago (MYA) in several protostome Bilateria, such as insects and nematodes. Afterwards, the number of GPCRs increased during evolution and the vertebrate GPCR numbers doubled comparing to invertebrates [3]. In other sub-families, especially the secretin-like receptors, their structure and evolution are not well characterized. PACAP, VIP and their receptors Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) are prominent neuropeptides that are structurally related [4]. They belong to the secretin/PACAP/VIP peptide family based on sequence identity. PACAP and VIP are widely distributed in the central nervous system and peripheral tissues. They have been found to exert many physiological and pathophysiological effects in development, growth, immune response, circadian rhythm, digestion, respiration, reproduction and heart functions. The actions of PACAP are mediated through the activation of specific receptors. There are three VIP/PACAP receptors in mammals: PAC1R, VPAC1R, and VPAC2R [6]. All these receptors are members of the secretin receptor family. The secretin receptor family can further be sub-divided into five branches via their interactions with the following peptides: a) Corticotrophin Releasing Factor (CRF); b) Secretin, VIP, PACAP, Growth Hormone-Releasing Hormone (GHRH) and recently characterized non-mammalian PACAP-related peptide (PRP); c) Glucagon, Glucagon-Like Peptides (GLP-1 or GLP-2), Glucose Insulinotropic Peptide (GIP); d) Parathyroid Hormone (PTH). e) Calcitonin and Calcitonin Gene-Related Peptide (CGRP). Members in these subfamilies share high degree of amino acid sequence similarity within the seven transmembrane domains. The receptors in the secretin family have rather long N-termini, often between 60 and 80 amino acids, with conserved disulfide bridges [5]. The evolution of PACAP, GHRH and VIP genes was previously proposed in accordance to their structures and genomic organizations [7]. It was hypothesized that the mammalian GHRHs were evolved from non-mammalian GHRH-like peptides which are encoded in the same gene with PACAP. Our recent study, however, provided new information regarding the evolution of PACAP, GHRH and VIP in non-mammalian vertebrates [8]. We convincingly showed that `a “real” GHRH, capable of stimulating growth hormone release, is distinctly present in another precursor protein, and hence in another gene, as in mammalian species. The so-called GHRH-like peptides are in fact PACAP-related peptides (PRPs). This conceptual revolution of the identities of GHRH and PRP in non-mammalian vertebrates sways the plausibility of the model previously proposed. For PACAP/VIP receptors, VPAC2R and PAC1R are located on the same chromosome in human and rat (human chromosome 7 and rat chromosome 4), whereas VPAC1R is located on human chromosome 3 and rat chromosome 8. Therefore, it was suggested that the evolution of PACAP/VIP receptors involved two rounds of gene duplication. In the first duplication, VPAC1R gene produced the common ancestor for VPAC2R/PAC1R, and in the second duplication, the ancestral gene of VPAC2R/PAC1R produced VPAC2R and PAC1R [9]. However, mapping of receptors in the chicken genome does not support this hypothesis. In the chicken genome, all PACAP/VIP receptors are located in chromosome 2, and PAC1R and VPAC1R are separated by our newly discovered PRPR gene. It is therefore possible that PAC1R is produced from duplicating VPAC1R directly. Meanwhile, as translocation of PACAP/VIP receptors are observed in some teleost models (zebrafish and fugu), information obtained from teleost is not suitable for investigating the evolution of PACAP/VIP receptors. Consequently, without information of PACAP/VIP receptors from lower vertebrates, especially from Agnathans, evolution of PACAP/VIP receptors remains to be established. Our Hypothesis: PACAP/VIP receptors are evolved from a common ancestor between tunicates and agnathans PACAP, VIP and their receptors are rarely studied in agnathans and invertebrates. The only published record is the identification of 2 PRP/PACAP genes in tunicate (Chelyosoma productum). It was thought that PACAP and their receptors are emerged at/before protochordate. A cephalochordate genome, amphioxus (Branchiostoma floridae) provided additional information to the story. Amphioxus is a cephalochordate that is regarded as one of the closest living relative of vertebrates. Amphioxus shares several structural features with vertebrates like a dorsal, hollow notochord, segmental muscles and pharyngeal gill slits. Amphioxus genome was recently released by Joint Genome Institute. From the genomic data, CRF, PTH and calcitonin secretin-like receptor are found in amphioxus [10]. No receptors from the PACAP/VIP/secretin receptor subgroup could be identified in amphioxus genome. Also, by bioinformatic analysis, no member of PACAP/VIP peptide family is found in amphioxus. As it has been recently been suggested that tunicates could be more closely related to vertebrates than cephalochordates based on phylogenetic analysis [11]. The missing PACAP/VIP receptors and ligands suggest strongly that the PACAP/VIP ligand-receptor were emerged after the separation of cephalochordates from tunicates and/or vertebrates. More important, genes encoding PACAP and VIP are predicted from the sea lamprey genome by our preliminary data. Together with our recently identified VIP-like receptor in hagfish (Figure 1). We hypothesize that PACAP/VIP and their receptors are emerged from a common ancestor in the time between tunicates and agnathans evolution. Objectives: 1. ptors in lamprey and hagfish 2. To functional characterize these PACAP/VIP receptors 3. To propose a hypothesis showing the origin and evolution of PACAP/VIP receptor family. |
| Project Title: | A role of secretin in the subfornical organ in sensing osmotic changes |
| Investigator(s): | Lee TO, Chow BKC |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | General Research Fund (GRF) |
| Start Date: | 01/2009 |
| Abstract: |
| (1) To investigate the role of secretin in SFO in regulating water intake; (2) To study the regulation of secretin gene in SFO under osmotic changes; (3) To substantiate the hypothesis that secretin is a part of the upstream pathway in SFO in Ang II-mediated control of volume homeostasis. |
| Project Title: | Interactions of secretin and orexin in controlling food intake |
| Investigator(s): | Lee TO, Chow BKC, Sun H |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Seed Funding Programme for Basic Research |
| Start Date: | 03/2009 |
| Abstract: |
| Appetite is regulated by a complex system of central and peripheral signals which interact to modulate the individual response to nutrient ingestion. During a meal, satiety signals originated from the gastrointestinal tract reach the nucleus tractus solitaries (NTS) through the vagus nerve.The NTS then projects the signal to the arcuate nucleus (ARC) where these peripheral signals are integrated with the central signals to control behavioral responses to a meal via downstream brain areas [1-3]. Lateral hypothalamuic area (LHA) is one of the brain areas controlled by ARC and is refereed as a feeding center in brain. Bilateral lesions in the LHA result in anorexia (lack of appetite) and animals may die of starvation after the lesions. It has been postulated that when body weight decreases or when the ARC senses “hunger”, LHA is activated to increase food intake. One of the mechanisms in LHA to increase appetite is to secret the orexigenic peptide, orexin. Two orexin peptides, orexin-A and orexin-B, are produced from a common prepro-orexin precursor [4, 5]. Orexin-A is a 33-residue peptide with two intrachain disulfide bonds, while orexin-B is a linear 28-residues peptide. Both peptides bind to two G protein-coupled receptors, orexin-1 (OX1R) and orexin-2 (OX2R) receptor. In vitro analysis showed that OX1R selectivity interacts with orexin-A while OX2R is able to interact with both orexins [6]. There are numerous studies suggesting that orexins could increase appetite. During fasting, the plasma orexin-A level and transcript level of prepro-orexin in LHA were increased significantly [4]. In passive immunoneutralization studies, infusion of antibodies of orexin-A reduced feeding in response to an overnight fast [7]. Moreover, the food intake induced by exogenously injection of orexin or fasting is decreased by anOX1R antagonist SB-334867 [8]. Orexin knockout mice (prepro-orexin gene deletion) ate less than wild-type mice, also suggesting a physiologically role for endogenous orexin in the regulation of appetite [9]. However, it is interesting to note that the orexin knockout mice grew normally. This observation indicating that some other determinant of food intake/body growth may be involved to compensate the lost of the orexin pathways [9]. On the basis of their hypothalamic localization and their sequence similarity to secretin, orexin is also named as hypocretins. In the alignment (Fig.1), orexin shares an identical region with the C-terminus of secretin. Also, additional sequence identities can be found between N-terminal residues of secretin and C-terminal residues of orexins. Therefore, it has been suggested that orexin was evolved from secretin by circular permutation. The structural similarity between orexin and secretin suggested the possible interaction between secretin, orexin and their receptors. However, secretin did not show significant binding to orexin receptors or stimulate cellular response in CHO cells expressing human OX1R or OX2R [10]. In receptor binding studies using rat hypothalamus, secretin was able to displace [125I]orexin-A significantly [11, 12]. Recently, we have shown that secretin receptor (SCTR) is also expressed in hypothalamus [13, 14], therefore, it is possible that secretin and orexin-A in hypothalamus are competing for SCTRs but not orexin receptors. The potential role of secretin in appetite control was also suggested in our studies. By intracerebroventricular (icv) injection of secretin into the lateral ventricle (LV), a significantly suppressed 24-hours food intake (Fig.2) was observed in both rats and wild-type (SCTR+/+) mice but not in SCTR knockout (SCTR-/-) mice. During fasting, the expression of both secretin and its receptor transcripts are decreased in rat hypothalamus (Fig.3). We have also shown the expressions of secretin and SCTR in LHA (Fig.4). These data indicated that secretin possesses an anorectic effect in rodents and this action may be involved the LHA. During fasting, the expression of orexin in LHA was increased while secretin expression was decreased. We propose here that the increased orexin not only stimulates the well known orexinergic pathways to enhance food intake, it may also inhibit the anorectic effect of secretin by competing with secretin on SCTR. To substantiate this hypothesis, we proposed to study the following: 1. To investigate interactions and signaling pathways of orexin and secretin with their receptors 2. To study structures responsible for ligand-receptor binding for orexin and secretin with their receptors 3. To evaluate the role of orexin in appetite control in SCTR knockout mice. |
| Project Title: | The 9th International Symposium on VIP, PACAP and Related Peptides Analysis of a putative VPAC2 receptor from sturgeon shed light on molecular and functional evolution of VPAC2R in vertebrates |
| Investigator(s): | Lee TO |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | URC/CRCG - Conference Grants for Teaching Staff |
| Start Date: | 10/2009 |
| Completion Date: | 10/2009 |
| Abstract: |
| N/A |
| Project Title: | Implications of PACAP/VIP receptor gene duplications in early vertebrates |
| Investigator(s): | Lee TO, Chow BKC |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | General Research Fund (GRF) |
| Start Date: | 01/2010 |
| Abstract: |
| 1) To consolidate a revised evolutionary scheme for PACAP/GHRH/SCT receptors by characterizing these receptors in several animal models that are crucial to the understanding of early vertebrate evolution; 2) To investigate if PACAP/VIP receptors were evolved from a common ancestral gene after the separation of vertebrate lineage from cephalochordate/urochordate; 3) To substantiate that PHIR shares the same origin with VPAC2R, and it was evolved only after the teleost-specific genome duplication event; 4) To characterize fish SCTR and to search for SCTR in early vertebrates. |
| Project Title: | The role of glucagon receptor family in the unique hyperglycermic activity of teleosts glucagon-like peptide 1. |
| Investigator(s): | Lee TO |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Seed Funding Programme for Basic Research |
| Start Date: | 06/2010 |
| Abstract: |
| Glucagon and the glucagon-like peptides 1 (GLP-1) are paramount regulators of carbohydrate, fat and protein metabolism, as well as blood glucose level. They are encoded by the proglucagon gene together with glucagon-like peptides 2 (GLP-2). Even though the structures of these peptides are highly comparable, in mammals, the functions of glucagon and GLP-1 oppose to each other in blood glucose regulation. In mammals, glucagon is produced and secreted by the pancreatic α cells in response to low blood glucose levels and it serves as the major counter-regulatory hormone to insulin(1). Among vertebrates, the major physiological functions of glucagon appear to be conserved. Both fish and mammalian glucagons elevate blood glucose level via inducing hepatic glycogenolysis and gluconeogeneisis(2-3). While the regulatory function of glucagon on insulin has been maintained from fish to mammals, GLP-1 has changed its physiological function. In mammals, GLP-1 is an incretin hormone that stimulates insulin secretion from pancreatic β cells. In disparity, fish GLP-1, produced in both pancreas and intestine, shows a glucagon-like activity contrasting the insulin-like activity of mammalian GLP-1(4-5). Bowfin fish (Amia calva) GLP-1 fails to stimulate insulin release from pancreas and it was found to be hyperglycermic and glucagon-like(6-7). Fish GLP-1 accelerates glucose transport and curtails glucose oxidation in enterocytes and it was found to elicit an increase in cAMP level in brain and enterocytes. GLP-1 also acts directly on the liver by supplementing the action of glucagon, including glycogenolysis, gluconeogenesis and lipolysis, but no such increase in cAMP level is noted in isolated hepatocytes in vitro(8). Therefore, GLP-1 is found to have acquired and subsequently lost gluconeogenic activity during the evolution as illustrated by its contrasting functions in fish and mammals. This leads to possibility of either the peptide or receptor being responsible. In the case of the proglucagon-derived peptides, the high conservation of primary structure indicates strong evolutionary pressure to preserve function. Hence, the receptor may play a greater contributing factor as to why there was a change in biological activity. To elicit their biological functions, glucagon and GLPs interact with corresponding receptors: glucagon receptor (Glu-R), GLP-1 receptor (GLP-1R) and GLP-2 receptor (GLP-2R). Collectively, these receptors belong to a class II B superfamily of G protein-coupled receptors family. To date, although a range of proglucagon ligand and receptor sequences are known, most research has been focused on mammals and the information regarding receptors in fish and frogs is relatively sparse. In fish, the goldfish (Carassius auratus) and zebrafish (Danio rerio) are the only species in which molecular cloning of glucagon and/or GLP-1 receptors has been successfully achieved(4, 9), whilst fugu (Takifugu rubripes), pufferfish (Tetraodon nigroviridis) and medaka (Oryzias latipes) comprise of the remaining species where receptor sequences have been predicted by data mining of their respective genomes. It was previously hypothesized that as a result of the fish-specific gene duplication event early in teleost evolution prior to species diversification, a duplicate Glu-R acquired GLP-1 binding ability whilst retaining glucagon binding ability, thus giving rise to receptors currently recognized as “fish GLP-1Rs” and are paralogous to Glu-Rs. The original GLP-1R in fish was also thought to have been lost from the fish lineage(10). However by extensive data mining of the fugu, pufferfish and medaka genomes, we have been able to predict sequences resembling those of known GLP-1Rs, forming a separate GLP-1R group. In our phylogenetic analysis, with the exception of fish GLP-1R-likes, all glucagon superfamily receptors can be grouped based on subtype. The results indicate fish GLP-1R-likes to be closely related to fish Glu-Rs and are clustered together within the Glu-R group. This suggests that a “true” GLP-1R in fish does indeed exist and was not lost. Based on this finding, we believe previously identified “fish GLP-1Rs” should be termed as “fish GLP-1R-likes”. Non-fish vertebrates have true GLP-1Rs exhibiting high resemblance in both phylogenetic analysis and physiological function. Therefore, this may suggest that the true fish GLP-1R is non-functional or functionally different to mammalian counterparts, thus resulting in GLP-1’s unique glucagon-like activity in fish. To substantiate this hypothesis, we proposed to study the following: 1. Isolate and express the Glu-R, GLP-1R, and GLP-1R-like receptors in three teleost animal models: goldfish (Carassius auratus), pufferfish (Tetraodon nigroviridis) and zebrafish (Danio rerio), 2. Evaluate the ligand-specificity and signaling properties of the identified teleost glucagon and GLP-1 receptors, 3. Study the role of the conserved motifs of the receptors by mutagenenesis-based strategy. |
| Researcher : Lee WWM |
| Project Title: | Interaction and ultrastructure of cell junction proteins in the testis |
| Investigator(s): | Lee WWM |
| Department: | Zoology |
| Source(s) of Funding: | General Research Fund (GRF) |
| Start Date: | 09/2006 |
| Completion Date: | 08/2009 |
| Abstract: |
| (1) What are the functional and structural relationship between tight junctions and basal ectoplasmic specialization (ES) structural protein complexes at the blood-testis barrier (BTB) ? Are there any common adaptors that structurally and functionally link the cadherin/catenin/c-Src/MTMR2, the nectin/afadin/ponsin, and the TJ protein complexes together to regulate BTB dynamics? (2) What are the molecular composition of the laminin α(?)β(?)γ3 receptors and its associated peripheral proteins, which interact with α6β1 integrin at the apical ES? |
| Project Title: | Extracellular matrix proteins and cell junction dynamics in the testis. |
| Investigator(s): | Lee WWM |
| Department: | Zoology |
| Source(s) of Funding: | General Research Fund (GRF) |
| Start Date: | 09/2007 |
| Abstract: |
| To investigate the role of collagens and collagen fragments in BTB junction dynamics. Can fragments of collagen IV perturb Sertoli cell functions, such as Sertoli cell tight junction-barrier, MMP-9 and TIMP-1 productions? To investigate the role of proteases and protease inhibitors in BTB junction dynamics. Can a disruption of the protease and protease inhibitor homeostasis modulate cytokine-induced transient BTB damage in the seminiferous epithelium? |
| Project Title: | Paracrine regulations of blood-testis barrier dynamics |
| Investigator(s): | Lee WWM |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Seed Funding Programme for Basic Research |
| Start Date: | 01/2009 |
| Completion Date: | 12/2009 |
| Abstract: |
| The blood-testis barrier (BTB) is formed by cell junctions between adjacent Sertoli cells in the testis. It is important for several reasons. First, it creates a microenvironment for germ cell development. Second, it insulates meiotic germ cells from the systemic circulation, thereby protecting them from potential cytotoxic substances and autoimmune recognition, and confers apical-basal polarity. Third, it must open and close at time intervals to allow the passage of preleptotene spermatocytes from the basal to adluminal compartment. Fourth, the delivery of drugs or contraceptives must take into account the constraints imposed by the BTB [1]. In this proposal, the P.I. seeks to examine how cytokines (e.g., tumor necrosis factor-a, TNF-a and transforming growth factor-b3, TGF-b3) are working in coordination with testosterone to regulate the BTB dynamics in adult rat testes. This allows the germ cells to traverse across the BTB, while at the same time still maintaining the barrier integrity. Recent studies from the P.I.’s laboratory have shown that TNFa and TGF-b3 disrupt the Sertoli cell tight junction (TJ)-permeability barrier in vitro and the BTB integrity in vivo [2] (for reviews, see [3]). It is plausible that cytokines (e.g., TNFa)secreted by Sertoli and germ cells into the BTB microenvironment induce the “opening” of the BTB to facilitate preleptotene spermatocyte migration that occurs at stages VII-VIII of the epithelial cycle. Along the line, it has also been shown that testosterone promotes the BTB integrity based on studies using the Sertoli cell specific androgen receptor knockout (SCAR KO) mouse model. For instance, SCAR KO mice are infertile due to spermatogenic arrest before the first meiosis [4] and the BTB in these mice was shown to be impaired plausibly as the result of reduced integral membrane proteins at the BTB. This promoting effect of testosterone on the BTB in vivo is also consistent with earlier in vitro studies that androgen promotes the Sertoli cell TJ-barrier and cell adhesion [5]. Furthermore, the expression of androgen receptor in adult testes is also stage-specific, being highest at stages VII-VIII at the time of preleptotene spermatocyte migration across the BTB, similar to TGF-b3 and TNF-a. The effects of cytokines and testosterone on testicular functions are multiple. However, in a latest study [6], the P.I. has shown that both cytokines and testosterone accelerate the kinetics of integral membrane protein endocytosis in Sertoli cells in vitro with functional BTB. In this instance, cytokines induce the endocytosed proteins (e.g., occludin) to late endosomes for degradation whereas testosterone promotes their recycling back to the cell surface. It is postulated that at defined stages of the spermatogenic cycle, cytokines contribute to the “destabilization” of the TJ fibrils at the apical portion of the migrating preleptotene spermatocytes, facilitating cell migration; whereas androgen promotes the “transcytosis” of the internalized integral membrane proteins from the apical to the basal region of the migrating germ cells to facilitate the assembly of new TJ fibrils, so as to maintain the immunological barrier during the epithelial cycle. How do testosterone and cytokines exert their differential actions on this junction dynamics is not known. However, this is likely mediated via their effects on extracellular matrix (ECM) components: collagens, proteases (e.g., matrix metalloproteases, MMPs, such as MMP-9), and protease inhibitors (e.g., tissue inhibitors of metalloproteases, TIMPs), thereby reducing the steady-state levels of integral membrane proteins at the BTB, and destabilizing the BTB to facilitate germ cell migration. The P.I. has the following specific aims to generate a complete study to vigorously examine the actions of testosterone and cytokines on BTB dynamics at the cellular and subcellular levels. In this seed funding period, work on Specific Aim 1 will be performed. After which, a full proposal (Specific Aims 1 and 2) with initial results and backup findings from [6] will be submitted for GRF funding in the 2010-2011 exercise. Specific Aim 1: To investigate how cytokines are working in concert with extracellular matrix (ECM) proteins, such as collagens, proteases (e.g., MMP-9), and protease inhibitors (e.g., TIMP-1), to determine the steady-state levels of integral membrane proteins at the BTB, inducing transient BTB “opening” to facilitate preleptotene spermatocyte migration during spermatogenesis. Specific Aim 2: To delineate the mechanism(s) utilized by the testis to maintain the BTB integrity regarding the opposing effects of cytokines (or NC1 domain) and testosterone on the barrier function during the seminiferous epithelial cycle of spermatogenesis. Literature cited: 1. Lui WY and Lee WM (2008) Mechanisms of reorganization of cell-cell junctions in the testis. Frontiers in Biosciences 13:6775-6786. 2. Li MWM, Xia WL, Mruk DD, Wang Q, Yan HHN, Siu MKY, Lui WY, Lee WM and Cheng CY (2006) TNFa can reversibly disrupt the blood-testis barrier (BTB) and impair Sertoli-germ cell adhesion – a novel mechanism to regulate junction dynamics during spermatogenesis. Journal of Endocrinology 190:313-329. 3. Xia WL, Mruk DD, Lee WM and Cheng CY (2005) Cytokines and junction restructuring during spermatogenesis – a lesson to learn from the testis. Cytokine & Growth Factor Reviews 16:469-493. 4. De Gendt K, Swinnen JV, Saunders PT, Schoonjans L, Dewerchin M, Devos A, Tan K, Atanassova N, Claessens F, Lecureuil C, Heyns W, Carmeliet P, Sharpe RM, Verhoeven GA (2004) Sertoli cell-selective knockout of the androgen receptor causes spermatogenic arrest in meiosis. Proc. Natl. Acad. Sci. USA 101: 1327-1332. 5. Zhang JY, Wong CH, Xia WL, Mruk DD, Lee NPY, Lee WM and Cheng CY (2005) Regulation of Sertoli-germ cell adherens junction dynamics via changes in protein-protein interactions of the N-cadherin-b-catenin protein complex which are possibly mediated by c-Src and MTMR2: an in vivo study using an androgen suppression model. Endocrinology 146:1268-1284. 6. Yan HHN, Mruk DD, Lee WM and Cheng CY (2008) Blood-testis barrier dynamics are regulated by testosterone and cytokines via their differential effects on the kinetics of protein endocytosis and recycling in Sertoli cells. FASEB J 22:1945-1959. |
| Project Title: | 34th FEBS (Federation of the European Biochemical Societies) Congress IFN-gamma and TNF-alpha downregulate the expression of Junctional Adhesion Molecule-C (JAM-C) through transcriptional and post-translational controls |
| Investigator(s): | Lee WWM |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | URC/CRCG - Conference Grants for Teaching Staff |
| Start Date: | 07/2009 |
| Completion Date: | 07/2009 |
| Abstract: |
| N/A |
| Project Title: | Cell junction dynamics at stage VIII of the seminiferous epithelial cycle -- coordination between spermiation and blood-testis barrier restructuring |
| Investigator(s): | Lee WWM |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Small Project Funding |
| Start Date: | 11/2009 |
| Abstract: |
| During spermatogenesis, preleptotene/leptotene spermatocytes at the basal compartment traverse the blood-testis barrier (BTB) at stages ~VIII-IX of the seminiferous epithelial cycle in adult rat testes, entering the adluminal compartment for further development [1]. This event takes place concurrently with spermiation wherein fully developed spermatids (i.e., spermatozoa) detach from the epithelium at the luminal edge, entering the tubule lumen for their eventual maturation in the epididymis. The P.I. seeks to study the mechanism that regulates and coordinates these events. The idea was based on a recent study in which a blockade of the laminin function at the apical ES by specific antibodies led to spermatid exfoliation and BTB restructuring [2] showing that a disruption of the apical ES may lead to a transient BTB disruption. This illustrates a plausible physiological link between these two ultrastructures. The apical ES is a testis-specific adherens junction (AJ) type that anchors developing spermatids to the Sertoli cell in the epithelium during spermatogenesis [3]. It has properties of both AJ and focal contacts. For instance, many proteins that are restricted to the cell-matrix interface at the focal contacts, such as integrins, laminins, and collagens are found at the apical ES. In adult rat testes, one of the major cell adhesion complexes at the apical ES is the laminin-333/alpha6beta1 integrin complex [3]. Indeed, laminin gamma3 chain was first identified as a non-basement laminin at the apical ES in mouse testes [4]. Subsequent studies in adult rat testes have shown that laminin gamma3, alpha3 and beta3 chains residing in the elongating/elongated spermatids form a protein complex known as laminin-333 (2), which is the bona fide partner of the alpha6beta1-integrin restricted to Sertoli cells [5,6] constituting the laminin-333/alpha6beta1-integrin adhesion complex at the apical ES. When the laminin-333 function was compromised by using blocking antibodies, spermatid sloughing from the epithelium was detected [3]. Furthermore, this laminin/integrin complex is associated with proteases: matrix metalloprotease-2 (MMP-2), membrane-type 1 (MT1)-MMP; and tissue inhibitor of metalloproteases-2 (TIMP-2) [7]. These findings suggested that the activation of MMP-2 by MT1-MMP and TIMP-2 at the apical ES during spermiation could cleave laminin-333 to generate the biologically active laminin fragments. Indeed, there are reports illustrating that fragments of laminin chains that arise under physiological and pathophysiological conditions are biologically active peptides, regulating cell migration, protein production and/or activation, inflammatory responses, and others [8-10]. Thus, it is possible that fragments of laminin chains released from the apical ES during spermiation could regulate the BTB in the testis. This has prompted us to study the effects of laminin fragments on BTB restructuring. Subsequently, our latest results published in PNAS [11] have shown that specific laminin fragments: laminin gamma3 chain domain IV (Lam g3DIV) and laminin beta3 chain domain I (Lamb3DI) are bioactive peptides regulating BTB restructuring. This study provides compelling evidence that during spermatogenesis there exists an autocrine-based regulatory axis (apical ES-BTB-hemidesmosome) coordinating spermiation and BTB restructuring. In this proposal, the PI provides a plan of follow-up studies to address the underlying mechanisms since this “apical ES-BTB-hemidesmosome” axis must be vigorously characterized, so that this information can be helpful to investigators in the field to study the biology and regulation of spermatogenesis. Additionally, this application will shed lights in the development of innovative male contraceptive, such as by using the laminin peptide to compromise spermatogenesis. This proposal is innovative, hypothesis driven, mechanistic in nature and based on recent findings published in leading journals. Specific Aim 1. Origin of the laminin fragments and their regulation: Is the production of laminin fragments stage-specific? Specific Aim 2. Mechanism(s) of action of the laminin fragment: (a) Is it mediated via integrin receptor and/or non-integrin-based proteins? Are non-receptor protein tyrosine kinases (e.g., FAK and c-Src) at the BTB and apical ES serve as the downstream signal transducers? Literature cited: 1. Russell LD. Movement of spermatocytes from the basal to the adluminal compartment of the rat testis. Am J Anat 1977; 148, 313-328. 2. Yan HHN, Cheng CY. Laminin a3 forms a complex with b3 and g3 chains that serves as the ligand for a6b1-integrin at the apical ectoplasmic specialization in adult rat testes. J Biol Chem 2006; 281 17286-17303. 3. Yan HHN, Mruk DD, Lee WM, Cheng CY. Ectoplasmic specialization: a friend or a foe of spermatogenesis? BioEssays 2007; 29, 36-48. 4. Koch M, Olson PF, Albus A, Jin W, Hunter DD, Brunken WJ, Burgeson RE, Champliaud MF. Characterization and expression of the laminin g3 chain: a novel, non-basement membrane-associated, laminin chain. J Cell Biol 1999; 145, 605-618. 5. Palombi F, Salanova M, Tarone G, Farini D, Stefanini M. Distribution of b1 integrin subunit in rat seminiferous epithelium. Biol Reprod 1992; 47, 1173-1182. 6. Salanova M, Stefanini M, De Curtis I, Palombi F. Integrin receptor a6b1 is localized at specific sites of cell-to-cell contact in rat seminiferous epithelium. Biol Reprod 1995; 52, 79-87. 7. Siu MKY, Cheng CY. Interactions of proteases, protease inhibitors, and the b1 integrin/laminin g3 protein complex in the regulation of ectoplasmic specialization dynamics in the rat testis. Biol Reprod 2004; 70, 945-964. 8. Koshikawa N, Schenk S, Moeckel G, Sharabi A, Miyazaki K, Gardner H, Zent R, Quaranta V. Proteolytic processing of laminin-5 by MT1-MMP in tissues and its effects on epithelial cell morphology. FASEB J 2004; 18, 364-366. 9. Ogawa T., Tsubota Y., Hashimoto J., Kariva Y., Miyazaki K. The short arm of laminin g2 chain of laminin-5 (laminin-332) binds syndecan-1 and regualtes cellular adhesion and migration by suppressing phosphorylation of integrin b4 chain. Mol Biol Cell 2007; 18, 1621-1633. 10. Remy L., Trespeuch C., Bachy S., Scoazec J.Y., Rousselle P. Matrilysin 1 influences colon carcinoma cell migration by cleavage of the laminin-5 b3 chain. Cancer Res 2006; 66, 11228-11237. 11. Yan HHY, Mruk DD, Wong EWP, Lee WM, Cheng CY. An autocrine axis in the testis that coordinates spermiation and blood-testis barrier restructuring during spermatogenesis. Proc Natl Acad Sci USA 2008; 105: 8950-8955. |
| Researcher : Leung FCC |
| Project Title: | Cloning of the viral genes from the newly identified SARS coronavirus |
| Investigator(s): | Leung FCC |
| Department: | Zoology |
| Source(s) of Funding: | VCO SARS Research Fund |
| Start Date: | 07/2003 |
| Abstract: |
| To clone all the viral gene into vector and these cloned genes will be then be used as reagents by us and other as the first step for investigation. |
| Project Title: | Development of a rapid high throughput RT-PCR assay to detect SARS-CoV |
| Investigator(s): | Leung FCC |
| Department: | Zoology |
| Source(s) of Funding: | VCO SARS Research Fund |
| Start Date: | 07/2003 |
| Abstract: |
| To develop a 96-wells RT-PCR platform assay for the detection of the coronavirus. |
| Project Title: | Isolation and characterization of a PCV2 virus causing postweaning multisystemic wasting syndrome in pigs |
| Investigator(s): | Leung FCC |
| Department: | Zoology |
| Source(s) of Funding: | Small Project Funding |
| Start Date: | 11/2003 |
| Abstract: |
| To isolate and characterize the PCV2 virus that causes the Postweaning multisystemic wasting syndrome (PMWS) in pigs. |
| Project Title: | International Conference on Farm Animal Endocrinology Characterization of the 5'-Flanking Transcriptional Regulatory Region of the Chicken Growth Hormone Gene |
| Investigator(s): | Leung FCC |
| Department: | Zoology |
| Source(s) of Funding: | URC/CRCG - Conference Grants for Teaching Staff |
| Start Date: | 07/2004 |
| Abstract: |
| N/A |
| Project Title: | Characterization of the angiotensin-converting enzyme 2 receptors from various animals and the use of pseudotyped virus to correlate the receptor-binding to susceptibility of SARS-CoV infection |
| Investigator(s): | Leung FCC |
| Department: | Zoology |
| Source(s) of Funding: | Research Fund for the Control of Infectious Diseases - Full Grants |
| Start Date: | 01/2007 |
| Abstract: |
| To identify the susceptible animals to severe acute respiratory syndrome associated coronavirus SARS-CoV and SARS-like bat CoV through a molecular approach. |
| Project Title: | 2009 Conference for Research Workers in Animal Diseases A Web-based Database and Phylogenetic Tools to Study Molecular Epidemiology and Evolution of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) |
| Investigator(s): | Leung FCC |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | URC/CRCG - Conference Grants for Teaching Staff |
| Start Date: | 12/2009 |
| Abstract: |
| N/A |
| Researcher : Li ETS |
| Project Title: | Nutritional benefits of dietary fiber supplementation in hospitalized geriatrics |
| Investigator(s): | Li ETS |
| Department: | Zoology |
| Source(s) of Funding: | Other Funding Scheme |
| Start Date: | 09/1997 |
| Abstract: |
| To improve gentle bowel fitness, relieve constipation and improve serum lipid profile of institutionalized geriatric patients via dietary fiber supplementation. |
| Project Title: | An evaluation on the antioxidant effects of Lycium barbarum L. and its supplementation on cataract formation in rats |
| Investigator(s): | Li ETS |
| Department: | Zoology |
| Source(s) of Funding: | Small Project Funding |
| Start Date: | 11/2003 |
| Abstract: |
| To systematically examine the antioxidant properties of Lycium barbarum L. and evaluate the effect of its supplementation on cataract development in rats. |
| Project Title: | Anti-cataract effects of Lycium barbarum L and Momordica charantia |
| Investigator(s): | Li ETS |
| Department: | Zoology |
| Source(s) of Funding: | Small Project Funding |
| Start Date: | 11/2004 |
| Abstract: |
| To systematically examine the antioxidant properties of Lycium barbarum L. and Momordica charantia and evaluate the effects of supplementation on cataract development in rats. |
| Project Title: | The CSCN 5th Annual Scientific Meeting Bitter Melon Supplementation Changes Expression of Genes Controlling Lipid Metabolism in Diet-induced Obese Rats |
| Investigator(s): | Li ETS |
| Department: | Zoology |
| Source(s) of Funding: | URC/CRCG - Conference Grants for Teaching Staff |
| Start Date: | 05/2006 |
| Abstract: |
| N/A |
| Project Title: | Offsetting the Adverse Effects of Maternal Overnutrition by Bitter Melon Supplementation |
| Investigator(s): | Li ETS |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Seed Funding Programme for Basic Research |
| Start Date: | 04/2009 |
| Abstract: |
| Metabolic syndrome (MS) posts a serious health concern worldwide. The etiology of MS is complex and a lot of attention has been given to the impact of perinatal nutrition because substantial evidence suggest maternal nutrition has a trans-generation effect on disease risk. In fact, both under- and overnutrition are known to predispose offspring to develop phenotypes characteristics of MS. This study will focus on examining in utero overnutrition as a predisposing factor. High fat intake is acknowledged to attribute to the high incident of chronic degenerative diseases. Fetal programming effect is well established. Offspring of rats fed a high fat diet during pregnancy had impaired glucose homeostasis. There is a positive association between birth weight and later BMI (and obesity). furthermore, macrosomic infants have higher cardiovascular disease risk. Hence, the relationship between birth weight and type 2 diabetes (T2D) is U-shape. It should be reminded that increasing prevalence and younger age of MS onset takes place at time when fat consumption follows a decreasing trend. However, the consumption of another macronutrient, fructose, had steadily increased in the past 30 years. High fructose diet is also known to precipitate MS. In addition to changes in energy and macronutrient supply, developmental programming can take place with perinatal exposure to certain dietary ingredients. For instance, isoflavones given to dams during pregnancy and lactation provide cardioprotection to offspring in adulthood. Reduced risk of obesity has also been demonstrated in Avy mouse. When genistein was given to a/a females two weeks prior to mating with Avy/a males, the coat color of the heterozygous yellow agouti pups was shifted to pseudoagouti. These results led us to reason that other bioactive ingredients, known to influence energy metabolism and glucose homeostasis, introduced at pregnancy and lactation might be beneficial. In this context, we have the experience in the use of botanical / herbal preparations in counteracting the phenotype characteristics of MS. We have documented the beneficial effects of bitter melon juice and its supplementation in the diet results in lower blood glucose and triglyceride; and less body fat accumulation. The molecular mechanisms involved include elevated expression of uncoupling protein in white adipose tissue and lipolytic enzymes in skeletal muscles. Whether bitter melon supplementation could offset the adverse metabolic effect of fructose has not be examined. The overall objective of this proposal is to test the hypothesis that supplementation of bitter melon extract from periconceptual through lactation period can alleviate the negative impact of maternal overnutrition induced by fructose on chronic disease risk of offspring. Specific objectives: a. To establish the effects of maternal bitter melon supplementation on fetal programming in offspring from fructose fed dams. c. To examine the molecular mechanisms that counteract metabolic syndrome imprinted by maternal overnutrition. |
| Researcher : Lie PPY |
| List of Research Outputs |
| Wong W.P.E., Yan H.N.H., Li W.M.M., Lie P.P.Y., Mruk D.D. and Cheng C.Y., Cell junctions in the testis as targets for toxicants, In: Charlene A. McQueen, Comprehensive Toxicology. Elservier Ltd, 2010, 11: 167-188. |
| Researcher : Lui WY |
| Project Title: | Function of CLMP in rat testis and its regulation |
| Investigator(s): | Lui WY |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Seed Funding Programme for Basic Research |
| Start Date: | 01/2008 |
| Completion Date: | 12/2009 |
| Abstract: |
| The BTB situated between adjacent Sertoli cells physically divides the seminferous epithelium into the basal and adluminal compartments. The BTB not only maintains cell polarity, it also segregates the post-meiotic germ cell development from the systemic circulation to avoid autoimmune response [1, 3]. However, this intact barrier must disassemble at stage VIII of the seminiferous cycle to allow pre-leptotene and leptotene spermatocytes residing outside the BTB (the basal compartment) to migrate across the barrier and enter the adluminal compartment. Recent studies have indicated that Coxsackie and Adenovirus Receptor-Like Membrane Protein (CLMP) is a novel member of the Cortical Thymocyte Marker in Xenopus (CTX) family and a new component of epithelial tight junction [6]. Exogenously expressed CLMP in polarized MDCK cells has shown that CLMP is restricted to the cell-cell interface and co-localized with ZO-1, a tight junction marker [6]. A significant increase in transepithelial electrical resistance (TER) has been found in cultured CLMP-expressing MDCK cells when compared to cells not expressing CLMP. CLMP has been found to associate with occludin in caco-2 cells and forced expression of CLMP in CHO cells induces cell aggregation [6]. Our recent findings have showed that testis expresses CLMP and CLMP is co-localized with occludin at the BTB [2]. We have also studied the transcriptional regulation of CLMP in Sertoli cells. It has noted that the expression of CLMP is mediated via the interaction of GATA and the Kruppel family proteins, KLF4 and Sp1. Moreover, our preliminary data showed that CLMP could be down-regulated by tumor necrosis factor alpha (TNF-alpha) via alteration of its mRNA turnover. In our laboratory in the past years, a series of in vitro and in vivo studies have clearly demonstrated that TNF alpha and TGF-beta3 are key cytokines that perturb BTB and exerts potent negative effect in the expression of several TJ proteins in the testis such as JAM-A, CAR and occludin [1, 4, 5, 7]. These results taken together suggest that CLMP is a novel TJ protein that is regulated by TNF alpha and TGF-beta and is also involved in BTB dynamics. The P.I. seeks to characterize the role of Coxsackie and Adenovirus Receptor-Like Membrane Protein (CLMP) in the testis. Specifically, the P.I. will unravel (i) the functional role of CLMP in the testis and (ii) the effects of germ cells, cytokines (TNF-alpha and TGF-beta 3) and hormones (FSH and testosterone) on the expression of CLMP in Sertoli cells and in the testis. 1. Lui WY* and Cheng CY* (2007) Regulation of cell junction dynamics by cytokines in the testis – a molecular and biochemical perspective. Cytokine and Growth Factor Reviews 18:299-311 *Corresponding authors 2. Sze KL, Lee WM, Lui WY* (2007) Expression of CLMP, a novel tight junction protein, is mediated via the interaction of GATA with the Kruppel Family proteins, KLF4 and Sp1, in mouse testis. Journal of Cellular Physiology (in press) 3. Lui WY and Lee WM (2006) Regulation of junction dynamics in the testis – Transcriptional and post-translational regulations of cell junction proteins. Molecular and Cellular Endocrinology 250:25-35 4. Li MW, Xia W, Mruk D, Wang CQ, Yan HH, Siu MK, Lui WY, Lee WM, Cheng CY (2006) Tumor necrosis factor alpha reversibly disrupts the blood-testis barrier and impairs Sertoli-germ cell adhesion in the seminiferous epithelium of adult rat testis. Journal of Endocrinology 190:313-329 5. Wong CH, Mruk DD, Lui WY, Cheng CY (2004) Regulation of the blood-testis barrier dynamics in the testis: an in vivo study. Journal of Cell Science 117:783-798 6. Raschperger E, Engstrom U, Pettersson RF, Fuxe J (2004) CLMP, a novel member of the CTX family and a new component of epithelial tight junctions. Journal of Biological Chemistry 279:796-804 7. Lui WY, Wong CH, Mruk DD, Cheng CY (2003) TGF-beta 3 regulates the blood-testis barrier dynamics via the p38 mitogen activated protein (MAP) kinase pathway: An in vivo study. Endocrinology 144:1139-1142 |
| Project Title: | Unraveling the mechanisms on cytokine-mediated regulation of Junctional Adhesion Molecules (JAM) in the testis |
| Investigator(s): | Lui WY, Lee WWM |
| Department: | Zoology |
| Source(s) of Funding: | General Research Fund (GRF) |
| Start Date: | 01/2008 |
| Abstract: |
| (1) Identification of the signaling pathway(s) in cytokine-mediated transcriptional regulation of JAMs in testicular cells; (2) Characterization of the cytokine-mediated post-transcriptional regulation of JAM transcripts; (3) Characterization of cytokine-mediated post-translational modification of JAM proteins. |
| Project Title: | Role of nectin-like molecules (necls) and its regulation in the testis |
| Investigator(s): | Lui WY |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Seed Funding Programme for Basic Research |
| Start Date: | 06/2009 |
| Abstract: |
| Background: Importance of junction restructuring in spermatogenesis Dynamic restructuring of adherens junctions (AJ) between Sertoli and germ cells is a crucial event for germ cell development. Migration of developing germ cells across the seminiferous epithelium requires timely disassembly and reassembly of AJs, so that germ cells are able to migrate towards the tubular lumen for further development, and at the same time they must remain physically attach onto Sertoli cells for nutritional and structural supports. The timely disassembly of AJs between Sertoli cells and mature spermatids is also important to allow the release of spermatozoa at spermiation. Any interruption of AJ restructuring could either lead to premature release of germ cells or blockage of germ cell migration along the epithelium, resulting in infertility. Therefore, it is apparent that precise control of AJ restructuring in the epithelium is crucial for the completion of germ cell development (1, 2). Biology and structural components of adherens junctions in the epithelium Actin-based cell-cell AJs are extensively localized at the Sertoli cell/germ cell interface and between adjacent Sertoli cells. Ectoplasmic specialization (ES) is a testis-specific AJ, namely basal ES and apical ES. Basal ES is limited to adjacent Sertoli cells at the blood-testis barrier while apical ES is restricted to Sertoli cells and elongating spermatids (step 8 and beyond). Basal ES is constituted by several AJ proteins such as cadherin, nectin-2, Junctional Adhesion Molecule-B (JAM-B). However, four specific interlocking protein complexes, namely nectin-2/nectin-3, β1-integrin/laminin, JAM-B/JAM-C and nectin-like molecule 2 (necl-2)/necl-5 interlocks, are found at the apical ES (for review, see 2). These multiple interactions of junction proteins in the seminiferous epithelium ensure a close interaction of Sertoli and germ cells, and avoid detachment of premature germ cells from Sertoli cells during translocation. Nectin-like molecules (necls) and its members in the testis Nectin-like molecules are immunoglobulin (Ig)-like adhesion molecule that has three extracellular Ig domains, one transmembrane domain and a cytoplasmic tail containing a type II PDZ-binding domain. The necl family comprises five members and they are ubiquitously expressed. Like nectins, necls mediates both homophilic and heterophilic cell adhesion in Ca2+-independent manner (3). Other than its function on cell-cell adhesion, recent studies have shown that necls are involved in other cellular activities, including cell polarization, differentiation, movement and survival (4, 5). Three necl members (necl-2, -3, -5) are expressed in the testis (6-8). In the mouse testis, necl-2 is expressed exclusively on germ cells (6). Necl-2 is found in spermatogenic cells from intermediate spermatogonia to pachytene spermatocytes and steps 7-16 spermatids (6). Necl-2-based cell adhesion is found to be essential for retaining spermatocytes and elongating spermatids in the Sertoli cells for their maturation and the translocation of mature spermatids to the adluminal surface for release since three separate gene knockout studies have shown that male mice lacking necl-2 are infertile (6, 9, 10). Recent studies have shown that necl-2 can form a ternary complex with JAM-C via interaction with PAR3 in elongating and elongated spermatids (7). For necl-5, it is expressed in Sertoli cells. Co-immprecipitation studies have been shown that necl-5 is an interacting partner of necl-2 (11), suggesting that necl-2/necl-5 protein complex is one of the essential interlocking protein complexes to mediate the interaction between spermatogenic cells and Sertoli cells for germ cell development. For necl-3, a high level of necl-3 mRNA was detected in the testis. However, the localization and its function in the testis remain enigmatic (8). Cytokines and junction restructuring There are accumulating evidences showing that TGF-βs (TGF-β2 and TGF-β3) and TNFα are actively involved in junction restructuring in the seminiferous epithelium, thus facilitating the movement of developing germ cells. TGF-β3 is a crucial cytokine that modulates the disassembly of blood-testis barrier (constituted by basal ES and tight junctions), apical ES and AJ by down-regulating the expression of integral membrane proteins such as occludin and N-cadherin via the activation of different signaling pathways such as p38 and ERK pathways (12-15). Studies from our laboratory have demonstrated that TGF-β2 reduces JAM-B protein level. TGF-β2 exerts its negative regulatory effects on JAM-B via activating Smad proteins. Activated Smads compete against and displace Sp1 proteins from the TGIF motif of JAM-B promoter, resulting in JAM-B repression (16). A recent study has shown that TGF-β2 accelerates the internalization of integral membrane proteins such as JAM-A and occludin via clathrin-coated pit and targets them into late endosomes for degradation by lysosomes, thereby reducing the level of proteins and leading to the disassembly of cell junctions (17). Apart from TGF-β, TNFα is also a potent cytokine in regulating junction restructuring. TNFα can downregulate occludin, ZO-1, CLMP protein levels in the testis (18). We have demonstrated that TNFα acts on the CLMP mRNA transcript and destabilize the transcript by promoting the binding of tristetraprolin (TTP) at the 3’UTR region under the activation of JNK pathway (19). Besides, TNFα is capable to induce the disorganization of actin bundles and the cisternae of ER at the apical ES, leading to the release of premature spermatids (18). Objectives: This proposal aims to investigate the role of necls and its regulation in the testis. 1. To identify the interacting partners of necls in the testis 2. To investigate the effects of cytokines on the expression of necls in the testis 3. To identify the localization of necl-3 in the testis References: 1. Lui WY, Mruk D, Lee WM, Cheng CY (2003) J Androl 24:1-14 2. Lui WY and Cheng CY (2007) Cytokine and Growth Factor Reviews 18:299-311 3. Fujita E, Soyama A, Momoi T (2003) Exp Cell Res 2003 287:57-66 4. Takai Y, Irie K, Shimizu K, Sakisaks T, Ikeda W (2003) Cancer Sci 94:655-667 5. Takai Y, Miyoshi, Ikeda W, Ogita H (2008) Nat Rev 9:603-615 6. Fujita E, Kouroku Y, Ozeki S, Tanabe Y, Toyama Y, et al. (2006) Mol Cell Biol 26:718-726 7. Fujita E, Tanabe Y, Hirose T, Aurrand-Lions M, Kasahara T, et al. (2007) Am J Pathol 171:1800-1810 8. Pellissier F, Gerber A, Bauer C, Ballivet M, Ossipow V (2007) BMC Neurosci 8:90-107 9. van der Weyden L, Arends MJ, Chausiaux OE, Ellis PJ, et al. (2006) Mol Cell Biol 26:3595-3609 10. Yamada D, Yoshida M, Williams YN, Fukami T, Kikuchi S, et al. (2006) Mol Cell Biol 26:3610-3624 11. Wakayama T, Sai Y, Ito A, Kato Y, Kurobo M, et al. (2007) Biol Reprod 76:1081-1090 12. Lui WY, Lee WM, Cheng CY (2001) Endocrinology 142:1865-1877 13. Lui WY, Lee WM, Cheng CY (2003) Biol Reprod 68:1597-1612 14. Xia W, Cheng CY (2005) Dev Biol 280:321-43 15. Xia W, Mruk DD, Lee WM, Cheng CY (2006) J Biol Chem 281:16799-813 16. Wang Y and Lui WY (2008) Endocrinology (accepted) 17. Yan HH, Mruk DD, Lee WM, Cheng CY. (2008) FASEB J 22:1945-1959 18. Li MW, Xia W, Mruk D, Wang CQ, et al. (2006) J Endocrinol 190:313-329 19. Sze KL, Lui WY, Lee WM (2008) Biochem J 410:575-583 |
| Project Title: | 34th FEBS Congress Itch interacts with the pre-initiation complex for gene transcription |
| Investigator(s): | Lui WY |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | URC/CRCG - Conference Grants for Teaching Staff |
| Start Date: | 07/2009 |
| Completion Date: | 07/2009 |
| Abstract: |
| N/A |
| Project Title: | Outstanding Young Researcher Award 2008-2009 |
| Investigator(s): | Lui WY |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Outstanding Young Researcher Award |
| Start Date: | 12/2009 |
| Abstract: |
| The Awards are intended to recognize, reward, and promote exceptional research accomplishments of academic and research staff. |
| Project Title: | Coxsackie and adenovirus receptor-like membrane protein (CLMP) is a new tight junction protein in the testis: its role and regulations |
| Investigator(s): | Lui WY, Lee WWM |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | General Research Fund (GRF) |
| Start Date: | 01/2010 |
| Abstract: |
| 1) To investigate the role of CLMP in BTB dynamics; 2) To identify CLMP-associating partners in the cytosol; 3) To assess the effect of cytokine on the expression of CLMP in Sertoli cells and in the testis. |
| Project Title: | Unraveling the role of Ets related molecule (ERM) in the testis |
| Investigator(s): | Lui WY |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Seed Funding Programme for Basic Research |
| Start Date: | 06/2010 |
| Abstract: |
| Background: Cell junctions in the seminiferous epithelium and their physiological significance in spermatogenesis Different types of cell junctions are found in the seminiferous epithelium. Apart from the tight junctions (TJ) that is localized at the blood-testis barrier (BTB), anchoring junctions are also found at the BTB and the Sertoli-germ cell interface in the seminiferous epithelium (1). These junctions are adherens junctions (AJ) including basal and apical ectoplasmic specializations (ES). ES is an actin-based testis-specific hybrid AJ type which is confined to the region between the plasma membrane of two adjacent Sertoli cells. Basal ES is limited to the BTB, coexisting with TJ, whereas apical ES is found between Sertoli cells and elongating/elongated spermatids (step 8 and beyond in adult rat testes). Knockout studies have shown that various TJ proteins including occludin, junctional adhesion molecule-A (JAM-A) claudin-11 and ZO-2 are essential for spermatogenesis. Knockout of these TJ proteins result in male infertility (2-4). Like TJ proteins, knockout of AJ proteins such as nectin-2, nectin-3 or JAM-C in mice cause severe disruption in spermatogenesis and all male mice are sterile (5-7). These results apparently illustrated the functional significance of junction proteins in spermatogenesis. Formation of the intact BTB between adjacent Sertoli cells and dynamic restructuring of cell junctions (such as ES) between Sertoli and germ cells are crucial events for germ cell development (1). The formation of the BTB allows physical segregation of meiotic germ cells from the systemic circulation that avoids autoimmune response. During spermatogenesis, developing germ cells must migrate across the seminiferous epithelium towards the tubular lumen for further development, and at the same time they must remain physically attach onto Sertoli cells for nutritional and structural supports, such movement requires precise disassembly and reassembly of cell junctions. In addition, the timely disassembly of ES between Sertoli cells and mature spermatids is also important to allow the release of spermatozoa at spermiation (1). Any interruption of junction restructuring could either lead to premature release of germ cells or blockage of germ cell migration along the epithelium, resulting in infertility. Therefore, it is apparent that the formation of the BTB and timely junction restructuring in the epithelium are crucial for the completion of germ cell development. Infertility in male Ets related molecule (ERM) knockout mice Male mice with targeted disruption of Ets related molecule (ERM) displayed testicular atrophy with tubules devoid of germ cells (8). Although ERM is a transcription factor exclusively expressed in Sertoli cells, changes in gene expression levels are not restricted to Sertoli cells. In fact, a plethora of genes in spermatogonial germ cells are found to be altered in their expressions in the knockout mice (8). It is believed that knockout of ERM alters the gene expression in Sertoli cells, which results in significant change in seminiferous epithelial microenvironment and halts germ cell development. A list of genes in Sertoli cells has been found to be down-regulated (9- to 25-fold reduction) in ERM knockout mice and they are stromal cell-derived factor (SDF-1), chemokine ligand 5 (CXCL5), chemokine ligand 7 (CCL7) and matrix metalloproteinase 12 (MMP-12) (8). Based on the studies from other epithelial cells, it is known that chemokines and MMPs are crucial molecules that regulate the barrier function. For instance, chemokine, CCL2 induces TJ disassembly in blood-brain barrier by inducing internalization of claudin and occludin (9), whereas MMP promotes extracellular matrix degradation and resulting in junction disruption (10). However, it remains unknown whether the knockout of ERM gene affects the integrity of the blood-testis barrier and cell junction restructuring in the testis and whether ERM acts directly to control the gene transcription of junction proteins. Regulation of junction dynamics via transcriptional and post-translational modifications Timely regulation of the expression of different junction proteins in the testis is crucial during spermatogenesis. Both transcriptional and post-translational modifications are major approaches to exhibit temporal and spatial expression of junction proteins in the testis (1, 11). Studies from our laboratory have demonstrated that cytokine exerts its negative regulatory effects on JAM-B transcription via activating Smad proteins (12). Activated Smads compete against and displace Sp1 proteins from the TGIF motif of JAM-B promoter, resulting in JAM-B repression. A recent study has shown that cytokine trigger the internalization of integral membrane proteins such as JAM-A and occludin via clathrin-coated pit and targets them into late endosomes for degradation by lysosomes (13), thereby reducing the level of proteins and leading to the disassembly of the BTB. Objectives: This proposal aims to investigate the role of ERM on junction restructuring in the testis 1. To generate two Sertoli cell cell lines, (1) stably-expressing ERM in TM4 cells; and (2) stably-expressing ERM shRNA in MSC-1 cells 2. To assess the effect of ERM on the expression of junction proteins Reference: 1. Lui WY and Cheng CY (2007) Cytokine and Growth Factor Reviews 18:299-311 2. Saitou M, Furuse M, Sasaki H, Schulzke JD, Fromm M, Takano H, Noda T and Tsukita S (2000) Mol Biol Cell 11:4131-4142 3. Xu J, Anuar F, Ali SM, Ng MY, Phua DC and Hunziker W (2009) Mol Biol Cell 20:4268-4277 4. Gow A, Southwood CM, Li JS, Pariali M, Riordan GP, Brodie SE, Danias J, Bronstein JM, Kachar B and Lazzarini RA (1999) Cell 99:649-659 5. Mueller S, Rosenquist TA, Takai Y, Bronson RA and Wimmer E (2003) Biol Reprod 69:1330-1340 6. Ozaki-Kuroda K, Nakanishi H, Ohta H, Tanaka H, Kurihara H, Mueller S, Irie K, Ikeda W, Sakai T, Wimmer E, Nishimune Y and Takai Y (2002) Curr Biol 12:1145-1150 7. Gliki G, Ebnet K, Aurrand-Lions M, Imhof BA and Adams RH (2004) Nature 431:320-324 8. Chen C, Ouyang W, Grigura V, Zhou Q, Carnes K, Lim H, Zhao GQ, Arber S, Kurpios N, Murphy TL, Cheng AM, Hassell JA, Chandrashekar V, Hofmann MC, Hess RA and Murphy KM (2005) Nature 436:1030-1034 9. Stamatovic SM, Keep RF, Wang MM, Jankovic I and Andjelkovic AV (2009) J Biol Chem 284:19053-19066 10. Klessner JL, Desai BV, Amargo EV, Getsios S and Green KJ (2009) Mol Biol Cell 20:328-337 11. Lui WY and Lee WM (2008) Front Biosci 13:6775-6786 12. Wang Y and Lui WY (2009) Endocrinology 150:2404-2012 13. Yan HH, Mruk DD, Lee WM and Cheng CY. (2008) FASEB J 22:1945-1959 |
| Researcher : Mruk DD |
| List of Research Outputs |
| Wong W.P.E., Mruk D.D., Lee W.W.M. and Cheng C.Y., Regulation of blood-testis barrier dynamics by TGF-ß3 is a Cdc42-dependent protein trafficking event. , Proc. Natl. Acad. Sci. U.S.A. . 2010, 107: 11399-11404. |
| Researcher : Sze KL |
| List of Research Outputs |
| Siu E.R., Wong W.P.E., Mruk D.D., Sze K.L., Porto C.S. and Cheng C.Y., An Occludin-focal Adhesion Kinase Protein Complex At The Blood-testis Barrier: A Study Using The Cadmium Model, Endocrinology. 2009, 150: 3336-3344. |
| Researcher : Wan JMF |
| Project Title: | Effects of tumor necrosis factor-[alpha]TNF-[alpha] on cyclins and related cell cycle proteins expressions in human tumor cell lines as determined |
| Investigator(s): | Wan JMF |
| Department: | Zoology |
| Source(s) of Funding: | Other Funding Scheme |
| Start Date: | 07/1995 |
| Abstract: |
| To investigate: a) the effect of TNF on cyclins: D, E, A, B expression by flow cytometry; b) the effect of TNF on P21, PCNA expression by cytometric studies; c) the effect of TNF on tumor cell lines proliferation and apoptosis studied by flow cytometry. |
| Project Title: | The role of free radicals and antioxidants in motor neuron degenerative disease |
| Investigator(s): | Wan JMF, Vacca-Galloway LL |
| Department: | Zoology |
| Source(s) of Funding: | Other Funding Scheme |
| Start Date: | 07/1995 |
| Abstract: |
| There are increasing evidence indicating the involvement of free radicals damage in many chronic diseases such as Parkinson's disease, Alzheimer's disease and neurondegenerative disease. By using a motor neuron degenerative disease mouse model, to investigate the roles of free radicals in the disease process and investigating whether antioxidants such as vitamins E and C can be any therapeutic use by naturalizing the free radicals. |
| Project Title: | The effects of antioxidants on small cell lung cancer cell line, NCI-H446 |
| Investigator(s): | Wan JMF |
| Department: | Zoology |
| Source(s) of Funding: | Other Funding Scheme |
| Start Date: | 09/1995 |
| Abstract: |
| Lung cancer is almost certainly the most common cancer in the world today. Over the past several years, work has focused on characterizing the prevention, inhibition and regression of lung cancer by [beta]-carotene, vitamin C and vitamin C which act as antioxidants. This study aims to investigate the antiproliferative potential of [beta]-carotene and retinoids by flow cytometry technology. The data will help us to understand how antioxidants prevent lung cancer formation and progression. |
| Project Title: | The effects of different dietary fatty acids on the development of mammary tumors in female fischer 344 rats |
| Investigator(s): | Wan JMF |
| Department: | Zoology |
| Source(s) of Funding: | Other Funding Scheme |
| Start Date: | 09/1995 |
| Abstract: |
| Exciting evidences demonstrated that the quality of dietary fatty acids, especially W-6 and W-3 polyunsaturated fatty acids affect the development of cancers such as the colon, breast, and prostate. This project aims to investigate the effect of saturated, monosaturated W-3, and W-6 polyunsaturated fatty acids on breast cancer cells proliferation by using flow cytometry technology. The data in this study will help us understand the mechanisms involved in more depth. |
| Project Title: | Molecular Structural Determination of Protein-bound Polysaccharide peptide (PSP) isolated from the Chinese Medicinal Mushroom Coriolus versicolor (Cov-1) |
| Investigator(s): | Wan JMF, Sze KH, Che CM |
| Department: | Zoology |
| Source(s) of Funding: | Seed Funding Programme for Basic Research |
| Start Date: | 05/2006 |
| Abstract: |
| Purpose: The goal of this project is to determine the molecular structure of Cov-1 PSP molecule dervies from the Chinese Medicine mushroom Yun Zhi. Key Issues: Polysaccharide peptide (PSP) isolated from the mycelia of fungus Coriolus versicolor (Cov-1 strain) or Yun Zhi is Chinese Medicine best known for its anticancer and immunomodulatory properties. PSP is classified as a biological response modifier (Ng TB 1998 review) with the ability to induce gamma-interferon, intelerukin-2 production, T-cell proliferation in cancer patients. A small peptide with a molecular weight of 16-18 kDa originating from PSP has been produced with antiproliferative and antitumor activities (Yang et al 1992). We have recently published the cellular and molecular detailed cell death induction pathways of PSP on human leukemic cells by flow cytometry (Yang &Wan 2005, Hui and Wan 2005) and cDNA arrays (Zeng, Leung &Wan 2005). The ability of the Cov-1 PSP to distinguish cancerous cells from non-cancerous cells as recently determined by us (Yang & Wan 2005) and previously by others (NT 1998 review), indeed suggesting its uniqueness potential in its development into anticancer agent. Cov-1 PSP possesses a molecular weight of approximately 100 kDa. The polysaccharide moiety is a heteropolysaccharide made up of monosccharides with alpha-1, 4 and beta-1, 3 glucosidic linkages consisting of glucose, glactose, mannose, xylose, arabinose and trace amount of rhamnose (NT 1998 review). The polypeptide unit contains glutamic and aspartic acids as the abundant amino acids. PSP is presently used as over-the-counter dietary health supplement with multiple health claims such as anti-cancer, anti-inflammatory, antioxidant, anti-allergic and anti-viral. Our preliminary work on separation and purification of PSP by HPLC technology has identified two factions: a small molecular fraction of < 5000 Da and a macro-molecular fraction of > 5000 Da. The former fraction exhibited anticancer effect onhuman leukemia and the latter fraction exhibited immunomodulatory effect on healthy normal human T-lymphocytes. Despite the promising potentials of PSP, pharmaceumatical industry is not willing to invest into its therapeutic development unless the molecular structural information is apparent. It is urgent to identify the molecular structure of the PSP molecules as soon as possible since this unqiue Cov-1 strain medicinal mushroom exhibits most promising anticancer and immunomodulatory properties. The Cov-1 PSP strain is currently in Phase III clinical testing for anti-cancer properties in China with sucessful outcomes. Up-to-now, there are no clear structural determination of the PSP parent molecule and its fractions. The present propoal thus sought to reveal the molecular structure of PSP. Issues to be addressed: Additional experiments are also required to more thoroughly assess the molecular structure of the compounds that are responsible for the immunomodulatory effect and that which posses anticancer activities. Purpose of this proposed project: The goal of this project is to determine the structure of Cov-1 PSP molecule by carrying out the following objectives: 1. to purify fractions of PSP by DEAE Anion-exchange HPLC system 2. to elucidate the molecular structure of PSP by spectroscpic analysis, NMR and/or X-ray crystallograpghy. We believe that the structural determination of the molecular structure of PSP is urgently needed as to provide important insight into its distinct functions/roles in treatment of cancer and infectious diseases. References Cited: Yang MM et al (1992). The anticancer effect of a small polypeptide from Coriolus versicolor. Am J Clin Med 20: 221-32. Ng TB (1998). A review or research on the protein-bound polysaccharide PSP from the mushroom Coriolus versiolor. Gen Pharmacol 30: 1-4. Zeng F, Leung F and Wan JMF (2005). Molecular characterization of Coriolus versicolor in human promyelotic leukemic HL-60 cells using cDNA microarray. In J of Oncology 26: 10-16. Hui K.P.Y& Wan JMF (2005) Induction of S phase cells arrest and caspase activation by polysaccharide peptide (PSP) isolated from coriolus versicolor enhanced the cell-cycle-dependent activity and apoptotic cell death of Doxorubicin and Etoposide but not Cytarabine in HL-60 cells. Oncology Reports. 14(1): 145-165. Yang X and Wan JMF (2005). The Cell Death Process of the Anticancer Agent Polysaccharide-peptide (PSP) in Human Promyelocytic Leukemic HL-60 Cells. Oncology Reports 13(6): 1201-1221. |
| Project Title: | The 5th International Medicinal Mushroom Conference Polysaccaropeptide of Coriolus versicolor Enhances the Anticancer Activity of Camptorhecin (CAM) on Human :eukemic HL-60 cells |
| Investigator(s): | Wan JMF |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | URC/CRCG - Conference Grants for Teaching Staff |
| Start Date: | 09/2009 |
| Completion Date: | 09/2009 |
| Abstract: |
| N/A |
| Researcher : Wong AOL |
| Project Title: | Novel actions of somatostatin in grass carp pituitary cells: inhibition of growth hormone synthesis through up-regulation of CREB Gene expression |
| Investigator(s): | Wong AOL |
| Department: | Zoology |
| Source(s) of Funding: | General Research Fund (GRF) |
| Start Date: | 12/2006 |
| Completion Date: | 12/2009 |
| Abstract: |
| To demonstrate the presence of somatostatin (SRIF) immunoreactivity in the grass carp pituitary; to confirm that SRIF can inhibit GH production and GH gene expression in grass carp pituitary cells; to test if SRIF can interact with other GH-releasing factors to regulate GH gene expression at the pituitary level; to study the role of GH transcript stability and gene transcription in SRIF inhibition of GH mRNA expression; to test if SRIF can modulate CREB production and CREB gene expression in grass carp pituitary cells; to examine the role of transcript stability and gene transcription in SRIF-induced CREB mRNA expression; to check for spatial and temporal correlations between CREB and GH mRNA expression after SRIF treatment; to elucidate the post-receptor signaling events mediating SRIF actions on CREB and GH mRNA expression; to test if SRIF treatment and CREB over-expression can affect the promoter activity of grass carp GH gene; to map the location(s) of SRIF and CREB responsive sequence(s) in grass carp GH promoter by 5' deletion; to identify the cis-acting element(s) in GH promoter for SRIF and CREB regulation of GH gene transcription. |
| Project Title: | CREB co-ordinates the differential actions of c-Fos and Jun-B in PACAP-induced growth hormone gene expression in grass carp. |
| Investigator(s): | Wong AOL |
| Department: | Zoology |
| Source(s) of Funding: | General Research Fund (GRF) |
| Start Date: | 12/2007 |
| Abstract: |
| To establish the spatial and temporal correlation of c-Fos and Jun-B expression with PACAP-induced CREB phosphorylation & GH gene expression in carp somatotrophs; to elucidate the functional role of cAMP/PKA, PLC/PKC, Ca2+/CaM cascades in PACAP-stimulated c-Fos and Jun-B gene expression in carp somatotrophs; to characterize the genomic organization and 5’promoters of grass carp c-Fos and Jun-B genes by molecular cloning and sequence matching; to confirm that c-Fos and Jun-B promoters can be activated by PACAP through appropriate signaling mechanisms and CREB phosphorylation; to test the functional role of CRE sites in c-Fos and Jun-B promoters in PACAP-induced c-Fos & Jun-B gene transcription via CREB activation. |
| Project Title: | Pituitary D1/D1A Receptor in Dopamine-Stimulated Growth Hormone Gene Expression in Grass Carp. |
| Investigator(s): | Wong AOL |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | General Research Fund (GRF) |
| Start Date: | 01/2009 |
| Abstract: |
| (1) To establish the structural identity of grass carp D1/D1A receptor by molecular cloning and confirm its expression in carp somatotrophs isolated by laser capture microdissection; (2) To characterize the receptor binding specificity and functional properties of grass carp D1/D1A receptor by expression studies in mammalian cell line; (3) To examine the local actions of GH and LH on both basal as well as DA D1-stimulated D1/D1A gene expression in carp somatotrophs; (4) To elucidate the functional coupling of the cAMP/PKA pathway with MAPK- and PI3K-dependent cascades in DA D1-stimulated GH gene expression in grass carp pituitary cells; (5) To unveil the functional role of cAMP/PKA-, ERK1/2-, P38 MAPK-, and PI3K-dependent cascades and CREB phosphorylation in DA induction of GH promoter activity through D1/D1A receptors. |
| Project Title: | Negative Modulation of Intrapituitary Feedback Loop in Grass Carp by Insulin-like Growth Factors through down-regulation of pituitary growth hormone receptor expression. |
| Investigator(s): | Wong AOL |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | General Research Fund (GRF) |
| Start Date: | 01/2010 |
| Abstract: |
| 1) To establish the structural identity of IGF-I & -II expressed in the carp pituitary and characterize their expression patterns in different pituitary cell types; 2) To examine the local actions of LH and GH on IGF-I & -II expression in grass carp pituitary cells, both at the transcript level and protein level; 3) To confirm the functional involvement of locally produced IGFs in LH- and GH-induced GHR down-regulation in carp pituitary cells; 4) To elucidate the functional role of ERK1/2-, P38 MAPK-, JNK- and PI3K-dependent cascades in IGF-I & -II modulation of GHR mRNA & protein expression in carp somatotrophs. |
| Project Title: | The 92th Annual Meeting & Expo of the Endocrine Society (ENDO 2010) Functional Interactions of Activin with PACAP and GnRH in regulating Grass Carp LH beta Gene Transcription. |
| Investigator(s): | Wong AOL |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | URC/CRCG - Conference Grants for Teaching Staff |
| Start Date: | 06/2010 |
| Completion Date: | 06/2010 |
| Abstract: |
| N/A |
| Researcher : Wong EWP |
| List of Research Outputs |
| Cheng C.Y., Wong E.W.P., Yan H.H.N. and Mruk D.D., Regulation of spermatogenesis in the microenvironment of the seminiferous epithelium: new insights and advances, Mol Cell Endocrinol. 2010, 315: 49-56. |
| Researcher : Xu RJ |
| Project Title: | A study on the physiological role of transforming growth factor [beta] in postnatal adaptation of the gastrointestinal tract in neonatal pigs |
| Investigator(s): | Xu RJ |
| Department: | Zoology |
| Source(s) of Funding: | Small Project Funding |
| Start Date: | 11/2003 |
| Abstract: |
| To investigate the possible physiological role of TGF-[beta] in regulation of postnatal adaptation of the gastrointestinal tract in neonatal animals. |
| Researcher : Yan HHN |
| Project Title: | Characterization of claudin-1 and its interacting partners in gastric cancer |
| Investigator(s): | Yan HHN, Leung SY |
| Department: | Pathology |
| Source(s) of Funding: | Small Project Funding |
| Start Date: | 01/2009 |
| Abstract: |
| Epithelial-mesenchymal transition (EMT) converts epithelial cells into mesenchymal migratory cells, which also plays a key role in tumor progression (1, 2). During tumor metastasis, EMT begins with a loss of cell adhesion and apical-basal polarity in carcinoma cells, to be followed by a shift in cytoskeletal dynamics, acquiring the mesenchymal phenotype. This can facilitate the migration of malignant cells to specific destinations. Disassembly of tight junctions (TJs) and adherens junctions (AJs) is believed to be a pre-requisite for tumor cells to gain motility, mobility and invasiveness. Whilst alterations in AJs, such as E-cadherin and β-catenin, are well-known to play critical roles in EMT, less is known about the role of TJ alteration in this process. In normal epithelia, TJ forms a selective permeability barrier between epithelial cells. TJ regulates the passage of ions and solutes across paracellular space and functions as a fence, which maintains cell polarity by preventing the diffusion of lipids and proteins between the apical and basolateral regions of the cell epithelium (3, 4). Three classes of transmembrane proteins are currently identified in TJ: occludins, claudins, and junctional adhesion molecules (JAMs). They are linked to actin cytoskeleton for scaffolding via the binding of cytoplasmic proteins, such as zonula occludens (ZO) 1, 2 and/or 3 at the C-terminus. Thus, the TJ barrier has to be compromised for a metastatic tumor cell to: (i) ‘escape’ from its primary site, and (ii) ‘transplant’ to the new site during tumorigenesis via changes in the integral membrane proteins at the site. If the biology of these events is understood, it is conceivable that better therapeutic approaches can be developed. Recent studies have demonstrated the aberrant up-regulation of several TJ proteins, especially claudins, in various types of cancers (5, 6). For instance, up-regulation of claudin-1 has been found in colorectal cancer via the β-catenin/Tcf signaling (7, 8). Transfection of wild-type adenomatous polyposis coli (APC) gene, a tumor suppressor gene, in APC-deficient colon cell line resulted in significant down-regulation of claudin-1 (7). Besides, enhanced expression of claudin-1 was found to be inversely correlated with another tumor suppressor gene, smad4 (9). Upon overexpression of claudin-1 in colon cancer cell lines, this led to an increase in tumor invasiveness (8). EMT was also observed, accompanied with a loss of E-cadherin and up-regulation of vimentin (8). On the contrary, ZO proteins have been implicated as tumor suppressors. For instance, ZO proteins share high homology to the tumor suppressor gene of Drosophila called discs-large-A (dlgA) at the src homology region 3 (SH3) and guanylate kinase (GUK) domains (10). ZO-1 was shown to induce EMT in Madin-Darby Canine Kidney (MDCK) cells when mutant ZO-1 lacking the GUK domain was expressed at cytosol instead of at the cell surface (11). Expression of ZO-1 was down regulated in poorly differentiated gastric and colon tumors (12). Besides, expression of ZO-1 in gastric is less frequent in diffuse type as compare with intestinal-type adenocarcinoma (13). When there is an open up of intestinal epithelial barrier upon inflammation, there was down-regulation of ZO-1 and 3, but an increase in expression of claudin-1 as demonstrated in Caco-2 cell line treated with mixture of cytokines (14). The changes closely parallel those observed in carcinogenic process. Additionally, claudin-1 is structurally associated with Pals1-assoicated tight junction protein (PATJ) via the PDZ domain (15). The PATJ, together with Crumbs (CRB) and protein associated with Lin seven1 (Pals1, or Stardust) form a polarity complex at the apical side of an epithelium with TJ (16). This complex was firstly identified in Drosophila and conserved among species. Knock-down of PATJ in polarized Caco-2 cell line resulted in mislocalization of occludin and ZO-3 (17), this suggests the importance of PATJ in maintaining TJ stability. Furthermore, PATJ was found to regulate the migration of MDCK cells by recruiting other polarity complex components to the leading edge of migrating cells, illustrating the role of the polarity complex in regulating cell migratory behavior such as those found in metastatic tumor cells (18). These findings thus illustrate: 1) overexpression of specific TJ proteins (e.g., claudin-1) facilitates tumorigenesis, 2) the opposing effects of claudin-1 and ZOs on tumorigenesis, and 3) TJ proteins are likely involved in tumor metastasis, other than their structural function. The regulation of claudin-1, ZOs as well as the CRB polarity complexes during gastric cancer development is poorly explored except the aberrant up-regulation of claudin-1,3,4 and 7 (13, 19). From our available microarray dataset that contains 90 gastric tumors, 14 tumors that had metastasized to lymph nodes, and 22 normal mucosa (20), we have detected up-regulation of claudin-1, 3 and 4 in gastric tumor, with reciprocal down-regulation of claudin-18. For ZO proteins, there are heterogeneous expressions among tumor samples (Fig 1A). Up-regulation of claudin-1 in gastric tumors versus normal mucosa was further confirmed by real-time quantitative PCR and immunoblotting (see Fig 1B-C). These preliminary findings thus support this application to examine the role of claudin-1 and other TJ proteins in gastric cancer. Aim: To understand the changes in protein levels and interactions between TJ proteins (claudin-1, ZOs and PATJ) in gastric cancer development and their contribution to Epithelial-Mesenchymal-Transition. This will be done by examining for morphological changes, cell mobility, invasive behaviour, alter in steady-state protein levels, subcellular localization, protein-protein interaction between claudin-1, ZOs and polarity complexes via forced overexpression and knockdown of claudin-1 in gastric cancer cell lines. |
| Project Title: | AACR 101st Annual Meeting 2010 Retinoic acid receptor responder 3 expression suppresses colorectal cancer cell growth in vitro and is a stage-independent prognostic marker in colorectal cancer patients in vivo |
| Investigator(s): | Yan HHN |
| Department: | Pathology |
| Source(s) of Funding: | URC/CRCG - Conference Grants for Teaching Staff |
| Start Date: | 04/2010 |
| Completion Date: | 04/2010 |
| Abstract: |
| N/A |
| List of Research Outputs |
| Cheng C.Y., Wong E.W.P., Yan H.H.N. and Mruk D.D., Regulation of spermatogenesis in the microenvironment of the seminiferous epithelium: new insights and advances, Mol Cell Endocrinol. 2010, 315: 49-56. |
| Researcher : Zhou H |
| List of Research Outputs |
| Tang K.S., Zhou H., Yam J.W.P. and Wong A.S.T., c-Met overexpression contributes to the acquired apoptosis resistance of non-adherent ovarian cancer cells through a cross-talk mediated by phosphatidylinositol 3-kinase and extracellular signal-regulated kinase 1/2, 101th Annual Meeting of the American Association for Cancer Research, Washington, DC, (Abstract No. 1722). 2010. |
| Tang K.S., Zhou H., Yam J.W.P. and Wong A.S.T., c-Met overexpression contributes to the acquired apoptotic resistance of ovarian cancer cells through a cross-talk mediated by phosphatidylinositol 3-kinase and extracellular-signal regulated kinase 1/2, Neoplasia. 2010, 12: 128-138. |