Unraveling G protein-coupled receptor endocytosis pathways using real-time monitoring of agonist-promoted interaction between beta-arrestins and AP-2

The most widely studied pathway underlying agonist-promoted internalization of G protein-coupled receptors (GPCRs) involves beta-arrestin and clathrin-coated pits. However, both beta-arrestin- and clathrin-independent processes have also been reported. Classically, the endocytic routes are characterized using pharmacological inhibitors and various dominant negative mutants, resulting sometimes in conflicting results and interpretational difficulties. Here, taking advantage of the fact that beta-arrestin binding to the beta2 subunit of the clathrin adaptor AP-2 (beta2-adaptin) is needed for the beta-arrestin-mediated targeting of GPCRs to clathrin-coated pits, we developed a bioluminescence resonance energy transfer-based approach directly assessing the molecular steps involved in the endocytosis of GPCRs in living cells. For 10 of the 12 receptors tested, including some that were previously suggested to internalize via clathrin-independent pathways, agonist stimulation promoted beta-arrestin 1 and 2 interaction with beta2-adaptin, indicating a beta-arrestin- and clathrin-dependent endocytic process. Detailed analyses of beta-arrestin interactions with both the receptor and beta2-adaptin also allowed us to demonstrate that recruitment of beta-arrestins to the receptor and the ensuing conformational changes are the leading events preceding AP-2 engagement and subsequent clathrin-mediated endocytosis. Among the receptors tested, only the endothelin A and B receptors failed to promote interaction between beta-arrestins and beta2-adaptin. However, both receptors recruited beta-arrestins upon agonist stimulation, suggesting a beta-arrestin-dependent but clathrin-independent route of internalization for these two receptors. In addition to providing a new tool to dissect the molecular events involved in GPCR endocytosis, the bioluminescence resonance energy transfer-based beta-arrestin/beta2-adaptin interaction assay represents a novel biosensor to assess receptor activation.     

Cell death induced by Pteris semipinnata L. is associated with p53 and oxidant stress in gastric cancer cells

In this study, we demonstrated that Ent-11alpha-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F) had stronger cytotoxicity against MKN-45, a gastric cancer cell line bearing wild-type p53 than MKN-28, another gastric cancer cell line containing missense mutation in p53. The rapid increase of ROS level was involved in the mechanism of cytotoxicity. Classical features of apoptosis induced by 5F were observed in MKN-45 cells only or more significant in MKN-45 cells than MKN-28 cells. Translocation of Bax from cytosol to mitochondria, reduction of delta psi m and DNA fragmentation were induced by 5F in the p53-dependent manner. We conclude that the expression of Bax and its downstream molecules requires the presentation of a wild-type p53 in the cells treated by 5F.     

香港的生物多样性及其保育工作

香港位于热带,属海洋性气候。地势崎岖多山,山地约占全港总面积的3/4。城市发展多集中在沿海平坦地带。目前香港的城市和乡镇面积约占总面积的20%,农地约占5%(当中大部份已遭荒废),余下的均为郊野地区,这包括天然林和人工林(约占14%)、灌丛(约占36%)及草地(约占17%)。由于良好的气候和地理条件,形成了众多不同的生态环境,使总面积仅1090 km²的弹丸之地孕育出种类多样的动植物,生物多样性十分丰富。香港约有2500种原生植物,包括被子植物约1900种,裸子植物7种,蕨类植物220多种及苔藓植物300多种。动物方面,已记录的野生哺乳类动物有40多种,鸟类超过459种,两栖类23种,爬行类70多种。昆虫种类繁多,其中蜻蜓目100多种,鳞翅目2200多种(蝴蝶200多种,蛾类2000多种)。有很多是国家保护物种和特有种。植物方面属国家一级保护的有1种——刺桫椤(Alsophila spinulosa);国家二级保护的有6种,如四药门花(Tetrathryrium subcordatum);国家三级保护的有8种,如穗花杉(Amentotaxus argotaenia)。此外,香港特有种有16种,例如紫萁科(Osmundaceae)的粤紫萁(Osmunda mildei)、马兜铃科(Aristolochiaceae)的香港细辛(Asarum hongkongense)和兰科(Orchidaceae)的谢氏卷瓣兰(Bulbophyllum tseanum)。动物方面有9种属国家一级保护,例如中华白海豚(Sousa chinensis);79种属国家二级保护。特有种则有卢氏小树蛙(Philautus romeri)、包氏双足蜥(Dibamus bogadeki)及多种昆虫。为了保护丰富的野生动植物及其栖息的环境,香港特别行政区政府制定了一些法例并推行了不少保护措施,例如设立了21个郊野公园和14个特别地区,占全港陆地总面积约38%。此外,还成立了2个禁区、3个海岸公园和1个海洋保护区。另一方面,政府还设立了59个“具特殊科学价值地点”,以保护及研究各种动植物、生态系统和特殊的地质地貌。香港地少人多,总人口超过600万,是世界上人口最稠密的地区之一。多年来香港这个生物宝库不断地遭受人类活动的威胁,近年来由于人口急剧上升,对土地需求迫切,不少郊野地区被开发利用,环境污染亦日益严重。此外,一些野生植物因具有药用价值、观赏价值或其他用途而遭盗伐或采集。上述种种因素已使香港野生动植物及其生境受到严重损害,一些物种更濒于灭绝,进行生物多样性的保育工作刻不容缓。因此,香港确实需要制定整体的生物多样性保护策略。有鉴于此,香港大学生态及分类学系于1996年展开了一项为期3年的香港生物多样性调查,以增加对动植物资源现况的了解,为保护香港的珍稀濒危物种和日益恶化的自然环境提出补救方案,并为制订长远的保育策略奠定基础。     

A comparison of two microbial detection methods used in aseptic processing of musculoskeletal allograft tissues

Tissues from 78 musculoskeletal donors were concurrently tested for microorganisms using both a swab and liquid culture method. An aggregate total of 20 organisms were detected by both methods. The swab detected 4/20 organisms while the liquid culture detected 18/20 organisms. The swab method yielded sensitivity and negative predictive values of 20 and 92.3%, respectively. Comparatively, the liquid culture displayed a sensitivity of 90% and a negative predictive value of 99%. These results clearly demonstrate that the liquid culture method is superior to swab cultures in microbial detection. Additional studies are necessary to determine the optimal culture conditions for different types of tissues when utilizing the liquid culture method.     

Rye Affects Bacterial Translocation,Intestinal Viscosity,Microbiota Composition and Bone Mineralization in Turkey Poults

Previously, we have reported that rye significantly increased both viscosity and Clostridium perfringens proliferation when compared with corn in an in vitro digestive model. Two independent trials were conducted to evaluate the effect of rye as a source of energy on bacterial translocation, intestinal viscosity, gut microbiota composition, and bone mineralization, when compared with corn in turkey poults. In each experiment, day-of-hatch, turkey poults were randomly assigned to either a corn or a rye diet (n = 0 /group). At 10 d of age, in both experiments, 12 birds/group were given an oral gavage dose of fluorescein isothiocyanate dextran (FITC-d). After 2.5 h of oral gavage, blood and liver samples were collected to evaluate the passage of FITC-d and bacterial translocation (BT) respectively. Duodenum, ileum and cecum gut sections were collected to evaluate intestinal viscosity and to enumerate gut microbiota. Tibias were collected for observation of bone parameters. Broilers fed with a rye diet showed increased (p<0.05) intestinal viscosity, BT, and serum FITC-d. Bacterial enumeration revealed that turkey poults fed with rye had increased the number of total lactic acid bacteria (LAB) in all three sections of the gastrointestinal tract evaluated when compared to turkey poults fed with corn. Turkey poults fed with rye also had significantly higher coliforms in duodenum and ileum but not in the ceca, whereas the total number of anaerobes increased only in duodenum. A significant reduction in bone strength and bone mineralization was observed in turkey poults fed with rye when compared with corn fed turkey poults. In conclusion, rye evoked mucosal damage in turkey poults that increased intestinal viscosity, increased leakage through the intestinal tract, and altered the microbiota composition and bone mineralization. Studies to evaluate dietary inclusion of selected Direct-Fed Microbial (DFM) candidates that produce exogenous enzymes in rye fed turkey poults are currently being evaluated.     

Stylet activity of cowpea aphid (Homoptera: Aphididae) on leaf extracts of resistant and susceptible cowpea cultivars

Electrical penetration graph recordings using direct current (DC-EPGs) were used to analyze aspects of the probing behavior of cowpea aphid,Aphis craccivora Koch, on intact plants and on hexane, ethyl acetate, and methanol extracts of leaves of aphid-resistant (ICV-12) and aphid-susceptible (ICV-1) cultivars of cowpeaVigna unguiculata (L.) Walp. In one set of experiments, recordings were done on plants with or without parafilm wrapping, or on plants painted with raw leaf juice and extracts of the two cultivars. In another study, recordings were done on leaf extracts homogenized in water or in 0.5M sucrose solution and then placed in parafilm membrane sachets. Electrodes were inserted into soil mix for the experiments on potted plants or into extract fractions and raw juice for the membrane feeding experiments on leaf extracts in parafilm sachets. Waveform signals were recorded from resistance fluctuations from interactions between aphids and substrates, and electromotive forces generated within each preparation. ICV-12 plants with or without parafilm wrapping, and ethyl acetate extracts and raw juice of that cultivar significantly (P≤0.05) reduced stylet penetration behavior. Thus, antixenosis as manifested by disruption of aphid stylet activity on host substrates, appeared to be a governing modality of aphid resistance in ICV-12.     

The Grp170 nucleotide exchange factor executes a key role during ERAD of cellular misfolded clients

When a protein misfolds in the endoplasmic reticulum (ER), it retrotranslocates to the cytosol and is degraded by the proteasome via a pathway called ER-associated degradation (ERAD). To initiate ERAD, ADP-BiP is often recruited to the misfolded client, rendering it soluble and translocation competent. How the misfolded client is subsequently released from BiP so that it undergoes retrotranslocation, however, remains enigmatic. Here we demonstrate that the ER-resident nucleotide exchange factor (NEF) Grp170 plays an important role during ERAD of the misfolded glycosylated client null Hong Kong (NHK). As a NEF, Grp170 triggers nucleotide exchange of BiP to generate ATP-BiP. ATP-BiP disengages from NHK, enabling it to retrotranslocate to the cytosol. We demonstrate that Grp170 binds to Sel1L, an adapter of the transmembrane Hrd1 E3 ubiquitin ligase postulated to be the retrotranslocon, and links this interaction to Grp170’s function during ERAD. More broadly, Grp170 also promotes degradation of the nonglycosylated transthyretin (TTR) D18G misfolded client. Our findings thus establish a general function of Grp170 during ERAD and suggest that positioning this client-release factor at the retrotranslocation site may afford a mechanism to couple client release from BiP and retrotranslocation.     

Expression of recombinant human glucose-dependent insulinotropic polypeptide in Escherichia coli by sequence-specific proteolysis of a protein A fusion protein

Glucose-dependent insulinotropic polypeptide (GIP) is a forty-two amino acid hormone that stimulates the secretion of insulin from the pancreatic B-cells in the presence of elevated glucose concentrations. The human GIP gene with the human A-fibrinopeptide sequence was synthesized and linked to the Staphylococcus aureus protein A gene in the vector pRIT2T. This plasmid was expressed in Escherichia coli, and the resulting fusion protein consisted of three domains: protein A for ease of purification, fibrinopeptide sequence for thrombin cleavage and human GIP. The GIP was subsequently cleaved from the fusion protein with -thrombin. The identity of the recombinant human GIP was confirmed by SDS-PAGE, ELISA, HPLC and amino-terminal amino acid sequence analysis. This recombinant product was shown to have comparable insulinotropic activity to porcine GIP in the isolated perfused pancreas.