Transcriptional Response of Selenopolypeptide Genes and Selenocysteine Biosynthesis Machinery Genes in Escherichia coli during Selenite Reduction

This work was supported by a United States Department of Agriculture-Cooperative State Research, Education, and Extension Service grant (no. 2009-35318-05032), a Biotechnology Research grant (no. 2007-BRG-1223) from the North Carolina Biotechnology Center, and a startup fund from the Golden LEAF Foundation to the Biomanufacturing Research Institute and Technology Enterprise (BRITE).Bacteria can reduce toxic selenite into less toxic, elemental selenium (Se0), but the mechanism on how bacterial cells reduce selenite at molecular level is still not clear. We used Escherichia coli strain K12, a common bacterial strain, as a model to study its growth response to sodium selenite (Na2SeO3) treatment and then used quantitative real-time PCR (qRT-PCR) to quantify transcript levels of three E. coli selenopolypeptide genes and a set of machinery genes for selenocysteine (SeCys) biosynthesis and incorporation into polypeptides, whose involvements in the selenite reduction are largely unknown. We determined that 5 mM Na2SeO3 treatment inhibited growth by ∼50% while 0.001 to 0.01 mM treatments stimulated cell growth by ∼30%. Under 50% inhibitory or 30% stimulatory Na2SeO3 concentration, selenopolypeptide genes (fdnG, fdoG, and fdhF) whose products require SeCys but not SeCys biosynthesis machinery genes were found to be induced ≥2-fold. In addition, one sulfur (S) metabolic gene iscS and two previously reported selenite-responsive genes sodA and gutS were also induced ≥2-fold under 50% inhibitory concentration. Our findings provide insight about the detoxification of selenite in E. coli via induction of these genes involved in the selenite reduction process.Publisher PDFPeer reviewe

Application of Scutellariae radix

Salmonella enterica serovar Choleraesuis, a host-adapted pathogen of swine, usually causes septicemia. Lactic acid bacteria (LAB) strains have been widely studied in recent years for their probiotic properties. In this study, a mouse infection model first screened for potential agents against infection, then a pig infection model evaluated effects of LAB strains and herbal plants against infection. Scutellariae radix (SR) and Gardeniae fructus (GF) showed abilities to reduce bacteria shedding and suppressing serum level of TNF-α induced by infection in swine. Bioactivities of SR and GF were enhanced by combining with LAB strains, which alone could speed up the bacteria elimination time in feces and boost immunity of infected pigs. Baicalein and genipin exhibited stronger cytotoxicity than baicalin and geniposide did, as well as prevent Salmonella from invading macrophages. Our study suggests LAB strains as exhibiting multiple functions: preventing infection, enhancing immunity to prepare host defenses against further infection, and adjusting intestinal microbes’ enzymatic activity in order to convert herbal compounds to active compounds. The SR/GF-LAB strain mixture holds potential infection-prevention agents supplied as feed additives

An Effective Way of Producing Fully Assembled Antibody in Transgenic Tobacco Plants by Linking Heavy and Light Chains via a Self-Cleaving 2A Peptide

Therapeutic monoclonal antibodies (mAbs) have evolved into an important class of effective medicine in treatment of various diseases. Since the antibody molecule consists of two identical heavy chains (HC) and two light chains (LC), each chain encoded by two different genes, their expressions at similar levels are critical for efficient assembly of functional recombinant mAbs. Although the plant-based expression system has been tested to produce fully assembled recombinant mAbs, coordinately expressing HC and LC at similar levels in a transgenic plant remains a challenge. A sequence coding for a foot-and-mouth disease virus (FMDV) 2A peptide has been successfully used to link two or more genes, which enable the translated polyprotein to be “self-cleaved” into individual protein in various genetically modified organisms. In the present study, we exploited the usage of F2A in Ebola virus monoclonal antibody (EBOV mAb) production. We found that transgenic tobacco plants carrying a transcription unit containing HC and LC linked by 2A not only produced similar levels of HC and LC but also rendered a higher yield of fully assembled EBOV mAb compared to those expressing HC and LC in two independent transcription units. Purified EBOV mAb bound to an Ebola epitope peptide with apparent Kd-values of 90.13–149.2 nM, indicating its proper assembly and high affinity binding to Ebola epitope peptide. To our knowledge, this is the first report showing mAb production by overexpressing a single transcription unit consisting of HC, LC and 2A in stable transformed tobacco plants

Transcriptional Response of Selenopolypeptide Genes and Selenocysteine Biosynthesis Machinery Genes in Escherichia coli during Selenite Reduction

This work was supported by a United States Department of Agriculture-Cooperative State Research, Education, and Extension Service grant (no. 2009-35318-05032), a Biotechnology Research grant (no. 2007-BRG-1223) from the North Carolina Biotechnology Center, and a startup fund from the Golden LEAF Foundation to the Biomanufacturing Research Institute and Technology Enterprise (BRITE).Bacteria can reduce toxic selenite into less toxic, elemental selenium (Se0), but the mechanism on how bacterial cells reduce selenite at molecular level is still not clear. We used Escherichia coli strain K12, a common bacterial strain, as a model to study its growth response to sodium selenite (Na2SeO3) treatment and then used quantitative real-time PCR (qRT-PCR) to quantify transcript levels of three E. coli selenopolypeptide genes and a set of machinery genes for selenocysteine (SeCys) biosynthesis and incorporation into polypeptides, whose involvements in the selenite reduction are largely unknown. We determined that 5 mM Na2SeO3 treatment inhibited growth by ∼50% while 0.001 to 0.01 mM treatments stimulated cell growth by ∼30%. Under 50% inhibitory or 30% stimulatory Na2SeO3 concentration, selenopolypeptide genes (fdnG, fdoG, and fdhF) whose products require SeCys but not SeCys biosynthesis machinery genes were found to be induced ≥2-fold. In addition, one sulfur (S) metabolic gene iscS and two previously reported selenite-responsive genes sodA and gutS were also induced ≥2-fold under 50% inhibitory concentration. Our findings provide insight about the detoxification of selenite in E. coli via induction of these genes involved in the selenite reduction process.Publisher PDFPeer reviewe

A virtual travel agent system for M-tourism with semantic Web service based design and implementation

With the recent advances in Internet and mobile technologies and infrastructures, there are increasing demands for ubiquitous access to tourist information systems for service coordination and integration. However, disparate tourist information and service resources such as airlines, hotels, tour operators, etc., make it difficult for tourist to use them effectively when planning their trips and/or during their trips. Motivated by the emerging technologies of Multi-Agent Information System (MAIS) and its ability to aid Internet and mobile users, together with semantic Web that can effectively organize information and service resources. In this paper, we propose a Virtual Travel Agent System (VTAS), which is built upon these technologies. In this paper, we formulate a scalable, flexible, and intelligent MAIS architecture for VTAS with agent clusters based on a case study of a large service-oriented travel agency. Agent clusters may comprise several types of agents to achieve the goals of the major processes of a tourist's trip. We show how agents can make use of ontology from the semantic web help tourists better plan, understand, and specify their requirements. We further illustrate how this can be successfully implemented with Web service technologies to integrate disparate Internet tourist resources. © 2007 IEEE

A Sharing Mind Map-oriented Approach to Enhance Collaborative Mobile Learning With Digital Archiving Systems

With the advances in mobile network technology, the use of portable devices and mobile networks for learning is not limited by time and space. Such use, in combination with appropriate learning strategies, can achieve a better effect. Despite the effectiveness of mobile learning, students’ learning direction, progress, and achievement may differ. Thus, the enhancement of learners’ opinions on the usability and interactivity during mobile learning are challenging issues to overcome. This study developed a sharing mind map-oriented mobile learning system integrated with valuable information preserved in a digital archiving system. In addition to the functions of traditional mind maps, this system also enabled students to complete and record relevant information that they had found onto the mind map and further improve the integrity of their own knowledge. To investigate the effectiveness of this teaching approach, this study added digital archive data and used mind map sharing to help learners develop knowledge.  By using the proposed approach, students were able to perform self-assessment on learning content, choose appropriate learning directions, and progress according to their level of learning. At the same time, they could collaboratively learn with peers to engage themselves more deeply in their learning. That is, their learning motivation could be constantly triggered through the observations and sharing of mind maps from one to another. This study selected sixth graders as its research subjects in two classes at the school where one researcher works. There were 31 and 30 valid samples in the experimental group and control group, respectively, with a total of 61 students. The experimental group was conducted by using sharing mind map with corresponding geographical archived information to investigate the effectiveness of sharing mind map (SMM) in mobile learning; on the other hand, the control group was conducted by using a traditional learning approach. The outcomes indicate that students’ learning performance could be enhanced by using archived information SMM mobile learning

Asialo-rhuEPO as a Potential Neuroprotectant for Ischemic Stroke Treatment

Neuroprotective drugs to protect the brain against cerebral ischemia and reperfusion (I/R) injury are urgently needed. Mammalian cell-produced recombinant human erythropoietin (rhuEPOM) has been demonstrated to have excellent neuroprotective functions in preclinical studies, but its neuroprotective properties could not be consistently translated in clinical trials. The clinical failure of rhuEPOM was thought to be mainly due to its erythropoietic activity-associated side effects. To exploit its tissue-protective property, various EPO derivatives with tissue-protective function only have been developed. Among them, asialo-rhuEPO, lacking terminal sialic acid residues, was shown to be neuroprotective but non-erythropoietic. Asialo-rhuEPO can be prepared by enzymatic removal of sialic acid residues from rhuEPOM (asialo-rhuEPOE) or by expressing human EPO gene in glycoengineered transgenic plants (asialo-rhuEPOP). Both types of asialo-rhuEPO, like rhuEPOM, displayed excellent neuroprotective effects by regulating multiple cellular pathways in cerebral I/R animal models. In this review, we describe the structure and properties of EPO and asialo-rhuEPO, summarize the progress on neuroprotective studies of asialo-rhuEPO and rhuEPOM, discuss potential reasons for the clinical failure of rhuEPOM with acute ischemic stroke patients, and advocate future studies needed to develop asialo-rhuEPO as a multimodal neuroprotectant for ischemic stroke treatment

A Universal Role for Inositol 1,4,5-Trisphosphate-Mediated Signaling in Plant Gravitropism

Inositol 1,4,5-trisphosphate (InsP(3)) has been implicated in the early signaling events of plants linking gravity sensing to the initiation of the gravitropic response. However, at present, the contribution of the phosphoinositide signaling pathway in plant gravitropism is not well understood. To delineate the role of InsP(3) in plant gravitropism, we generated Arabidopsis (Arabidopsis thaliana) plants constitutively expressing the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme that specifically hydrolyzes InsP(3). The transgenic plants show no significant differences in growth and life cycle compared to wild-type plants, although basal InsP(3) levels are reduced by greater than 90% compared to wild-type plants. With gravistimulation, InsP(3) levels in inflorescence stems of transgenic plants show no detectable change, whereas in wild-type plant inflorescences, InsP(3) levels increase approximately 3-fold within the first 5 to 15 min of gravistimulation, preceding visible bending. Furthermore, gravitropic bending of the roots, hypocotyls, and inflorescence stems of the InsP 5-ptase transgenic plants is reduced by approximately 30% compared with the wild type. Additionally, the cold memory response of the transgenic plants is attenuated, indicating that InsP(3) contributes to gravisignaling in the cold. The transgenic roots were shown to have altered calcium sensitivity in controlling gravitropic response, a reduction in basipetal indole-3-acetic acid transport, and a delay in the asymmetric auxin-induced β-glucuronidase expression with gravistimulation as compared to the controls. The compromised gravitropic response in all the major axes of growth in the transgenic Arabidopsis plants reveals a universal role for InsP(3) in the gravity signal transduction cascade of plants