Oxygen radical-mediated oxidation reactions of an alanine peptide motif - density functional theory and transition state theory study

<p>Abstract</p> <p>Background</p> <p>Oxygen-base (O-base) oxidation in protein backbone is important in the protein backbone fragmentation due to the attack from reactive oxygen species (ROS). In this study, an alanine peptide was used model system to investigate this O-base oxidation by employing density functional theory (DFT) calculations combining with continuum solvent model. Detailed reaction steps were analyzed along with their reaction rate constants.</p> <p>Results</p> <p>Most of the O-base oxidation reactions for this alanine peptide are exothermic except for the bond-breakage of the C_α-N bond to form hydroperoxy alanine radical. Among the reactions investigated in this study, the activated energy of OH α-H abstraction is the lowest one, while the generation of alkylperoxy peptide radical must overcome the highest energy barrier. The aqueous situation facilitates the oxidation reactions to generate hydroxyl alanine peptide derivatives except for the fragmentations of alkoxyl alanine peptide radical. The C_α-C_βbond of the alkoxyl alanine peptide radical is more labile than the peptide bond.</p> <p>Conclusion</p> <p>the rate-determining step of oxidation in protein backbone is the generation of hydroperoxy peptide radical via the reaction of alkylperoxy peptide radical with HO₂. The stabilities of alkylperoxy peptide radical and complex of alkylperoxy peptide radical with HO₂are crucial in this O-base oxidation reaction.</p

Development of recombinant baculoviruses for insect pest control and foreign protein expression

重組桿狀病毒在蟲害防治及外源蛋白表現的開發 中文摘要 桿狀病毒(baculoviruses)已被成功的發展為生物農藥，其中以加州苜蓿夜蛾核多角體病毒(Autographa californica multiple nuclear polyhedrosis viruses ; AcMNPVs)最常被應用；但由於緩慢的殺蟲效果限制了桿狀病毒在農業害蟲的防治應用。此外，以安全評估的考量，如何追蹤重組桿狀病毒之基因在環境中的穩定性與存在亦是十分重要的議題。因此，vAcPhsp70EGFP/Ppag90IT2重組加州苜蓿夜蛾核多角體病毒被構築，使其同時表現綠螢光蛋白(EGFP)和以色列蠍毒殺蟲蛋白(LqhIT2)，並分別以Phsp70及Ppag90啟動子啟動。結果顯示此重組病毒除能有效防治擬尺蠖幼蟲外，還可表現EGFP進行偵測已感染之病蟲。另一方面，由於珊瑚紅螢光蛋白(DsRed)能於接收太陽光後釋放出較長波長的光；因此在本論文研究中構築可同時表現EGFP 和DsRed的重組桿狀病毒vAcR-IR-G，以進一步探討EGFP 和DsRed二螢光蛋白在作為重組桿狀病毒(GMBV)追蹤劑的比較。結果顯示此重組病毒vAcR-IR-G在感染擬尺蠖、甜菜夜蛾和斜紋夜蛾幼蟲後，當其內含之DsRed蛋白表現，於日照下且不需任何其他輔助儀器，即可釋放紅螢光。因此，我們認為將此DsRed蛋白應用於偵測田間的重組桿狀病毒，將是一個有效的追蹤劑。自1983年起，桿狀病毒/昆蟲細胞表現系統即被廣泛的用來生產許多不同的生物醫療蛋白。故本研究以此系統來表現松杉靈芝(Ganoderma tsugae)中所分離出的免疫調節蛋白FIP-gts，結果依生物活性來評估，由Sf21昆蟲細胞所產生的rFIP-gts比由E.coli細胞所產生的rFIP-gts佳。Development of Recombinant Baculoviruses for Insect Pest Control and Foreign protein expression Abstract Baculoviruses have been successfully developed as biological control agents, especially Autographa californica multiple nucleopolyhedrosis viruses (AcMNPVs), which is used most frequently. However, the slow effectiveness limits their application in agriculture. In a safety standpoint, it is also important to track the genetic stability of recombinant baculoviruses and their presence in the environments. Thus, a polyhedrin-positive recombinant AcMNPV vAcPhsp70EGFP/Ppag90IT2 was constructed for larvae to express the EGFP and scorpion neurotoxin LqhIT2 under the control of Phsp70 and Ppag90 promoter, respectively. These results revealed that recombinant AcMNPV vAcPhsp70EGFP/Ppag90IT2 may significantly increase the insecticidal potency against Trichoplusia ni when additional EGFP was expressed as a visible marker. On the other hand, the red-protein pigment of DsRed, a red fluorescence protein, may convert the short wavelength light component of the solar radi&not;ation into a longer wavelength light. Thus, the EGFP and DsRed fluorescent genes are also expressed simultaneously in insect larva to evaluate which fluorescent protein is relatively bright and is suitable to act as GMBV tracer. Results demonstrate the insect larvae, including Tri&not;choplusia ni, Spodoptera exigua, and Spodoptera litura, infected by the genetic modified baculovirus (GMBV) containing DsRed gene can emit red fluorescence under sun light without any prosthetic apparatus. So, DsRed may serve as a powerful tracer to evaluate the efficiency of GMBV as an insecticide in the field. Since 1983, the baculovirus-insect cell expression system has been widely used to produce recombinant proteins for many different biomedical applications. In this study, the baculovirus/insect cell expression system is utilized to produce active FIP-gts, a fungal immunomodulatory protein from Song-Shan Lingzhi (Ganoderma tsugae). As expected, rFIP-gts produced in Sf21 cells is a better source than that produced in E. coli cells for the application of oral administration.Table of Contents English abstract ⅰ Chinese abstract ⅲ List of Tables and Figures ⅵ Research background 1 Reference 11 Figures 16 Chapter1. Enhancing insecticidal efficacy of baculovirus by early expressing an insect neurotoxin, LqhIT2, in infected Trichoplusia ni larvae Abstract 27 Introduction 28 Materials and Methods 31 Results 35 Discussion 38 References 40 Tables 44 Figures 46 Chapter2. Coral red fluorescence protein as tracer Abstract 51 Introduction 52 Materials and Methods 54 Results and Discussion 56 References 59 Figures 61 Chapter3. Functional expression of FIP-gts, a fungal immunomodulatory protein from Ganoderma tsugae, in Sf21 insect cells Abstract 63 Introduction 64 Materials and Methods 67 Results 73 Discussion 76 References 79 Table 84 Figures 85 Conclusion and Future Prospects 90 Reference 9

The Structural Study of Copper-binding Peptides: Implication in the Aggregation of Amyloid-β Peptides

There is evidence that copper is bound to amyloid-β peptide (Aβ) in senile plaque of Alzheimer's disease. Copper also plays a role in the neurotoxicity of the Aβ aggregates associated with free radical damage. In this study, we used L-histidyl-L-histidine peptide (di-histidine) to represent Aβ along with ab initio calculation to characterize the probable coordination between Cu(II) and Aβs to explore the Aβ aggregate structures. Sixteen models of copper-di-histidine complexes were proposed to investigate the copper-induced aggregation of Aβs through copper ionic coordination. All structures of the proposed models were optimized at the M05-2X/LanL2DZ level, and an Aβ aggregation formation mechanism was proposed

Identification of Rhopalosiphum Padi Virus 5′ Untranslated Region Sequences Required for Cryptic Promoter Activity and Internal Ribosome Entry

The 579-nucleotide 5′ untranslated region (5′UTR) of the Rhopalosiphum padi virus (RhPV) possesses a cross-kingdom internal ribosome entry site (IRES) activity that functions in insect, mammalian, and plant-derived in vitro translation systems, and six TAAG motifs within the DNA fragment encoding the RhPV 5′UTR were previously found to confer the RhPV 5′UTR with late promoter activity in baculovirus. In the present study, various truncated RhPV 5′UTR sequences were produced, and among them, a fragment of 110 bp ranging from nucleotides 309 to 418 was identified to be the shortest fragment responsible for the late promoter activity in baculovirus infected Sf21 cells. This 110 bp fragment contains a TAAG tandem repeat that retains more than 60% of the late promoter activity of the full length RhPV 5′UTR sequence. Further, IRES activity remained unchanged in all truncated RhPV 5′UTR constructs. Taken together, this novel 110 bp fragment having late promoter activity in baculovirus as well as IRES activity in mammalian cell, renders it a useful tool for the development of a “shuttle” bi-cistronic baculovirus gene expression and/or delivery vector

Inhibition of Klebsiella pneumoniae Growth and Capsular Polysaccharide Biosynthesis by Fructus mume

Klebsiella pneumoniae is the predominant pathogen isolated from liver abscess of diabetic patients in Asian countries. With the spread of multiple-drug-resistant K. pneumoniae, there is an increasing need for the development of alternative bactericides and approaches to block the production of bacterial virulence factors. Capsular polysaccharide (CPS), especially from the K1 and K2 serotypes, is considered the major determinant for K. pneumoniae virulence. We found that extracts of the traditional Chinese medicine Fructus mume inhibited the growth of K. pneumoniae strains of both serotypes. Furthermore, Fructus mume decreased the mucoviscosity, and the CPS produced in a dose-dependent manner, thus reducing bacterial resistance to serum killing. Quantitative reverse transcription polymerase chain reaction analyses showed that Fructus mume downregulated the mRNA levels of cps biosynthesis genes in both serotypes, possibly by increasing the intracellular iron concentration in K. pneumoniae. Moreover, citric acid, a major organic acid in Fructus mume extracts, was found to have an inhibitory effect on growth and CPS biosynthesis in K. pneumoniae. Taken together, our results indicate that Fructus mume not only possesses antibacterial activity against highly virulent K. pneumoniae strains but also inhibits bacterial CPS biosynthesis, thereby facilitating pathogen clearance by the host immune system

IscR Regulation of Capsular Polysaccharide Biosynthesis and Iron-Acquisition Systems in <i>Klebsiella pneumoniae</i> CG43

<div><p>IscR, an Fe–S cluster-containing transcriptional factor, regulates genes involved in various cellular processes. In response to environmental stimuli such as oxidative stress and iron levels, IscR switches between its holo and apo forms to regulate various targets. IscR binding sequences are classified into two types: the type 1 IscR box that is specific for holo-IscR binding, and the type 2 IscR box that binds holo- and apo-IscR. Studying <i>Klebsiella pneumoniae</i> CG43S3, we have previously shown that iron availability regulates capsular polysaccharide (CPS) biosynthesis and iron-acquisition systems. The present study investigated whether IscR is involved in this regulation. Compared with that in CG43S3, the amount of CPS was decreased in AP001 (Δ<i>iscR</i>) or AP002 (<i>iscR</i>_3CA), a CG43S3-derived strain expressing mutated IscR mimicked apo-IscR, suggesting that only holo-IscR activates CPS biosynthesis. Furthermore, a promoter-reporter assay verified that the transcription of <i>cps</i> genes was reduced in AP001 and AP002. Purified IscR::His₆, but not IscR_3CA::His₆, was also found to bind the predicted type 1 IscR box specifically in the <i>cps</i> promoter. Furthermore, reduced siderophore production was observed in AP004 (Δ<i>fur</i>-Δ<i>iscR</i>) but not in AP005 (Δ<i>fur</i>-<i>iscR</i>_3CA), implying that apo-IscR activates iron acquisition. Compared with those in AP004, mRNA levels of three putative iron acquisition systems (<i>fhu</i>, <i>iuc</i>, and <i>sit</i>) were increased in AP005, and both purified IscR::His₆ and IscR_3CA::His₆ bound the predicted type 2 IscR box in the <i>fhuA</i>, <i>iucA</i>, and <i>sitA</i> promoters, whereas IscR_3CA::His₆ displayed a lower affinity. Finally, we analyzed the effect of external iron levels on <i>iscR</i> expression. The transcription of <i>iscR</i> was increased under iron-depleted conditions as well as in AP001 and AP002, suggesting an auto-repression exerted by apo-IscR. Our results show that in <i>K. pneumoniae</i>, IscR plays a dual role in the regulation of CPS biosynthesis and iron-acquisition systems in response to environmental iron availability.</p></div

qRT-PCR analyses of the expression of iron-acquisition genes in <i>K. pneumoniae</i> strains.

a<p> AP004, CG43S3Δ<i>fur</i>-Δ<i>iscR</i>; AP005, CG43S3Δ<i>fur-iscR</i>_3CA.</p>b<p> Mean expression ratio (±SD) of AP005 relative to AP004, AP004 [pIscR_3CA] relative to AP004 [pACYC184], or AP004 [pIscR] relative to AP004 [pACYC184].</p>c<p> ND, not determined.</p><p>qRT-PCR analyses of the expression of iron-acquisition genes in <i>K. pneumoniae</i> strains.</p