The Functional Motif of SARS-CoV S Protein Involved in the Interaction with ACE2

SARS-CoV is a newly discovery pathogen causing severe acute respiratory problems. It has been established that the S protein in this pathogen plays an important rule in the adsorption and penetration of SARS-CoV into the host cell by interaction with the ACE2 receptor. To determinant which functional motif of the S protein was involved in the interaction with ACE2, seven truncated S proteins deleted from the N or C terminal were obtained by an E.coli expression system and purified by column chroma-tography to homogeneity. Each truncated S protein was fixed on to the well of an ELISA plate and an interaction was initiated with the ACE2 protein. The adsorption were quantified by ELISA, and the results indicated that amino acids from 388 to 496 of the S protein was responsible for the interaction with the ACE2 receptor, and the interaction could be completely disrupted by an antibody specific to these amino acids. Deletions adjacent to this domain did not appear to have a significant impact on the interaction with ACE2, suggesting that the S protein of SARS-CoV could be developed as a vaccine to prevent the spread of SARS-CoV.     

Effect of different immunosuppressive drugs on calcineurin and its mutants

Several mutants in Loop7 region and near Loop7 region of calcineurin A (CN A) subunit have been constructed and purified using site-directed mutagenesis. Their phosphatase activity and the corresponding solution conformation were examined. Their phosphatase activities between wild-type CN and mutants were compared to identify the interaction of different immuno-suppressive drugs with CN. The results showed that the phosphatase activities of the mutants at Loop7 were much higher than the one of wild-type CN. Furthermore, circular dichroism spectra of the mutants revealed that their solution conformations gave rise in changes in native structure of the protein. Cyclophilin-CyclosporinA (CyP-CsA) significantly inhibited the phosphatase activity of wild-type CN, and had no effects on the phosphatase activity of mutants in Loop7 region, which indicates that the site-directed mutagenesis at Loop7 region made a significant change in the interaction between CyP-CsA and CN. Examination of the activities of these     

A new theoretical model for the origin of amino acid homochirality

Amino acid homochirality, as a unique behavior of life, could have originated synchronously with the genetic code. In this paper, phosphoryl amino-acid -5′-nucleosides with P－N bond are postulated to be a chiral origin model in prebiotic molecular evolution. The enthalpy change in the intramolecular interaction between the nucleotide base and the amino-acid side-chain determines the sta-bility of the particular complex, resulting in a preferred conformation associated with the chirality of amino acids. Based on the theoretical model, our experiments and calculations show that the chiral selection of the earliest amino acids for L-enantiomers seems to be a strict stereochemi-cal/physicochemical determinism. As other amino acids developed biosynthetically from the earliest amino acids, we infer that the chirality of the later amino acids was inherited from the precursor amino acids. This idea probably goes far back in history, but it is hoped that it will be a guide for further ex-periments in this area.     

Sequence Diversity and Enzyme Activity of Ferric-Chelate Reductase LeFRO1 in Tomato

Ferric-chelate reductase which functions in the reduction of ferric to ferrous iron on root surface is a critical protein for iron ho- meostasis in strategy I plants. LeFROI is a major ferric-chelate reductase involved in iron uptake in tomato. To identify the natural variations of LeFRO1 and to assess their effect on the ferric-chelate reductase activity, we cloned the coding sequences of LeFRO1 from 16 tomato varieties collected from different regions, and detected three types of LeFRO1 （LeFRO1MM, LeFRO1Ailsa and LeFRO1Monita） with five amino acid variations at the positions 21, 24, 112, 195 and 582. Enzyme activity assay revealed that the three types of LeFRO1 possessed different ferric-chelate reductase activity （LeFRO1AiISa 〉 LeFRO1MM 〉 LeFRO1M~nita）. The 112th amino acid residue Ala of LeFRO1 is critical for maintaining the high activity of ferric-chelate reductase, because modification of this amino acid resulted in a significant reduction of enzyme activity. Further, we showed that the combination of the amino acid residue lie at the site 24 with Lys at the site 582 played a positive role in the enzyme activity of LeFRO1. In conclusion, the findings are helpful to understand the natural adaptation mechanisms of plants to iron-limiting stress, and may provide new knowledge to select and manipulate LeFRO1 for improving the iron deficiency tolerance in tomato.     

Compensation effects of coated cysteamine on meat quality,amino acid composition,fatty acid composition,mineral content in dorsal muscle and serum biochemical indices in finishing pigs offered reduced trace minerals diet

<正>Dear Editor.Trace mineral elements (ME) and amino acids have been demonstrated to be essential bioelements in animal nutrition.If there is a deficiency in ME,a number of biological functions in animals may be affected,including physical growth,psychomotor development,and immunity.Supplementation of pig diets with ME is known to improve the animals'growth,reproduction,and their immunity against oxidative stress and cell damage.Pigs that are fed diets with high-dose supplemental Zn or Cu have been shown to have an increased ADG and a decreased F:G ratio (Zhu et al.,     

Two observed regions in B lymphocyte stimulator important for its biological activity

B lymphocyte stimulator (BLyS),a member of the tumor necrosis factor superfamily ofligands,is a crucial survival factor for B cells.We successfully constructed seven mutants of the functionalsoluble fragment of human BLyS (named cBLyS,amino acid 134-285),including three deletion mutants andfour site-directed mutants.All the mutant proteins were expressed in Escherichia coli and purified by Ni-NTA affinity chromatography.The biological activities of these mutants were assessed by the ligand-recep-tor binding assay,B cell proliferation assay and immune effect response in vivo.Our results indicated thatfour residues,H~(218),F~(220),T~(228) and L~(229),are indispensable for the biological activity of cBLyS,whereas tworegions,amino acid 134-148 and amino acid 271-285,are related to the biological activity of BLyS.Theprotein of deletion of amino acid 134-148 leads to a complete defection in raising the antigen-specific IgMtiter.The deletion of amino acid 271-285 reduces the effectiveness compared with the native cBLyS.Thisindicates that the region of amino acid 134-148 is indispensable for cBLyS to function normally.     

Single amino-acid substitution in the N-terminal arm altered the tetramer stability of rat muscle lactate dehydrogenase A

Lactate dehydrogenase A (LDHA) is a well-characterized tetrameric enzyme. Its N-terminal arm, comprised of an α-helix and a p-strand, was suggested to be essential for subunit interactions. To examine the critical amino acid residues in the N-terminus involved in the subunit association, two single-point mutants, Leu3Pro (L3P) and IIe8GIu (I8E), have been constructed. We compared the stability of WT-LDHA (WT) and its variants by unfolding experiments. For WT, a dimeric but inactive intermediate was observed by size-exclusion chromatography at 0.6-0.8 mol/L GdmCI. Leu3Pro exists in an active tetrameric structure in aqueous solution as WT does, but it dissociates into dimers under lower concentration of GdmCI (0.2 mol/L). In aqueous solution, the lle8GIu variant exists predominantly in the dimeric form with increased KM and decreased kcat as compared with those of WT and L3P. However, the activity of lle8GIu increases significantly in the presence of sodium sulfate. In conclusion, two mutants are less sta     

Two Potent α3/5 Conotoxins from Piscivorous Conus achatinus

Every cone snail produces a mixture of different conotoxins and secretes them to immobilize their prey and predators. α3/5 Conotoxins, isolated from fish-hunting cone snails, target muscle nicotinic acetylcholine receptors. The structure and function of α3/5 conotoxin from the piscivorous Conus achatinus have not been studied. We synthesized two pentadecamer peptides, Ac 1.1 a and Ac 1.1 b, with appropriate disulfide bonding, based on cDNA sequences of α3/5 conotoxins from C. achatinus. Ac 1.1 a and Ac 1.1 b differ by only one amino acid residue. They have similar potency on blocking recombinant mouse muscle acetylcholine receptor expressed in Xenopus laevis oocytes, with IC_(50) values of 36 nM and 26 nM, respectively. For Ac 1.1b, deletion of the first three N-terminal amino acids did not change its activity, indicating that the Nterminus is not involved in the interaction with its receptor. Furthermore, our experiments indicate that both toxins strongly prefer the α1-δ subunit interface instead of the α1-γ binding site on the mouse muscle nicotinic acetylcholine receptor. These peptides provide additional tools for the study of the structure and function of nicotinic receptor.     

Discovery of a Homolog of Siderophilin in a Plant

Members belonging to the siderophilin family are iron-binding and iron-transporting proteins, which includes transferrin and lactoferrin. They have only been found in animals previously. If siderophilin could be found in and isolated from a plant, its production and subsequent extensive application could be increased. The present study is the first to report the discovery of a homolog of siderophilin in a plant. In order to purify antifreeze proteins from Ammopiptanthus mongolicus （Maxim.） Cheng f., the authors processed the proteins from the leaves using techniques such as column chromatography using DEAE-Cellulose-52, gel filtration via Sephacryl S-100 HR medium, hydrophobic interaction chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mass spectroscopy was performed on the three proteins purified and the sequence of one of the proteins （containing 32 amino acids） was found to have 97% homology with the corresponding part of one type of human lactoferrin. Moreover, one of the two peptides belongs to an iron-binding domain. So, it is possible that siderophilin also exists in plants and plays a role as an antibacterial and antifungal, among other actions.     

Identification and genetic mapping of four novel genes that regulate leaf development in Arabidopsis

Molecular and genetic characterizations of mutants have led to a better understanding of many developmental processes in the model system Arabidopsis thaliana.However,the leaf development that is specific to plants has been little studies.With the aim of contributing to the genetic dissection of leaf development,we have performed a large-scare screening for mutants with abnormal leaves.Among a great number of leaf mutants we have generated by T-DNA and transposon tagging and ethylmethae sulfonate (EMS) mutagenesis,four independent mutant lines have been identified and studied genetically.Phenotypes of these mutant lines represent the defects of four novel muclear genes designated LL1(LOTUS LEAF 1),LL2(LOTUS LEAF2),URO(UPRIGHT ROSETTE),and EIL(ENVIRONMENT CONDITION INDUCED LESION).The phenotypic analysis indicates that these genes play important roles during leaf development.For the further genetic analysis of these genes and the map-based cloning of LL1 and LL2,we have mapped these genes to chromosome regions with an efficient and rapid mapping method.     

Mutation of Residue Arginine^18 of Cytochrome b559 α-Subunit and its Effects on Photosystem Ⅱ Activities in Chlamydomonas reinhardtii

It has been known that arginine is used as the basic amino acid in the α-subunit of cytochrome bsss （Cyt bsss） except histidine. However, previous studies have focused on the function of histidine in the activities of photosystem （PS） Ⅱ and there are no reports regarding the structural and/or functional roles of arginine in PSll complexes. In the present study, two arginine18 （R18） mutants of Chlamydomonas reinhardtii were constructed using site-directed mutagenesis, in which R18 was replaced by glutamic acid （E） and glycine （G）. The results show that the oxygen evolution of the PSII complex in the R18G and R18E mutants was approximately 60% of wild-type （WT） levels and that, after irradiation at high light intensity, oxygen evolution for the PSll of mutants was reduced to zero compared with 40% in WT cells. The efficiency of light capture by PSll （Fv/Fm） of R18G and R18E mutants was approximately 42%-46% that of WT cells. Furthermore, levels of the α-subunit of Cyt bsss and PsbO proteins were reduced in thylakoid membranes compared with WT. Overall, these data suggest that R18 plays a significant role in helping Cyt bss9 maintain the structure of the PSll complex and its activity, although it is not directly bound to the heme group.     

A computational approach to explore the functional missense mutations in the spindle check point protein Mad1

In this work, the most detrimental missense mutations of Madl protein that cause various types of cancer were identified computationally and the substrate binding efficiencies of those missense mutations were analyzed. Out of 13 missense mutations, I Mutant 2.0, SIFT and PolyPhen programs identified 3 variants that were less stable, deleterious and damaging respectively. Subsequently, modeling of these 3 variants was performed to understand the change in their conformations with respect to the native Madl by computing their root mean squared deviation （RMSD）. Furthermore, the native protein and the 3 mutants were docked with the binding partner Mad2 to explain the substrate binding efficiencies of those detrimental missense mutations. The docking studies identified that all the 3 mutants caused lower binding affinity for Mad2 than the native protein. Finally, normal mode analysis determined that the loss of binding affinity of these 3 mutants was caused by altered flexibility in the amino acids that bind to Mad2 compared with the native protein. Thus, the present study showed that majority of the substrate binding amino acids in those 3 mutants displayed loss of flexibility, which could be the theoretical explanation of decreased binding affinity between the mutant Madl and Mad2.     

Selection of HBV preSl-binding penta-peptides by phage display

Chronic hepatitis B virus （HBV） infection can lead to liver cirrhosis and hepatocellular carcinoma. Current therapies have a very limited efficacy in virus clearance. New anti- viral targets and agents are urgently needed. The envelope of HBV virion contains three surface glycoproteins, namely the large （LHBs）, middle （MHBs）, and small （SHBs） pro- teins. LHBs has an amino terminal preS which is composed of the preS1 and preS2 domains. The amino half of preS1 which is myristoylated plays a pivotal role in HBV entry, which can be exploited as an antiviral target. A common motif of five amino acids had been previously discovered to bind preSll_~s and HBV particles. In this study, we used preSl 1-65 to screen a phage display library of random penta-peptides to select the penta-peptides possessing a high preSl-binding affinity. After nine rounds of panning, we obtained one peptide designated as A5 which could bind preS1 with a high affinity. By systematically substitut- ing each residue of A5 with the other 19 amino acids, we identified a novel peptide with an increased preSl-binding affinity. Both peptides could inhibit HBV attachment to HepG2 cells, making them be potential candidates for HBV entry inhibitors.     

Mutation of Residue Arginine~(18)of Cytochrome b_(559)a-Subunit and its Effects on Photosystem II Activities in Chlamydomonas reinhardtii

It has been known that arginine is used as the basic amino acid in the a-subunit of cytochrome b_(559)(Cyt b_(559)) excepthistidine. However, previous studies have focused on the function of histidine in the activities of photosystem (PS) II andthere are no reports regarding the structural and/or functional roles of arginine in PSII complexes. In the present study,two arginine (R18) mutants of Chlamydomonas reinhardtii were constructed using site-directed mutagenesis, in whichR18 was replaced by glutamic acid (E) and glycine (G). The results show that the oxygen evolution of the PSII complexin the R18G and R18E mutants was approximately 60% of wild-type (WT) levels and that, after irradiation at high lightintensity, oxygen evolution for the PSII of mutants was reduced to zero compared with 40% in WT cells. The efficiency oflight capture by PSII (F_v/F_m) of R18G and R18E mutants was approximately 42%-46% that of WT cells. Furthermore, levelsof the a-subunit of Cyt b_(559) and PsbO proteins were reduced in thylakoid membranes compared with WT. Overall, thesedata suggest that R18 plays a significant role in helping Cyt b_559 maintain the structure of the PSII complex and its activity,although it is not directly bound to the heme group.     

Mutational Analysis of Bacterial NAD^＋-dependent DNA Ligase： Role of Motif IV in Ligation Catalysis

The bacterial DNA ligase as a multiple domain protein is involved in DNA replication, repair and recombination. Its catalysis of ligation can be divided into three steps. To delineate the roles of amino acid residues in motif IV in ligation catalysis, site-directed mutants were constructed in a bacterial NAD^＋-dependent DNA ligase from Thermus sp. TAK16D. It was shown that four conserved residues （D286, G287, V289 and K291） in motif IV had significant roles on the overall ligation. Under single turnover conditions, the observed apparent rates of D286E, G287A, V289I and K291R mutants were clearly reduced compared with that of WT ligase on both match and mismatch nicked substrates. The effects of D286E mutation on overall ligation may not only be ascribed to the third step. The G287A mutation has a major effect on the second step. The effects of V289I and K291R mutation on overall ligation are not on the third step, perhaps other aspects, such as conformation change of ligase protein in ligation catalysis, are involved. Moreover, the amino acid substitutions of above four residues were more sensitive on mismatch nicked substrate, indicating an enhanced ligation fidelity.