Molecular engineering of biotin-glutamic acid decarboxylase 65 fusion protein (Biotin-GAD65) for non-radioactive GAD65 antibody assay

We constructed biotinylated fusion proteins that linked to three detection tags to GAD65 at the N-terminus, and expressed them in an E. coli expression system. The Biotin14-GAD65 protein exhibited the strongest binding to both the GAD65 antibody and the streptavidin among the three constructs. We describe the optimal conditions using a Biotin14-GAD65-based immunoassay for the detection of GAD65 antibody.     

Molecular recognition between 4a<Emphasis Type="Italic">S/R</Emphasis>-galanthamine diastereoisomers and α-cyclodextrin

Molecular recognition between 4aS/R-galanthamine diastereoisomers (1: 4aS-galanthamine; 2: 4aR-galanthamine) and -cyclodextrin (-CD) were studied by use of docking and molecular dynamics (MD) simulation approaches. The binding energy of constructed 2···-CD complexes is ~17 kcal mol^–1 lower than that of 1···-CD, implying a stronger binding ability of 2 with -CD than that of 1. The theoretical modeling result is consistent with our previous CZE result, which demonstrated that -CD is an efficient chiral additive for separating 1 and 2. The modeling result also indicates that both hydrophobic interaction and H-bond force may work as major factors for molecular recognition between the galanthamine diastereoisomers and -CD. Figure Chemical structures of 4aS-galanthamine (left) and 4aR-galanthamine (right)Abbreviations Galanthamine 4aS,6R,8aS-4a,5,9,10,11,12-Hexahydroxy-3-methoxy-11-methyl-6H-benzofuro[3a,3,2-e,f]benzazepin-6-ol     

Functional genomics of wood quality and properties

Genomics promises to enrich the investigations of biology and biochemistry. Current advancements in genomics have major implications for genetic improvement in animals, plants, and microorganisms, and for our understanding of cell growth, development, differentiation, and communication. Significant progress has been made in the understanding of plant genomics in recent years, and the area continues to     

Kinetic study on the enzymatic resolution of homophenylalanine ester using ionic liquids

Two ionic liquids (ILs) were investigated as novel media for the enzymatic resolution of amino acid ester to obtain enantiomeric amino acid homophenylalanine. The effects of solvent nature, polarity, and concentration on the kinetic resolution were investigated. With change in solvent concentration, a systematic study shows that an improved enzyme activity can be obtained by adjusting these solvent parameters.     

Molecular cloning, expression, purification, and mass spectrometric characterization of 3C-like protease of SARS coronavirus

Severe acute respiratory syndrome (SARS) is an acute respiratory illness, which has broken out in China. It has been known that SARS coronavirus (SARS_CoV) is a novel human coronavirus and is responsible for SARS infection. Belonging to one of the major proteins associated with SARS_CoV, SARS 3C-like protease (SARS_3CL(pro)) functions as a cysteine protease engaging in the proteolytic cleavage of the viral precursor polyprotein to a series of functional proteins required for coronavirus replication and is considered as an appealing target for designing anti-SARS agents. To facilitate the studies regarding the functions and structures of SARS_3CL(pro), in this report the synthetic genes encoding 3CL(pro) of SARS_CoV were assembled, and the plasmid was constructed using pQE30 as vector and expressed in Escherichia coli M15 cells. The highly yielded ( approximately 15mg/L) expressed protease was purified by use of NTA-Ni(2+) affinity chromatography and FPLC system, and its sequence was determined by LC/MS with the residue coverage of 46.4%.     

Cloning, purification, and characterization of thermostable hypoxanthine-guanine phosphoribosyltransferase from Thermoanaerobacter tengcongensis

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8) from a newly characterized thermophile Thermoanaerobacter tengcongensis was expressed in Escherichia coli and purified. Analytical gel filtration suggested that the enzyme exist as a homotetramer in solution. The optimal pH for the forward reaction was found to be 8.0 and the optimal temperature 70 degrees C. The steady-state kinetic characteristics suggest that hypoxanthine is the most effective substrate. This enzyme showed a half-life of 75min at 50 degrees C and no apparent loss of activity after 3 months at 4 degrees C.     

PCAS – a precomputed proteome annotation database resource

Background  

Many model proteomes or "complete" sets of proteins of given organisms are now publicly available. Much effort has been invested in computational annotation of those "draft" proteomes. Motif or domain based algorithms play a pivotal role in functional classification of proteins. Employing most available computational algorithms, mainly motif or domain recognition algorithms, we set up to develop an online proteome annotation system with integrated proteome annotation data to complement existing resources.     

Psychophysical study of image orientation perception

The experiment reported here investigates the perception of orientation of color photographic images. A collection of 1000 images (mix of professional photos and consumer snapshots) was used in this study. Each image was examined by at least five observers and shown at varying resolutions. At each resolution, observers were asked to indicate the image orientation, the level of confidence, and the cues they used to make the decision. The results show that for typical images, accuracy is close to 98% when using all available semantic cues from high-resolution images, and 84% when using only low-level vision features and coarse semantics from thumbnails. The accuracy by human observers suggests an upper bound for the performance of an automatic system. In addition, the use of a large, carefully chosen image set that spans the 'photo space' (in terms of occasions and subject matter) and extensive interaction with the human observers reveals cues used by humans at various image resolutions: sky and people are the most useful and reliable among a number of important semantic cues.     

Promoter analysis in transient assays using a GUS reporter gene construct in creeping bentgrass (Agrostis palustris)

Transient expression profiles for several chimeric beta-glucuronidase (GUS) gene constructs were determined in tissues (young leaves, mature leaves and roots) of creeping bentgrass (Agrostis palustris, cv. Penn A4) following microprojectile bombardment. The constructs analyzed consisted of the uidA (GUS) reporter gene driven by four different promoters (ubiquitin 3-potato, ubiquitin corn, ubiquitin rice and CaMV 35S). The total number of GUS hits (or transient expression units; TEUs) were determined manually under a dissecting scope after histochemical staining for GUS. Results suggest that the ubiquitin rice promoter is most active in cells of turfgrass, regardless of the developmental stage or tissue-type. The ubiquitin corn promoter was the next best. Of the four promoter used, except for ubiquitin 3-potato, reporter gene activity was dramatically higher in mature leaves compared to young leaves. The relative efficiency of each promoter was about the same in roots and leaves. We have also analyzed uidA (GUS) reporter gene activity following microprojectile bombardment in transient expression assays with callus from two cultivars (Providence or Penn A4) of creeping bentgrass. Differences in the frequency of GUS positive hits were observed between cultivars up to 72 hours post-bombardment. However, this difference between cultivars disappeared after 72 hours post-bombardment. This information describing promoter functionality in bentgrass will be important when designing gene constructs for trait modification and when choosing appropriate cultivars for improvement through gene transfer experiments. This is the first in depth report on organ-specific and developmental gene expression profiles for transgenes in a turfgrass species.