bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phenylalanine containing hydrophobic nanospheres for antibody purification
Authors: Türkmen Deniz , Öztürk Nevra , Akgöl Sinan , Elkak Assem , Denizli Adil 
Published: December 2008

AbstractIn this study, novel hydrophobic nanospheres with an average size of 158 nm utilizing N‐methacryloyl‐(L)‐phenylalanine methyl ester (MAPA) as a hydrophobic monomer were produced by surfactant free emulsion polymerization of 2‐hydroxyethyl methacrylate (HEMA) and MAPA conducted in an aqueous dispersion medium. MAPA was synthesized using methacryloyl chloride and L‐phenylalanine methyl ester. Specific surface area of the nonporous nanospheres was found to be 1874 m2/g. Poly(HEMA–MAPA) nanospheres were characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Average particle size, size distribution, and surface charge measurements were also performed. Elemental analysis of MAPA for nitrogen was estimated as 0.42 mmol/g polymer. Then, poly(HEMA–MAPA) nanospheres were used in the adsorption of immunoglobulin G (IgG) in batch system. Higher adsorption values (780 mg/g) were obtained when the poly (HEMA–MAPA) nanospheres were used from both aqueous solutions and human plasma. The adsorption phenomena appeared to follow a typical Langmuir isotherm. It was observed that IgG could be repeatedly adsorbed and desorbed without significant loss in adsorption amount. These findings show considerable promise for this material as a hydrophobic support in industrial processes.
Evaluation of the antimicrobial activity of cationic polyethylenimines on dry surfaces
Authors: Koplin Stephen A. , Lin Shirley , Domanski Tammy 
Published: December 2008

AbstractThe antimicrobial activity of cationic N‐dodecyl‐N‐methylpolyethylenimine (PEI+) against S. aureus, A. baumannii, and E. coli was evaluated when the polymer was applied as a coating to various surfaces using a paint‐like procedure. Antimicrobial activity of PEI+ as a function of time was determined using an assay for long‐term survival involving placement of single drops of various bacterial concentrations on dry surfaces. These data were compared with an assay method where bacteria were applied by spraying and surfaces were incubated overnight under agar. PEI+‐coated surfaces were found to be highly bactericidal after 30 min when bacteria were sprayed onto surfaces. However, when bacteria were applied as single drops, PEI+‐coated surfaces were less biocidal at short contact times particularly for A. baumannii and E. coli. The observations are explained in the context of the difference in drying time between drops deposited on uncoated surfaces and PEI+‐coated surfaces and the sensitivity of bacterial survival to dehydration. These results demonstrate that PEI+‐coated surfaces are not effectively biocidal for some types of bacteria under certain conditions and that the method of assaying bactericidal efficiency can greatly affect the results obtained.
The characterization of pretreated lignocellulosic substrates prior to enzymatic hydrolysis, part 1: A modified Simons' staining technique
Authors: Chandra Richard , Ewanick Shannon , Hsieh Carmen , Saddler Jack N. 
Published: December 2008

AbstractTo date, there is limited knowledge available regarding the key features of pretreated lignocellulosic substrates that promote the effective enzymatic hydrolysis of the cellulose component to glucose during bioconversion processes to produce ethanol. Fundamentally, cellulase enzymes require access to the cellulose to carry out effective hydrolysis. Porosity and the overall surface area of substrates have major structural features influencing the hydrolysis of pretreated substrates by cellulases. Simons' Stain (SS) is a potentially useful semiquantitative method for estimating the available surface area of lignocellulosic substrates. In this study, a modified, rapid SS method was developed, where the processing time was decreased from >50 to 6 h and the maximum dye adsorbed on the substrate was calculated using the adsorption isotherm for the orange and blue components of the dye mixture. The modified SS test readily measures the decrease in accessibility and hydrolyzability of a steam pretreated substrate that had been dried under three different drying regimes. For each of the lignocellulosic substrates, the total dye adsorption correlated well with the hydrolysis yields resulting in a correlation coefficient of r2 = 0.95. The modified SS procedure is an effective tool for assessing how lignocellulosic substrates might be potentially hydrolyzed by cellulases.
Complete sequence of biosynthetic gene cluster responsible for producing triostin A and evaluation of quinomycin‐type antibiotics from Streptomyces triostinicus
Authors: Praseuth Alex P. , Wang Clay C. C. , Watanabe Kenji , Hotta Kinya , Oguri Hiroki , Oikawa Hideaki 
Published: December 2008

AbstractStreptomyces triostinicus produces triostin A, an antitumor antibiotic, as its major secondary metabolite. Surprisingly, this strain also produced a trace amount of echinomycin. We sequenced the entire triostin A biosynthetic gene cluster from S. triostinicus, and found that this 36 kilobase‐long gene cluster contained an ORF homologous to ecm18 that encodes a thioacetal‐forming enzyme responsible for the triostin A‐to‐echinomycin bioconversion. These findings indicate that, unlike previously thought, S. triostinicus is capable of producing not only triostin A but also echinomycin. Our observation suggests potential value in careful re‐analysis for metabolites from previously characterized natural product producers with the current technologies.
Microcontact printing of P‐selectin increases the rate of neutrophil recruitment under shear flow
Authors: Lee Dooyoung , King Michael R. 
Published: December 2008

AbstractThe local variation of P‐selectin expression on inflamed endothelial layers affects leukocyte recruitment in vivo. As an initial study of the spatially heterogeneous presentation of P‐selectin in vitro, the influence of microcontact printing (μCP) of P‐selectin on a planar surface in neutrophil recruitment was investigated using a parallel‐plate flow chamber. Microline patterned and nonpatterned P‐selectin were prepared using PDMS, Poly(dimethylsiloxane) (PMDS) stamps and isolated neutrophils perfused over the surface to quantify the level of neutrophil recruitment. We first found a significant increase in cell rolling flux and a decrease in cell rolling velocity on the microcontact printed P‐selectin‐surfaces compared with a randomly adsorbed P‐selectin‐surface. However, the increase in rolling adhesion under shear on the surfaces prepared by μCP was not proportional to the number of functional sites of P‐selectin transferred using immunofluorescent labeling. Interestingly, the relative immunofluorescent intensities of the nonfunctional regions of microcontact printed P‐selectin‐surfaces were substantially lower than that that of randomly adsorbed P‐selectin. Taken together, these data indicate that the μCP of selectin increases the transfer rate of the adhesion molecule on a surface in the functionally correct orientation and, consequently, improves the recruitment of leukocytes to the selectin surface under flow. It is concluded that μCP may be a more general technique to control protein orientation on a substrate.
A novel three‐stage light irradiation strategy in the submerged fermentation of medicinal Mushroom Ganoderma lucidum for the efficient production of ganoderic acid and Ganoderma polysaccharides
Authors: Zhang Wei , Tang Ya‐Jie 
Published: December 2008

AbstractA novel three‐stage light irradiation strategy in the submerged fermentation of medicinal mushroom Ganoderma lucidum for the efficient production of bioactive metabolites ganoderic acid (GA) and Ganoderma polysaccharides was developed. Significance of light quality, i.e., blue light (390–500 nm, λmax = 470 nm), red light (560–700 nm, λmax = 625 nm), and white light (400–740 nm, λmax = 550 nm), was studied at first. Interestingly, there was a gradual decrease trend of GA content after the culture of day 2 when the maximal GA content was obtained, while GA content decreased slowly under white light irradiation after day 6. The dark environment was favorable to the specific GA biosynthesis (i.e., GA content) before day 6, and after that the optimum was white light irradiation. A relatively lower irradiation density of white light (i.e., 0.94 and 2.82 W/m2) was beneficial for the specific GA biosynthesis before day 6, while GA content was higher under higher irradiation density of white light (i.e., 4.70 and 9.40 W/m2) at the later‐stage of cultivation. 4.70 W/m2 white light irradiation culture was the best from the viewpoint of GA accumulation. Therefore, a two‐stage light irradiation strategy by combing the first 2 days dark culture with the following 4.70 W/m2 white light irradiation culture was developed. The highest GA production in the two‐stage culture was 276.0 ± 12.5 mg/L, which was increased by 19% compared to 4.70 W/m2 white light irradiation culture (i.e., 232.4 ± 15.8 mg/L) and by 178% compared to the dark culture (i.e., 99.4 ± 1.0 mg/L). Although there still existed a gradual decrease trend of GA content after day 2 when the maximal GA content was obtained in the two‐stage culture. Following three‐stage light irradiation strategy was further demonstrated in order to turn around the sharp decrease of GA content after day 2. The first‐stage was the 2‐day dark culture; the second‐stage was the following six‐day 0.94 W/m2 white light irradiation culture, and the third‐stage was 4.70 W/m2 white light irradiation culture until the end of fermentation. During the three‐stage culture of G. lucidum, the gradual decrease trend of GA content after day 2 was turned around, which suggested that 0.94 W/m2 white light irradiation was beneficial for the metabolic flux towards the GA biosynthesis. The maximal GA content of 3.1 ± 0.1 mg/100 mg DW was obtained, which was higher by 41% compared to the two‐stage culture. The maximal GA production (i.e., 466.3 ± 24.1 mg/L) and productivity (i.e., 38.9 mg/L per day) in the three‐stage culture were 69 and 101% higher than those obtained in the two‐stage culture. This is the first report investigating the significance of light irradiation on the medicinal mushroom submerged fermentation. Such work is very helpful to other mushroom fermentations for useful metabolite production.
Homogeneous esterification by lipase from Burkholderia cepacia in the fluorinated solvent
Authors: Shipovskov S. 
Published: December 2008

AbstractThe formation of noncovalent complexes between lipase from Burkholderia cepacia and the fluorinated ionic surfactant Kryto Development Product 4606 KDP was shown to promote the solubilization of the enzyme in the fluorinated solvent perfluoro(methylcyclohexane) (PFMC) and its operation as a catalyst in the fluorous PFMC/hexane biphasic system (FBS). In the reaction of esterification of 1‐phenylethanol and vinyl acetate, the solubilized lipase showed high stereospecificity (ca. 99%) with a catalytic efficiency up to 56 nmol/(U h) with a high operational and storage stability. Temperature modulation of the FBS miscibility allowed the separation and recovery of the solubilized lipase for at least three times. The results are of practical importance for the development of recoverable biocatalysts soluble and active in the FBS.
Thiopropyl‐agarose as a solid phase reducing agent for chemical modification of IgG and F(ab′)2
Authors: Ferraz Natalia , Leverrier Juliana , Batista‐Viera Francisco , Manta Carmen 
Published: December 2008

AbstractSelective reduction of native disulfide bonds in immunoglobulins is one of the best methods for introducing reactive groups on to the protein surface. Additionally, the thiol groups so generated may allow oriented conjugation at a specific site of the immunoglobulin. Solid‐phase reducing agents have many advantages over soluble ones (including ease of separation of excess reagent from reduced protein by filtration, and the potential for regeneration and multiple reuse). In this work we report a comparative study of the reduction of rabbit IgG and its F(ab′)2 fragments, with mercaptohydroxypropylether‐agarose (thiopropyl‐agarose), a solid phase reducing agent, and dithiothreitol. The effect of different parameters on the process, such as the amount of reducing agent, incubation period, and temperature, was assessed by titration of thiol groups and SDS‐PAGE analysis. Optimized reduction with thiopropyl‐agarose introduced six thiol groups in the F(ab′)2 fragment (mol/mol). Native IgG was less reactive, probably due to steric effects, as only an average of three thiol groups were introduced. However, by increasing reaction temperature from 22 to 37°C, six thiol groups could be introduced in native IgG (mol/mol). Reduction with dithiothreitol also introduced six thiol groups in F(ab′)2 fragments (mol/mol) but led to higher thiol content for the whole IgG. These results demonstrated that thiopropyl‐agarose can be a very useful tool for exercising more precise control over the reduction treatment, and for selecting which disulfide bridges are to be broken. After 6 h incubation with reducing agent containing 8 and 16 μmoles SH per mg of protein, the resulting reduced IgG retained the same biological activity as the native immunoglobulin. The controlled modification of native disulfides achieved with thiopropyl‐agarose will be useful for the development of soluble and insoluble immunoglobulin conjugates.
Facile evaluation of cell disruption efficiency using pH‐controlled fluorescence resonance energy transfer
Authors: Kim Young Soo , Cha Hyung Joon 
Published: December 2008

AbstractThe evaluation of cell disruption efficiency is important in monitoring durability and diverse cellular physiologies of host cells and improving recovery yield of intracellular products. On the basis of our previously suggested pH‐controlled fluorescence resonance energy transfer (FRET) system, we herein demonstrated that easy and effective estimation of the degree of cell disruption under certain stress environments is possible using a simple measurement of changes in fluorescence spectra of two fused fluorescent proteins, because its unique linear correlation was established with cell disruption efficiency from the conventional colony counting method.
Augmentation of EB‐directed hepatocyte‐specific function via collagen sandwich and SNAP
Authors: Novik Eric I. , Barminko Jeffery , Maguire Tim J. , Sharma Nripen , Wallenstein Eric J. , Schloss Rene S. , Yarmush Martin L. 
Published: December 2008

AbstractThe development of implantable engineered liver tissue constructs and ex vivo hepatocyte‐based therapeutic devices are limited by an inadequate hepatocyte cell source. In our previous studies, embryoid body (EB)‐mediated stem cell differentiation spontaneously yielded populations of hepatocyte lineage cells expressing mature hepatocyte markers such as albumin (ALB) and cytokeratin‐18 (CK18). However, these cultures neither yielded a homogenous hepatocyte lineage population nor exhibited detoxification function typical of a more mature hepatocyte lineage cell. In this study, secondary culture configurations were used to study the effects of collagen sandwich culture and oncostatin‐M (OSM) or S‐nitroso‐N‐acetylpenicillamine (SNAP) supplementation of EB‐derived hepatocyte‐lineage cell function. Quantitative immunofluorescence and secreted protein analyses were used to provide insights into the long‐term maintenance and augmentation of existing functions. The results of these studies suggest that SNAP, independent of the collagen supplementation, maintained the highest levels of ALB expression, however, mature liver‐specific CK18 was only expressed in the presence of gel sandwich culture supplemented with SNAP. In addition, albumin secretion and cytochrome P450 detoxification studies indicated that this condition was the best for the augmentation of hepatocyte‐like function. Maintenance and augmentation of hepatocyte‐like cells isolated from heterogeneous EB cell populations will be a critical step in generating large numbers of functional differentiated cells for therapeutic use.
Preparation and characterization of polysulfone affinity membranes bearing a synthetic peptide ligand for the separation of murine immunoglobulins
Authors: Boi Cristiana , Algeri Cristian , Sarti Giulio C. 
Published: December 2008

AbstractAffinity membranes have been prepared by immobilizing D‐PAM, a synthetic ligand that exhibits affinity for the Fc portion of antibodies, onto poliethersulfone microporous membranes. The ligand density has been measured and the ligand utilization was evaluated and compared with literature data available for chromatographic beads. The resulting new affinity membranes have been experimentally characterized and tested by using pure murine IgG solutions and mouse serum. Equilibrium and kinetic parameters have been obtained in batch experiments using pure protein solutions. The highest binding capacity measured for murine IgG was 45 μg/cm2 obtained at 1.2 mg/mL protein concentration at equilibrium, while the maximum static binding capacity calculated with the Langmuir model was 81 μg/cm2. The adsorption of murine IgG on the affinity membranes was described using different isotherms: Freundlich and Temkin models have been considered and critically compared with the Langmuir adsorption model. A dynamic binding capacity of 21 μg/cm2 was obtained by feeding a solution of 0.3 mg/mL of murine IgG, confirming the results obtained in batch experiments at the same concentration. The affinity membranes considered are endowed with good binding capacity for murine IgG and good selectivity for immunoglobulins and can be considered for the capturing step of an antibody production process.
Microbeads display of proteins using emulsion PCR and cell‐free protein synthesis
Authors: Gan Rui , Yamanaka Yumiko , Kojima Takaaki , Nakano Hideo 
Published: December 2008

AbstractWe developed a method for coupling protein to its coding DNA on magnetic microbeads using emulsion PCR and cell‐free protein synthesis in emulsion. A PCR mixture containing streptavidin‐coated microbeads was compartmentalized by water‐in‐oil (w/o) emulsion with estimated 0.5 template molecules per droplet. The template molecules were amplified and immobilized on beads via bead‐linked reverse primers and biotinylated forward primers. After amplification, the templates were sequentially labeled with streptavidin and biotinylated anti‐glutathione S‐transferase (GST) antibody. The pool of beads was then subjected to cell‐free protein synthesis compartmentalized in another w/o emulsion, in which templates were coupled to their coding proteins. We mixed two types of DNA templates of Histidine6 tag (His6)‐fused and FLAG tag‐fused GST in a ratio of 1:1,000 (His6: FLAG) for use as a model DNA library. After incubation with fluorescein isothiocyanate (FITC)‐labeled anti‐His6 (C‐term) antibody, the beads with the His6 gene were enriched 917‐fold in a single‐round screening by using flow cytometry. A library with a theoretical diversity of 106 was constructed by randomizing the middle four residues of the His6 tag. After a two‐round screening, the randomized sequences were substantially converged to peptide‐encoding sequences recognized by the anti‐His6 antibody.
Towards area‐based in vitro metabolic engineering: Assembly of Pfs enzyme onto patterned microfabricated chips
Authors: Lewandowski Angela T. , Bentley William E. , Yi Hyunmin , Rubloff Gary W. , Payne Gregory F. , Ghodssi Reza 
Published: December 2008

AbstractWe report an approach for spatially selective assembly of an enzyme onto selected patterns of microfabricated chips. Our approach is based on electrodeposition of the aminopolysaccharide chitosan onto selected electrode patterns and covalent conjugation of a target enzyme to chitosan upon biochemical activation of a genetically fused “pro‐tag.” We report assembly of S‐adenosylhomocysteine nucleosidase (Pfs) fused with a C‐terminal pentatyrosine pro‐tag. Pfs is a member of the bacterial autoinducer‐2 biosynthesis pathway, catalyzing the irreversible cleavage of S‐adenosylhomocysteine. The assembled Pfs retains its catalytic activity and structure, as demonstrated by retained antibody recognition. Assembly is controlled by the electrode area, resulting in reproducible rates of catalytic conversion for a given area, and thus allowing for area‐based manipulation of catalysis and small molecule biosynthesis. Our approach enables optimization of small molecule biosynthesis in 1‐step as well as multistep enzymatic reactions, including entire metabolic pathways, and we envision a wide variety of potential applications.
Production of a His‐tagged canecystatin in transgenic sugarcane and subsequent purification
Authors: Ribeiro Carolina W. , Soares‐Costa Andrea , Falco Maria Cristina , Chabregas Sabrina M. , Ulian Eugênio C. , Cotrin Simone S. , Carmona Adriana K. , Santana Lucimeire A. , Oliva Maria Luiza V. , Henrique‐Silva Flavio 
Published: December 2008

AbstractTransgenic plants have been used widely as expression systems of recombinant proteins in recent years. This process can be an efficient alternative for the large‐scale production of proteins. In this work, we present the establishment of transgenic sugarcane expressing a His‐tagged canecystatin under the control of the maize ubiquitin promoter. A number of studies have demonstrated that cystatins, which are natural inhibitors of cysteine proteinases, can be used for protection against insect attacks. A transformed sugarcane plant that presented high levels of HISCaneCPI‐1 expression, was selected for the purification of this protein through affinity chromatography in a nickel column. This purified HISCaneCPI‐1 was immunodetected using a polyclonal antibody, which was also able to detect the HISCaneCPI‐1 in a crude extract from transgenic plant leaves. Assays of inhibitory activity performed with the purified HISCaneCPI‐1 revealed its ability to inhibit the catalytic activity of midgut cysteine proteinase partially purified from the sugarcane weevil Sphenophorus levis and human cathepsin L in nanomolar order. These studies demonstrate that sugarcane is a viable expression system for recombinant protein production.