bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hyaluronan and morphogenesis
Authors: Spicer Andrew P. , Tien Janet Y. L. 
Published: March 2004

AbstractIn the past decade, there has been an explosion of interest in hyaluronan, an often misunderstood, biochemically simple, yet functionally complex carbohydrate polymer that is a resident of many extracellular matrices. Previously thought of as a passive, space‐filling component of the extracellular matrix, the so‐called “goo” concept, hyaluronan has risen to a much higher regard in recent years, even being called “magic glue” in a recent perspective. Hyaluronan is likely to be the common thread in many morphogenetic processes, including condensation events and epithelial‐to‐mesenchymal transformation. Hyaluronan is comparatively unique as a component of the extracellular matrix as it is solely composed of carbohydrate. In order to truly understand this biopolymer, one must first understand its biosynthesis, then understand its uptake and turnover, then identify its binding proteins and receptors. Major advances have been made in all of these arenas within the past decade. Hyaluronan synthases, hyaluronidases, and the hyaladherins have been molecularly identified and cloned. Furthermore, many have now been inactivated, employing gene targeting strategies, to create mice deficient in the respective gene product function. Collectively, huge strides have been made in our understanding of the diverse biological functions for this fascinating molecule. Hyaluronan appeared in metazoans immediately prior to the arrival of the vertebrates, and may be required for the differentiation, development, and/or function of most cell lineages, structures, and tissues that we associate with vertebrates, such as the neural crest, the skeleton, including the teeth, skin, and hair, and the chambered heart. In this review, we will update the reader on the advances of the past decade and provide insight into those morphogenetic processes through which hyaluronan regulates vertebrate development. Birth Defects Research (Part C) 72:89–108, 2004. © 2004 Wiley‐Liss, Inc.
Aqueous solubility and membrane interactions of hydrophobic peptides with peptoid tags
Authors: Tang Yan‐Chun , Deber Charles M. 
Published: March 2004

AbstractLysine tagging of hydrophobic peptides of parent sequence KKAAALAAAAALAAWAALAAAKKKK‐NH2 has been shown to facilitate their synthesis and purification through water solubilization, yet not impact on the intrinsic properties of the hydrophobic core sequence with respect to its insertion into membranes in an α‐helical conformation. However, due to their positively charged character, such peptides often become bound to phospholipid head groups in membrane surfaces, which inhibits their transbilayer insertion and/or prevents their transport across cellular bilayers. We sought to develop more neutral peptides of membrane‐permeable character by replacing most Lys residues with uncharged peptoid [N‐(R)glycyl] residues, which might similarly confer water solubility while retaining membrane‐interactive properties of the hydrophobic core. Several “peptoid‐tagged” derivatives of the parent peptide were prepared with varying peptoid content, with five of the six Lys residues replaced with peptoids Nala and/or Nval. Conformations of these peptides measured by circular dichroism spectroscopy demonstrated that these water‐soluble peptides retain the α‐helix structure in micelles (lysophosphatidylcholine and sodium dodecyl sulfate) notwithstanding the known helix‐breaking capacity of the peptoid tags. Blue shifts in Trp fluorescence spectra and quenching experiments with acrylamide confirmed that peptoid‐tagged peptides insert spontaneously into micellar membranes. Results suggest that upon introduction of uncharged tags, the interaction between the membrane and the peptides is dominated by the hydrophobicity of the peptide core rather than the electrostatic interactions between the Lys and the head groups of the lipids. The overall findings indicate that peptoid residues are effective surrogates for Lys as uncharged water‐solubilizing tags and, as such, provide a potentially valuable feature of design of membrane‐interactive peptides. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004
Synthesis of fluorine‐containing bioisosteres corresponding to phosphoamino acids and dipeptide units
Authors: Otaka Akira , Mitsuyama Etsuko , Watanabe Junko , Watanabe Hideaki , Fujii Nobutaka 
Published: March 2004

AbstractIt has been shown that fluorinated analogues of naturally occurring biological active compounds including amino acids often exhibit unique physiological activity. Among wide varieties of fluorine‐containing amino acids, nonhydrolyzable phosphoamino acids possessing a substituent of the difluoromethylene (CF2) unit for the phosphoryl ester oxygen are of value in the medicinal and biological fields. We have engaged in the synthesis of these classes of nonhydrolyzable phosphoamino acids corresponding to pTyr 3, pSer 4, and pThr 5 with their incorporation into peptides using newly developed deprotecting procedures. In this article, stereoselective synthesis of the CF2‐substituted pThr mimetics and development of a two‐step deprotecting methodology for the nonhydrolyzable analogues are reviewed. In the course of the above synthetic study, we found that γ,γ‐difluoro‐α,β‐enoates were reduced to γ‐fluoro‐β,γ‐enoates by organocopper reagents and then applied to the synthesis of (Z)‐fluoroalkene dipeptide isosteres, which have served as potential dipeptide mimetics having structural as well as electrostatic similarity to the parent peptide bonds. Furthermore, mechanistic investigation of the organocopper‐mediated reduction led us to development of a SmI2‐mediated approach toward the synthesis of the fluoroalkene isosteres. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004
Recognition and fluorescence sensing of specific amino acid residue on protein surface using designed molecules
Authors: Ojida Akio , Miyahara Yoshifumi , Kohira Takahiro , Hamachi Itaru 
Published: March 2004

AbstractMany biological processes are mediated by surface recognition between proteins. Small molecules that recognize and bind a specific region of a protein surface may be promising agents for disrupting certain protein–protein surface interactions, which consequently leads to regulation of cellar functions. This article describes our recent efforts toward the development of the designed small molecules, which can recognize histidine or phosphorylated amino acid residues on peptide surfaces in a sequence‐selective manner. These results demonstrate that cooperative metal–ligand interaction is powerful for tight and selective binding to the specific amino acid residues of proteins in aqueous medium. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004
Molecular spacers for physicochemical investigations based on novel helical and extended peptide structures
Authors: Toniolo Claudio , Crisma Marco , Formaggio Fernando , Peggion Cristina , Broxterman Quirinus B. , Kaptein Bernard 
Published: March 2004

AbstractA proper understanding of the detailed nature and mechanism of physicochemical interactions depends heavily upon our ability to design and synthesize conformationally constrained 3D structures whose intercomponent geometry (either rigorously rigid or able to undergo destructuration, if required, but in all cases precisely tunable) would be well defined. To this end we have recently reported a few initial studies and we are currently working on the exploitation of stable, short, helical peptide spacers based on achiral and/or chiral Cα‐tetrasubstituted α‐amino acids. These building blocks are known to force the peptides either to predominantly fold into a 310‐helical structure or to adopt a fully extended, planar 2.05‐helix. The systems under investigation involve a donor and an acceptor moiety linked to the N‐ and C‐termini of the oligopeptide spacer main chain. By increasing the number of intervening residues the donor···acceptor separation can be easily modulated. This review highlights details of these two novel peptide secondary structures and their use as molecular spacers in physicochemical investigations. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004
Enhancing multiple disulfide bonded protein folding in a cell‐free system
Authors: Yin Gang , Swartz James R. 
Published: March 2004

AbstractA recombinant plasminogen activator (PA) protein with nine disulfide bonds was expressed in our cell‐free protein synthesis system. Due to the unstable and reducing environment in the initial E. coli‐based cell‐free system, disulfide bonds could not be formed efficiently. By treating the cell extract with iodoacetamide and utilizing a mixture of oxidized and reduced glutathione, a stabilized redox potential was optimized. Addition of DsbC, replacing polyethylene glycol with spermidine and putrescine to create a more natural environment, adding Skp, an E. coli periplasmic chaperone, and expressing PA at 30°C increased the solubility of the protein product as well as the yield of active PA. Taken together, the modifications enabled the production of more than 60 μg/mL of bioactive PA in a simple 3‐h batch reaction. © 2004 Wiley Periodicals, Inc.
Screening of large protein libraries by the “cell immobilized on adsorbed bead” approach
Authors: Freeman Amihay , Cohen‐Hadar Noa , Abramov Simona , Modai‐Hod Ronit , Dror Yael , Georgiou George 
Published: March 2004

AbstractScreening of mutant libraries for in vitro enzyme evolution is carried out primarily by physical separation of the cells, followed by growth of individual clones and screening of biocatalytic activity on the basis of color or fluorescence signal development. Currently, most frequently employed methods are labor‐intensive or require robotic equipment, resulting in screening limited to a relatively small fraction of the potential inherent in a given library. In this study we present a design, development, and feasibility demonstration of a new screening approach, providing convenient handling of large libraries consisting of 106 to 107 clones and screening based on a simultaneous enzymatic assay with commercially available substrates. This new screening method is based on the “cell immobilized on adsorbed bead” approach: the cell population to be screened is mixed with an excess of medium pre‐equilibrated polyacrylamide beads, chemically derivatized to affect quantitative cell immobilization by adsorption. The resulting bead population, comprising of single cell on a bead or blank beads, is then immobilized on a solid glass support. After removal of the freely flowing liquid, the cells immobilized on the adsorbed beads are allowed to grow into microcolonies, utilizing the medium retained within the supporting hydrogel matrix. These colonies are subsequently equilibrated with chromogenic or fluorogenic substrate and screening is affected under a stereomicroscope, resulting in readily retrieved of the most active colonies. This technique may be particularly useful when the screened mutants are expressed and displayed on the cell surface, providing an active and homogeneous “naturally immobilized” enzyme population with minimal substrate diffusion limitations. © 2004 Wiley Periodicals, Inc.
A novel system for expressing recombinant proteins over a wide temperature range from 4 to 35°C
Authors: Nakashima Nobutaka , Tamura Tomohiro 
Published: March 2004

AbstractEscherichia coli cells are the most commonly used host cells for large‐scale production of recombinant proteins, but some proteins are difficult to express in E. coli. Therefore, we tested the nocardioform actinomycete Rhodococcus erythropolis, which grows at temperatures ranging from 4 to 35°C, as an expression host cell. We constructed inducible expression vectors, where the expression of the target genes could be controlled with the antibiotic thiostrepton. Using these expression vectors, several milligrams of reporter proteins could be isolated from 1 liter of culture of R. erythropolis cells grown at a temperature range from 4 to 35°C. Moreover, we successfully purified serum amyloid A1, NADH dehydorogenase 1 α subcomplex 4, cytochrome b5‐like protein, apolipoprotein A‐V, cathepsin D, pancreatic Rnase, and HMG‐1 that are all difficult to express in E. coli. In the case of kallikrein 6, mouse deoxyribonuclease I and Kid1, which are also difficult to express in E. coli, the expression level of each protein increased when proteins were expressed at low temperature (4°C). Based on these results, we conclude that a recombinant protein expression system using R. erythropolis as the host cell is superior to respective E. coli systems. © 2004 Wiley Periodicals, Inc
New‐generation multicistronic expression platform: pTRIDENT vectors containing size‐optimized IRES elements enable homing endonuclease‐based cistron swapping into lentiviral expression vectors
Authors: Fux Cornelia , Langer Dominik , Kelm Jens M. , Weber Wilfried , Fussenegger Martin 
Published: March 2004

AbstractCapitalizing on a proven multicistronic expression vector platform we have designed novel pTRIDENT vectors which (1) enable coordinated expression of three desired transgenes, (2) are size‐optimized, (3) take advantage of small highly efficient internal ribosome entry sites of the GTX or Rbm3 type, (4) harbor various sites specific for homing endonucleases facilitating promoter/multicistronic expression unit/polyadenylation site swapping as well as (5) straightforward integration into human HIV‐l‐based lentiviral expression vectors tailored to contain compatible homing endonucleases. Multicistronic expression profiles of novel pTRIDENT vectors engineered for different tricistronic expression configurations encoding human low‐molecular‐weight urokinase‐type plasminogen activator (u‐PALMW) or Bacillus stearothermophilus‐derived α‐amylase (SAMY), human vascular endothelial growth factor (hVEGF), and human placental secreted alkaline phosphatase (SEAP) have been quantified in Chinese hamster ovary cells (CHO‐K1), mouse fibroblasts (NIH/3T3), and/or human fibrosarcoma (HT‐1080) cells. In addition, a pTRIDENT‐derived SAMY‐VEGF‐SEAP expression cassette transferred into a compatible lentiviral expression vector enabled simultaneous high‐level transgene expression following transduction of transgenic lentiviral particles into primary human chondrocytes. © 2004 Wiley Periodicals, Inc.
Bulking sludge in biological nutrient removal systems
Authors: Martins Antönio M. P. , Heijnen Joseph J. , van Loosdrecht Mark C. M. 
Published: March 2004

AbstractBulking sludge problems are commonly reported in biological nutrient removal (BNR) systems. This has led to the general belief that intrinsic BNR conditions favor the growth of undesirable and excessive filamentous bacteria. The present study shows that other factors have a major role in bulking, and not the BNR conditions. These factors have been verified in well‐controlled, strictly anoxic–aerobic and strictly anaerobic–aerobic sequencing batch reactor systems. The experimental results show that conditions known to be responsible for bulking sludge in aerobic systems (i.e., low concentration of electron donor and/or electron acceptor) did not lead to bulking. Even when acetate was present at very low concentrations in the aerobic stage of an anaerobic–aerobic bio‐P system, the sludge settleability remained very good. This clearly demonstrates that good bio‐P activity can stabilize and improve sludge settleability. The presence of microaerophilic conditions in the anoxic stage of the anoxic–aerobic system was the only factor leading to worsening sludge settling characteristics. The results are discussed in light of our previous hypothesis about the importance of diffusion‐limited substrate uptake for the development of filamentous structures in biological flocs. The hypothesis is extended to anaerobic–aerobic and anoxic–aerobic conditions, typical of BNR‐activated sludge systems. Taking into account the effect of feeding patterns on biochemical rates and on the development of filamentous bacterial structures, we recommend the adoption of plug‐flow selector configurations, with strictly anaerobic and/or strictly anoxic conditions, wherein microaerophilic conditions are excluded, in order to maintain reliable and robust BNR performance. © 2004 Wiley Periodicals, Inc.
Catalytic antibody light chain capable of cleaving a chemokine receptor CCR‐5 peptide with a high reaction rate constant
Authors: Mitsuda Yukie , Hifumi Emi , Tsuruhata Kumi , Fujinami Hiroko , Yamamoto Naoki , Uda Taizo 
Published: March 2004

AbstractA monoclonal antibody (MAb), ECL2B‐2, was obtained by immunizing a peptide possessing a part of a sequence of a chemokine receptor, CCR‐5, which is present as a membrane protein on the macrophage surface, and which plays an important role in human immunodeficiency virus (HIV) infection. From the DNA and the deduced amino acid sequences of the light and heavy chains of ECL2B‐2 MAb, molecular modeling was conducted to calculate the steric conformation of the antibody. Modeling suggested that the structure of ECL2B‐2 could possess one or two catalytic triad(s), composed of Asp1, Ser27a (or Ser27e), and His93 (or His27d), in the light chain of ECL2B‐2. The three amino acid residues, Asp1, Ser27a, and His93, are identical to those of catalytic antibody light chains such as VIPase and i41SL1‐2. The light chain of ECL2B‐2 MAb degraded the antigenic peptide CCR‐5 within about 100 h. Surprisingly, the light chain had a very high catalytic reaction rate constant (kcat) of 2.23 min−1, which is greater by factors of tens to hundreds than those of natural catalytic antibodies obtained previously. The heavy chain of ECL2B‐2 MAb, which has no catalytic triad because of a lack of His residue, did not degrade the CCR‐5 peptide. © 2004 Wiley Periodicals, Inc.
Impact of transamination reactions and protein turnover on labeling dynamics in 13C‐labeling experiments
Authors: Grotkjær Thomas , Åkesson Mats , Christensen Bjarke , Gombert Andreas K. , Nielsen Jens 
Published: March 2004

AbstractA dynamic model describing carbon atom transitions in the central metabolism of Saccharomyces cerevisiae is used to investigate the influence of transamination reactions and protein turnover on the transient behavior of 13C‐labeling chemostat experiments. The simulations performed suggest that carbon exchange due to transamination and protein turnover can significantly increase the required time needed for metabolites in the TCA cycle to reach isotopic steady state, which is in agreement with published experimental observations. On the other hand, transamination and protein turnover will speed‐up the net rate of incorporation of labeled carbon into some free and protein‐bound amino acids. The simulation results indicate that the pattern of labeled carbon incorporation into amino acids obtained from biomass hydrolysate shows significant deviation from the commonly assumed first‐order kinetics behavior until after three residence times. These observations suggest that greater caution should be used while also pointing to new opportunities in the design and interpretation of 13C‐labeling experiments. © 2004 Wiley Periodicals, Inc.
High‐rate sulfate reduction at high salinity (up to 90 mS.cm−1) in mesophilic UASB reactors
Authors: Vallero Marcus V. G. , Sipma Jan , Lettinga Gatze , Lens Piet N. L. 
Published: March 2004

AbstractSulfate reduction in salt‐rich wastewaters using unadapted granular sludge was investigated in 0.9 L UASB reactors (pH 7.0 ± 0.2; hydraulic retention time from 8–14 h) fed with acetate, propionate, or ethanol at organic loading rates up to 10 gCOD.L−1.day−1 and in excess sulfate (COD/SO of 0.5). High‐rate sulfate reduction rates (up to 3.7 gSO42‐.L−1.day−1) were achieved at salinities exceeding 50 gNaCl.L−1 and 1 gMgCl2.L−1. Sulfate reduction proceeded at a salinity of up to 70 gNaCl.L−1 and 1 gMgCl2.L−1 (corresponding to a conductivity of about 85–90 mS.cm−1), although at lower rates compared to a conductivity of 60–70 mS.cm−1. Ethanol as well as propionate were suitable substrates for sulfate reduction, with acetate and sulfide as the end products. The successful high‐rate treatment was due to the proliferation of a halotolerant incomplete oxidizing SRB population present in the unadapted inoculum sludge. Bioaugmentation of this sludge with the acetate oxidizing halotolerant SRB Desulfobacter halotolerans was unsuccessful, as the strain washed out from the UASB reactor without colonizing the UASB granules. © 2004 Wiley Periodicals, Inc.
Digital quantitative measurements of gene expression
Authors: Mikkilineni Venugopal , Mitra Robi D. , Merritt Joshua , DiTonno Jason R. , Church George M. , Ogunnaike Babatunde , Edwards Jeremy S. 
Published: March 2004

AbstractOne of the primary goals of functional genomics is to provide a quantitative understanding of gene function. However, the success of this enterprise is dependent on the accuracy and precision of the functional genomic data. A novel approach, digital analysis of gene expression (DAGE) described herein, is an accurate and precise technology for measuring digital gene expression on a relative or absolute scale by simply counting the number of transcripts of a gene being expressed at a given time. The result is a greatly improved technology sensitive enough for identifying and quantifying small (but biologically important and statistically relevant) changes in gene expression. Fourteen genes involved in galactose metabolism in Saccharomyces cerevisiae were analyzed for their expression levels in glucose and galactose minimal media. The quantitative expression results were characterized in terms of distributional and accuracy attributes; they were also in general agreement (in terms of direction of change) with corresponding results obtained using microarray technology. DAGE is likely to have profound implications in the field of functional genomics because the gene expression measurements are digital in nature and therefore more accurate than any other technologies. © 2004 Wiley Periodicals, Inc.
Optimization of rifamycin B fermentation in shake flasks via a machine‐learning‐based approach
Authors: Bapat Prashant M. , Wangikar Pramod P. 
Published: March 2004

AbstractRifamycin B is an important polyketide antibiotic used in the treatment of tuberculosis and leprosy. We present results on medium optimization for Rifamycin B production via a barbital insensitive mutant strain of Amycolatopsis mediterranei S699. Machine‐learning approaches such as Genetic algorithm (GA), Neighborhood analysis (NA) and Decision Tree technique (DT) were explored for optimizing the medium composition. Genetic algorithm was applied as a global search algorithm while NA was used for a guided local search and to develop medium predictors. The fermentation medium for Rifamycin B consisted of nine components. A large number of distinct medium compositions are possible by variation of concentration of each component. This presents a large combinatorial search space. Optimization was achieved within five generations via GA as well as NA. These five generations consisted of 178 shake‐flask experiments, which is a small fraction of the search space. We detected multiple optima in the form of 11 distinct medium combinations. These medium combinations provided over 600% improvement in Rifamycin B productivity. Genetic algorithm performed better in optimizing fermentation medium as compared to NA. The Decision Tree technique revealed the media–media interactions qualitatively in the form of sets of rules for medium composition that give high as well as low productivity. © 2004 Wiley Periodicals, Inc.