bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthetic heterotrimeric collagen peptides as mimics of cell adhesion sites of the basement membrane
Authors: Renner Christian , Saccà Barbara , Moroder Luis
Published: February 2004

Collagen type IV forms a network in the basement membrane into which other constituents of the tissue are incorporated. It also provides cell‐adhesion sites that are specifically recognized by cell‐surface receptors, i.e., the integrins. Different from the ubiquitous sequential RGD adhesion motif found in most of the matrix proteins, in collagen type IV, the responsible binding sites for α1β1 integrin have been identified as Asp461 of the two α1 chains and Arg461 of the α2 chain. Because of the heterotrimeric character of this collagen, the spatial geometry of the binding epitope depends not only on the triple‐helical fold, but decisively even on the stagger of the chains. To investigate the effects of chain registration on the conformational properties and binding affinities of this adhesion epitope, two synthetic heterotrimeric collagen peptides consisting of the identical three chains were assembled by an artificial cystine knot in two different registers, i.e., in the most plausible α2α1α1′ and less probable α1α2α1′ chain alignment. A detailed conformational characterization of both trimers allowed to correlate their different binding affinities for α1β1 integrin with the degree of local plasticity of the two different triple helices. Optimal local breathing of the rod‐shaped collagens is apparently crucial for selective recognition by proteins interacting with these main components of the extracellular matrix. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004
Ribonucleopeptides: Functional RNA–peptide complexes
Authors: Hagihara Masaki , Hasegawa Tetsuya , Sato Shin‐ichi , Yoshikawa Susumu , Ohkubo Katsutoshi , Morii Takashi
Published: February 2004

Selection of functional RNAs from randomized pool of RNA molecules successfully affords RNA aptamers that specifically bind to small molecules, and that have catalytic activities. Recent structural analyses of the ribosomal RNA complex suggest that the RNA–protein complex would be a new structural candidate for the design of tailor‐made receptors and enzymes. We have designed an ATP binding domain that consists of an RNA subunit and a peptide subunit by means of structure‐based design approach and successive in vitro selection method. The RNA subunit is designed to consist of two functional domains; an ATP binding domain with 20 randomized nucleotides and an adjacent stem region that serves as a binding site for the RNA‐binding peptide. The randomized nucleotide region was placed next to the HIV‐1 Rev response element to enable the formation of “ribonucleopeptide” pools in the presence of the Rev peptide. In vitro selection of RNA oligonucleotides from the randomized pool afforded a ribonucleopeptide receptor specific for ATP. The ATP‐binding ribonucleopeptide did not share the known consensus nucleotide sequence for ATP aptamers, and completely lost its ATP‐binding ability in the absence of the Rev peptide. The ATP‐binding activity of the ribonucleopeptide was increased by a substitution of the N‐terminal amino acid of the Rev peptide. These results demonstrate that the peptide stabilizes the functional structure of RNA and suggest that amino acids outside the RNA binding region of the peptide participate in the ATP binding. Our approach would provide a new strategy for the design of tailor‐made ribonucleopeptide receptors. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004
Molecular design of functional peptides by utilizing unnatural amino acids: Toward artificial and photofunctional protein
Authors: Ishida Hitoshi , Kyakuno Masato , Oishi Shigero
Published: February 2004

Unnatural amino acids are effective as building blocks to design functional peptides from the following two points: (1) utilization of rigid unnatural amino acids for the incorporated peptides to control the conformation to appear the function, and (2) incorporation of functional and unnatural amino acids into peptides resulting in appearance of the inherent functions. As a combined strategy, molecular design of artificial metalloproteins utilizing 5′‐amino‐2,2′‐bipyridine‐5‐carboxilic acid (H–5Bpy–OH) as an unnatural amino acid is proposed. The peptide containing three residues of the unnatural amino acid would fold through coordination to a metal ion. In particular, ruthenium(II) ion would yield a ruthenium tris(bipyridine) derivative as the core complex of the artificial protein, which would appear the similar photochemical functions as that of ruthenium(II) tris(bipyridine) complex. The central complex could form two isomers, fac and mer. For selective synthesis of the mer complex, which is expected as the core complex in the artificial protein, dicyclohexylamide as a bulky group is introduced at the C‐terminal of the unnatural amino acid to destabilize the fac complex due to steric hindrance. Furthermore, in order to know the photochemical properties and function of the protein mimics, ruthenium(II) tris(2,2′‐bipyridine) complexes bearing amide groups at 5,5′ positions have been synthesized as the model complexes. As a result, the direction of amide groups (RNHCOor RCONH) in ruthenium complexes is found to significantly affect the emission efficiency: the former reduces the quantum yield and the latter enhances it, respectively. The ruthenium(II) tris(5,5′‐diamide‐2,2′‐bipyridine) complexes are also found to strongly bind with various anions [e.g., halogen ions (Cl−, Br−) and acetate anion] in acetonitrile and to detect these anions through the emission spectral changes under air. The molecular design of artificial protein is expected to develop new fields among peptide, organic, inorganic, and physical chemistry. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004
Chain‐length dependence of α‐helix to β‐sheet transition in polylysine: Model of protein aggregation studied by temperature‐tuned FTIR spectroscopy
Authors: Dzwolak Wojciech , Muraki Takeshi , Kato Minoru , Taniguchi Yoshihiro
Published: February 2004

The chain‐length dependence of the α‐helix to β‐sheet transition in poly(L‐lysine) is studied by temperature‐tuned FTIR spectroscopy. This study shows that heterogeneous samples of poly(L‐lysine), comprising polypeptide chains with various lengths, undergo the α–β transition at an intermediate temperature compared to homogenous ingredients. This holds true as long as each individual fraction of the polypeptide is capable of adopting an antiparallel β‐sheet structure. The tendency is that the longer chain is, the lower the α–β transition temperature is, which has been linked to the presence of distorted or solvated helices with turns or β sheets in elongating chains of poly(L‐lysine). As such helical structures are apparently conducive to the α–β transition, this draws a comparison to the hypothesis of metastable protein conformational states being a common stage in amyloid‐formation pathways. The antiparallel architecture of the β sheet is likely to reflect the pretransition interhelical interactions in poly(L‐lysine). Namely, the chains are arranged in an antiparallel manner because of energetically favored antiparallel preassembly of dipolar α helices. © 2004 Wiley Periodicals, Inc. Biopolymers, 2004
Synthesis of novel basic head‐to‐side‐chain cyclic dynorphin A analogs: Strategies and side reactions
Authors: Vig Balvinder S. , Murray Thomas F. , Aldrich Jane V.
Published: February 2004

Novel N‐terminus‐to‐side‐chain cyclic analogs of the opioid peptide dynorphin (Dyn) A–(1–11)NH2 were prepared that retain the basicity of the N‐terminal amine and restrict the backbone conformation around the important Tyr1 residue. Cyclic peptides were synthesized in which the N‐terminal amine and the Nε‐amine of a Lys at position 3 or 5 were attached to the α‐carbon and carbonyl of an acetyl group, respectively. Several synthetic strategies were explored with detailed analysis of the side reactions in order to obtain the desired cyclic peptides. One of the side reactions observed involved premature loss of the N‐terminal 9‐fluorenylmethoxycarbonyl (Fmoc) group during the neutralization step following deprotection of the Mtt (4‐methyltrityl) protecting group from the side chain of Lys. The successful strategy involved the synthesis of the linear peptide up through Gly2 and functionalization through the Nε‐amine of Lys. A linear N‐terminal alkylated analog was prepared by alkylation of the peptide on the resin with an equimolar amount of bromoacetamide, followed by treatment of the peptide with Fmoc–OSu prior to cleavage from the resin to facilitate separation by reversed phase high performance liquid chromatography of unreacted peptide from the desired alkylated product. The novel N‐terminal cyclic Dyn A analogs and the linear analog were evaluated for their opioid receptor affinities. These peptides exhibited large losses in affinity for opioid receptors; the low affinity of the linear N‐terminal alkylated peptide suggested that the α‐acetamide group on the N‐terminal amine resulted in unfavorable interactions with opioid receptors. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2003
Local sequence information in cellular retinoic acid‐binding protein I: Specific residue roles in β‐turns*
Authors: Rotondi Kenneth S. , Gierasch Lila M.
Published: February 2004

We have recently shown that two of the β‐turns (III and IV) in the ten‐stranded, β‐clam protein, cellular retinoic acid‐binding protein I (CRABP I), are favored in short peptide fragments, arguing that they are encoded by local interactions (K. S. Rotondi and L. M. Gierasch, Biochemistry, 2003, Vol. 42, pp. 7976–7985). In this paper we examine these turns in greater detail to dissect the specific local interactions responsible for their observed native conformational biases. Conformations of peptides corresponding to the turn III and IV fragments were examined under conditions designed to selectively disrupt stabilizing interactions, using pH variation, chaotrope addition, or mutagenesis to probe specific side‐chain influences. We find that steric constraints imposed by excluded volume effects between near neighbor residues (i,i+2), favorable polar (i,i+2) interactions, and steric permissiveness of glycines are the principal factors accounting for the observed native bias in these turns. Longer‐range stabilizing interactions across the β‐turns do not appear to play a significant role in turn stability in these short peptides, in contrast to their importance in hairpins. Additionally, our data add to a growing number of examples of the 3:5 type I turn with a β‐bulge as a class of turns with high propensity to form locally defined structure. Current work is directed at the interplay between the local sequence information in the turns and more long‐range influences in the mechanism of folding of this predominantly β‐sheet protein. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2003
Side‐chain anchoring strategy for solid‐phase synthesis of peptide acids with C‐terminal cysteine
Authors: Barany George , Han Yongxin , Hargittai Balazs , Liu Rong‐Qiang , Varkey Jaya T.
Published: February 2004

Many naturally occurring peptide acids, e.g., somatostatins, conotoxins, and defensins, contain a cysteine residue at the C‐terminus. Furthermore, installation of C‐terminal cysteine onto epitopic peptide sequences as a preliminary to conjugating such structures to carrier proteins is a valuable tactic for antibody preparation. Anchoring of Nα‐Fmoc, S‐protected C‐terminal cysteine as an ester onto the support for solid‐phase peptide synthesis is known to sometimes occur in low yields, has attendant risks of racemization, and may also result in conversion to a C‐terminal 3‐(1‐piperidinyl)alanine residue as the peptide chain grows by Fmoc chemistry. These problems are documented for several current strategies, but can be circumvented by the title anchoring strategy, which features the following: (a) conversion of the eventual C‐terminal cysteine residue, with Fmoc for Nα‐amino protection and tert‐butyl for Cα‐carboxyl protection, to a corresponding S‐xanthenyl (2XAL4) preformed handle derivative; and (b) attachment of the resultant preformed handle to amino‐containing supports. This approach uses key intermediates that are similar to previously reported Fmoc–XAL handles, and builds on earlier experience with Xan and related protection for cysteine. Implementation of this strategy is documented here with syntheses of three small model peptides, as well as the tetradecapeptide somatostatin. Anchoring occurs without racemization, and the absence of 3‐(1‐piperidinyl)alanine formation is inferred by retention of chains on the support throughout the cycles of Fmoc chemistry. Fully deprotected peptides, including free sulfhydryl peptides, are released from the support in excellent yield by using cocktails containing a high concentration (i.e., 80–90%) of TFA plus appropriate thiols or silanes as scavengers. High‐yield release of partially protected peptides is achieved by treatment with cocktails containing a low concentration (i.e., 1–5%) of TFA. In peptides with two cysteine residues, the corresponding intramolecular disulfide‐bridged peptide is obtained by either (a) oxidation, in solution, of the dithiol product released by acid; (b) simultaneous acidolytic cleavage and disulfide formation, achieved by addition of the mild oxidant DMSO to the cleavage cocktail; or (c) concomitant cleavage/cooxidation (involving a downstream S‐Xan protected cysteine), using reagents such as iodine or thallium tris(trifluoroacetate) in acetic acid. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2003
New tools for the control of peptide conformation and supramolecular chemistry: Crown‐carrier, Cα‐methyl L‐DOPA amino acids
Authors: Formaggio Fernando , Oancea Simona , Peggion Cristina , Crisma Marco , Toniolo Claudio , Wright Karen , Wakselman Michel , Mazaleyrat Jean‐Paul
Published: February 2004

The preferred conformation of five, terminally protected, model peptide series to the hexamer level, based on three novel crowned, Cα‐methyl L‐DOPA amino acids combined with either L‐Ala/Aib or Gly/Aib, were assessed in structure supporting solvents using FT‐IR absorption, 1H NMR, and CD techniques. The FT‐IR absorption spectra strongly suggest that the contribution of the crowned Cα‐tetrasubstituted residue to intramolecular H‐bonding is equivalent to that of Aib and is much more significant than that of either L‐Ala or Gly. In addition, the 1H NMR titrations and the CD patterns resemble those typically exhibited by (right‐handed) 310‐helical structures. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2003
Polar residue tagging of transmembrane peptides
Authors: Melnyk Roman A. , Partridge Anthony W. , Yip Jeannie , Wu Yanqiu , Goto Natalie K. , Deber Charles M.
Published: February 2004

Studies that focus on packing interactions between transmembrane (TM) helices in membrane proteins would greatly benefit from the ability to investigate their association and packing interactions in multi‐spanning TM domains. However, the production, purification, and characterization of such units have been impeded by their high intrinsic hydrophobicity. We describe the polar tagging approach to biophysical analysis of TM segment peptides, where incorporation of polar residues of suitable type and number at one or both peptide N‐ and C‐termini can serve to counterbalance the apolar nature of a native TM segment, and render it aqueous‐soluble. Using the native TM sequences of the human erythrocyte protein glycophorin A (GpA) and bacteriophage M13 major coat protein (MCP), properties of tags such as Lys, His, Asp, sarcosine, and Pro‐Gly are evaluated, and general procedures for tagging a given TM segment are presented. Gel‐shift assays on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) establish that various tagged GpA TM segments spontaneously insert into micellar membranes, and exhibit native TM dimeric states. Sedimentation equilibrium analytical centrifugation is used to confirm that Lys‐tagged GpA peptides retain the native dimer state. Two‐dimensional nuclear magnetic resonance (NMR) spectroscopy studies on Lys‐tagged TM MCP peptides selectively enriched with N‐15 illustrate the usefulness of this system for evaluating monomer–dimer equilibria in micelle environments. The overall results suggest that polar‐tagging of hydrophobic (TM) peptides approach constitutes a valuable tool for the study of protein–protein interactions in membranes. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2003
Solution structures of cyclic melanocortin agonists and antagonists by NMR
Authors: Ying Jinfa , Kövér Katalin E. , Gu Xuyuan , Han Guoxia , Trivedi Dev B. , Kavarana Malcolm J. , Hruby Victor J.
Published: February 2004

The melanocortin receptors are involved in many physiological functions, including pigmentation, sexual function, feeding behavior, and energy homeostasis, making them potential targets for drugs to treat obesity, sexual dysfunction, etc. Understanding the conformational basis of the receptor–ligand interactions is crucial to the design of potent and selective ligands for these receptors. The solution structures of the cyclic melanocortin agonists, partial agonist, and antagonists MTII, VJH085, SHU9119, MK5, and MK9 were determined by two‐dimensional nuclear magnetic resonance (2D NMR) spectroscopy at pH 4.5 and 25 °C in water (90% H2O/10% D2O). The overall backbone structures of these cyclic α‐melanocyte‐stimulating hormone (α‐MSH) analogues around the message sequence (His6‐D‐Phe7/D‐Nal(2′)7‐Arg8‐Trp9) were similar and reasonably well defined. β‐Turns spanning His6 and D‐Phe7/D‐Nal(2′)7 were identified in all analogues, and an amphiphilic molecular surface was obtained for the message sequence residues in most structures within the NMR ensembles. The β‐turn, which most closely resembles a type II β‐turn, leads to stacking between the aromatic rings of His6 and D‐Phe7 in MTII and VJH085. However, no aromatic stacking between His6 and D‐Nal(2′)7 was found in structures of the D‐Nal(2′)7‐containing analogues. The difference in the side‐chain dispositions of His6 and D‐Nal(2′)7 may be responsible for the reduced potency or antagonist activity of the D‐Nal(2′)7‐containing analogues. In addition, our results suggest that the side‐chain orientations may also modulate the receptor selectivity. The information found in this study will be useful for the further design of ligands for melanocortin receptors. © 2003 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2003
Dendritic oligoguanidines as intracellular translocators
Authors: Chung Hyun‐Ho , Harms Guido , Min Seong Churl , Choi Byung Hyune , Min Changhee , Taulane Joseph P. , Goodman Murray
Published: February 2004

A series of polyguanidylated dendritic structures that can be used as molecular translocators have been designed and synthesized based on nonpeptide units. The dendritic oligoguanidines conjugated with fluorescein or with a green fluorescent protein (GFP) mutant as cargos were isolated and characterized. Quantification and time‐course analyses of the cellular uptake of the conjugates using HeLa S3 and human cervical carcinoma cells reveal that the polyguanidylated dendrimers have comparable translocation efficiency to the Tat(49–57) peptide. Furthermore, the deconvolution microscopy image analysis shows that they are located inside the cells. These results clearly show that nonlinear, branched dendritic oligoguanidines are capable of translocation through the cell membrane. This work also demonstrates the potential of these nonpeptidic dendritic oligoguanidines as carriers for intracellular delivery of small molecule drugs, bioactive peptides, and proteins. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004
Targeting duplex DNA with DNA‐PNA chimeras? Physico‐chemical characterization of a triplex DNA‐PNA/DNA/DNA
Authors: Petraccone L. , Erra E. , Messere A. , Montesarchio D. , Piccialli G. , De Napoli L. , Barone G. , Giancola C.
Published: February 2004

Targeting double‐stranded DNA with homopyrimidine PNAs results in strand displacement complexes PNA/DNA/PNA rather than PNA/DNA/DNA triplex structures. Not much is known about the binding properties of DNA‐PNA chimeras. A 16‐mer 5′‐DNA‐3′‐p‐(N)PNA(C) has been investigated for its ability to hybridize a complementary duplex DNA by DSC, CD, and molecular modeling studies. The obtained results showed the formation of a triplex structure having similar, if not slightly higher, stability compared to the same all‐DNA complex. © 2004 Wiley Periodicals, Inc. Biopolymers, 2004
Structure‐based design of novel nonpeptide inhibitors of the Src SH2 domain: Phosphotyrosine mimetics exploiting multifunctional group replacement chemistry*
Authors: Sundaramoorthi Raji , Kawahata Noriyuki , Yang Michael G. , Shakespeare William C. , Metcalf Chester A. , Wang Yihan , Merry Taylor , Eyermann Charles J. , Bohacek Regine S. , Narula Surinder , Dalgarno David C. , Sawyer Tomi K.
Published: February 2004

A series of novel nonpeptide inhibitors of the pp60c‐Src (Src) SH2 domain is described that exploit multifunctional group replacement of the phenylphosphate moiety of phosphotyrosine (pTyr). Relative to an x‐ray structure of citrate complexed to the pTyr binding site of the Src SH2 domain, these nonpeptide ligands illustrate the systematic replacement of the phosphate group by multiple nonhydrolyzable, mono‐ or dianionic functionalities. Specifically, several phenylalanine (Phe) analogs incorporating key 4′ and 3′ substituents were synthesized and incorporated into a bicyclic benzamide template previously reported (W. C. Shakespeare et al., Proceedings of the National Academy of Science USA , 2000, Vol. 97, pp. 9373–9378). These pTyr mimetics included 4′,3′‐diphosphono‐Phe (Dpp), 4′,3′‐dicarboxymethyloxy‐Phe (Dcp), and 4′‐phosphono‐3′‐carboxymethyloxy‐Phe (Cpp). Noteworthy were nonpeptide inhibitors 8–11 that were 5‐ to 10‐fold more potent than the cognate tetrapeptide ligand Ac–pTyr–Glu–Glu–Ile–NH2 in binding to the Src SH2 domain. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2003
Binding of isofraxidin to bovine serum albumin
Authors: Liu Jiaqin , Tian Jianniao , Hu Zhide , Chen Xingguo
Published: February 2004

The binding of isofraxidin to bovine serum albumin (BSA) was studied under physiological conditions with BSA concentration of 1.5×10−6 mol · L−1 and drug concentration in the range of 1.67×10−6 mol · L−1 to 2.0×10−5 mol · L−1. Fluorescence quenching spectra in combination with uv absorption spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and CD spectroscopy was used to determine the drug‐binding mode, binding constant, and the protein structure changes in the presence of isofraxidin in aqueous solution. The linearity of Scatchard plot indicates that isofraxidin binds to a single class of binding sites on BSA and the values given for the binding constants agree very closely with those obtained by the modified Stern‐Volmer equation. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS), were calculated to be −17.63 kJ · mol−1 and 51.38 J · mol−1 · K−1 according to the van't Hoff equation, which indicated that hydrophobic interaction played a main role in the binding of isofraxidin to BSA. © 2004 Wiley Periodicals, Inc. Biopolymers, 2004
Micellar environments induce structuring of the N‐terminal tail of the prion protein
Authors: Renner Christian , Fiori Stella , Fiorino Ferdinando , Landgraf Dirk , Deluca Dominga , Mentler Matthias , Grantner Klaus , Parak Fritz G. , Kretzschmar Hans , Moroder Luis
Published: February 2004

In the physiological form, the prion protein is a glycoprotein tethered to the cell surface via a C‐terminal glycosylphosphatidylinositol anchor, consisting of a largely α‐helical globular C‐terminal domain and an unstructured N‐terminal portion. This unstructured part of the protein contains four successive octapeptide repeats, which were shown to bind up to four Cu2+ ions in a cooperative manner. To mimic the location of the protein on the cell membrane and to analyze possible structuring effects of the lipid/water interface, the conformational preferences of a single octapeptide repeat and its tetrameric form, as well of the fragment 92–113, proposed as an additional copper binding site, were comparatively analyzed in aqueous and dodecylphosphocholine micellar solution as a membrane mimetic. While for the downstream fragment 92–113 no conformational effects were detectable in the presence of DPC micelles by CD and NMR, both the single octapeptide repeat and, in an even more pronounced manner, its tetrameric form are restricted into well‐defined conformations. Because of the repetitive character of the rigid structural subdomain in the tetrarepeat molecule, the spatial arrangement of these identical motifs could not be resolved by NMR analysis. However, the polyvalent nature of the repetitive subunits leads to a remarkably enhanced interaction with the micelles, which is not detectably affected by copper complexation. These results strongly suggest interactions of the cellular form of PrP (PrPc) N‐terminal tail with the cell membrane surface at least in the octapeptide repeat region with preorganization of these sequence portions for copper complexation. There are sufficient experimental facts known that support a physiological role of copper complexation by the octapeptide repeat region of PrPc such as a copper‐buffering role of the PrPc protein on the extracellular surface. © 2004 Wiley Periodicals, Inc. Biopolymers, 2004
Aerobic phosphorus release linked to acetate uptake in bio‐P sludge: Process modeling using oxygen uptake rate
Authors: Guisasola Albert , Pijuan Maite , Baeza Juan A. , Carrera Julián , Casas Carles , Lafuente Javier
Published: February 2004

The main processes involved in enhanced biological phosphorus removal (EBPR) under anaerobic and subsequently aerobic conditions are widely described in the literature. Polyphosphate accumulating organisms (PAO) are the organisms responsible for this process. However, the mechanisms of PAO are not fully established yet under conditions that differ from the classical anaerobic/aerobic conditions. In this work, we made a comparison between the behavior of PAO under classical EBPR conditions and its behavior when consuming substrate under only aerobic conditions. In addition, oxygen uptake rate (OUR) was measured in the set of experiments under aerobic conditions to improve the characterization of the process. A kinetic and stoichiometric model based on Activated Sludge Model No.2 (ASM2) and including glycogen economy (AnOx model), calibrated for classical anaerobic/aerobic conditions, was not able to describe the experimental data since it underestimated the acetate consumption, the PHB storage, and the OUR. Two different hypotheses for describing the experimental measurements were proposed and modeled. Both hypotheses considered that PAO, under aerobic conditions, uptake acetate coupled to PHB storage, glycogen degradation, and phosphorus release as in anaerobic conditions. Moreover, the first hypothesis (PAO‐hypothesis) considered that PAO were able to store acetate as PHB linked to oxygen consumption and the second one (OHO hypothesis) considered that this storage was due to ordinary heterotrophic organisms (OHO). Both hypotheses were evaluated by simulation extending the AnOx model with additional equations. The main differences observed were the predictions for PHB degradation during the famine phase and the OUR profile during both feast and famine phases. The OHO hypothesis described the experimental profiles more accurately than the PAO hypothesis. © 2004 Wiley Periodicals, Inc.
Mechanical properties of desiccated ragweed pollen grains determined by micromanipulation and theoretical modelling
Authors: Liu T. , Zhang Z.
Published: February 2004

The mechanical properties of desiccated ragweed pollen grains were determined using a micromanipulation technique and a theoretical model. Single pollen grains with a diameter of approximately 20 μm were compressed and held, compressed and released, and compressed to rupture at different speeds between two parallel surfaces. Simultaneously, the force being imposed on the pollen grains was measured. It has been found that the rupture force of pollen grains increased linearly with their displacement at rupture on average, but was independent of their diameter. The mean rupture force was 1.20 ± 0.03 mN, and mean deformation (the ratio between the displacement and diameter) at rupture was 22 ± 0.6%. Single pollen grains were modeled as a capsule with a core full of air and a non permeable wall. A constitutive equation based on Hookean law was used to determine the mechanical property parameters Eh (product of the Young's modulus and wall thickness), and the mean value of Eh of desiccated pollen gains was estimated to be 1653 ± 36 N/m © 2004 Wiley Periodicals, Inc.
Oxidative burst, jasmonic acid biosynthesis, and taxol production induced by low‐energy ultrasound in Taxus chinensis cell suspension cultures
Authors: Wu Jianyong , Ge Xiuchun
Published: February 2004

This work aims to detect the two signal events in the elicitation of plant defense responses and secondary metabolism in plant cell cultures by low‐energy ultrasound (US), transient production of reactive oxygen species (ROS) or the oxidative burst and jasmonic acid (JA) biosynthesis, and examine their influence on secondary metabolism. Experiments were carried out in Taxus chinensis cell suspension culture which produces the anticancer diterpenoid Taxol (paclitaxel). The culture was exposed to low‐frequency US for a short period of time (2 min). At sufficiently high US power levels the US exposure significantly enhanced the Taxol production and slightly depressed cell growth and viability. The US exposure induced transient production of O and H2O2 and an increase in the intracellular JA level as well as the activities of enzymes for JA synthesis, lipoxygenase (LOX), and allene oxide synthase (AOS). Inhibition of the ROS production by putative ROS scavengers or the JA accumulation by LOX inhibitors effectively suppressed the US‐stimulated Taxol production. Inhibition of the ROS production also suppressed the US‐induced JA accumulation. These results suggest that oxidative burst is an upstream event to JA accumulation, and both ROS from the oxidative burst and JA from the LOX pathway are key signal elements in the elicitation of Taxol production of T. chinensis cells by low‐energy US. © 2004 Wiley Periodicals, Inc.
High‐rate ferric sulfate generation by a Leptospirillum ferriphilum‐dominated biofilm and the role of jarosite in biomass retainment in a fluidized‐bed reactor
Authors: Kinnunen Päivi H.‐M. , Puhakka Jaakko A.
Published: February 2004

The aims of this work were to develop a high‐rate fluidized‐bed bioprocess for ferric sulfate production, to characterize biomass retainment, and to determine the phylogeny of the enrichment culture. After 7 months of continuous enrichment and air aeration at 37°C, the iron oxidation rate of 8.2 g Fe2+ L−1h−1 (4.5·10−12 g Fe2+ cell‐1 h−1) was obtained at a hydraulic retention time (HRT) of 0.6 h. However, oxygen supply became the rate‐limiting factor. With gas mixture (99.5% O2/0.5% CO2 (vol/vol)) aeration and HRT of 0.2 h, the iron oxidation rate was 26.4 g Fe2+ L−1h−1 (1.0·10−11 g Fe2+ cell−1 h−1). Leptospirillum sp. was predominant in the mesophilic fluidized‐bed reactor (FBR) enrichment culture as determined by fluorescent in situ hybridization, while Acidithiobacillus ferrooxidans was not detected. Denaturing gradient gel electrophoresis (DGGE) of the amplified partial 16S rDNA showed only three bands, indicating a simple microbial community. DGGE fragment excision and sequencing showed that the populations were related to L. ferriphilum (100% similarity in sequence) and possibly to the genus Ferroplasma (96% similarity to F. acidiphilum). Jarosite precipitates accumulated on the top of the activated carbon biomass carrier material, increasing the rate of iron oxidation. The activated carbon carrier material, jarosite precipitates, and reactor liquid contained 59% (or 3.71·109 cells g−1), 31% (or 3.12·1010 cells g−1) and 10% (or 1.24·108 cells mL−1) of the total FBR microbes, respectively, demonstrating that the jarosite precipitates played an important role in the FBR biomass retainment. © 2004 Wiley Periodicals, Inc.
Optimization of reovirus production from mouse L‐929 cells in suspension culture
Authors: Jung Sunghoon , Behie Leo A. , Lee Patrick W. K. , Farrell Patrick J.
Published: February 2004

Reovirus serotype 3 Dearing (T3D) has shown potential as a novel cancer therapy. To support the increasing demand for reovirus, a two‐stage perfusion mode scheme is proposed for cell growth and reovirus production. Mouse L‐929 cells were used as the host for reovirus infection due to their ability to grow well in suspension culture. Several L‐929 cell growth and reovirus infection characteristics were investigated and optimized in spinner flask batch cultures. For the growth of L‐929 cells, a balanced nutrient‐fortification of SMEM medium increased the maximum cell density by 30%, compared to normal SMEM; however, ammonia and lactate accumulations were found to inhibit further cell growth. For the production of reovirus, ∼90% increase in viral yield resulted when the infection temperature was reduced from 37 to 33°C. Infectious reovirus particles were shown to be stable in conditioned medium at 37 and 33°C. The final virus titer was dependent on the multiplicity of infection (MOI) and the host cell density at the time of infection. A combination of an MOI of 0.1 pfu/cell and an initial host cell density of 1.0 × 106 cells/mL in fortified medium resulted in a maximum virus titer of (4.59 ± 0.16) × 109 pfu/mL and a specific yield of (2.34 ± 0.08) × 103 pfu/cell. At an optimal harvest time of the infection process, 99% of the virus was associated with the cellular debris. Finally, the presence of 5.0 mM ammonia in the culture medium was shown to seriously inhibit the reovirus yield, whereas lactate concentrations up to 20 mM had no effect. © 2004 Wiley Periodicals, Inc.
Whole cell–enzyme hybrid amperometric biosensor for direct determination of organophosphorous nerve agents with p‐nitrophenyl substituent
Authors: Lei Yu , Mulchandani Priti , Chen Wilfred , Wang Joseph , Mulchandani Ashok
Published: February 2004

In this paper, we reported the construction of a hybrid biosensor for direct, highly selective, sensitive, and rapid quantitative determination of organophosphate pesticides with p‐nitrophenyl substituent using purified organophosphorus hydrolase (OPH) for the initial hydrolysis and Arthrobacter sp. JS443 for subsequent p‐nitrophenol oxidation. The biocatalytic layer was prepared by co‐immobilizing Arthrobacter sp. JS443 and OPH on a carbon paste electrode. OPH catalyzed the hydrolysis of organophosphorus pesticides with p‐nitrophenyl substituent such as paraoxon and methyl parathion to release p‐nitrophenol that was oxidized by the enzymatic machinery of Arthrobacter sp. JS443 to carbon dioxide through electroactive intermediates 4‐nitrocatechol and 1,2,4‐benzenetriol. The oxidization current of the intermediates was measured and correlated to the concentration of organophosphates. The best sensitivity and response time were obtained using a sensor constructed with 0.06 mg dry weight of cell and 965 IU of OPH operating at 400 mV applied potential (vs. Ag/AgCl reference) in 50 mM citrate‐phosphate pH 7.5 buffer at room temperature. Using these conditions, the biosensor measured as low as 2.8 ppb (10 nM) of paraoxon and 5.3 ppb (20 nM) of methyl parathion without interference from phenolic compounds, carbamate pesticides, triazine herbicides, and organophosphate pesticides that do not have the p‐nitrophenyl substituent. The biosensor had excellent operational life‐time stability with no decrease in response for more than 40 repeated uses over a 12‐h period when stored at room temperature, while its storage life was approximately 2 days when stored in the operating buffer at 4°C. © 2004 Wiley Periodicals, Inc.
Biocatalytic transformation of petroporphyrins by chemical modified cytochrome C
Authors: García‐Arellano Humberto , Buenrostro‐Gonzalez Eduardo , Vazquez‐Duhalt Rafael
Published: February 2004

A semi‐synthetic biocatalyst was prepared by a double chemical modification of cytochrome c. Free amino groups were modified with poly(ethylene glycol) while free carboxylic groups were alkylated to form methyl esters. The double chemically modified protein, PEG‐Cyt‐Met, oxidized synthetic porphyrins in a ternary solvent mixture composed by methylene chloride, methanol, and phosphate buffer. The highest activity was found in the ternary systems with low water content (5%). The use of relatively hydrophobic peroxides, such as tert‐butyl and cumene hydroperoxides, extended the operational life of the biocatalyst, which, in turn, resulted in an extended oxidation of the substrates tested. PEG‐Cyt‐Met is able to transform asphaltenes, a highly recalcitrant petroleum fraction. The huge energetic resource found as asphaltene‐rich deposits is the driving force to investigate and to innovate upgrading technologies, including biotechnological strategies. © 2004 Wiley Periodicals, Inc.
Uncoupling of cell growth and proliferation results in enhancement of productivity in p21CIP1‐arrested CHO cells
Authors: Bi Jing‐Xiu , Shuttleworth John , Al‐Rubeai Mohamed
Published: February 2004

Chinese hamster ovary cells have been engineered to inducibly over‐express the p21CIP1 cyclin‐dependent kinase inhibitor, to achieve cell cycle arrest and increase cell productivity. In p21CIP1‐arrested cells production of antibody from a stably integrated lgG4 gene, was enhanced approximately fourfold. The underlying physiological basis for enhanced productivity was investigated by measuring a range of cellular and metabolic parameters. Interestingly, the average cell volume of arrested cells was approximately fourfold greater than that of proliferating cells. This was accompanied by significant increases in mitochondrial mass, mitochondrial activity, and ribosomal protein S6 levels. Our results suggest that p21CIP1‐induced cell cycle arrest uncouples cell growth from cell‐cycle progression, and provides new insight into how improved productivity can be achieved in a cell line commonly used for large‐scale production of pharmaceutical proteins. © 2004 Wiley Periodicals, Inc.
Expression of a single‐chain human interleukin‐12 gene in transgenic tobacco plants and functional studies
Authors: Gutiérrez‐Ortega Abel , Ávila‐Moreno Federico , Saucedo‐Arias Luis Jorge , Sánchez‐Torres Carmen , Gómez‐Lim Miguel Ángel
Published: February 2004

Interleukin 12 (IL‐12) is a key heterodimeric cytokine produced by a variety of antigen‐presenting cells, including dendritic cells, macrophages, and B cells. It displays a potent array of biological activities affecting natural killer (NK) and T cells. These activities include promotion of cell‐mediated or type 1 T helper cell responses (Th1). Due to that property, IL‐12 has been employed in cancer immunotherapy, in mouse models of infectious diseases and in airway inflammation, and it may also have utility as a vaccine adjuvant. Transgenic plants are being used in many laboratories around the world for the production of therapeutically valuable proteins and as vehicles for oral vaccines. Here we present the expression of a single‐chain human interleukin‐12 in transgenic tobacco plants. The biological activity of plant‐produced IL‐12 was determined by interferon gamma (IFN‐γ) production by natural killer (NK) cells, and the level of production was comparable to that obtained with commercially available recombinant IL‐12. The potential use of this recombinant protein is discussed. © 2004 Wiley Periodicals, Inc.
Redundant men: constraints on identity change
Authors: Willott Sara , Griffin Christine
Published: February 2004

In this article the constraints on identity change for a group of working class men experiencing long‐term unemployment in the UK is examined. Despite their lack of access to paid employment, these men continued to construct their gender identity around the breadwinner persona. The theoretical framework employed concepts from both Connell and Bourdieu. Based on discourse analysed data collected from a series of four loosely structured group discussions, it is argued here that these men had (i) less access to capital generally and (ii) reduced access to ‘useful’ types of capital resources. In other words, these groups of men have fewer resources to hand in constructing masculine identities that reflect the changing structural and ideological demands of the current social‐historical period. Copyright © 2004 John Wiley & Sons, Ltd.
You still feel different: the experience and meaning of women's self‐injury in the context of a lesbian or bisexual identity
Authors: Alexander Natasha , Clare Linda
Published: February 2004

This article reports an intensive qualitative study of the subjective experience and meaning of self‐injury for 16 women who identified as lesbian or bisexual and who had deliberately self‐injured on repeated occasions. In individual interviews, the women talked about their experiences of self‐injury and the role it played in their lives as lesbian or bisexual women. Interpretative Phenomenological Analysis (IPA) was used to elicit themes arising within their accounts. These highlighted a number of ways in which social and contextual factors contributed to the development of self‐injury. Although many of these factors seemed applicable to any woman who self‐injures, there were some aspects that were specific to the experience of lesbian and bisexual women. In addition, the women's accounts raised a number of important issues about the way in which mental health services respond to lesbian and bisexual women who self‐injure. It is argued that self‐injury can be understood as a coping response that arises within a social context characterized by abuse, invalidation, and the experience of being regarded as different or in some way unacceptable. These factors are especially salient in the lives of women, and they emerge particularly strongly as part of the experience of women who are developing a lesbian or bisexual identity. Copyright © 2004 John Wiley & Sons, Ltd.
Radical psychology networks: a review and guide
Authors: Burton Mark
Published: February 2004

Movements of psychologists with a liberatory agenda have emerged in a number of countries. A guide and overview is offered here. These networks have not yet developed into a particularly strong movement. Some of the reasons for this will be discussed in relation to the contradictory role of mainstream psychology as both loyal critic and as resource for the ruling order. Copyright © 2004 John Wiley & Sons, Ltd.
Different Roles of Electrostatics in Heat and in Cold: Adaptation by Citrate Synthase
Authors: Kumar Sandeep , Nussinov Ruth
Published: February 2004

Electrostatics plays a major role in heat adaptation by thermophilic proteins. Here we ask whether electrostatics similarly contributes to cold adaptation in psychrophilic proteins. We compare the sequences and structures of citrate synthases from the psychrophile Arthobacter Ds2–3R, from chicken, and from the hyperthermophile Pyrococcus furiosus. The three enzymes share similar packing, burial of nonpolar surface area, and main‐chain hydrogen bonding. However, both psychrophilic and hyperthermophilic citrate synthases contain more charged residues, salt bridges, and salt‐bridge networks than the mesophile. The electrostatic free‐energy contributions toward protein stability by individual charged residues show greater variabilities in the psychrophilic citrate synthase than in the hyperthermophilic enzyme. The charged residues in the active‐site regions of the psychrophile are more destabilizing than those in the active‐site regions of the hyperthermophile. In the hyperthermophilic enzyme, salt bridges and their networks largely cluster in the active‐site regions and at the dimer interface. In contrast, in the psychrophile, they are more dispersed throughout the structure. On average, salt bridges and their networks provide greater electrostatic stabilization to the thermophilic citrate synthase at 100 °C than to the psychrophilic enzyme at 0 °C. Electrostatics appears to play an important role in both heat and cold adaptation of citrate synthase. However, remarkably, the role may be different in the two types of enzyme: In the hyperthermophile, it may contribute to the integrity of both the protein dimer and the active site by possibly countering conformational disorder at high temperatures. On the other hand, in the psychrophile at low temperatures, electrostatics may contribute to enhance protein solvation and to ensure active‐site flexibility.
Structural Models of the Photointermediates in the Rhodopsin Photocascade, Lumirhodopsin, Metarhodopsin I, and Metarhodopsin II
Authors: Ishiguro Masaji , Oyama Yoshiaki , Hirano Takahiro
Published: February 2004

Model building of the two photointermediates, lumirhodopsin and metarhodopsin I, and the activated form of rhodopsin, metarhodopsin II, is described. An outward swing of the C‐terminal portion of transmembrane segment 3, pivoting on Cys110 at the N‐terminal end of transmembrane segment 3, led to structural models of lumirhodopsin and metarhodopsin I. The conformation of the chromophore in the lumirhodopsin and metarhodopsin I models is controlled by the motion of transmembrane segment 3 and agreed closely with the hydrogen‐bonding states of the protonated Schiff base in lumirhodopsin and metarhodopsin I as deduced from their FTIR and resonance Raman spectra and with the negative and positive CD bands of lumirhodopsin and metarhodopsin I, respectively. The structure of metarhodopsin II was constructed by an outward swing of transmembrane segment 3 and the rigid‐body motion of transmembrane segment 6. The arrangement of the entire transmembrane segment of the metarhodopsin II model closely agreed with the electron paramagnetic resonance spectra of spin‐labeled rhodopsin mutants and provided a structural basis for the protonation of Glu134, which is a key process in transducin activation.
Surface‐Functionalized Nanoparticle Library Yields Probes for Apoptotic Cells
Authors: Schellenberger Eyk A. , Reynolds Fred , Weissleder Ralph , Josephson Lee
Published: February 2004

We have developed techniques for the efficient synthesis and screening of small libraries of surface‐functionalized nanoparticles for the recognition of specific types of cells. To illustrate this concept we describe the development of a nanoparticle that preferentially recognizes apoptotic Jurkat cells in a manner similar to the apoptosis‐recognizing protein annexin V. The nanoparticle, which is detectable by fluorescence or NMR relaxometry, was analyzed for the ability to recognize normal and apoptotic cells by fluorescence‐activated cell sorting (FACS) analysis and fluorescence microscopy. The capability to develop nanoparticles which interact with specific target cells could be applied to the design of materials for diverse applications including quantum dots, which serve as fluorescence tracers, colloidal gold, which serves as a tracer for electron micrographs, or the crystalline forms of drugs.
Use of Magnetic Nanoparticles as Nanosensors to Probe for Molecular Interactions
Authors: Perez J. Manuel , Josephson Lee , Weissleder Ralph
Published: February 2004

Biocompatible magnetic nanosensors have been designed to detect molecular interactions in biological media. Upon target binding, these nanosensors cause changes in the spin–spin relaxation times of neighboring water molecules, which can be detected by magnetic resonance (NMR/MRI) techniques. These magnetic nanosensors have been designed to detect specific mRNA, proteins, enzymatic activity, and pathogens (e.g., virus) with sensitivity in the low femtomole range (0.5–30 fmol).