bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemiluminescence Assay Improves Specificity of Hepatitis C Antibody Detection
Authors: Dufour D Robert , Talastas Mageli , Fernandez Maria D A , Harris Barbara
Published: February 2004

Background: Antibodies to hepatitis C virus (anti-HCV) have typically been detected by enzyme immunoassay (EIA). A chemiluminescence assay (CA) for anti-HCV is now commercially available.

Methods: We compared the positive rate for a CA in a HCV screening program for veterans with historical rates obtained with EIA. We also compared results in 2824 samples tested by both methods and assessed the significance of low signal-to-cutoff (S/C) ratios.

Results: The frequency of CA-positive results was significantly lower than with EIA (12.6% vs 16.0%; P <0.0001). The frequency of low S/C ratios was also significantly lower with CA (11.5% vs 20.0%; P <0.0001). Among low-positive values, samples positive by CA were significantly less likely to be recombinant immunoblot assay (RIBA)-negative (64% vs 84%; P <0.0005). In parallel testing, results for 111 samples (3.9%) were discrepant between the two assays; all but 6 had low S/C ratios, and confirmatory testing was performed on all but 8 samples. Of 56 EIA-positive, CA-negative samples tested by RIBA, only 1 was positive. Of 24 CA-positive, EIA-negative samples, 62% were RIBA-negative. Using a negative RIBA result as an indication of false-positive anti-HCV results, the positive predictive value of EIA was 93% compared with 98% with CA. HCV RNA was positive in 90% of samples high-positive by both CA and EIA. Only 2 of 30 (7%) low-positive CA samples were RNA-positive.

Conclusions: CA produces fewer false-positive and fewer low-positive results that require confirmatory RIBA testing. The S/C ratio remains useful for characterizing positive results.
Urinary Cannabinoid Detection Times after Controlled Oral Administration of Δ9-Tetrahydrocannabinol to Humans
Authors: Gustafson Richard A , Levine Barry , Stout Peter R , Klette Kevin L , George M P , Moolchan Eric T , Huestis Marilyn A
Published: February 2004

Background: Urinary cannabinoid excretion and immunoassay performance were evaluated by semiquantitative immunoassay and gas chromatography–mass spectrometry (GC/MS) analysis of metabolite concentrations in 4381 urine specimens collected before, during, and after controlled oral administration of tetrahydrocannabinol (THC).

Methods: Seven individuals received 0, 0.39, 0.47, 7.5, and 14.8 mg THC/day in this double-blind, placebo-controlled, randomized, clinical study conducted on a closed research ward. THC doses (hemp oils with various THC concentrations and the therapeutic drug Marinol®) were administered three times daily for 5 days. All urine voids were collected over the 10-week study and later tested by Emit II®, DRI®, and CEDIA® immunoassays and by GC/MS. Detection rates, detection times, and sensitivities, specificities, and efficiencies of the immunoassays were determined.

Results: At the federally mandated immunoassay cutoff (50 μg/L), mean detection rates were <0.2% during ingestion of the two low doses typical of current hemp oil THC concentrations. The two high doses produced mean detection rates of 23–46% with intermittent positive tests up to 118 h. Maximum metabolite concentrations were 5.4–38.2 μg/L for the low doses and 19.0–436 μg/L for the high doses. Emit II, DRI, and CEDIA immunoassays had similar performance efficiencies of 92.8%, 95.2%, and 93.9%, respectively, but differed in sensitivity and specificity.

Conclusions: The use of cannabinoid-containing foodstuffs and cannabinoid-based therapeutics, and continued abuse of oral cannabis require scientific data for accurate interpretation of cannabinoid tests and for making reliable administrative drug-testing policy. At the federally mandated cannabinoid cutoffs, it is possible but unlikely for a urine specimen to test positive after ingestion of manufacturer-recommended doses of low-THC hemp oils. Urine tests have a high likelihood of being positive after Marinol therapy. The Emit II and DRI assays had adequate sensitivity and specificity, but the CEDIA assay failed to detect many true-positive specimens.
Distribution of 3-Hydroxy Fatty Acids in Tissues after Intraperitoneal Injection of Endotoxin
Authors: Szponar Bogumiła , Kraśnik Leonard , Hryniewiecki Tomasz , Gamian Andrzej , Larsson Lennart
Published: February 2004

Background: 3-Hydroxy fatty acids (3-OH FAs) with 10- to 18-carbon chain lengths are constituents of the endotoxin [lipopolysaccharide (LPS)] of gram-negative bacteria. We investigated whether these FAs may be used as chemical markers in measuring endotoxin concentrations in mammalian tissue samples.

Methods: We used gas-liquid chromatography–tandem mass spectrometry to measure 3-OH FAs in serum and tissues (heart, liver, and skeletal muscles) of rats after intraperitoneal injection of Escherichia coli LPS. One group of rats (group I) received a single LPS dose of 20 mg/kg of body weight; group II rats received the same total dose but over the course of 10 days (2 mg/kg each day). Rats receiving saline (group III) were used as controls.

Results: 3-OH FAs with chain lengths of 10, 12, 14, 16, and 18 carbons were detected in all studied types of samples. Group I rats had 50-fold and group II rats had 3-fold higher serum concentrations of 3-hydoxytetradecanoic acid (3-OH 14:0, the predominant 3-OH FA of E. coli LPS) than group III rats. Concentrations of 3-OH 14:0 in livers from group I and II rats were similar and fourfold higher than in group III rats, whereas concentrations of the same acid in skeletal and heart tissues did not differ among the three groups of rats. 3-OH 14:0 dominated in heart and liver of group III rats, whereas 3-OH 16:0 (followed by 3-OH 14:0) dominated in skeletal muscles and blood.

Conclusions: 3-OH FAs 10–18 carbons in length, probably originating from endotoxin and mitochondrial β-oxidation, are abundant in rat liver, skeletal muscles, and heart and can also be detected in blood. The widespread presence of these compounds in mammals limits their usefulness as LPS markers for endotoxin in clinical samples.
Detection of Cystic Fibrosis Mutations by Peptide Mass Signature Genotyping
Authors: Malehorn David E , Telmer Cheryl A , McEwen Sherri B , An Jiyan , Kinsey Ashley D , Retchless Adam C , Mason Christopher , Vieta William M , Jarvik Jonathan W
Published: February 2004

Background: The diversity of genetic mutations and polymorphisms calls for the development of practical detection methods capable of assessing more than one patient/one nucleotide position per analysis.

Methods: We developed a new method, based on peptide mass signature genotyping (PMSG), for the detection of DNA mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Exons of the gene were amplified, cloned, and expressed in Escherichia coli as peptide fusions, in natural as well as unnatural reading frames. Peptide analytes were purified by immobilized metal affinity chromatography and analyzed by matrix-assisted, laser desorption/ionization time-of-flight mass spectrometry. Synthetic and natural DNA samples with the 25 mutations recommended for CFTR carrier screening (Grody et al. Genet Med 2001;3:149–54) were assessed using the PMSG test for the CFTR gene.

Results: Peptide analytes ranged from 6278 to 17 454 Da and varied 30-fold in expression; highly expressing peptides were observed by electron microscopy to accumulate as inclusion bodies. Peptides were reliably recovered from whole-cell lysates by a simple purification method. CFTR mutations caused detectable changes in resulting mass spectrometric profiles, which were >95% reliably detected in blinded testing of replicate synthetic heterozygous DNA samples. Mutation detection was possible with both sample pooling and multiplexing. The PMSG CFTR test was used to determine compound heterozygous mutations in DNA samples from cystic fibrosis patients, which were confirmed by direct DNA sequencing.

Conclusions: The PMSG test of the CFTR gene demonstrates unique capabilities for determining the sequence status of a DNA target by sensitively monitoring the mass of peptides, natural or unnatural, generated from that target.
Frequent Misdiagnosis and Mismanagement of Hyperprolactinemic Patients before the Introduction of Macroprolactin Screening: Application of a New Strict Laboratory Definition of Macroprolactinemia
Authors: Suliman Abdulwahab M , Smith Thomas P , Gibney James , McKenna T Joseph
Published: February 2004

Background: Macroprolactin (big big prolactin) has reduced bioactivity and is measured by immunoassays for prolactin when it accumulates in the plasma of some individuals. We applied normative data for serum prolactin after treatment of sera to remove macroprolactin to elucidate the contribution of macroprolactin to misleading diagnoses, inappropriate investigations, and unnecessary treatment.

Methods: We reviewed records of women attending a tertiary referral center who had prolactin >1000 mIU/L. Application of a reference interval to polyethylene glycol (PEG)-treated hyperprolactinemic sera identified 21 patients in whom hyperprolactinemia was accounted for entirely by the presence of macroprolactin. Presenting clinical features, diagnoses, and treatment were compared in these patients and 42 age-matched true hyperprolactinemic patients.

Results: Prolactin concentrations in sera of 110 healthy individuals ranged from 78 to 564 mIU/L. The range of values for the sera after PEG treatment was 70–403 mIU/L. For macroprolactinemic samples, PEG treatment decreased mean (SD) prolactin from 1524 (202) mIU/L to 202 (27) mIU/L but decreased it only from 2096 (233) mIU/L to 1705 (190) mIU/L in true hyperprolactinemic patients (P <0.01 between groups). Oligomenorrhea or amenorrhea and galactorrhea were the most common clinical features in both groups, although they occurred more frequently in true hyperprolactinemic patients (P <0.05). Serum estradiol and luteinizing hormone concentrations were significantly higher in participants with macroprolactinemia than in those with true hyperprolactinemia (P <0.05). Among participants with retrospectively identified macroprolactinemia, pituitary imaging was performed in 93% and treatment with dopamine agonist was prescribed in 87%.

Conclusions: Macroprolactin is a significant cause of misdiagnosis, unnecessary investigation, and inappropriate treatment. The use of an appropriate reference interval for the PEG immunoprecipitation procedure may be of particular importance in those patients who have an excess of both macroprolactin and monomeric prolactin.
Der Weg in die Brücke: ein neuer Bindungstyp für tertiäre Phosphane, Arsane und Stibane
Authors: Werner Helmut
Published: February 2004

Die lange Zeit gehegte Annahme, dass tertiäre Phosphane, Arsane und Stibane nur als terminale Liganden an Übergangsmetalle koordinieren können, wurde erst in jüngster Zeit korrigiert. Bei Untersuchungen zur Reaktivität quadratisch‐planarer Carbenrhodium(I)‐Komplexe trans‐[RhCl(CRR′)(L)2] wurde gefunden, dass Verbindungen mit L=SbiPr3 thermisch in Zweikernkomplexe [Rh2Cl2(μ‐CRR′)2(μ‐SbiPr3)] überführt werden können, in denen sowohl die Carbeneinheiten als auch der Stibanligand verbrückende Positionen einnehmen. Während Versuche scheiterten, das Stiban in diesen Komplexen durch tertiäre Arsane oder Phosphane zu ersetzen, gelang die Synthese der Phosphan‐ bzw. Arsan‐verbrückten Verbindungen [Rh2(acac)2(μ‐CRR′)2(μ‐PR3)] und [Rh2(acac)2(μ‐CRR′)2(μ‐AsMe3)] durch Austausch der Brückenliganden nach Substitution der Chloroliganden durch Acetylacetonat. Die Acetylacetonatoliganden können durch anionische Lewis‐Basen ersetzt werden, was zu Monomeren [Rh2X2(μ‐CRR′)2(μ‐ER3)] oder kettenförmigen Dimeren [XRh(μ‐CRR′)2(μ‐ER3)Rh(μ‐X)2Rh(μ‐CRR′)2(μ‐ER3)RhX] führt.
Rationale Synthese kovalent gebundener organisch‐anorganischer Hybdridverbindungen
Authors: Peng Zhonghua
Published: February 2004

Kovalent über organische Einheiten mit delokalisiertem π‐Elektronensystem verbundene Polyoxometallat(POM)‐Hybridmaterialien haben zunehmendes Interesse geweckt. Diese Hybride wurden traditionell durch das nicht kontrollierbare Verknüpfen von Clustern synthetisiert, das Ergebnis war jedoch nicht vorhersagbar. Jetzt lassen sich solche Hybride durch etablierte organische Reaktionen herstellen, bei denen mit organischen Gruppen funktionalisierte POM‐Cluster als Bausteine eingesetzt werden. In diesem Kurzaufsatz werden einige aktuelle Fortschritte bei der Synthese von Hybriden vorgestellt, in denen organische und anorganische Einheiten über Imidobrücken verknüpft sind.
Titelbild: Schrittweise Bindung zweiwertiger Kationen an Phospholipid‐Membranen: eine Moleküldynamikstudie (Angew. Chem. 8/2004)
Authors: Böckmann Rainer A. , Grubmüller Helmut
Published: February 2004

Membran‐Kationen‐Wechselwirkungen haben großen Einfluss auf die Eigenschaften von biologischen Membranen. H. Grubmüller und R. Böckmann beschreiben in ihrer Zuschrift auf S. 1039 ff. die Simulation der Bindung von Natrium‐ und Calciumionen an neutrale, zwitterionische Lipidmembranen. Das Titelbild zeigt eine Momentaufnahme der Simulation eines Membranbereichs mit Ca2+‐Ionen.
The Way into the Bridge: A New Bonding Mode of Tertiary Phosphanes, Arsanes, and Stibanes
Authors: Werner Helmut
Published: February 2004

Until recently, tertiary phosphanes, arsanes, and stibanes were considered to bind to transition‐metal centers only in a terminal coordination mode. Investigations on the reactivity of square‐planar trans‐[RhCl(CRR′)(L)2] compounds revealed that compounds in which L=SbiPr3 can be converted upon heating into dinuclear complexes [Rh2Cl2(μ‐CRR′)2(μ‐SbiPr3)] with the carbene and stibane ligands in bridging positions. Although attempts to replace the stibane in these complexes with a tertiary arsane or phosphane failed, substitution of the chloro ligands for acetylacetonates followed by bridge–ligand exchange allowed the preparation of the phosphane‐ and arsane‐bridged compounds [Rh2(acac)2(μ‐CRR′)2(μ‐PR3)] and [Rh2(acac)2(μ‐CRR′)2(μ‐AsMe3)]. The acac ligands can be replaced by anionic Lewis bases to give either monomeric [Rh2X2(μ‐CRR′)2(μ‐ER3)] or dimeric chain‐like [XRh(μ‐CRR′)2(μ‐ER3)Rh(μ‐X)2Rh(μ‐CRR′)2(μ‐ER3)RhX] molecules.
Rational Synthesis of Covalently Bonded Organic–Inorganic Hybrids
Authors: Peng Zhonghua
Published: February 2004

Hybrid materials based on covalently linked inorganic polyoxometalates (POMs) and organic species containing a delocalized π system have drawn increasing attention. These hybrids, traditionally prepared by cluster assembly approaches that lack predictability and controllability, can now be synthesized through common organic reactions by using organically functionalized POM clusters as building blocks. This minireview highlights some of the most recent advances on a particular type of hybrids where the organic and inorganic components are connected by an imido linkage.
Cover Picture: Multistep Binding of Divalent Cations to Phospholipid Bilayers: A Molecular Dynamics Study (Angew. Chem. Int. Ed. 8/2004)
Authors: Böckmann Rainer A. , Grubmüller Helmut
Published: February 2004

Membrane–cation interactions can greatly affect the properties of biological membranes. In their Communication on page 1021 ff. H. Grubmüller and R. A. Böckmann describe the simulation of the binding of sodium and calcium ions to neutral, zwitterionic lipid membranes. The cover picture shows a snapshot from the molecular dynamics simulation of a membrane with Ca2+ ions.
Text mining: Generating hypotheses from MEDLINE
Authors: Srinivasan Padmini
Published: February 2004

Hypothesis generation, a crucial initial step for making scientific discoveries, relies on prior knowledge, experience, and intuition. Chance connections made between seemingly distinct subareas sometimes turn out to be fruitful. The goal in text mining is to assist in this process by automatically discovering a small set of interesting hypotheses from a suitable text collection. In this report, we present open and closed text mining algorithms that are built within the discovery framework established by Swanson and Smalheiser. Our algorithms represent topics using metadata profiles. When applied to MEDLINE, these are MeSH based profiles. We present experiments that demonstrate the effectiveness of our algorithms. Specifically, our algorithms successfully generate ranked term lists where the key terms representing novel relationships between topics are ranked high.
Making digital libraries effective: Automatic generation of links for similarity search across hyper‐textbooks
Authors: Melucci Massimo
Published: February 2004

Textbooks are more available in electronic format now than in the past. Because textbooks are typically large, the end user needs effective tools to rapidly access information encapsulated in textbooks stored in digital libraries. Statistical similarity‐based links among hyper‐textbooks are a means to provide those tools. In this paper, the design and the implementation of a tool that generates networks of links within and across hyper‐textbooks through a completely automatic and unsupervised procedure is described. The design is based on statistical techniques. The overall methodology is presented together with the results of a case study reached through a working prototype that shows that connecting hyper‐textbooks is an efficient way to provide an effective retrieval capability.
Constructing an associative concept space for literature‐based discovery
Authors: van der Eijk C. Christiaan , van Mulligen Erik M. , Kors Jan A. , Mons Barend , van den Berg Jan
Published: February 2004

Scientific literature is often fragmented, which implies that certain scientific questions can only be answered by combining information from various articles. In this paper, a new algorithm is proposed for finding associations between related concepts present in literature. To this end, concepts are mapped to a multidimensional space by a Hebbian type of learning algorithm using co‐occurrence data as input. The resulting concept space allows exploration of the neighborhood of a concept and finding potentially novel relationships between concepts. The obtained information retrieval system is useful for finding literature supporting hypotheses and for discovering previously unknown relationships between concepts. Tests on artificial data show the potential of the proposed methodology. In addition, preliminary tests on a set of Medline abstracts yield promising results.
Unlocking the museum: A manifesto
Authors: Jörgensen Corinne
Published: February 2004

At the same time that the Internet is becoming more accessible to large numbers of people and information consumers are becoming information producers, traditional methods of organizing, describing, and providing access to “documents” are being overwhelmed by the ever‐increasing number of digitized materials. Another parallel occurrence is the disappearance of cultural and indigenous knowledge as environments and peoples cease to exist. Therefore, the knowledge and ability to build and describe collections needs to be spread among a larger distributed group of participants. Three mechanisms are needed to facilitate this “unlocking” of collections and their management: the distributed description and annotation of documents, distributed collection building, and distributed knowledge creation.
Time‐tracking of the research profile of a drug using bibliometric tools
Authors: Bordons María , Bravo Carmen , Barrigón Santos
Published: February 2004

This study explores the usefulness of bibliometric analyses to detect trends in the research profile of a therapeutic drug, for which Aspirin was selected. A total of 22,144 documents dealing with Aspirin and published in journals covered by MEDLINE during the years 1965–2001 are studied. The research profile of Aspirin over the 37‐year period is analyzed through Aspirin subheadings and MeSH indexing terms. Half of the documents had Aspirin as a major indexing term, being the main aspects studied therapeutic uses (28% of the documents), pharmacodynamics (26%), adverse effects (18%), and administration and dosage (10%). A frequency data table crossing indexing terms × years is examined by correspondence analysis to obtain time trends, which are shown graphically in a map. Four time periods with a different distribution of indexing terms are identified through cluster analysis. The indexing term profile of every period is obtained by comparison of the distribution of indexing terms of each cluster with that of the whole period by means of the Chi‐2 test. The research profile of the drug tends to change faster with time. The most relevant finding is the expanding therapeutic profile of Aspirin over the period. The main advantages and limitations of the methodology are pointed out.
Process strategies to enhance pyruvate production with recombinant Escherichia coli: From repetitive fed‐batch to in situ product recovery with fully integrated electrodialysis
Authors: Zelić Bruno , Gostović Srđan , Vuorilehto K. , Vasić‐Rac̆ki Đurđa , Takors Ralf
Published: February 2004

Using the pyruvate production strain Escherichia coli YYC202 ldhA::Kan different process alternatives are studied with the aim of preventing potential product inhibition by appropriate product separation. This strain is completely blocked in its ability to convert pyruvate into acetyl‐CoA or acetate, resulting in acetate auxotrophy during growth in glucose minimal medium. Continuous experiments with cell retention, repetitive fed‐batch, and an in situ product recovery (ISPR) process with fully integrated electrodialysis were tested. Although the continuous approach achieved a high volumetric productivity (QP) of 110 g L−1 d−1, this approach was not pursued because of long‐term production strain instabilities. The highest pyruvate/glucose molar yield of up to 1.78 mol mol−1 together with high QP 145 g L−1 d−1 and high pyruvate titers was achieved by the repetitive fed‐batch approach. To separate pyruvate from fermentation broth a fully integrated continuous process was developed. In this process electrodialysis was used as a separation unit. Under optimum conditions a (calculated) final pyruvate titer of >900 mmol L−1 (79 g L−1) was achieved. © 2004 Wiley Periodicals, Inc.
Immobilization and biocatalytic activity of fungal protease on gold nanoparticle‐loaded zeolite microspheres
Authors: Phadtare Sumant , Vinod V.P. , Mukhopadhyay Kausik , Kumar Ashavani , Rao Mala , Chaudhari Raghunath V. , Sastry Murali
Published: February 2004

Gold nanoparticles are excellent biocompatible surfaces for the immobilization of enzymes. However, separation of the gold nanoparticle–enzyme bioconjugate material from the reaction medium is often difficult. In this study, we investigate the assembly of the gold nanoparticles on the surface of the amine‐functionalized zeolite microspheres in the formation of zeolite–gold nanoparticle “core‐shell” structures and, thereafter, the use of this structure in immobilization of fungal protease. The assembly of gold nanoparticles on the zeolite surface occurs through the amine groups present in 3‐aminopropyltrimethoxysilane (3‐APTS). The fungal proteases bound to the massive “core‐shell” structures were easily separated from the reaction medium by mild centrifugation and exhibited excellent reuse characteristics. The biocatalytic activity of fungal protease in the bioconjugate was marginally enhanced relative to the free enzyme in solution. The bioconjugate material also showed significantly enhanced pH and temperature stability and a shift in the optimum temperature of operation. © 2004 Wiley Periodicals, Inc.
Application of Plackett‐Burman design and response surface methodology to achieve exponential growth for aggregated shipworm bacterium
Authors: Ahuja S. K. , Ferreira G. M. , Moreira A. R.
Published: February 2004

Here we report the successful implementation of the Plackett‐Burman multifactorial design to screen the limiting components for growth and subsequent use of the response surface methodology (RSM) to design a medium that supported exponential growth of the aggregated morphology of the shipworm bacterium,Teredinobacter turnirae. The results obtained with the help of Plackett‐Burman design indicated limitations of three components in the growth medium, MnCl2.4H2O, Na2CO3, and K2HPO4. The concentrations of these three components were further optimized using RSM. By increasing the concentrations of the above‐mentioned components by 4‐fold, 12‐fold, and 12‐fold, respectively, it became possible to achieve exponential growth of the culture. © 2004 Wiley Periodicals, Inc.
Optimized procedure for renaturation of recombinant human bone morphogenetic protein‐2 at high protein concentration
Authors: Vallejo Luis Felipe , Rinas Ursula
Published: February 2004

The human gene encoding the mature form of bone morphogenetic protein‐2 (hBMP‐2), a dimeric disulfide‐bonded protein of the cystine knot growth factor family, was expressed in recombinant Escherichia coli using a temperature‐inducible expression system. The recombinant protein was produced in the form of cytoplasmic inclusion bodies and the effect of different variables on the renaturation of rhBMP‐2 was investigated. In particular, variables such as pH, redox conditions, protein concentration, temperature, the presence of different types of aggregation suppressors, and host cell contaminants were studied with respect to their effect on aggregation during refolding and on the final renaturation yield of rhBMP‐2. It is shown that the renaturation yield is particularly sensitive to pH, temperature, protein concentration, and the presence of aggregation suppressors. In contrast, little effect of the redox conditions and the ionic strength on the renaturation yield was observed, as equal yields were obtained in a broad range of reduced to oxidized glutathione ratios and concentrations of NaCl, respectively. The aggregation suppressor 2‐(cyclohexylamino)ethanesulfonic acid (CHES) proved to be superior with respect to the final renaturation yield, although, in comparison to the more common arginine, it was less efficient in preventing aggregation of rhBMP‐2 during refolding. Detergent washing of inclusion bodies was sufficient, as further purification of rhBMP‐2 prior to refolding was without effect on the final renaturation yield. An increase in the concentration of renatured rhBMP‐2 was achieved by a pulsed refolding procedure by which up to a total amount of 2.1 mg mL−1 rhBMP‐2 could be transferred in seven pulses into the renaturation buffer with an overall refolding yield of 38%, corresponding to 0.8 mg mL−1 renatured dimeric rhBMP‐2. Furthermore, a simplified purification procedure is presented that also includes freeze‐drying for long‐term storage of biologically active rhBMP‐2. Finally, it is shown that the appearance of rhBMP‐2 variants could be avoided by using a host strain overexpressing rare codon tRNAs. © 2004 Wiley Periodicals, Inc.
MIRACLE: mass isotopomer ratio analysis of U‐13C‐labeled extracts. A new method for accurate quantification of changes in concentrations of intracellular metabolites
Authors: Mashego M. R. , Wu L. , Van Dam J. C. , Ras C. , Vinke J. L. , Van Winden W. A. , Van Gulik W. M. , Heijnen J. J.
Published: February 2004

First, we report the application of stable isotope dilution theory in metabolome characterization of aerobic glucose limited chemostat culture of S. cerevisiae CEN.PK 113‐7D using liquid chromatography‐electrospray ionization MS/MS (LC‐ESI‐MS/MS). A glucose‐limited chemostat culture of S. cerevisiae was grown to steady state at a specific growth rate (μ) = 0.05 h−1 in a medium containing only naturally labeled (99% U‐12C, 1% U‐13C) carbon source. Upon reaching steady state, defined as 5 volume changes, the culture medium was switched to chemically identical medium except that the carbon source was replaced with 100% uniformly (U) 13C labeled stable carbon isotope, fed for 4 h, with sampling every hour. We observed that within a period of 1 h ∼80% of the measured glycolytic metabolites were U‐13C‐labeled. Surprisingly, during the next 3 h no significant increase of the U‐13C‐labeled metabolites occurred. Second, we demonstrate for the first time the LC‐ESI‐MS/MS‐based quantification of intracellular metabolite concentrations using U‐13C‐labeled metabolite extracts from chemostat cultivated S. cerevisiae cells, harvested after 4 h of feeding with 100% U‐13C‐labeled medium, as internal standard. This method is hereby termed “Mass Isotopomer Ratio Analysis of U‐13C Labeled Extracts” (MIRACLE). With this method each metabolite concentration is quantified relative to the concentration of its U‐13C‐labeled equivalent, thereby eliminating drawbacks of LC‐ESI‐MS/MS analysis such as nonlinear response and matrix effects and thus leads to a significant reduction of experimental error and work load (i.e., no spiking and standard additions). By coextracting a known amount of U‐13C labeled cells with the unlabeled samples, metabolite losses occurring during the sample extraction procedure are corrected for. © 2004 Wiley Periodicals, Inc.
Biodegradable polymers from organic acids by using activated sludge enriched by aerobic periodic feeding
Authors: Dionisi Davide , Majone Mauro , Papa Viviana , Beccari Mario
Published: February 2004

This article describes a new process for the production of biopolymers (polyhydroxyalkanoates, PHAs) based on the aerobic enrichment of activated sludge to obtain mixed cultures able to store PHAs at high rates and yields. Enrichment was obtained through the selective pressure established by feeding the carbon source in a periodic mode (feast and famine regime) in a sequencing batch reactor. A concentrated mixture of acetic, lactic, and propionic acids (overall concentration of 8.5 gCOD L−1) was fed every 2 h at 1 day−1 overall dilution rate. Even at such high organic load (8.5 gCOD L−1 day−1), the selective pressure due to periodic feeding was effective in obtaining a biomass with a storage ability much higher than activated sludges. The immediate biomass response to substrate excess (as determined thorough short‐term batch tests) was characterized by a storage rate and yield of 649 mgPHA (as COD) g biomass (as COD)−1 h−1 and 0.45 mgPHA (as COD) mg removed substrates (as COD−1), respectively. When the substrate excess was present for more than 2 h (long‐term batch tests), the storage rate and yield decreased, whereas growth rate and yield significantly increased due to biomass adaptation. A maximum polymer fraction in the biomass was therefore obtained at about 50% (on COD basis). As for the PHA composition, the copolymer poly(β‐hydroxybutyrate/β‐hydroxyvalerate) with 31% of hydroxyvalerate monomer was produced from the substrate mixture. Comparison of the tests with individual and mixed substrates seemed to indicate that, on removing the substrate mixture for copolymer production, propionic acid was fully utilized to produce propionylCoA, whereas the acetylCoA was fully provided by acetic and lactic acid. © 2004 Wiley Periodicals, Inc.
Aven and Bcl‐xL enhance protection against apoptosis for mammalian cells exposed to various culture conditions
Authors: Figueroa Bruno , Chen Sulin , Oyler George A. , Hardwick J. Marie , Betenbaugh Michael J.
Published: February 2004

A balance between proliferation and cell death is critical for achieving desirable high cell densities in mammalian cell culture. In this study, we evaluate a recently discovered anti‐apoptotic gene, aven, and examine its effectiveness alone and in combination with a member of the Bcl‐2 family, bcl‐xL. The commercially popular cell line, Chinese hamster ovary (CHO), was genetically modified to constitutively express aven, bcl‐xL, and the two genes in combination. Cells were exposed to several model insults that simulate severe bioreactor environments, including serum deprivation, spent medium, and Sindbis virus infection, as well as staurosporine, a known chemical inducer of apoptosis. CHO cells exhibited DNA fragmentation, a hallmark of apoptosis, after exposure to these model insults. After exposure to serum deprivation, 4‐ and 5‐day spent medium, and staurosporine, cells expressing Aven provided limited protection against cell death when compared with the protection afforded by cells expressing Bcl‐xL alone. However, the highest survival levels for all insults were achieved when Aven was expressed in combination with Bcl‐xL. In fact, Aven appeared to act synergistically to enhance the protective function of Bcl‐xL for several insults, because the protective function of the two genes expressed together in one cell line often exceeded the additive protective levels of each anti‐apoptosis gene expressed alone. Surprisingly, Aven expression provided a mildly pro‐apoptotic response in CHO isolates infected with Sindbis virus. However, CHO cells expressing both Bcl‐xL and Aven showed protection against Sindbis virus infection due to the inhibitory properties of the bcl‐xL anti‐apoptosis gene. This study shows that combinatorial anti‐apoptosis cell engineering strategies may be the most effective mechanisms for providing extended protection against cell death in mammalian cell culture. © 2004 Wiley Periodicals, Inc.
High‐level secretion of functional green fluorescent protein from transgenic tobacco cell cultures: Characterization and sensing
Authors: Su Wei Wen , Guan Peizhu , Bugos Robert C.
Published: February 2004

Green fluorescent protein (GFP) is useful for studying protein trafficking in plant cells. This utility could potentially be extended to develop an efficient secretory reporter system or to enable on‐line monitoring of secretory recombinant protein production in plant cell cultures. Toward this end, the aim of the present study was to: (1) demonstrate and characterize high levels of secretion of fluorescent GFP from transgenic plant cell culture; and (2) examine the utility of GFP fluorescence for monitoring secreted recombinant protein production. In this study we expressed in tobacco cell cultures a secretory GFP construct made by splicing an Arabidopsis basic chitinase signal sequence to GFP. Typical extracellular GFP accumulation was 12 mg/L after 10 to 12 days of culture. The secreted GFP is functional and it accounts for up to 55% of the total GFP expressed. Findings from culture treatments with brefeldin A suggest that GFP is secreted by the cultured tobacco cells via the classical endoplasmic reticulum–Golgi pathway. Over the course of flask cultures, medium fluorescence increased with the secreted GFP concentrations that were determined using either Western blot or enzyme‐linked immunoassay. Real‐time monitoring of secreted GFP in plant cell cultures by on‐line fluorescence detection was verified in bioreactor cultures in which the on‐line culture fluorescence signals showed a linear dependency on the secreted GFP concentrations. © 2004 Wiley Periodicals, Inc.
Biodegradation kinetics of volatile hydrophobic organic compounds in cultures with variable fractional volumes
Authors: Hong Tien Truong , Katayama Arata
Published: February 2004

An extension of the models developed by Guha and Jaffé (Biotechnol Bioeng [1996] 50:693–699) to describe the phenanthrene biodegradation kinetics for the cultures with variable fractional volumes is presented. Batch experiments were conducted with a culture capable of degrading the phenanthrene using a single culture vessel from which samples were withdrawn over time to monitor the disappearance of phenanthrene. For accurate measurement of phenanthrene concentrations, a sampling procedure designed for quantifying the sorption of phenanthrene onto glassware was also introduced. The Monod parameters were estimated by nonlinear regression analyses of simultaneous solutions to the substrate utilization/volatilization and Monod equations for growth of the cell mass. The results demonstrate that the models were able to be extended to phenanthrene‐degrading cultures with variable fractional volumes. When the ratio between sampling volume and volume of the culture medium was relatively small, the parameters obtained were similar to those which would be obtained using constant fractional volumes of culture medium. It was also found that the model's fit to the phenanthrene disappearance data in this study were better than those obtained by Guha and Jaffé, implying that the sorption process of phenanthrene during the sampling period could significantly affect the measurement of phenanthrene concentrations. Failing to account for these losses led to less accurate measurements of substrate concentrations, which in turn resulted in a poor estimation of the parameters. The findings of this study reduce considerably the experimental work necessary in the estimation of Monod kinetic parameters for the purpose of modeling. © 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.
Hyperactivation of Rhizomucor miehei lipase by hydrophobic xerogels
Authors: Aucoin Marc G. , Erhardt Frank A. , Legge Raymond L.
Published: February 2004

Although a variety of approaches exist for the immobilization of enzymes, the “science” of enzyme immobilization is still in its infancy. In recent years, considerable interest has developed regarding the use of xerogels for enzyme immobilization. There are several advantages to xerogels for enzyme immobilization, including the opportunity to produce them in defined shapes or thin films and the ability to manipulate their physical characteristics (e.g., porosity, hydrophobicity, and optical properties). In this study we examined the effect of xerogel hydrophobicity on the activity of lipase (EC 3.2.2.3) from Rhizomucor miehei. The hydrophobicity of the xerogels was manipulated by generating xerogels with various molar ratios of propyltrimethoxysilane (PTMS) to tetramethoxysilane (TMOS), from 1:1 to 10:1. The belief was that, by increasing the proportion of propyl groups, the hydrophobicity of the resulting xerogel would be increased. Differences in the hydrophobicity of the resulting xerogels were confirmed using water‐affinity studies. Two approaches were taken for water‐affinity determinations by examining the ability of the xerogels to remove water from air (controlled humidity) and from water‐saturated isopropyl ether. Xerogels with higher propyl content showed a reduced affinity for water. A crude lipase preparation from Rhizomucor miehei was then contacted with sized xerogel particulates and the effect of the xerogel on lipase activity was determined. The presence of the xerogel resulted in hyperactivation of the lipase. Analysis of the protein adsorption revealed changes in the profile of proteins adsorbed to the xerogel based on the hydrophobicity of the xerogel. Based on estimations of the specific activity of the hyperactivated lipase, a minimum hyperactivation of 207% was observed. Part of the hyperactivation may be attributable to xerogel–lipase interactions, but also to the adsorption of a component from the crude lipase preparation that may complex with the lipase and the xerogel producing a stabilizing effect. Further improvements in hyperactivation and selectivity of the xerogels is likely possible by working at lower PTMS:TMOS ratios than those investigated in this study. © 2004 Wiley Periodicals, Inc.
Hollow fiber bioreactor: New development for the study of contrast agent transport into hepatocytes by magnetic resonance imaging
Authors: Planchamp Corinne , Ivancevic Marko K. , Pastor Catherine M. , Vallée Jean‐Paul , Pochon Sibylle , Terrier François , Mayer Joachim M. , Reist Marianne
Published: February 2004

The aim of our study was to develop a magnetic resonance (MR)‐compatible in vitro model containing freshly isolated rat hepatocytes to study the transport of hepatobiliary contrast agents (CA) by MR imaging (MRI). We set up a perfusion system including a perfusion circuit, a heating device, an oxygenator, and a hollow fiber bioreactor (HFB). The role of the porosity and surface of the hollow fiber (HF) as well as the perfusate flow rate applied on the diffusion of CAs and O2 was determined. Hepatocytes were isolated and injected in the extracapillary space of the HFB (4 × 107 cells/mL). The hepatocyte HFB was perfused with an extracellular CA, gadopentetate dimeglumine (Gd‐DTPA), and gadobenate dimeglumine (Gd‐BOPTA), which also enters into hepatocytes. The HFB was imaged in the MR room using a dynamic T1‐weighed sequence. No adsorption of CAs was detected in the perfusion system without hepatocytes. The use of a membrane with a high porosity (0.5 μm) and surface (420 cm2), and a high flow rate perfusion (100 mL/min) resulted in a rapid filling of the HFB with CAs. The cellular viability of hepatocytes in the HFB was greater than 85% and the O2 consumption was maintained over the experimental period. The kinetics of MR signal intensity (SI) clearly showed the different behavior of Gd‐BOPTA that enters into hepatocytes and Gd‐DTPA that remains extracellular. Thus, these results show that our newly developed in vitro model is an interesting tool to investigate the transport kinetics of hepatobiliary CAs by measuring the MR SI over time. © 2004 Wiley Periodicals, Inc.
Pediocin production by Pediococcus acidilactici in solid state culture on a waste medium: Process simulation and experimental results
Authors: Vázquez J.A. , González M.P. , Murado M.A.
Published: February 2004

The production of pediocin by Pediococcus acidilactici was comparatively studied in submerged and solid‐state culture, using polyurethane foam particles soaked in commercial (MRS) and waste media with various supplements, where product concentrations were 15 times higher in MRS medium. For the solid state analysis, cultures were treated by successive compression and refilling of tubular minireactors equipped with a piston, without the need for reinoculation. This method was found to be simple, reproducible, and easily controllable, allowing culture productivity to be maintained for long periods of time without alterations in the basic properties of the system. In addition, yields were found to be superior compared to those from submerged culture. The system kinetics were modeled on the basis of widely accepted assumptions with a good fit to the experimental results and observed biomass fluctuations less evident than those predicted by the kinetic model. © 2004 Wiley Periodicals, Inc.
Computational aspects of protein functionality
Authors: Paton R. C. , Toh C.‐H.
Published: February 2004

The purpose of this short article is to examine certain aspects of protein functionality with relation to some key organizing ideas. This is important from a computational viewpoint in order to take account of modelling both biological systems and knowledge of these systems. We look at some of the lexical dimensions of the function and how certain constructs can be related to underlying ideas. The pervasive computational metaphor is then discussed in relation to protein multifunctionality, and the specific case of von Willebrand factor as a ‘smart’ multifunctional protein is briefly considered. Some diagrammatic techniques are then introduced to better articulate protein function. Copyright © 2004 John Wiley & Sons, Ltd.
MicroCore: mapping genome expression to cell pathways and networks
Authors: Patel Manish , Nagl Sylvia
Published: February 2004

The MicroCore toolkit is a suite of analysis programs for microarray and proteomics data that is open source and programmed exclusively in Java. MicroCore provides a flexible and extensible environment for the interpretation of functional genomics data through visualization. The first version of the application (downloadable from the MicroCore website: http://www.ucl.ac.uk/oncology/MicroCore/microcore.htm), implements two programs—PIMs (protein interaction maps) and MicroExpress—and is soon to be followed by an extended version which will also feature a fuzzy k‐means clustering application and a Java‐based R plug‐in for microarray analysis. PIMs and MicroExpress provide a simple yet powerful way of graphically relating large quantities of expression data from multiple experiments to cellular pathways and biological processes in a statistically meaningful way. Copyright © 2004 John Wiley & Sons, Ltd.
Rewriting systems and the modelling of biological systems
Authors: Giavitto Jean‐Louis , Malcolm Grant , Michel Olivier
Published: February 2004

This paper gives a brief survey of the use of algebraic rewriting systems for modelling and simulating various biological processes, particularly at the cellular level. Copyright © 2004 John Wiley & Sons, Ltd.
Development of a grounded‐trench MOS‐assisted bipolar‐mode FET
Authors: Murakami Yoshinori , Asada Kunihiro
Published: February 2004

The grounded‐trench MOS‐assisted bipolar‐mode FET, or GTBT, is made by introducing an insulated trench electrode into a bipolar‐mode JFET. This resulting device has normally‐off characteristics which are retained until avalanche breakdown, a high current gain, high rate switching, and so on. This paper summarizes GTBT's structure, mechanism, our design approach to enhance its functionality, and the various characteristics of the chips we fabricated. © 2004 Wiley Periodicals, Inc. Electron Comm Jpn Pt 2, 87(3): 62–73, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/ecjb.10105
An 85‐Mbit/s DQPSK MODEM‐LSI using a ROM storage capacity reducing technique based on phase plane symmetry
Authors: Yamagishi Akihiro , Tsukahara Tsuneo , Muraguchi Masahiro
Published: February 2004

As a configuration for a baseband delayed detection circuit for the phase modulation scheme widely used in wireless communication systems, a technique is proposed for reducing the capacity of the ROM used for phase detection by means of symmetry in the phase plane. With this technique, the capacity of the ROM needed can be reduced to 1/5. A MODEM‐LSI is fabricated in a one‐chip format for trial by the present technique with 0.5‐µm CMOS. A bandwidth of more than 85 Mbit/s and a low power consumption of 23 mA in 80 Mbit/s operation are achieved. Also, by means of the present LSI, a DQPSK modulator and demodulator are configured and a fixed deterioration of 2 dB at BER = 1e−5 is obtained by folding measurement. © 2004 Wiley Periodicals, Inc. Electron Comm Jpn Pt 2, 87(3): 12–20, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/ecjb.10194