bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection and identification ofEscherichia coli, Shigella,andSalmonellaby microarrays using thegyrBgene
Authors: Kakinuma Kenichi , Fukushima Masao , Kawaguchi Ryuji
Published: July 2003

Commonly, 16S ribosome RNA (16S rRNA) sequence analysis has been used for identifying enteric bacteria. However, it may not always be applicable for distinguishing closely related bacteria. Therefore, we selectedgyrBgenes that encode the subunit B protein of DNA gyrase (a topoisomerase type II protein) as target genes. The molecular evolution rate ofgyrBgenes is higher than that of 16S rRNA, andgyrBgenes are distributed universally among bacterial species. Microarray technology includes the methods of arraying cDNA or oligonucleotides on substrates such as glass slides while acquiring a lot of information simultaneously. Thus, it is possible to identify the enteric bacteria easily using microarray technology. We devised a simple method of rapidly identifying bacterial species through the combined use ofgyrBgenes and microarrays. Closely related bacteria were not identified at the species level using 16S rRNA sequence analysis, whereas they were identified at the species level based on the reaction patterns of oligonucleotides on our microarrays usinggyrBgenes. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 721–728, 2003.
Scale‐up of Artemisia annua L. hairy root cultures produces complex patterns of terpenoid gene expression
Authors: Souret Frédéric F. , Kim Yoojeong , Wyslouzil Barbara E. , Wobbe Kristin K. , Weathers Pamela J.
Published: July 2003

Hairy roots grow quickly, reach high densities, and can produce significant amounts of secondary metabolites, yet their scale‐up to bioreactors remains challenging. Artemisia annua produces a rich array of terpenoids, including the sesquiterpene, artemisinin, and transformed roots of this species provide a good model for studying terpenoid production. These cultures were examined in shake flasks and compared with cultures grown in two types of bioreactors, a mist reactor and a bubble column reactor, which provide very different environments for the growing roots. Mist reactors have been shown previously to result in cultures that produce significantly more artemisinin per gram fresh weight of culture, while bubble column reactors have produced greater biomass. We have compared expression levels of four key terpenoid biosynthetic genes: 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase (HMGR), 1‐deoxy‐D‐xylulose‐5‐phosphate synthase (DXS), 1‐deoxy‐D‐xylulose‐5‐phosphate reductoisomerase (DXR), and farnesyl diphosphate synthase (FPS) in the three culture conditions. In shake flasks we found that although all four genes showed temporal regulation, only FPS expression correlated with artemisinin production. Light also affected the transcription of all four genes. Although expression in reactors was equivalent to or greater than that of roots grown in shake flasks, no correlation was found between expression level within six different zones of each reactor and their respective oxygen levels, light, and root‐packing density. Surprisingly, transcriptional regulation of HMGR, DXS, DXR, and FPS was greatly affected by the position of the roots in each reactor. Thus, relying on a single reactor sample to characterize the gene activity in a whole reactor can be misleading, especially if the goal is to examine the difference between reactor types or operating parameters, steps essential in scaling up cultures for production. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 653–667, 2003.
Chitosan scaffolds for biomolecular assembly: Coupling nucleic acid probes for detecting hybridization
Authors: Yi Hyunmin , Wu Li‐Qun , Sumner James J. , Gillespie James B. , Payne Gregory F. , Bentley William E.
Published: July 2003

Chitosan, a naturally occurring biopolymer, was used as a scaffold for the covalent binding of single‐stranded DNA oligonucleotide probes in a fluorescence‐based nucleic acid hybridization assay. Chitosan's pH dependent chemical and electrostatic properties enable its deposition on electrodes and metal surfaces, as well as on the bottom of microtiter plates. A combinatorial 96‐well microtiter plate format was used to optimize chemistries and reaction conditions leading to hybridization experiments. We found the coupling of oligonucleotides using relatively common glutaraldehyde chemistry was quite robust. Our hybridization results for complementary ssDNA oligonucleotides (E. coli dnaK sequences) demonstrated linear fluorescence intensity with concentration of E. coli dnaK‐specific oligonucleotide from 0.73 μM to 6.6 μM. Moreover, hybridization assays were specific as there was minimal fluorescence associated with noncomplementary groEL oligonucleotide. Finally, these results demonstrate the portability of a DNA hybridization assay based on covalent coupling to chitosan, which, in turn, can be deposited onto various surfaces. More arduous surface preparation techniques involving silanizing agents and hazardous washing reagents are eliminated using this technique. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 646–652, 2003.
The effects of metabolites from the indigenous Acidithiobacillus thiooxidans and temperature on the bioleaching of cadmium from soil
Authors: Liu Hsuan‐Liang , Chiu Chi‐Wei , Cheng Yang‐Chu
Published: July 2003

The effect of metabolites from the indigenous Acidithiobacillus thiooxidans and temperature on the bioleaching of cadmium from soil was investigated in the present study. Bioleaching was found to be more effective than chemical leaching of cadmium. The metabolite, mainly sulfuric acid, which was shown to be growth‐associated in the exponential phase, plays a major role in bioleaching. The maximum amount of cadmium leached was obtained after 8 days of precultivation when cells were directly involved in the leaching process. It indicates that cells in the exponential growth phase exhibit higher activity toward bioleaching. In contrast, the maximum amount of cadmium leached and the maximum initial rate for bioleaching were reached after 16 days of precultivation when only metabolites were involved in the bioleaching process. It implies that higher sulfuric acid concentration results in higher leaching efficiency. In addition, higher temperature leads to higher leaching efficiency. The optimal operation condition for bioleaching was determined to be a two‐stage process: The first stage involves the precultivation of the indigenous A. thiooxidans at 30°C for 8 days followed by 20 minutes of centrifugation to discard cells. The second stage involves the bioleaching with the subsequent supernatant at 50°C. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 638–645, 2003.
Global incorporation of norleucine in place of methionine in cytochrome P450 BM‐3 heme domain increases peroxygenase activity
Authors: Cirino Patrick C. , Tang Yi , Takahashi Katsuyuki , Tirrell David A. , Arnold Frances H.
Published: July 2003

In this study we have replaced all 13 methionine residues in the cytochrome P450 BM‐3 heme domain (463 amino acids) with the isosteric methionine analog norleucine. This experiment has provided a means of testing the functional limits of globally incorporating into an enzyme an unnatural amino acid in place of its natural analog, and also an efficient way to test whether inactivation during peroxide‐driven P450 catalysis involves methionine oxidation. Although there was no increase in the stability of the P450 under standard reaction conditions (in 10 mM hydrogen peroxide), complete substitution with norleucine resulted in nearly two‐fold‐increased peroxygenase activity. Thermostability was significantly reduced. The fact that the enzyme can tolerate such extensive amino acid replacement suggests that we can engineer enzymes with unique chemical properties via incorporation of unnatural amino acids while retaining or improving catalytic properties. This system also provides a platform for directing enzyme evolution using an extended set of protein building blocks. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 729–734, 2003.
A novel method of simulating oxygen mass transfer in two‐phase partitioning bioreactors
Authors: Nielsen David R. , Daugulis Andrew J. , Mclellan P. James
Published: July 2003

An empirical correlation, based on conventional forms, has been developed to represent the oxygen mass transfer coefficient as a function of operating conditions and organic fraction in two‐phase, aqueous–organic dispersions. Such dispersions are characteristic of two‐phase partitioning bioreactors, which have found increasing application for the biodegradation of toxic substrates. In this work, a critical distinction is made between the oxygen mass transfer coefficient, kLa, and the oxygen mass transfer rate. With an increasing organic fraction, the mass transfer coefficient decreases, whereas the oxygen transfer rate is predicted to increase to an optimal value. Use of the correlation assumes that the two‐phase dispersion behaves as a single homogeneous phase with physical properties equivalent to the weighted volume‐averaged values of the phases. The addition of a second, immiscible liquid phase with a high solubility of oxygen to an aqueous medium increases the oxygen solubility of the system. It is the increase in oxygen solubility that provides the potential for oxygen mass transfer rate enhancement. For the case studied in which n‐hexadecane is selected as the second liquid phase, additions of up to 33% organic volume lead to significant increases in oxygen mass transfer rate, with an optimal increase of 58.5% predicted using a 27% organic phase volume. For this system, the predicted oxygen mass transfer enhancements due to organic‐phase addition are found to be insensitive to the other operating variables, suggesting that organic‐phase addition is always a viable option for oxygen mass transfer rate enhancement. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 735–742, 2003.
Resistance of Lactobacillus casei in plastic‐composite‐support biofilm reactors during liquid membrane extraction and optimization of the lactic acid extraction system
Authors: Demirci Ali , Cotton Julie C. , Pometto Anthony L. , Harkins Kristi R. , Hinz Paul N.
Published: July 2003

Lactic acid fermentations were performed with plastic‐composite‐support (PCS) disks in solvent‐saturated media with Lactobacillus casei subsp. rhamnosus (ATCC 11443). The PCS disks contained 50% (w/w) polypropylene, 35% (w/w) ground soybean hulls, 5% (w/w) yeast extract, 5% (w/w) soybean flour, and 5% (w/w) bovine albumin. Bioassays were performed by growing L. casei in solvent‐saturated media after soaking the PCS disks. Eighteen different solvent and carrier combinations were evaluated. Overall, L. casei biofilm fermentation demonstrated the same lactic acid production in solvent‐saturated medium as suspended cells in medium without solvents (control). To evaluate PCS solvent‐detoxifying properties, two bioassays were developed. When solvent‐saturated medium in consecutive equal volumes (10 mL then 10 mL) was exposed to PCS, both media demonstrated lactic acid fermentation equal to the control. However, when solvent‐saturated medium with two consecutive unequal volumes (10 mL then 90 mL) was exposed to PCS, some degree of toxicity was observed. Furthermore, iso‐octane, tributylphosphate (TBP), and Span 80 were optimized for recovery as 91%, 5%, and 4% (v/v), respectively, with a 1:1 ratio of 1.2 M Na2CO3 stripping solution. Also, recovery by emulsion liquid extraction in the hollow‐fiber contactor was minimal due to low recovery at pH 5.0 and incompatibility of the solvent and hollow‐fiber material. These results suggest that PCS biofilm reactors can benefit lactic acid fermentation by eliminating the toxic effect from solvent leakage into the fermentation medium from liquid–liquid extractive integrated fermentations. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 749–759, 2003.
Three‐dimensional numerical approach to investigate the substrate transport and conversion in an immobilized enzyme reactor
Authors: Esterl Stefan , Özmutlu Özlem , Hartmann Christoph , Delgado Antonio
Published: July 2003

This numerical study evaluates the momentum and mass transfer in an immobilized enzyme reactor. The simulation is based on the solution of the three‐dimensional Navier‐Stokes equation and a scalar transport equation with a sink term for the transport and the conversion of substrate to product. The reactor consists of a container filled with 20 spherical enzyme carriers. Each of these carriers is covered with an active enzyme layer where the conversion takes place. To account for the biochemical activity, the sink term in the scalar transport equation is represented by a standard Michaelis‐Menten approach. The simulation gives detailed information of the local substrate and product concentrations with respect to external and internal transport limitations. A major focus is set on the influence of the substrate transport velocity on the catalytic process. For reactor performance analysis the overall and the local transport processes are described by a complete set of dimensionless variables. The interaction between substrate concentration, velocity, and efficiency of the process can be studied with the help of these variables. The effect of different substrate inflow concentrations on the process can be seen in relation to velocity variations. The flow field characterization of the system makes it possible to understand fluid mechanical properties and its importance to transport processes. The distribution of fluid motion through the void volume has different properties in different parts of the reactor. This phenomenon has strong effects on the arrangement of significantly different mass transport areas as well as on process effectiveness. With the given data it is also possible to detect zones of high, low, and latent enzymatic activity and to determine whether the conversion is limited due to mass transfer or reaction resistances. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 780–789, 2003.
Modeling chromate reduction in Shewanella oneidensis MR‐1: Development of a novel dual‐enzyme kinetic model
Authors: Viamajala Sridhar , Peyton Brent M. , Petersen James N.
Published: July 2003

Chromate (Cr(VI)) reduction tests were performed with nitrate‐ and fumarate‐grown stationary phase cultures of Shewanella oneidensis MR‐1 (henceforth referred to as MR‐1) and disappearance of Cr(VI) was monitored over time. A rapid initial decrease in Cr(VI) concentration was observed, which was followed by a slower, steady decrease. These observations appear to be consistent with our previous results indicating that Cr(VI) reduction in MR‐1 involves at least two mechanisms (Viamajala et al., 2002b). Modeling of metal reduction kinetics is often based on single‐enzyme Michaelis‐Menten equations. However, these models are often developed using initial rates and do not always match actual reduction profiles. Based on the hypothesis that multiple Cr(VI) reduction mechanisms exist in MR‐1, a model was developed to describe the kinetics of Cr(VI) reduction by two parallel mechanisms: (1) a rapid Cr(VI) reduction mechanism that was deactivated (or depleted) quickly, and (2) a slower mechanism that had a constant activity and was sustainable for a longer duration. Kinetic parameters were estimated by fitting experimental data, and model fits were found to correspond very closely to quantitative observations of Cr(VI) reduction by MR‐1. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 790–797, 2003.
Cometabolism of Cr(VI) by Shewanella oneidensis MR‐1 produces cell‐associated reduced chromium and inhibits growth
Authors: Middleton Sarah S. , Latmani Rizlan Bencheikh , Mackey Mason R. , Ellisman Mark H. , Tebo Bradley M. , Criddle Craig S.
Published: July 2003

Microbial reduction is a promising strategy for chromium remediation, but the effects of competing electron acceptors are still poorly understood. We investigated chromate (Cr(VI)) reduction in batch cultures of Shewanella oneidensis MR‐1 under aerobic and denitrifying conditions and in the absence of an additional electron acceptor. Growth and Cr(VI) removal patterns suggested a cometabolic reduction; in the absence of nitrate or oxygen, MR‐1 reduced Cr(VI), but without any increase in viable cell counts and rates gradually decreased when cells were respiked. Only a small fraction (1.6%) of the electrons from lactate were transferred to Cr(VI). The 48‐h transformation capacity (Tc) was 0.78 mg (15 μmoles) Cr(VI) reduced · [mg protein]−1 for high levels of Cr(VI) added as a single spike. For low levels of Cr(VI) added sequentially, Tc increased to 3.33 mg (64 μmoles) Cr(VI) reduced · [mg protein]−1, indicating that it is limited by toxicity at higher concentrations. During denitrification and aerobic growth, MR‐1 reduced Cr(VI), with much faster rates under denitrifying conditions. Cr(VI) had no effect on nitrate reduction at 6 μM, was strongly inhibitory at 45 μM, and stopped nitrate reduction above 200 μM. Cr(VI) had no effect on aerobic growth at 60 μM, but severely inhibited growth above 150 μM. A factor that likely plays a role in Cr(VI) toxicity is intracellular reduced chromium. Transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS) of denitrifying cells exposed to Cr(VI) showed reduced chromium precipitates both extracellularly on the cell surface and, for the first time, as electron‐dense round globules inside cells. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 627–637, 2003.
Elevated temperature effects on the oxidant/antioxidant balance in submerged batch cultures of the filamentous fungus Aspergillus niger B1‐D
Authors: Bai Zhonghu , Harvey Linda M. , McNeil Brian
Published: July 2003

In the present study the relationship between oxidative stress and elevated culture temperature was examined in an industrially relevant fungal culture, Aspergillus niger B1‐D. For the first time, both the intracellular levels of the main stressor species (superoxide radical [O2.−]) and activities of cellular defensive enzymes (superoxide dismutase [SOD], catalase [CAT], and glutathione peroxide [GPx]) were quantified at varying temperature (25, 30, 35, 40°C) to more fully characterize culture response in different growth phases. Elevated culture temperature led to increased O2.− levels in various culture phases. In the exponential phase this was due to an enhanced generation of O2.−, whereas in stationary phase a decreased dismutation rate may also have contributed. CAT activities generally increased with culture temperature, whereas GPx activity changed little as temperature rose, indicating that GPx played only a minor role in destroying H2O2 in this A. niger. The combination of elevated temperature (35°C) and increased O2 supply (50% enrichment) led to decreased levels of O2.− compared to the cultivation at 35°C gassed with air, probably due to enhanced activity of the alternative fungal respiratory pathway. Our findings indicate that while elevated cultivation temperature does clearly induce oxidative stress events, mechanistically, it does so by a rather more complex route than previous studies indicate. Elevated temperature caused a marked disparity in the activities of SOD and CAT, very distinct from the integrated increase in activity of these enzymes in response to oxidative stress. © 2003 Wiley Periodicals. Biotechnol Bioeng 83: 772–779, 2003.
Clonal expansion of adult rat hepatic stem cell lines by suppression of asymmetric cell kinetics (SACK)
Authors: Lee Hsuan‐Shu , Crane Gracy G. , Merok Joshua R. , Tunstead James R. , Hatch Nicole L. , Panchalingam Krishnanchali , Powers Mark J. , Griffith Linda G. , Sherley James L.
Published: July 2003

Adult stem cells have potential use for several biomedical applications, including cell replacement therapy, gene therapy, and tissue engineering. However, such applications have been limited due to difficulties encountered in expanding functional adult stem cells. We have developed a new approach to the problem of adult stem cell expansion based on the suppression of asymmetric cell kinetics (SACK). We postulated that asymmetric cell kinetics, required for adult stem cell function, were a major barrier to their expansion in culture. As such, conversion of adult stem cells from asymmetric cell kinetics to symmetric cell kinetics would promote their exponential expansion and longterm propagation in culture. The purine nucleoside xanthosine (Xs), which promotes guanine ribonucleotide biosynthesis, can be used to reversibly convert cells from asymmetric cell kinetics to symmetric cell kinetics. We used Xs supplementation to derive clonal epithelial cell lines from adult rat liver that have properties of adult hepatic stem cells. The properties of two Xs‐derived cell lines, Lig‐8 and Lig‐13, are described in detail and compared to properties of adult rat hepatic cell lines derived without Xs supplementation. The Xs‐derived cell lines exhibit Xs‐dependent asymmetric cell kinetics and Xs‐dependent expression of mature hepatic differentiation markers. Interestingly, Lig‐8 cells produce progeny with properties consistent with hepatocyte differentiation, while Lig‐13 progeny cells have properties consistent with bile duct epithelium differentiation. A stable adult cholangiocyte stem cell line has not been previously described. Consistent with the principles of their derivation, the SACK‐derived hepatic cell lines exhibit neither senescence nor tumorigenic properties, and their differentiation properties are stable after longterm culture. These characteristics of SACK‐derived stem cell lines underscore asymmetric cell kinetics as an essential adult stem cell property with potential to be the basis for a general approach to expansion and propagation of diverse adult stem cells. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 760–771, 2003.
Acetohydroxyacid synthase: A new enzyme for chiral synthesis of R‐phenylacetylcarbinol
Authors: Engel Stanislav , Vyazmensky Maria , Geresh Shimona , Barak Ze'ev , Chipman David M.
Published: July 2003

We have found that acetohydroxyacid synthase (AHAS) is an efficient catalyst for the enantiospecific (≥98% enantiomeric excess) synthesis of (R)‐phenylacetylcarbinol (R‐PAC) from pyruvate and benzaldehyde, despite the fact that its normal physiological role is synthesis of (S)‐acetohydroxyacids from pyruvate and a second ketoacid. (R)‐phenylacetylcarbinol is the precursor of important drugs having α and β adrenergic properties, such as L‐ephedrine, pseudoephedrine, and norephedrin. It is currently produced by whole‐cell fermentations, but the use of the isolated enzyme pyruvate decarboxylase (PDC) for this purpose is the subject of active research and development efforts. Some of the AHAS isozymes of Escherichia coli have important advantages compared to PDC, including negligible acetaldehyde formation and high conversion of substrates (both pyruvate and benzaldehyde) to PAC. Acetohydroxyacid synthase isozyme I is particularly efficient. The reaction is not limited to condensation of pyruvate with benzaldehyde and other aromatic aldehydes may be used. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 833–840, 2003.
Substrate inhibition kinetics for toluene and benzene degrading pure cultures and a method for collection and analysis of respirometric data for strongly inhibited cultures
Authors: Alagappan Gunaseelan , Cowan Robert
Published: July 2003

We present an evaluation of the qualitative and quantitative effects that high concentrations of benzene and toluene have on the growth rate of several pure cultures that use these compounds as their sole carbon and energy source. The cultures employed were five widely studied environmental isolates: Pseudomonas putida F1, P. putida mt2, P. mendocina KR, Ralstonia pickettii PKO1, and Burkholderia cepacia G4. Three cultures degraded toluene following a pattern consistent with the kinetic model of Wayman and Tseng (1976) while the other two followed a modification of this model introduced by Alagappan and Cowan (2001). The pattern followed for benzene degradation was different than that for toluene degradation for all four capable pure cultures and consistent with that described by the model of Luong (1987). Mechanisms of substrate inhibition and solvent toxicity are discussed, used to conceptually evaluate the reasons for the differences in inhibition behavior, and used to support a call for more widespread use of the empirical, terminal substrate concentration inhibition models employed here. We also present the methodology developed to overcome a limitation commonly encountered when attempting to collect oxygen uptake data for use in quantifying substrate inhibition kinetics. The experimental method was effective for use in the collection of high quality data and the substrate inhibition models most useful in representing the growth of bacteria on these solvents are those that show a complete loss of activity at high concentration rather than the more popular asymptotic inhibition models. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 798–809, 2003.
Evaluation of effective diffusion coefficient and intrinsic kinetic parameters on azo dye biodegradation using PVA‐immobilized cell beads
Authors: Chen Kuo‐Cheng , Wu Jane‐Yii , Yang Wen‐Bin , Hwang Sz‐Chwun John
Published: July 2003

An immobilized mixed culture (Aeromonas hydrophila, Comamonas testosteroni, and Acinetobacter baumannii) was prepared by entrapment into phosphorylated polyvinyl alcohol (PVA) gel beads. The unsteady‐state diffusion mechanism in a gel bead was applied to estimate the effective diffusion coefficients (De) and the partition coefficients (Kp) of azo dye. In addition, a simple method was developed to determine the intrinsic kinetic parameters of immobilized cells from observed reaction rates and the intrinsic kinetic parameters were then verified by fitting the experimental data into the reaction‐diffusion model in a batch reactor running at a well‐stirred state. The calculated effectiveness factor (ηcal) approached unity at Thiele modulus (Φ) < 0.3 (i.e., dp < 0.475 mm). The experimental effectiveness factor (ηexp) was in the range of 0.71–0.45 for a corresponding sphere diameter (dp) of 1.91 ± 0.16 to 4.43 ± 0.07 mm at an initial dye concentration of 200 mg/L. The results show that intraparticle diffusion resistance has a significant effect on the azo dye biodegradation rate. © 2003 Wiley Periodicals. Biotechnol Bioeng 83: 821–832, 2003.
Artificial regulatory networks and cascades for discrete multilevel transgene control in mammalian cells
Authors: Kramer Beat P. , Weber Wilfried , Fussenegger Martin
Published: July 2003

Prototype drug‐adjustable heterologous transcription control systems designed for gene therapy applications typically show sigmoid dose–response characteristics and enable fine‐tuning of therapeutic transgenes only within a narrow inducer concentration range of a few nanograms. However, the design of clinical dosing regimes which achieve tissue‐specific concentrations with nanogram precision is yet a “mission impossible.” Therefore, most of today's transcription control systems operate as ON/OFF switches and not in a true adjustable mode. The availability of robust transcription control configurations which lock expression of a single therapeutic transgene at desired levels in response to fixed clinical doses of different inducers rather than minute concentration changes of a single inducer would be highly desirable. Based on in silico predictions, we have constructed a variety of mammalian artificial regulatory networks by interconnecting the tetracycline‐ (TETOFF), streptogramin‐ (PIPOFF), and macrolide‐ (EOFF) repressible gene regulation systems as linear (auto)regulatory cascades. These networks enable multilevel expression control of several transgenes in response to different antibiotics or allow titration of a single transgene to four discrete expression levels by clinical dosing of a single antibiotic: 1) high expression in the absence of any antibiotic (+++), 2) medium level expression following addition of tetracycline (++), 3) low level expression in response to the macrolide erythromycin (+), and 4) complete repression by streptogramins such as pristinamycin (–). The first‐generation artificial regulatory networks exemplify modular interconnections of different heterologous gene regulations systems to achieve multigene expression, fine‐tuning, or to design novel control networks with unprecedented transgene regulation properties. Such higher‐level transcription control modalities will lead the way towards composite artificial regulatory networks able to effect complex therapeutic interventions in future gene therapy and tissue engineering scenarios. © 2003 Wiley Periodicals. Biotechnol Bioeng 83: 810–820, 2003.
Comparisons of oxidative stress response genes in aerobic Escherichia coli fermentations
Authors: Lu Canghai , Bentley William E. , Rao Govind
Published: July 2003

The promoter regions of five SoxRS regulon genes (sodA, fumC, zwf, acnA, and acrAB) and one SoxRS regulatory protein gene (soxS) were inserted upstream of the gene of green fluorescent protein (GFP) in pGlow‐TOPO. These promoter probe plasmids were transformed into Escherichia coli Top10 resulting in six strains that produce GFP in response to superoxide‐induced stresses. Initial characterization from paraquat insults revealed significant induction of all six genes, with sodA, fumC, zwf, and soxS leading to the others in time and strength. These stress probe strains were then grown under similar conditions in fermentors and systematically exposed to varying durations of pure oxygen. Significant stimulation of the regulon was observed and quantitatively and temporally characterized by online monitoring of GFP fluorescence production (with transcriptional rate sodA > fumC > soxS, zwf > acnA, acrAB = 0). Interestingly, SoxRS regulon response occurred in typical E. coli fermentations where DO is maintained approximately 30% with increased agitation speed (with transcriptional rate acnA > sodA > zwf > acrAB > soxS, fumC = 0). These results also suggest that different molecular responses occur under different aeration schemes, all of which are intended to combat oxidative damage. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 864–870, 2003.
In silico prediction and validation of the importance of the Entner–Doudoroff pathway in poly(3‐hydroxybutyrate) production by metabolically engineered Escherichia coli
Authors: Hong Soon Ho , Park Si Jae , Moon Soo Yun , Park Jong Pil , Lee Sang Yup
Published: July 2003

The metabolic network of Escherichia coli was constructed and was used to simulate the distribution of metabolic fluxes in wild‐type E. coli and recombinant E. coli producing poly(3‐hydroxybutyrate) [P(3HB)]. The flux of acetyl‐CoA into the tricarboxylic acid (TCA) cycle, which competes with the P(3HB) biosynthesis pathway, decreased significantly during P(3HB) production. It was notable to find from in silico analysis that the Entner–Doudoroff (ED) pathway flux increased significantly under P(3HB)‐accumulating conditions. To prove the role of ED pathway on P(3HB) production, a mutant E. coli strain, KEDA, which is defective in the activity of 2‐keto‐3‐deoxy‐6‐phosphogluconate aldolase (Eda), was examined as a host strain for the production of P(3HB) by transforming it with pJC4, a plasmid containing the Alcaligenes latus P(3HB) biosynthesis operon. The P(3HB) content obtained with KEDA (pJC4) was lower than that obtained with its parent strain KS272 (pJC4). The reduced P(3HB) biosynthetic capacity of KEDA (pJC4) could be restored by the co‐expression of the E. coli eda gene, which proves the important role of ED pathway on P(3HB) synthesis in recombinant E. coli as predicted by metabolic flux analysis. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 854–863, 2003.
Down‐regulation of acetate pathway through antisense strategy in Escherichia coli: Improved foreign protein production
Authors: Kim Jaoon Y. H. , Cha Hyung Joon
Published: July 2003

A problem with the use of Escherichia coli to produce foreign proteins is that although endogenously produced acetate is physiologically indispensable, it inhibits protein expression. Here we firstly employed an antisense RNA strategy as an elaborate metabolic engineering tool to partially block biosynthesis of two major acetate pathway enzymes, phosphotransacetylase (PTA) and acetate kinase (ACK). Three recombinant plasmids containing antisense genes targeting either or both of pta and ackA were constructed, and their effects on the acetate pathway and foreign protein productivity compared to control plasmid without any antisense genes were determined in E. coli BL21. Green fluorescent protein (GFP) was employed as a model foreign protein, and timing of antisense expression was controlled by using the intrinsic ackA promoter. We found that the antisense method partially reduced mRNA levels of target enzyme genes and, over time, lowered the concentration of acetate in culture media in all antisense‐regulated strains. Notably, total production of GFP was enhanced 1.6‐ to 2.1‐fold in antisense‐regulated strains, even though the degree of acetate reduction was not significantly large. It was revealed that the acetate pathway has more critical roles in cellular physiology than expected in the previous reports. When the scale of culture was increased, enhancement of protein production became larger, demonstrating that this antisense strategy can be successfully applied to practical large‐scale protein production processes. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 841–853, 2003.
Coronary stenting in patients with depressed left ventricular function: Acute and long‐term results in a selected population
Authors: Di Sciascio Germano , Patti Giuseppe , D'Ambrosio Andrea , Nusca Annunziata
Published: July 2003

Percutaneous coronary angioplasty (PTCA) in patients with depressed left ventricular ejection fraction (LVEF) is associated with increased acute and late mortality; in contrast to plain PTCA, results of stenting in these patients have not been characterized. To assess the current outcome of stenting in patients with LV dysfunction, results from 80 patients procedures were analyzed. Intervention for acute myocardial infarction (MI) was excluded; 21% of patients had unstable angina and 30% had a recent MI. Mean LVEF was 40 ± 9% (range, 25–45%). Multivessel revascularization was done in 25 patients (31%), with a total of 114 lesions treated. Prophylactic intra‐aortic balloon pump was used in only two patients. Angiographic and clinical success was achieved in 79/80 patients (99%). There were no in‐hospital deaths, one patient (1%) had a non‐Q‐wave MI, and no patients required emergency bypass surgery (CABG). All patients completed at least 6 months follow‐up (mean, 30 ± 14 months): 64 patients (80%) remained asymptomatic, 4 (5%) had acute MI, and 5 (6%) died. In‐stent restenosis occurred in five patients (6%); of these, three required repeat PTCA, three patients (4%) underwent subsequent elective CABG. Including patients with repeat intervention, 67 patients (84%) are clinically improved; actuarial event‐free survival was 87% at 56‐month follow‐up. Thus, stenting in patients with impaired LVEF is associated with excellent outcome and lower mortality than previously reported for balloon angioplasty alone. Whether coronary stenting may be a therapeutic strategy equivalent to surgery in selected patients needs to be investigated in prospective randomized trials. Cathet Cardiovasc Intervent 2003;59:429–433. © 2003 Wiley‐Liss, Inc.
Percutaneous intervention for unprotected left main disease prior to explantation of a left ventricular assist device
Authors: Butman Samuel M. , Jamison Karen , Slepian Marvin , Edling Nancy , Arabia Francisco , Copeland Jack G.
Published: July 2003

Percutaneous coronary intervention of unprotected left main coronary arterial disease is an alternative to surgical revascularization in selected patients. In this report, a patient with an implanted left ventricular assist device (LVAD) underwent successful coronary intervention prior to its planned removal. The implanted LVAD clearly assisted the technical performance of the intervention. Cathet Cardiovasc Intervent 2003;59:471–474. © 2003 Wiley‐Liss, Inc.
Aortic dissection after stent dilatation for coarctation of the aorta: A case report and literature review
Authors: Varma C. , Benson L.N. , Butany J. , McLaughlin Peter R.
Published: July 2003

A case of stenting for native coarctation is described in a 65‐year‐old female with a fatal dissection after implantation. The histology of the aorta in coarctation and in the elderly is described. The experience of stenting in older patients is reviewed and discussed. Cathet Cardiovasc Intervent 2003;59:528–535. © 2003 Wiley‐Liss, Inc.
Conflicting functional assessment of stenoses in patients with previous myocardial infarction
Authors: Leesar Massoud A. , Abdul‐Baki Talal , Yalamanchili Vankata , Hakim John , Kern Morton
Published: July 2003

The utility of fractional flow reserve, absolute and relative flow reserve, and intravascular ultrasound may have an impact on decision‐making for percutaneous coronary intervention in patients with previous myocardial infarction and microvascular dysfunction. The role for fractional flow reserve, absolute and relative flow reserve, and intravascular ultrasound is discussed. Cathet Cardiovasc Intervent 2003;59:489–495. © 2003 Wiley‐Liss, Inc.
New technique for superior guiding catheter support during advancement of a balloon in coronary angioplasty: The anchor technique
Authors: Fujita Shinya , Tamai Hideo , Kyo Eisho , Kosuga Kunihiko , Hata Tatsuhiko , Okada Masaharu , Nakamura Takuji , Tsuji Takafumi , Takeda Shinsaku , bin Hu Fang , Masunaga Nobutoyo , Motohara Seiichiro , Uehata Hiromu
Published: July 2003

To get superior guiding catheter support, we tried a new method called the anchor technique. By inflating a balloon in a nontarget vessel and holding its shaft with backward force while advancing another balloon, the anchor effect for the guiding catheter could be obtained and it appeared to be helpful for a balloon or a stent to cross the target lesion. Cathet Cardiovasc Intervent 2003;59:482–488. © 2003 Wiley‐Liss, Inc.
Does gender affect the clinical outcome of patients with acute myocardial infarction complicated by cardiogenic shock who undergo percutaneous coronary intervention?
Authors: Antoniucci David , Migliorini Angela , Moschi Guia , Valenti Renato , Trapani Maurizio , Parodi Guido , Bolognese Leonardo , Santoro Giovanni M.
Published: July 2003

This study sought to determine the impact of female gender on clinical outcome in patients with acute myocardial infarction (AMI) complicated by cardiogenic shock (CS) due to predominant ventricular failure undergoing percutaneous coronary intervention (PCI). We analyzed gender‐related differences in procedural, angiographic, and clinical outcomes in 208 consecutive patients with AMI complicated by CS. Out of 208 patients with CS, 65 were women and 143 men. Women were older than men (74 ± 10 years vs. 66 ± 12 years; P < 0.001) and had a greater incidence of a history of hypertension (43% vs. 29%; P = 0.041). The 6‐month mortality rate was 42% in women and 31% in men (P = 0.157). There were no differences between groups in reinfarction rate and target vessel revascularization rate. Multivariate analysis showed age as the only variable independently related to the 6‐month mortality, while female gender was not related to the risk of death. The benefit of early PCI is similar in women and men, and any potential referral bias in the use of PCI based on gender differences should be avoided. Cathet Cardiovasc Intervent 2003;59:423–428. © 2003 Wiley‐Liss, Inc.
Percutaneous stenting of anastomotic stenoses in tubular interposition grafts used to repair aortic coarctation in adults
Authors: Mustapha Jihad , Sandhu Satinder K. , Khuri Bahij N. , Glancy D. Luke
Published: July 2003

Stenting is the usual treatment for recoarctation following resection with direct end‐to‐end anastomosis. We describe for the first time stenting to relieve anastomotic stenoses in tubular interposition grafts, which are used to repair approximately 10% of coarctations. Success in these two adults expands further the spectrum of large conduits that may be relieved of stenosis by stenting. Cathet Cardiovasc Intervent 2003;59:544–546. © 2003 Wiley‐Liss, Inc.
Transcatheter closure of very large (≥ 25 mm) atrial septal defects using the Amplatzer septal occluder
Authors: Kannan Bhava Ramalingam Jawahar , Francis Edwin , Sivakumar Kothandam , Anil Sivadasan Radha , Kumar Raman Krishna
Published: July 2003

Between June 1999 and September 2002, 45 patients (age, 34 ± 13 years; mean shunt ratio, 2.6 ± 0.6) underwent transcatheter atrial septal defect (ASD) closure at our institution with the Amplatzer septal occluder (mean device size, 31.4 ± 3 mm). Patients were selected by transesophageal echocardiography. The mean ASD dimension was 25.3 ± 3.7 mm and 33 (73%) patients had deficient anterior rim. Specific procedural details included the use of 13 or 14 Fr introducer sheaths and the right upper pulmonary vein approach if the conventional approach failed. There were two procedural failures, with device embolization in both (surgical retrieval in one, catheter retrieval in one). During follow‐up (3–30 months; median, 16 months), one patient (59 years) with previous atrial flutter had pulmonary embolism and was managed with anticoagulation. Two patients developed symptomatic atrial flutter. Fluoroscopy time was 31.6 ± 19.5 min for the first 22 cases and 19.6 ± 11.4 min for the rest (P = 0.04). Transcatheter closure of large ASDs is technically feasible but careful long‐term follow‐up is needed to document its safety. Cathet Cardiovasc Intervent 2003;59:522–527. © 2003 Wiley‐Liss, Inc.
Platelet glycoprotein IIb/IIIa inhibition with eptifibatide: Prolongation of inhibition of aggregation in acute renal failure and reversal with hemodialysis
Authors: Sperling Robert T. , Pinto Duane S. , Ho Kalon K.L. , Carrozza Joseph P.
Published: July 2003

We report the time course to the restoration of normal platelet aggregation in three patients who were treated with eptifibatide in the setting of severe renal failure. There was prolonged platelet inhibition in the first patient who developed acute renal failure. The second patient with acute on chronic renal failure developed an intracerebral hemorrhage. Normal platelet aggregation was restored after acute hemodialysis. In a third patient with end‐stage renal disease, platelet function returned to the normal range following hemodialysis. There is a prolonged time course to the restoration of normal platelet aggregation in patients with severe renal dysfunction who are treated with eptifibatide. Acute renal hemodialysis may reverse the inhibitory effect of eptifibatide on platelet aggregation in these patients. Cathet Cardiovasc Intervent 2003;59:459–462. © 2003 Wiley‐Liss, Inc.
Preliminary experience with a new 18 mm Amplatzer PFO occluder for small persistent foramen ovale
Authors: Ewert Peter , Kretschmar Oliver , Peters Bjoern , Nuernberg Jan H. , Khaliq Hashim Abdul , Nagdyman Nicole , Lange Peter E.
Published: July 2003

We report our initial experience with a new 18 mm Amplatzer PFO device for the closure of small PFO. Patients were selected by a semiquantitative sizing procedure using the circle of a diagnostic pigtail catheter instead of a sizing balloon. In all eight patients, the device was easy to implant without problems. Due to its size, it has the potential to minimize the risk of inadvertent atrial erosion or perforation after deployment and thus will enhance the safety of interventional PFO closure. Cathet Cardiovasc Intervent 2003;59:518–521. © 2003 Wiley‐Liss, Inc.
Direct stenting with a combined intravascular ultrasound‐coronary stent delivery platform: A feasibility trial
Authors: Eeckhout Eric , Berger Alexandre , Roguelov Christan , Lyon Xavier , Imsand Christophe , Fivaz‐Arbane Malika , Girod Grégoire , De Benedetti Edoardo
Published: July 2003

IVUS is considered as the most accurate tool for the assessment of optimal stent deployment. Direct stenting has shown to be a safe, efficient, and resource‐saving procedure in selected patients. In a prospective 1‐month feasibility trial, a new combined IVUS‐coronary stent delivery platform (Josonics Flex, Jomed, Helsingborn, Sweden) was evaluated during direct stenting in consecutive patients considered eligible for direct stenting. The feasibility endpoint was successful stent deployment without any clinical adverse event, while the efficacy endpoint was strategic adaptation according to standard IVUS criteria for optimal stent deployment at the intermediate phase (after a result considered angiographically optimal) and at the end of the intervention (after optimization according to IVUS standards). A total of 16 patients were successfully treated with this device without any major clinical complication. At the intermediate phase, optimal stent deployment was achieved in four patients only, while at the end only one patient had nonoptimal IVUS stent deployment. In particular, the minimal in‐stent cross‐section area increased from 6.3 ± 1.2 to 8.3 ± 2.5 mm2. These preliminary data demonstrate the feasibility of direct stenting with a combined IVUS‐stent catheter in selected patients and confirm the results from larger randomized trials on the impact of IVUS on strategic adaptations during coronary stent placement. Cathet Cardiovasc Intervent 2003;59:451–454. © 2003 Wiley‐Liss, Inc.
Clinical outcomes following stent implantation in internal mammary artery grafts
Authors: Sharma Arvind K. , McGlynn Scott , Apple Sue , Pinnow Ellen , Canos Daniel A. , Gevorkian Natalie , Tebeica Mihaela , Gruberg Luis , Pichard Augusto D. , Lindsay Joseph
Published: July 2003

We evaluated our experience with percutaneous coronary intervention (PCI) of internal mammary artery (IMA) grafts. From the institution's database we identified 288 patients with 311 IMA lesions. Of these, 82 (26.4%) had stents placed during PCI. Angiographic success was 92%. Mortality at 1 month was 1.7%, myocardial infarction (MI) 15.7%, and target lesion revascularization (TLR) 0.4%. Cumulative 1‐year event rates were mortality 6.4%, MI 20.4%, and TLR 8.0%. TLR rates were significantly higher in the stented lesions than lesions treated with angioplasty alone (19.2% vs. 4.9%; P = 0.004). The higher TLR rate in stented lesions was most apparent at the anastomotic site (25.0% vs. 4.2%; P = 0.006). Percutaneous revascularization of IMA grafts can be performed safely with high procedural success and excellent short‐ and long‐term results. Stenting, particularly at the anastomotic site, was associated with significantly greater rates of TLR than angioplasty alone. Cathet Cardiovasc Intervent 2003;59:436–441. © 2003 Wiley‐Liss, Inc.