bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular and pharmacological properties of insect biogenic amine receptors: Lessons from Drosophila melanogaster and Apis mellifera
Authors: Blenau Wolfgang , Baumann Arnd
Published: August 2002

In the central nervous system (CNS) of both vertebrates and invertebrates, biogenic amines are important neuroactive molecules. Physiologically, they can act as neurotransmitters, neuromodulators, or neurohormones. Biogenic amines control and regulate various vital functions including circadian rhythms, endocrine secretion, cardiovascular control, emotions, as well as learning and memory. In insects, amines like dopamine, tyramine, octopamine, serotonin, and histamine exert their effects by binding to specific membrane proteins that primarily belong to the superfamily of G�protein–coupled receptors. Especially in Drosophila melanogaster and Apis mellifera considerable progress has been achieved during the last few years towards the understanding of the functional role of these receptors and their intracellular signaling systems. In this review, the present knowledge on the biochemical, molecular, and pharmacological properties of biogenic amine receptors from Drosophila and Apis will be summarized. Arch. Insect Biochem. Physiol. 48:13–38, 2001. © 2001 Wiley‐Liss, Inc.
Intracellular freezing, viability, and composition of fat body cells from freeze‐intolerant larvae of Sarcophaga crassipalpis
Authors: Davis Diana J. , Lee Richard E.
Published: August 2002

Although it is often assumed that survival of freezing requires that ice formation must be restricted to extracellular compartments, fat body cells from freeze‐tolerant larvae of the gall fly, Eurosta solidaginis (Diptera, Tephritidae) survive intracellular freezing. Furthermore, these cells are highly susceptible to inoculative freezing by external ice, undergo extensive lipid coalescence upon thawing, and survive freezing better when glycerol is added to the suspension medium. To determine whether these traits are required for intracellular freeze tolerance or whether they are incidental and possessed by fat body cells in general, we investigated the capacity of fat body cells from nondiapause‐destined and diapause‐destined (i.e., cold‐hardy) larvae of the freeze‐intolerant flesh fly Sarcophaga crassipalpis (Diptera, Sarcophagidae) to survive intracellular freezing. Fat body cells from both types of larvae were highly susceptible to inoculative freezing; all cells froze between –3.7 to –6.2°C. The highest rates for survival of intracellular freezing occurred at –5°C. The addition of glycerol to the media markedly increased survival rates. Upon thawing, the fat body cells showed little or no lipid coalescence. Fat body cells from E. solidaginis had a water content of only 35% compared to cells from S. crassipalpis larvae that had 52–55%; cells with less water may be less likely to be damaged by mechanical forces during intracellular freezing. Arch. Insect Biochem. Physiol. 48:199–205, 2001. © 2001 Wiley‐Liss, Inc.
Role of initial NSAID choice and patient risk factors in the prevention of NSAID gastropathy: A decision analysis
Authors: Fendrick A. Mark , Bandekar Rajesh R. , Chernew Michael E. , Scheiman James M.
Published: August 2002

Objective

To investigate the role of initial nonsteroidal antiinflammatory drug (NSAID) choice in the prevention of NSAID gastropathy, based on relative clinical and economic effects.

Methods

To mimic clinical practice, a symptom‐driven decision analytic model was constructed to compare 2 treatment strategies for long‐term users of NSAIDs over a 1‐year period: Strategy 1—generic NSAID used initially, and safer, more expensive NSAID reserved for treatment failures due to symptomatic gastropathy; and Strategy 2—safer, more expensive NSAID used in all instances. The only distinction between the strategies was the choice of initial NSAID. NSAIDs differed in gastrointestinal safety profiles and acquisition costs. The use and impact of antisecretory medications were included in the model. Because published data on patients' ulcer risk and relative NSAID safety show considerable variability, sensitivity analyses were used to evaluate the key clinical outcomes and costs.

Result

For patients without risk factors for NSAID ulcers (average risk), the model estimated that the strategy restricting use of the safer NSAID resulted in more symptomatic ulcers (Strategy 1, 2.58; Strategy 2, 0.73) and ulcer‐related complications (Strategy 1, 1.18; Strategy 2, 0.23) per 100 patient years. The restricted strategy led to a significantly lower cost per patient treated (Strategy 1, $239; Strategy 2, $831 per year). In the principal analysis, the incremental costs to prevent symptomatic and complicated ulcers were $31,900 and $56,700, respectively. The estimated incremental cost per ulcer avoided was sensitive to the relative protection provided by the safer NSAID and fell dramatically as the patients' ulcer risk was increased above average risk.

Conclusion

Unrestricted use of NSAIDs that reduce the risk of symptomatic ulcers has the potential to produce important clinical benefits at incremental cost. The impressive impact of ulcer risk on the incremental cost per ulcer prevented warrants increased attention to risk factor identification when NSAIDs are prescribed.
Design and quality considerations for randomized controlled trials in systemic sclerosis
Authors: Kyriakidi Maria , Ioannidis John P. A.
Published: August 2002

Objective

To appraise systematically randomized controlled trials (RCTs) on systemic sclerosis (SSc) in order to determine whether the parameter of study design and its quality may influence the reporting of efficacy for tested interventions.

Methods

Seventy RCTs were analyzed (1965–2000) in terms of design, patient characteristics, outcomes, and reported results.

Results

Median sample size was 28 patients. Fifty‐nine trials were double blind, but only 16 mentioned the randomization mode and only 7 described allocation concealment. There was sufficient information on withdrawals in 37 trials. Larger trials with longer followup scored higher on quality characteristics, but had higher withdrawal rates. Only 8 trials had a followup of more than 1 year. Significant efficacy was less likely to be reported in double‐blind studies (P = 0.029) and in studies with larger rates of withdrawal (P = 0.032). Specification of the following parameters improved over time: power calculations (P = 0.0003), outcomes (P = 0.001), and sample size per arm (P = 0.011).

Conclusions

Several aspects of the quality of design and conduct of SSc RCTs can be improved. Adequately powered trials with longer followup and clear outcomes are needed.
Convex upper and lower bounds for present value functions
Authors: Vyncke D. , Goovaerts M. , Dhaene J.
Published: August 2002

In this paper we present an efficient methodology for approximating the distribution function of the net present value of a series of cash‐flows, when discounting is presented by a stochastic differential equation as in the Vasicek model and in the Ho–Lee model. Upper and lower bounds in convexity order are obtained. The high accuracy of the method is illustrated for cash‐flows for which no analytical results are available. Copyright © 2001 John Wiley & Sons, Ltd.
Sequential sampling schemes and other statistical techniques in quality of service business surveys
Authors: Bar‐Lev Shaul K. , Fuchs Camil
Published: August 2002

While there is a general consensus in the business community regarding the importance of quality service surveys in improving the company's business results, relatively little attention was devoted to the theoretical aspects of data analysis and modelling which can increase substantially the effectiveness of those surveys. Using the data from a real‐life project, we present here the developments and improvements achieved in the three stages that ultimately yield the conclusions from the survey: the analysis of the historical data, the design of the sampling method for the current survey and the development of specific indices for differentiating among the units of the financial institution. The statistical techniques fitted to historical data helped both in assessing the relative contributions of the various aspects of service, the overall perceived quality of service, as well as in providing the theoretical grounds for reducing the size of the questionnaire and thus increasing the reliability of the received replies. The sequential sampling scheme designed for this survey was particularly suitable to address the managements concerns. Finally, the classes of quality service based on the quality indices developed in the project contributed substantially to the ability of the management to rank and reward differentially the branches of the financial institution. Copyright © 2002 John Wiley & Sons, Ltd.
Paclitaxel pharmacodynamics: application of a mechanism‐based neutropenia model
Authors: Fetterly Gerald J. , Tamburlin Judith M. , Straubinger Robert M.
Published: August 2002

Antineoplastic agents exert adverse effects that impact both dose and scheduling of drug administration. Our objective was to develop a quantitative relationship between paclitaxel (taxol) exposure and pharmacodynamic endpoints, such as neutropenia or body weight loss. Paclitaxel in liposomes or Cremophor EL was administered to rats at doses of 20 or 40 mg/kg. Body weight and absolute neutrophil count were determined daily. The decrease in body weight was greater for paclitaxel in Cremophor EL than for liposomal paclitaxel, but hematological toxicity was similar. The hematological data was fit using a pharmacodynamic model to investigate the temporal delay between drug exposure and neutropenia. From the model, the lifespan of neutrophils (TN), of surviving precursor cells in bone marrow (TP), and a killing rate constant (K) were determined. The values of TN, TP, and K for liposomal paclitaxel were 95 h, 82 h, and 0.735 (μM h)−1, respectively, and for paclitaxel in Cremophor EL, 86 h, 78 h, and 0.475 (μM h)−1, respectively. Simulations of various doses indicated a dependency of the neutropenia time course on paclitaxel exposure. The entire time course of changes in neutrophil count is more informative than a single measurement if myelosuppression is prolonged and at a level associated with increased incidence of clinical adverse effects. Copyright © 2001 John Wiley & Sons, Ltd.
Long‐term exposure of male and female mice to 50 Hz magnetic field: Effects on fertility
Authors: Elbetieha Ahmed , AL‐Akhras Moh'd‐Ali , Darmani Homa
Published: August 2002

The effect of an extremely low frequency (ELF) magnetic field on the fertility of adult male and female Swiss mice was investigated. Adult male and female mice were exposed to a 50 Hz sinusoidal magnetic field at ≈,25 μT (rms) for 90 days before they were mated with unexposed counterparts. There were no exposure related effects on the fertility of male or female mice. The number of implantation sites, viable fetuses, and the total number of resorptions were not significantly affected in females impregnated by males exposed to the 50 Hz magnetic field as compared with the control group. The number of implantation sites, viable fetuses and the total number of resorptions in exposed females were also not statistically different from the control group. There were no significant effects on the weights of the testes, seminal vesicles, preputial gland or body weights of males exposed to 50 Hz magnetic field. Furthermore, body and uterine weights were not affected in females exposed to 50 Hz field; however, ovarian weight was significantly increased in females exposed to the same field. These results suggest that exposure of male and female mice to low frequency magnetic field had no adverse effects on fertility and reproduction in mice. Bioelectromagnetics 23:168–172, 2002. © 2002 Wiley‐Liss, Inc.
Human exposure to 2450 MHz CW energy at levels outside the IEEE C95.1 standard does not increase core temperature†
Authors: Adair Eleanor R. , Mylacraine Kevin S. , Cobb Brenda L.
Published: August 2002

Permission was received from the Brooks AFB Institutional Review Board and the AF Surgeon General's Office to exceed the peak power density (PD = 35 mW/cm2) we had previously studied during partial body exposure of human volunteers at 2450 MHz. Two additional peak PD were tested (50 and 70 mW/cm2). The higher of these PD (normalized peak local SAR = 15.4 W/kg) is well outside the IEEE C95.1 guidelines for partial body exposure, as is the estimated whole body SAR ≈ 1.0 W/kg. Seven volunteers (four males, three females) were tested at each PD in three ambient temperatures (Ta = 24, 28, and 31°C) under our standard protocol (30 min baseline, 45 min RF exposure, 10 min baseline). The thermophysiological data (esophageal and six skin temperatures, metabolic heat production, local sweat rate, and local skin blood flow) were combined with comparable data at PD = 0, 27, and 35 mW/cm2 from our 1999 study to generate response functions across PD. No change in esophageal temperature or metabolic heat production was recorded at any PD in any Ta. At PD = 70 mW/cm2, skin temperature on the upper back (irradiated directly) increased 4.0°C in Ta = 24°C, 2.6°C in Ta = 28°C, and 1.8°C in Ta = 31°C. These differences were primarily due to the increase in local sweat rate, which was greatest in Ta = 31°C. Also at PD = 70 mW/cm2, local skin blood flow on the back increased 65% over baseline levels in Ta = 31°C, but only 40% in Ta = 24°C. Although Ta becomes an important variable when RF exposure exceeds the C95.1 partial body exposure limits, vigorous heat loss responses of blood flow and sweating maintain thermal homeostasis efficiently. It is also clear that strong sensations of heat and thermal discomfort will motivate a timely retreat from a strong RF field, long before these physiological responses are exhausted. Bioelectromagnetics 22:429–439, 2001. Published 2001 Wiley‐Liss, Inc.
Empirical validation of SAR values predicted by FDTD modeling*†
Authors: Gajsek P. , Walters T.J. , Hurt W.D. , Ziriax J.M. , Nelson D.A. , Mason P.A.
Published: August 2002

Rapid increase in the use of numerical techniques to predict current density or specific absorption rate (SAR) in sophisticated three dimensional anatomical computer models of man and animals has resulted in the need to understand how numerical solutions of the complex electrodynamics equations match with empirical measurements. This aspect is particularly important because different numerical codes and computer models are used in research settings as a guide in designing clinical devices, telecommunication systems, and safety standards. To ensure compliance with safety guidelines during equipment design, manufacturing and maintenance, realistic and accurate models could be used as a bridge between empirical data and actual exposure conditions. Before these tools are transitioned into the hands of health safety officers and system designers, their accuracy and limitations must be verified under a variety of exposure conditions using available analytical and empirical dosimetry techniques. In this paper, empirical validation of SAR values predicted by finite difference time domain (FDTD) numerical code on sphere and rat is presented. The results of this study show a good agreement between empirical and theoretical methods and, thus, offer a relatively high confidence in SAR predictions obtained from digital anatomical models based on the FDTD numerical code. Bioelectromagnetics 23:37–48, 2002. © 2002 Wiley‐Liss, Inc.
Derangement of growth and differentiation control in oncogenesis
Authors: Corn Paul G. , El‐Deiry Wafik S.
Published: August 2002

Human neoplasms develop following the progressive accumulation of genetic and epigenetic alterations to oncogenes and tumor suppressor genes. These alterations confer a growth advantage to the cancer cell, leading to its clonal proliferation, invasion into surrounding tissues, and spread to distant organs. Genes that are altered in neoplasia affect three major biologic pathways that normally regulate cell growth and tissue homeostasis: the cell cycle, apoptosis, and differentiation. While each of these pathways can be defined by a unique set of molecular events, they are not biologically separate. Rather, they function more as an integrated molecular network, and perturbations in one pathway can have profound consequences on another. Insights into what distinguishes the regulation of growth and differentiation in a normal cell versus a cancer cell have led to the development of novel anticancer therapies. BioEssays 24:83–90, 2002. © 2002 John Wiley & Sons, Inc.
Embryonic origin of the eyes in teleost fish
Authors: Chuang Jui Chang , Raymond Pamela A.
Published: August 2002

The developmental history of the vertebrate eye begins at an early embryonic stage, with the formation of the body axes and induction of neural tissue. Several recent experimental embryological and genetic studies in teleost fish have produced new insights into the morphogenetic and molecular regulation of eye formation. Molecular signaling pathways and patterned expression of transcription factors implicated in eye determination are discussed, and the importance of morphogenetic cell movements is emphasized. BioEssays 24:519–529, 2002. © 2002 Wiley Periodicals, Inc.
An analysis of the selective effects of NCR with punishment targeting problem behavior associated with positive affect
Authors: Rush Karena S. , Crockett Jennifer L. , Hagopian Louis P.
Published: August 2002

Among individuals with developmental disabilities and behavioral problems, self‐injury and aggression are often associated with negative affect such as crying. In the current report, we present data on two children who displayed problem behaviors (screaming and self‐injury) that were often associated with positive affect. In contrast to their other problem behaviors that were socially mediated, these behaviors were maintained independent of social consequences. One concern about treating problem behavior associated with positive affect is that the treatment may produce generalized reductions in positive affect. In both cases presented in the current study, the reductive effects of a treatment targeting these behaviors were highly selective, producing decreases in screaming and self‐injury, while minimally affecting affect. Copyright © 2001 John Wiley & Sons, Ltd.
Fluorescence quenching properties and chemiluminescence responses of α‐ketothiols derivatized with o‐phthalaldehyde and primary amino compounds
Authors: Sano Akira , Nakamura Hiroshi
Published: August 2002

Fluorescence and chemiluminescence responses of thiols derivatized with o‐phthalaldehyde (OPA) and primary amines have been studied. Among thiols tested, those having an α‐ketothiol structure [R‐CH(SH)‐CO‐] were found to give extremely low fluorescence intensities when derivatized with OPA and L‐valine. The relative fluorescence intensities of α‐ketothiols, such as mercaptoacetic acid (MAA), 2‐mercaptopropionic acid (2‐MPA), mercaptosuccinic acid (MSA) and N‐(2‐mercaptopropionyl)glycine (MPG), were only below 3% of that of N‐acetyl‐L‐cysteine (NAC), having no α‐ketothiol structure. The addition of detergents such as sodium dodecylsulphate had no effect on the fluorescence and an aprotic solvent (dimethylsulphoxide) showed little effect, whereas these thiols chemiluminesced intensely when derivatized with OPA and N‐(4‐aminobutyl)‐N‐ethylisoluminol as a primary amine and successively detected by high‐performance liquid chromatography (HPLC) with postcolumn chemiluminescence reactions using hydrogen peroxide and haematin. The relative chemiluminescence intensities of MSA, 2‐MPA and MPG to NAC were 41.0%, 27.8% and 117%, respectively. MPG was estimated to be detectable at a concentration level of at least 10 fmol per injection. In conclusion, the weak fluorescence of the α‐ketothiols derivatized with OPA and primary amines is probably due to the low fluorescence quantum yields of the derivatives, and HPLC with a chemiluminescence detection system is useful for the sensitive detection of thiols. Copyright © 2001 John Wiley & Sons, Ltd.
Kinetics studies of ultraweak luminescence induced by ascorbic acid in Characeae cells and their structures
Authors: Jaśkowska A. , Borc R. , Milczarek I. , Dudziak A. , Śpiewla E.
Published: August 2002

Native non‐stressed Nitellopsis obtusa cells emit low‐intensity (10–104 photons s−1 cm−2) electromagnetic radiation within the visible range. The intensity of emission by these cells is several times higher than the intensity of the environmental medium. This luminescence appears as a long‐lived (observable not for hours but for a few days) spontaneous light emission, sensitized by chlorophyll. Cells exposed to ascorbic acid action emit ultraweak radiation, dependent on reagent concentrations. Ultraweak luminescence (UL) intensity increases with rise in concentration, reaching maximum for higher concentrations faster and also changing the kinetics of UL decay. Studies of UL kinetics for different substructures and fractions, as well as for intact cells, allow us to consider single‐ and double‐exponential functions as decay curves, which could suggest two ways of ascorbic acid action in the cells. In the spectral distribution, the bands of chlorophyll, dimoles of singlet oxygen and NADH, FMN and Q10, which are electron carriers of the respiration chain, can be taken into consideration. Ascorbic acid action shows an increase in the red part of spectrum, except 627 nm, and in these bands of emission, which can be associated with the electron carriers of the respiration chain and lipid peroxidation. Copyright © 2001 John Wiley & Sons, Ltd.
Properties of firefly luciferase immobilized through a biotin carboxyl carrier protein domain
Authors: Eu Jiyang , Andrade Joseph
Published: August 2002

A fusion protein, consisting of biotin carboxyl carrier protein (BCCP) domain from Escherichia coli and firefly luciferase (FL) from Photinus pyralis, was immobilized through the biotin–avidin interaction on 6% cross‐linked agarose beads. Several properties of the immobilized BCCP–FL were studied. Immobilized and free enzymes showed no significant difference in thermal stability; both retained at least 91% activity after incubation at 4°C and 25°C for 22 h. Incubation at 37°C for 22 h caused significant activity loss. KM and kcat values were determined for both free and immobilized enzymes. KM values were similar between free and immobilized enzymes; however, kcat of immobilized BCCP–FL was one‐third of the kcat of the free enzyme. 294 µmol/L Co‐enzyme A (CoA) and 44 mmol/L dithiothreitol (DTT) enhanced the total bioluminescence output. Triton X‐100, Tween 20, PEG 8,000, PVP 40,000 and PVP 360,000 did not enhance the bioluminescence reaction of immobilized BCCP–FL. Copyright © 2001 John Wiley & Sons, Ltd.
Homonuclear 1H‐NMR assignment and structural characterization of human immunodeficiency virus type 1 Tat Mal protein
Authors: Grégoire Catherine , Péloponèse Jean‐Marie , Esquieu Didier , Opi Sandrine , Campbell Grant , Solomiac Murielle , Lebrun Evelyne , Lebreton Jacques , Loret Erwann P.
Published: August 2002

The transacting transcriptional activator (Tat) is a viral protein essential for activation of the human immunodeficiency virus (HIV) genes, and it plays an important role in HIV induced immunodeficiency. We report the NMR structural characterization of the active Tat Mal variant that belongs to a highly virulent D‐subtype HIV type‐1 (HIV‐1) strain (Mal) found mainly in Africa. A full Tat Mal protein (87 residues) is synthesized. This synthetic protein is active in a transactivation assay with HeLa cells infected with the HIV long terminal repeated noncoding sequences of the HIV‐1 provirus (LTR) lac Z gene. Homonuclear 1H‐NMR spectra allows the sequential assignment of the Tat Mal spin systems. Simulating annealing generates 20 conformers with similar folding. The geometry of the mean structure is optimized with energy minimization to obtain a final structure. As the European variant (Tat Bru) the N‐terminal region of Tat Mal constitutes the core, and there is a hydrophobic pocket composed of the conserved Trp 11 interacting with several aromatic residues. The two functional regions of Tat (basic and the cysteine‐rich regions) are well exposed to the solvent. A short α‐helix is observed in region V adjacent to the basic region. This α helix induces local structural variations compared to the NMR structure of Tat Bru, and it brings the cysteine‐rich and basic regions closer. This study suggests that similar folding exists among Tat variants. © 2001 John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 62: 324–335, 2001
In vitro adaptation of a ligase ribozyme for activity under a low‐pH condition
Authors: Miyamoto Yoshitaka , Teramoto Naozumi , Imanishi Yukio , Ito Yoshihiro
Published: August 2002

A ligase ribozyme accelerating a ligation reaction with oligonucleotide under a low‐pH condition was selected by in vitro adaptation. A ribozyme active at pH 7 was randomly mutated, and the resultant RNA library was subjected to in vitro adaptation under a low‐pH reaction condition. At pH 4, the adapted RNAs reacted with the oligonucleotide substrates about 200 times faster than the original ribozyme. When the ribozyme was cloned and sequenced, 10 of the 30 clones sequenced had identical sequences. The differences in sequence from the original ribozyme were found at four positions in the middle region and at the 3′ end. A few sequential differences dominated the activity of the ribozyme under the extreme condition. The adapted ribozyme had one repeating sequence that was critical for the activity. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 590–596, 2001.
Acetate‐inducible protein overexpression from the glnAp2 promoter of Escherichia coli
Authors: Farmer William R. , Liao James C.
Published: August 2002

The Ntr regulon in Escherichia coli has previously been engineered to control the expression of a heterologous metabolic pathway. In this study, we reengineered the same system for protein production. In the absence of NRII (glnL gene product), we showed that glnAp2 can be an effective promoter for protein production that is inducible by exogenous acetate, but both the induction ratio and the range of modulation are low. To deal with this issue, we inactivated phosphotransacetylase (pta gene product), which disrupts the acetate pathway and denies the cell the ability to synthesize acetate. With this additional modification, gene expression from glnAp2 can be controlled by directly adding acetate into the growth medium. Using a lacZ reporter fusion, we found that glnAp2 induction was modulatable over a range of potassium acetate concentrations, and the induction/noninduction ratio increased to 77 in the absence of pta. The extracellular acetate required for maximal induction is lower than the concentration that causes toxicity, and thus growth inhibition by acetate addition was not a matter of concern. Furthermore, compared to the Ptac promoter, overexpression of a model protein using the modified glnAp2 promoter system did not cause significant growth inhibition, although a higher level of protein expression was achieved. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 504–509, 2001.
Automatic inducer addition and harvesting of recombinant Escherichia coli cultures based on indirect on‐line estimation of biomass concentration and specific growth rate
Authors: Eriksen Niels Thomas , Kratchmarova Irina , Neve Søren , Kristiansen Karsten , Iversen Jens Jørgen Lønsmann
Published: August 2002

This article describes a novel bioreactor configuration for production optimization of recombinant proteins in Escherichia coli. Inducer addition and harvesting are controlled on‐line based on indirect estimation of biomass concentration and specific growth rate from addition of NaOH to maintain constant pH. When either a predetermined biomass concentration is reached or the cultures have obtained, a constant specific growth rate inducer is introduced automatically. The induction period is ended by automatic harvesting of the cultures either at a predetermined biomass concentration or when substrate (in this study glucose) is depleted, detected as an increase of pH, or dissolved oxygen tension. During harvesting, metabolic activities are quenched within 3 min by cooling of the cell suspension. The system has been used to optimize expression of glutathione S‐transferase (GST) fusion protein of the ligand binding domain of mouse peroxisome proliferator‐activated receptor, GST‐PPARα LBD. Total yield of GST‐PPARα LBD was independent of the time of inducer addition as long as the length of induction period corresponded to at least 0.25 cell divisions while the yield of soluble GST‐PPARα LBD, the only active form, increased with the length of induction period. Highest yields were obtained when the inducer was added at low cell concentration as soon as constant specific growth rate was detected, resulting in induction periods corresponding to 3.4 ± 0.4 cell divisions. The specific growth rate remained almost constant for one cell division after inducer addition, whereafter it decreased. No decrease of specific growth rate was observed when inducer was added in the lag‐phase, and no soluble protein was produced. These results suggest that solely soluble GST‐PPARα LBD acts as a growth inhibitor and that GST‐PPARα LBD is expressed predominantly as inclusion bodies immediately after inducer addition whereas the proportion expressed as soluble protein is increased after 1 h of induction. Compared to the procedures, which are generally used for protein expression in the laboratory, this system is less labor intensive, it automatically provides recording of biomass concentration and specific growth rate, and it allows direct comparisons between expression of different proteins and performance of different constructs since the induction period is linked to growth. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 355–361, 2001.
Toluene bioconversion to p‐hydroxybenzoate by fed‐batch cultures of recombinant Pseudomonas putida
Authors: Miller Edward S. , Peretti Steven W.
Published: August 2002

A microbial oxidation process for the production of p‐hydroxybenzoate (HBA) from toluene is reported. The oxidation reaction was studied in fed‐batch fermentations using a recombinant Pseudomonas putida grown on glutamate as the sole carbon and energy source with salicylate and IPTG induction of tmoABCDE, and pchCF and phbz pathway genes, respectively. An average volumetric HBA productivity of 13.4 mg HBA L−1 h−1 was obtained under rapid growth conditions (glutamate excess), giving an HBA titer of 132 mg L−1 after 9.8 h of fermentation. This corresponded to an average specific HBA productivity of 7.2 μg HBA (mg total protein)−1 h−1. In contrast, maximum HBA titers of 35 mg HBA L−1 were achieved in 27 h in comparative studies employing glutumate limited fed‐batch cultures. A specific productivity of 4.1 μg HBA (mg total protein)−1 h−1 and volumetric productivity of 1.3 mg HBA L−1 h−1 were calculated for the growth‐rate restricted cultures. The differences in HBA production between the two cultures could be correlated to the levels of specific toluene‐4‐monooxygenase (T4MO) polypeptides. T4MO catalyzes the rate‐limiting step in the pathway. Using experimental data, the half‐life value of TmoA was calculated to be approximately 28 h. Assuming linear, monomolecular decay of TmoA, a specific degradation constant of 0.025 h−1 was calculated, which placed the stability of recombinant TmoA in the range of relatively stable proteins, even in the absence of co‐expression of tmoF, the terminal oxidoreductase subunit of T4MO. © 2002 John Wiley & Sons, Inc. Biotechnol Bioeng 77: 340–351, 2002.
Substrate‐permeable encapsulation of enzymes maintains effective activity, stabilizes against denaturation, and protects against proteolytic degradation
Authors: Nasseau Mathieu , Boublik Yvan , Meier Wolfgang , Winterhalter Mathias , Fournier Didier
Published: August 2002

How can enzymes be protected against denaturation and proteolysis while keeping them in a fully functional state? One solution is to encapsulate the enzymes into liposomes, which enhances their stability against denaturation and proteases. However, the permeability barrier of the lipid membrane drastically reduces the activity of enzyme entrapped in the liposome by reducing the internal concentration of the substrate. To overcome this problem, we permeabilized the wall of the liposome by reconstitution of a porin from Escherichia coli. In this way, we recovered the full functionality of the enzyme while retaining the protection against denaturation and proteolytic enzymes. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 615–618, 2001.
Calculation of effective diffusivities for biofilms and tissues
Authors: Wood Brian D. , Quintard Michel , Whitaker Stephen
Published: August 2002

In this study we describe a scheme for numerically calculating the effective diffusivity of cellular systems such as biofilms and tissues. This work extends previous studies in which we developed the macroscale representations of the transport equations for cellular systems based on the subcellular‐scale transport and reaction processes. A finite‐difference model is used to predict the effective diffusivity of a cellular system on the basis of the subcellular‐scale geometry and transport parameters. The effective diffusivity is predicted for a complex three‐dimensional structure that is based on laboratory observations of a biofilm, and these numerical predictions are compared with predictions from a simple analytical solution and with experimental data. Our results indicate that, under many practical circumstances, the simple analytical solution can be used to provide reasonable estimates of the effective diffusivity. © 2002 John Wiley & Sons, Inc. Biotechnol Bioeng 77:495–516, 2002; DOI 10.1002/bit.10075
Decoupling cell growth and product formation in Chinese hamster ovary cells through metabolic control
Authors: Altamirano C. , Cairó J. J. , Gòdia F.
Published: August 2002

The development of a strategy for the culture of Chinese hamster ovary (CHO) cells producing tissue plasminogen activator (t‐PA) is investigated. This strategy is based on the replacement of the main carbon source, glucose, by another compound that is slowly metabolizable, particularly galactose. The introduction of this change allows for acute change in cell behavior at various levels. Cell growth is stopped after this nutrient shift, and the cells can be kept in long‐duration culture at a low growth rate and high viability as compared with a culture strategy based solely on glucose utilization. Moreover, the capability of cells to produce recombinant proteins (t‐PA in this work) can be maintained over the entire period of galactose feeding. From the metabolic point of view, use of a slowly metabolizable carbon source (galactose) introduces important changes in the production of lactate, ammonia, and some amino acids. The use of this metabolic shift enables the generation of biphasic processes, with a first phase with cell growth on glucose and a second stationary phase on galactose, which is particularly suited to perfusion systems. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 76: 351–360, 2001.
A combined method for determining inhibition type, kinetic parameters, and inhibition coefficients for aerobic cometabolism of 1,1,1‐trichloroethane by a butane‐grown mixed culture
Authors: Kim Young , Arp Daniel J. , Semprini Lewis
Published: August 2002

A combined method for determining inhibition type, kinetic parameters, and inhibition coefficients is developed and presented. The method was validated by applying it to data obtained from batch kinetics of the aerobic cometabolism of 1,1,1‐trichloroethane (1,1,1‐TCA) by a butane‐grown mixed culture. The maximum degradation rates (kmax) and half‐saturation coefficients (Ks) were independently determined in single compound tests, and compared with those obtained from inhibition tests. The inhibition type was determined using direct linear plots at various substrate and inhibitor concentrations. Kinetic parameters (kmax and Ks) and inhibition coefficients (Kic and Kiu) were determined by nonlinear least squares regression (NLSR) fits of the inhibition model determined from the direct linear plots. Initial guesses of the kinetic parameters for NLSR were determined from linearized inhibition equations that were derived from the correlations between apparent maximum degradation rates (k) and/or the apparent half‐saturation coefficient (K) and the kmax, Ks, and inhibitor concentration (IL) for each inhibition equation. Two different inhibition types were indicated from the direct linear plots: competitive inhibition of 1,1,1‐TCA on butane degradation, and mixed inhibition of 1,1,1‐TCA transformation by butane. Good agreement was achieved between independently measured kmax and Ks values and those obtained from both NLSR and the linearized inhibition equations. The initial guesses of all the kinetic parameters determined from linear plots were in the range of the values estimated from NLSR analysis. Overall the results show that use of the direct linear plot method to identify the inhibition type, coupled with initial guesses from linearized plots for NLSR analysis, results in an accurate method for determining inhibition types and coefficients. Detailed studies with pure cultures and purified enzymes are needed to further demonstrate the utility of this method. © 2002 John Wiley & Sons, Inc. Biotechnol Bioeng 77: 564–576, 2002; DOI 10.1002/bit.10145
Connection between gene dosage and protein stability revealed by a high‐yield production of recombinant proteins in an E. coli LexA1(Ind−) background
Authors: Medina Manel G. , Carbonell Xavier , Villaverde Antonio
Published: August 2002

Bacterial production of a plasmid‐encoded bacteriophage P22 tailspike protein shows different yield and impact on cell viability in RecA+ LexA+, RecA− LexA+ and RecA+ LexA1(Ind−) backgrounds. In a LexA1(Ind−) context, we have observed lesser toxicity and higher productivity than in the wild‐type strain, in which the bacterial growth was inhibited after induction of recombinant gene expression. Also, a negative effect of the incubation temperature on the growth of producing cells was also detected. By exploring the molecular basis of these inhibitory events, we found a connection between the dosage of the recombinant gene and the proteolytic stability of the encoded protein. Under both genetic and environmental conditions favoring higher plasmid copy number and consequently increasing the synthesis rate of the recombinant protein, enhanced protein degradation was observed in parallel with an important growth inhibition. Altogether, the obtained data suggest the existence of a critical concentration of recombinant protein over which cell proteolysis is stimulated at rates not compatible with optimal physiological conditions for bacterial growth. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 722–730, 2002.
Controlled shear affinity filtration (CSAF): A new technology for integration of cell separation and protein isolation from mammalian cell cultures
Authors: Vogel Jens H. , Anspach Birger , Kroner Karl‐Heinz , Piret James M. , Haynes Charles A.
Published: August 2002

Controlled shear affinity filtration (CSAF) integrates animal cell separation and product isolation in a single unit operation through the use of a specifically designed rotating disk filter with incorporated membrane chromatography column. Because of the decoupling of shear force and pressure generation and the specific hydrodynamics of the system, shear rates can be easily optimized and precisely controlled to maximize filtration performance while viability of the shear sensitive animal cells is maintained. In this study, the general methodology is demonstrated using the integration of Chinese hamster ovary cell separation and isolation of recombinant tissue plasminogen activator (t‐PA) as a model example. Direct capture of t‐PA from cell culture broth was realized by using custom‐made affinity membranes with lysine as a robust, small molecular weight affinity ligand. Small‐scale t‐PA adsorption experiments, as well as microfiltration experiments, were used to design the integrated CSAF process. A Chinese hamster ovary batch culture was processed with a lab‐scale prototype, yielding 86% of the t‐PA in the concentrated, particle‐free eluate, whereas 95% of the bulk protein was removed. Because the viability of the cells is not significantly affected and high specific flux rates can be achieved, the CSAF technology should also be well suited for continuous perfusion with integrated product isolation. A truly continuous operation could be realized with two systems in tandem configuration. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 806–814, 2002.
Scale‐down model to simulate spatial pH variations in large‐scale bioreactors
Authors: Amanullah A. , McFarlane C. M. , Emery A. N. , Nienow A. W.
Published: August 2002

For the first time a laboratory‐scale two‐compartment system was used to investigate the effects of pH fluctuations consequent to large scales of operation on microorganisms. pH fluctuations can develop in production‐scale fermenters as a consequence of the combined effects of poor mixing and adding concentrated reagents at the liquid surface for control of the bulk pH. Bacillus subtilis was used as a model culture since in addition to its sensitivity to dissolved oxygen levels, the production of the metabolites, acetoin and 2,3‐butanediol, is sensitive to pH values between 6.5 and 7.2. The scale‐down model consisted of a stirred tank reactor (STR) and a recycle loop containing a plug flow reactor (PFR), with the pH in the stirred tank being maintained at 6.5 by addition of alkali in the loop. Different residence times in the loop simulated the exposure time of fluid elements to high values of pH in the vicinity of the addition point in large bioreactors and tracer experiments were performed to characterise the residence time distribution in it. Since the culture was sensitive to dissolved oxygen, for each experiment with pH control by adding base into the PFR, equivalent experiments were conducted with pH control by addition of base into the STR, thus ensuring that any dissolved oxygen effects were common to both types of experiments. The present study indicates that although biomass concentration remained unaffected by pH variations, product formation was influenced by residence times in the PFR of 60 sec or longer. These changes in metabolism are thought to be linked to both the sensitivity of the acetoin and 2,3‐butanediol‐forming enzymes to pH and to the inducing effects of dissociated acetate on the acetolactate synthase enzyme. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 73: 390–399, 2001.
Encapsulation of proteins by layer‐by‐layer adsorption of polyelectrolytes onto protein aggregates: Factors regulating the protein release
Authors: Balabushevitch Nadejda G. , Sukhorukov Gleb B. , Moroz Natalia A. , Volodkin Dmitry V. , Larionova Natalia I. , Donath Edwin , Mohwald Helmuth
Published: August 2002

A novel method of protein encapsulation is proposed. Preformed protein aggregates are covered with polyelectrolyte layers by means of layer‐by‐layer adsorption. The polyelectrolyte membrane prevents protein leakage out of the capsule. Using chymotrypsin as a model enzyme the capsule wall selective permeability was demonstrated for substrates and inhibitors of different molecular weight and solubility. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 76: 207–213, 2001.
Water‐based nanoparticulate polymeric system for protein delivery: permeability control and vaccine application
Authors: Prokop Ales , Kozlov Evgenii , Newman Gale W. , Newman Mark J.
Published: August 2002

The idea of using polymeric nanoparticles as drug carriers is receiving an increasing amount of attention both in academia and industry, Nanoparticles have a number of potential applications in protein, drug and vaccine delivery, as well as gene therapy applications. In this article, we focus on this unique drug delivery technology as a method to control the release rate of substances, not only for protein delivery but also for delivering an experimental vaccine immunogen. Nanoparticles were assembled on the basis of ionic interaction between water‐soluble polymers so that the resulting particles were stable in physiologic media. Among the typical polymers used to assemble nanoparticles, different polysaccharides, natural amines, and poly‐amines were investigated. The entrapped substances tested included a protein and antigens. Polydextran aldehyde was incorporated into the particle core, to enable physiologic cross‐linking as a method to control permeability. This resulted in long‐term retention of substances that would otherwise rapidly leak out of the nanoparticles. Results of cross‐linking experiments clearly demonstrated that the release rate could be substantially reduced, depending on the degree of cross‐linking. For vaccine antigen delivery tests, we measured an antibody production after subcutaneous and oral administration. The data indicated that only the cross‐linked antigen was immunogenic when the oral route of administration was used. The data presented in this article address primarily the utility of nanoparticulates for oral delivery of vaccine antigen. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 459–466, 2002.
Disposition of triazolam in the rat by brain microdialysis and semi‐micro column high‐performance liquid chromatography with UV absorbance detection
Authors: Nakashima Kenichiro , Yamamoto Kazumi , Al‐Dirbashi Osama Y. , Nakashima Mihoko N.
Published: August 2002

A semi‐micro column high‐performance liquid chromatography with ultraviolet detection for the determination of triazolam is described. The method was applied to determine plasma and brain microdialysate concentrations of triazolam after single intravenous bolus of 2.5 mg/kg to rat. The separation was achieved on a 250 × 1.5 mm i.d. C18 column and the column effluent was monitored at 222 nm. The detection limits at a signal‐to‐noise ratio of 3 obtained using spiked plasma and artificial cerebrospinal fluid were 2.1 and 0.7 ng/mL, respectively. The intra‐ and inter‐day reproducibility of the present method were satisfactory with the highest relative standard deviation of 9.1 (n ≥ 5). The present method was successfully applied to study the disposition of triazolam in rat (n = 5) by analyzing plasma and brain microdialysate samples. Copyright © 2002 John Wiley & Sons, Ltd.