bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of the quality of radiotherapy randomized trials for painful bone metastases
Authors: Shakespeare Thomas P. , Thiagarajan Anuradha , Gebski Val
Published: March 2005

BACKGROUND

The quality of randomized radiotherapy studies investigating the palliation of painful bone metastases has been questioned, with some authors recognizing the potential impact of bias on result interpretation. However, there has been no published comprehensive evaluation of quality assessment. The goals of the current study were to evaluate the quality of randomized studies using a validated checklist and to discuss implications and future directions.

METHODS

The authors performed a search for studies that could be reliably assessed using the validated quality assessment instrument. Independent assessors scored study quality using the instrument.

RESULTS

The median quality score of the 17 identified randomized studies was 1 of 5 (range, 0–3). The majority (71%) of points were awarded for the authors describing the study as “randomized.” The method of randomization and description of withdrawals and dropouts were scored poorly for most studies. None of the studies were awarded points for allocation concealment (blinding). The overall quality was deemed poor (a score of 0–2) for 16 of 17 (94%) studies.

CONCLUSIONS

The quality of published randomized evidence comparing efficacy of fractionation schedules for the palliation of bone metastases was suboptimal. As a result of the potential biases present, subjective end points (e.g., retreatment rates) cannot be reliably evaluated. Greater efforts are required by radiation oncology trial groups to improve quality, with a particular focus on developing methods of allocation concealment and comprehensively reporting results. Cancer 2005. © 2005 American Cancer Society.
Early brain lesions and face‐processing development
Authors: de Schonen Scania , Mancini Josette , Camps Roselyne , Maes Emanuelle , Laurent Agathe
Published: March 2005

Studies of functional plasticity after pre‐ or perinatal brain damage can tell us whether the neural substrate normally involved in the development of a given ability is specific and, if so, when it becomes functionally specified and unique. Development of face processing was investigated in 5‐ to 17‐year‐old children who had a unilateral brain injury in the pre‐, peri‐, or postnatal period. In Studies 1 and 2, patients with a posterior injury involving the temporal regions exhibited a face‐processing deficit that was independent of their age at test time. Even though differences were observed between the two hemispheres in face processing during infancy as well as in adults in cases of normal development, no clear differences between right and left injury were observed here in face‐processing deficit. Poor postlesional face‐processing plasticity seems to contrast with results of several studies on speech development after early unilateral injury. If the difference in the time window for postlesional plasticity between these two areas of competency is confirmed, it would suggest that the two kinds of abilities rely on neural cells which are sensitive to different plasticity factors. © 2005 Wiley Periodicals, Inc. Dev Psychobiol 46: 184–208, 2005.
Are there critical periods for musical development?
Authors: Trainor Laurel J.
Published: March 2005

A critical period can be defined as a developmental window during which specific experience has a greater effect than at other times. Musical behavior involves many skills, including the basic encoding of pitch and time information, understanding scale and harmonic structure, performance, interpretation, and composition. We review studies of genetics, behavior, and brain structure and function in conjunction with the experiences of auditory deprivation and musical enrichment, and conclude that there is more supporting evidence for critical periods for basic than for more complex aspects of musical pitch acquisition. Much remains unknown about the mechanisms of interaction between genetic and experiential factors that create critical periods, but it is clear that there are multiple pathways for achieving musical expertise. © 2005 Wiley Periodicals, Inc. Dev Psychobiol 46: 262–278, 2005.
Use of Polished and Mercury Film‐Modified Silver Solid Amalgam Electrodes in Electrochemical Analysis of DNA
Authors: Fadrná Renata , Cahová‐Kucharíková Kateřina , Havran Luděk , Yosypchuk Bogdan , Fojta Miroslav
Published: March 2005

Polished and mercury film‐modified silver solid amalgam electrodes (p‐AgSAE and MF‐AgSAE, respectively) were used for the measurements of intrinsic redox (faradic) and tensammetric voltammetric signals of single‐ (ss) and double‐stranded, linear (lin) or supercoiled (sc) DNAs, synthetic polynucleotides and free adenine base. At the MF‐AgSAE, all of these species yielded signals similar to those previously obtained on the hanging mercury drop electrode (HMDE) or on solid silver amalgam electrode modified with mercury meniscus (m‐AgSAE). Good measurable anodic peak G (due to guanine residues) and tensammetric signals of ssDNA were obtained also with the p‐AgSAE (albeit the latter appeared at significantly less negative potentials than respective signals measured at HMDE). In contrast, the cathodic DNA peak CA (due to reduction of cytosine and adenine residues) was not detected at the polished electrode. For the free adenine base, at least an order of magnitude lower sensitivity (compared to HMDE or MF‐AgSAE) was achieved when its reduction signal was measured. Double‐stranded DNAs (including sc and linDNA) yielded no measurable tensammetric signals at the p‐AgSAE. Similarly as with the HMDE and m‐AgSAE, measurements at the MF‐AgSAE allowed differentiation between sc and linDNA and were successfully applied for the detection of DNA strand breaks induced by ionizing radiation. The p‐AgSAE could be used for detection of the DNA strand breaks too, but selective denaturation of the damaged DNA (converting it into ssDNA detectable at the polished electrode) was necessary prior to the voltammetric analysis. Our results suggest that the different behavior of DNA at the p‐AgSAE and at the MF‐AgSAE (HMDE) is probably related to a considerably weaker adsorption of the DNA molecules on the polished solid amalgam surface, compared to the surface of mercury (or liquid silver amalgam).
Time‐varying reconstruction in single photon emission computed tomography
Authors: Kervinen M. , Vauhkonen M. , Kaipio J. P. , Karjalainen P. A.
Published: March 2005

We propose a new method for reconstruction of images in single photon emission computed tomography (SPECT), when the activity distribution of the object is time‐varying. The activity evolution is modeled with the first‐order Markov model, and linear observation model is used to characterize the measurement system. The state‐space representation of the measurement sequence reduces to an ill‐conditioned state estimation problem, which is solved recursively using the Kalman filter and smoother algorithms. Two special models, the compartmental model and the diffusion model, for the time variation are discussed. The method is evaluated using simulations. © 2005 Wiley Periodicals, Inc. Int J Imaging Syst Technol, 14, 186–197, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/ima.20023
SAR imaging using multidimensional continuous wavelet transform and applications to polarimetry and interferometry
Authors: Colin E. , Tria M. , Titin‐Schnaider C. , Ovarlez J.P. , Benidir M.
Published: March 2005

Usual SAR imaging process makes the assumption that the reflectors are isotropic and white (i.e., they behave in the same way regardless the angle from which they are viewed and the emitted frequency within the bandwidth). The multidimensional continuous wavelet transform (CWT) in radar imaging was initially developed to highlight the image degradations due to these assumptions. In this article the wavelet transform method is widened to polarimetry and interferometry fields. The wavelet tool is first used for polarimetric image enhancement, then for coherence optimization in interferometry. This optimization by wavelets, compared with the polarimetric one, gives better results on the coherence level. Finally, a combination of both methods is proposed. © 2005 Wiley Periodicals Inc. Int J Imaging Syst Technol, 14, 206–212, 2004; Published online in Wiley Inter‐Science (www.interscience.wiley.com). DOI 10.1002/ima.20025
In vitro transfer of keratinocytes: Comparison of transfer from fibrin membrane and delivery by aerosol spray
Authors: Duncan Christian O. , Shelton Richard M. , Navsaria Harshad , Balderson Debra S. , Papini Remo P. G. , Barralet Jake E.
Published: March 2005

There are a variety of approaches for the delivery of autologous keratinocytes to restore epithelial coverage of burns wounds that include utilizing cultured keratinocyte sheets, transfer of cultured keratinocytes using a membrane and more recently aerosol spraying of a keratinocyte suspension. The purpose of this study was to compare the effectiveness of direct aerosol delivery of a keratinocyte suspension with a fibrin transfer system to an in vitro wound model consisting of organotypical deepidermalized dermis (DED). A comparison was made between the number of keratinocytes delivered to DED with time, either by transfer from a fibrin membrane or using an aerosol. Additionally, the effect of application time of fibrin membranes to DED, on the number of keratinocytes delivered was investigated and compared with keratinocytes delivered by aerosol at the same time points. After 2 days culture little transfer of keratinocytes had occurred from the fibrin membrane to the DED, whereas 20% more cells were present on the DED than were initially delivered by aerosol spraying. After 7 days, aerosol‐delivered cells were found to express cytokeratin K6, indicating a proliferative phenotype. The results from this study show that preconfluent keratinocytes can be delivered by aerosol, and thus may well find application clinically. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater
On stability of the hamiltonian index under contractions and closures
Authors: Xiong Liming , Ryjáček Zdeněk , Broersma Hajo
Published: March 2005

The hamiltonian index of a graph G is the smallest integer k such that the k‐th iterated line graph of G is hamiltonian. We first show that, with one exceptional case, adding an edge to a graph cannot increase its hamiltonian index. We use this result to prove that neither the contraction of an AG(F)‐contractible subgraph F of a graph G nor the closure operation performed on G (if G is claw‐free) affects the value of the hamiltonian index of a graph G. AMS Subject Classification (2000): 05C45, 05C35. © 2005 Wiley Periodicals, Inc. J Graph Theory
Kinetics and mechanism of the reaction of benzyl bromide with copper in hexamethylphosphoramide
Authors: Egorov Anatoly M. , Matyukhova Svetlana A. , Anisimov Alexander V.
Published: March 2005

The reaction of copper with benzyl bromides in hexamethylphosphoramide has been studied. The kinetic and thermodynamic parameters of the reaction have been obtained. Hammett plots of log (k/ko) vs the substituent constant σ gave good correlations (ρ = 0.15, Sρ = 0.02, r = 0.954). The structure of the organic group has little effect on the rate of reaction of benzyl bromide with copper. In the absence of atmospheric oxygen, the oxidative dissolution of copper occurred by the mechanism of single‐electron transfer with the formation of 1,2‐diphenylethane and copper(I) complexes. The stereochemistry and intermediates compound was also investigated.

The reaction mechanism is discussed. © 2005 Wiley Periodicals, Inc. Int J Chem Kinet 37: 296–305, 2005
Kinetics and mechanisms for reactions of HNO with CH3 and C6H5 studied by quantum‐chemical and statistical‐theory calculations
Authors: Choi Y. M. , Lin M. C.
Published: March 2005

Kinetics and mechanisms for the reactions of HNO with CH3 and C6H5 have been investigated by ab initio molecular orbital (MO) and transition‐state theory (TST) and/or Rice‐Ramsperger‐Kassel‐Marcus/Master Equation (RRKM/ME) calculations. The G2M(RCC, MP2)//B3LYP/6‐31G(d) method was employed to evaluate the energetics for construction of their potential energy surfaces and prediction of reaction rate constants. The reactions R + HNO (R = CH3 and C6H5) were found to proceed by two key product channels giving (1) RH + NO and (2) RNO + H, primarily by direct abstraction and indirect association/decomposition mechanisms, respectively. As both reactions initially occur barrierlessly, their rate constants were evaluated with a canonical variational approach in our TST and RRKM/ME calculations. For practical applications, the rate constants evaluated for the atmospheric‐pressure condition are represented by modified Arrhenius equations in units of cm3 mol−1 s−1 for the temperature range 298–2500 K: κ1A = 1.47 × 1011T0.76 exp[−175/T], κ2A = 8.06 × 103T2.40 exp[−3100/T], κ1B = 3.78 × 105T2.28 exp[230/T], and κ2B = 3.79 × 109T1.19 exp[−4800/T], where A and B represent CH3 and C6H5 reactions, respectively. Based on the predicted rate constant at 1 atm pressure for R + HNO → RNO + H, we estimated their reverse rate constants for R + HNO production from H + RNO in units of cm3 mol−1 s−1: κ−2A′ = 7.01 × 1010 T0.84 exp[120/T] and κ−2B′ = 2.22 × 1019 T−1.01 exp[−9700/T]. The heats of formation at 0 K for CH3NO, CH3N(H)O, CH3NOH, C6H5N(H)O, and C6H5NOH have been estimated to be 18.6, 18.1, 22.5, 47.2, and 50.7 kcal mol−1 with an estimated ±1 kcal mol−1 error. © 2005 Wiley Periodicals, Inc. Int J Chem Kinet 37: 261–274, 2005
β‐chemokine receptor expression in idiopathic inflammatory myopathies
Authors: De Paepe Boel , De Bleecker Jan L.
Published: March 2005

β‐chemokines attract and activate T cells and monocytes and have a key role in chronic inflammation. Certain β‐chemokines, such as monocyte chemoattractant protein‐1 (MCP‐1), have been reported to be upregulated in the idiopathic inflammatory myopathies (IIM). We studied the distribution of β‐chemokine receptors in polymyositis (PM), sporadic inclusion‐body myositis (sIBM), dermatomyositis (DM), and control samples. CCR1–5 were localized to blood vessels in all samples. In addition, increased endothelial expression of CCR2A was observed in IIM. Subsets of inflammatory cells, identified as macrophages and T cells, in all three types of IIM expressed CCR2A, CCR2B, CCR3, CCR4, and CCR5. In contrast to an earlier report, we found CCR2B to be the most prominent MCP‐1 receptor on inflammatory cells in IIM, especially in PM and sIBM. Strong CCR4 expression was present on myonuclei of regenerating muscle fibers. The prominence of the CCR2 receptors further underlines the importance of the interaction with their ligand MCP‐1 in the immunopathogenesis of IIM and puts CCR2B forward as a potential target for future therapeutic intervention. Muscle Nerve, 2005
Proteomic analysis of differential protein expression in human atherosclerotic plaque progression
Authors: Donners Marjo MPC , Verluyten Monique J , Bouwman Freek G , Mariman Edwin CM , Devreese Bart , Vanrobaeys Frank , van Beeumen Jozef , van den Akker Luc HJM , Daemen Mat JAP , Heeneman Sylvia
Published: March 2005

In this study, differential protein expression was assessed during human atherosclerotic plaque progression. A multifaceted approach was used in which differential protein expression was studied by two‐dimensional (2D) gel electrophoresis and validated in individual patients using western blotting and immunohistochemistry. 2D profiles of whole‐mount advanced stable lesions were compared to those of plaques containing a thrombus. Mass spectrometry analysis identified vinexin‐β and α1‐antitrypsin (AAT) in the same spot that was differentially expressed in plaques with a thrombus. Immunohistochemistry and western blotting showed limited expression of both vinexin‐β and AAT in early lesions, whereas high expression of both proteins was found in advanced lesions. Differential expression of vinexin‐β in lesions with a thrombus compared to stable plaques could not be confirmed, indicating the importance of validation of proteomic analysis. For AAT, western blotting of 2D gels revealed expression of six isoforms in advanced plaques, one of which was confirmed to be solely expressed in thrombus‐containing plaques. In conclusion, vinexin‐β is expressed in advanced human atherosclerotic plaques, but differential expression of this protein in lesions with a thrombus versus stable plaques could not be confirmed. However, this analysis revealed expression of six isoforms of AAT in advanced plaques, one of which was uniquely expressed in thrombus‐containing plaques. Copyright © 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Anaplastic transformation from papillary thyroid carcinoma with increased serum CA19‐9
Authors: Ogawa Masahiro , Hori Hiroki , Hirayama Masahiro , Kobayashi Minako , Shiraishi Taizou , Watanabe Yuri , Komada Yoshihiro
Published: March 2005

A 24‐year‐old woman presented with anaplastic transformation from papillary thyroid carcinoma with increased serum CA19‐9. The patient had been diagnosed as having papillary thyroid carcinoma with lung metastasis at 11 years of age. She received a total thyroidectomy with cervical lymph node dissection followed by iodine‐131 (131I) therapy over 12 years, but died due to sudden onset of rapid dissemination. Elevated serum CA19‐9 was detected in the terminal stage, and anaplastic transformation was confirmed by post‐mortem examination. Although there are few clinical reports suggesting a prognostic indicator for anaplastic thyroid carcinoma, CA19‐9 may be a useful serum marker for this tumor. Pediatr Blood Cancer 2005; 45:64–67. © 2005 Wiley‐Liss, Inc.
Trustees and staff: Building effective fundraising teams
Authors: Seiler Timothy L. , Tempel Eugene R.
Published: March 2005

A series of interviews with staff and trustees of not‐for‐profits with a record of successful fundraising offers a model for staff and trustee interaction in the fundraising process.
Five reasons for nonprofit organizations to be inclusive
Authors: Gough Samuel N.
Published: March 2005

Nonprofit organizations should be governed and managed by representative numbers of their constituents but not limited to certain groups of people. Even organizations founded to empower or serve specific groups should have governing boards and managers that can look at issues from a range of different points of view.
Calculation of polymer blend compositions from vibrational spectra: A simple method
Authors: Tomba J. Pablo
Published: March 2005

In this work, the application of a new approach for quantitative analysis, originally developed for Raman spectroscopy, is extended to IR spectroscopy. The attractive features of this methodology are its simplicity and ease of use in comparison with traditional approaches. Unlike other methods, rich spectral information containing several overlapped peaks can be used in the calculations. A robust and well‐conditioned calculation scheme renders precise results, which are independent of the operator's decisions. The method was applied to study the chemical compositions of homogeneous polymer blends made of polystyrene and poly(vinyl methyl ether). Raman and IR blend spectra were acquired with confocal Raman microspectroscopy and attenuated total reflection/Fourier transform infrared, respectively. The blend compositions were calculated from the corresponding vibrational spectra with the proposed strategy, and excellent agreement between those values and the true ones was found for both techniques. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 1144–1151, 2005
Do Intraoperative Total Serum and Ionized Calcium Levels, Like Intraoperative Intact PTH Levels, Correlate with Cure of Hyperparathyroidism?
Authors: Quiros Roderick M. , Pesce Catherine E. , Djuricin Goldie , Prinz Richard A.
Published: March 2005

Intraoperative parathyroid hormone (ioPTH) monitoring is useful in the operative management of hyperparathyroidism. Measurement of intraoperative total serum calcium (TSC) and ionized calcium (ICa) levels may be less expensive and more readily available methods of intraoperative guidance during neck dissection than ioPTH levels, the gold standard. We compared the accuracy of monitoring intraoperative TSC and ICa to that of ioPTH for predicting surgical cure during parathyroidectomy. Over a 10‐month period, 47 parathyroidectomies were performed, during which ioPTH, TSC, and ICa were measured. Samples were obtained at the start of the operation and 5 and 10 minutes after gland removal. Data were compared and trends analyzed with respect to removal of abnormal parathyroid tissue as confirmed by pathology. The Wilcoxon signed rank test was used to determine if decreases in TSC and ICa were significant. The mean baseline ioPTH level (253 ± 247 pg/ml) dropped by 70% at 5 minutes after removal of the abnormal glands (68 ± 85 pg/ml) and by 83% at 10 minutes (32 ± 25 pg/ml). The mean baseline TSC level (10.1 ± 0.9 mg/dl) dropped by 4% at 5 minutes after removal of the abnormal glands (9.7 ± 0.8 mg/dl) and remained at 4% at 10 minutes (9.6 ± 0.7 mg/dl). The mean baseline ICa level (1.4 ± 0.1 mmol/dl) also dropped by 4% at 5 minutes after removal of the abnormal glands (1.3 ± 0.1 mmol/dl) and remained at 4% at 10 minutes (1.3 ± 0.1 mg/dl). ioPTH dropped by ≥ 50% in 39 patients (83%) at 5 minutes and in 46 patients (98%) at 10 minutes after gland resection. TSC decreased below baseline at 5 minutes and remained below baseline at 10 minutes in only 37 patients (79%). In the remaining 21% of patients, TSC decreased inconsistently, if at all, with respect to baseline at both the 5‐ and 10‐minute time points. ICa decreased below baseline at 5 minutes and remained below baseline at 10 minutes in only 35 patients (77%). In the remaining 23% of patients, ICa, like TSC, changed inconsistently at 5 and 10 minutes after parathyroidectomy with respect to baseline levels. Decreases in TSC and ICa during parathyroidectomy, if present, are thus minimal. Unlike ioPTH levels, TSC and ICa levels do not consistently decrease at 5 and 10 minutes after gland resection. Although inexpensive and readily available, monitoring the intraoperative TSC and ICa is not clinically reliable for confirming removal of hyperfunctioning parathyroid glands.
Evaluation of nuclear transfer and transcription of plasmid DNA condensed with protamine by microinjection: The use of a nuclear transfer score
Authors: Masuda Tomoya , Akita Hidetaka , Harashima Hideyoshi
Published: March 2005

In the present study, the nuclear delivery of a green fluorescence protein (GFP)‐encoding pDNA condensed by protamine was investigated in terms of trans‐gene expression after cytoplasmic (E(cyt)) and nuclear (E(nuc)) microinjection. To compare the nuclear transfer process, a novel parameter; the nuclear transfer (NT) score was introduced. The E(cyt) value for protamine/pDNA particles increased in a charge ratio‐dependent manner. The calculated NT score showed that this increase results from an enhancement in nuclear transfer efficiency, which was also quantitatively confirmed by a recently developed confocal image‐assisted three‐dimensionally integrated quantification (CIDIQ) method. Moreover, E(nuc) for protamine/pDNA particles was significantly higher than that for poly‐l‐lysine/pDNA particles, suggesting that pDNA, when condensed with protamine, is more accessible to intra‐nuclear transcription. Collectively, protamine is an excellent DNA condenser, with bi‐functional advantages: improvement in nuclear delivery and efficient intra‐nuclear transcription.
Effect of Acetaldehyde on Growth, Substrates, and Products by Leuconostoc mesenteroides ssp cremoris
Authors: Schmitt P. , Diviès C.
Published: March 2005

Addition of 5.6 mmol/L of acetaldehyde to glucose broth led to an increase in the specific growth rate, a decrease in the specific glucose consumption rate, and an increase in the specific lactate production rate of Leuconostoc mesenteroides ssp cremoris. The amount of acetate produced was enhanced. For cells growing in broth containing glucose plus citrate, addition of acetaldehyde did not stimulate growth and did not significantly change the maxima of specific consumption rate of citrate and glucose; however, a change in kinetics was observed: the maxima appeared earlier in the presence of acetaldehyde than in its absence. In the absence of acetaldehyde, the specific citrate utilization rate was higher than the specific glucose consumption rate.
Optimal Chemostat Cascades for Periplasmic Protein Production
Authors: Davis Robert H. , Ramirez W. Fred , Chatterjee Ashish
Published: March 2005

This theoretical work predicts the optimal system design for the steady‐state production of secreted protein in a chemostat cascade, using bakers' yeast (Saccharomyces cerevisiae) as the host organism. The protein of interest, mutant invertase, is secreted to the periplasmic space instead of the culture medium on account of its large size. This work uses the secretion model developed and tested by Park and Ramirez (1988). It is shown that the highest productivity is achieved when the chemostat cascade contains two stages, although the improvement over the single‐stage productivity is small. When no recycle is used, the advantage of two stages results from the tradeoff between maximizing the cell concentration and maximizing the rate of protein production per cell. When recycle is used, the cell concentration and protein productivity are increased, and the advantage of two stages results from the tradeoff between maximizing the specific protein production rate and maximizing the specific protein secretion rate. Cascades with three stages were also investigated, but these were found to have no improvement over the corresponding two‐stage cascades.
Overloaded Hollow‐Fiber Liquid Chromatography
Authors: Ding Hongbing , Cussler E. L.
Published: March 2005

Liquid chromatography in hollow fibers can separate solutes like flavors and proteins by using a stationary phase of organic solvent, sometimes containing reversed micelles. Such separations, which have a much smaller pressure drop than equivalent separations in packed beds, show dispersion consistent with chromatographic theories at low flows and dilute feeds. These separations behave less predictably at high flows and concentrated feeds, which overload the hollow fibers. The results for flavors correlate well with the Graetz number, consistent with available theories of chromatography and adsorption. The results for proteins correlate poorly with the Graetz number but better with a dimensionless flux based on facilitated diffusion in the stationary phase.
Fractionation of Recombinant Tumor Necrosis Factor Using Hydrophobic and Hydrophilic Membranes
Authors: Dorin Glenn , Thomson James , Hanisch Wolf
Published: March 2005

Recombinant tumor necrosis factor (TNF) is expressed in Escherichia coli as a soluble intracellular protein. A purification process is described that incorporates a hydrophilic membrane (cellulosic) separation followed by a hydrophobic one (PTFE). The hydrophilic step is a traditional one in that species are separated primarily on the basis of size. The hydrophobic step separates species on the basis of parameters apparently not related to size. By combining these two steps, an increase in TNF purity of 7‐10‐fold can be achieved with a yield of 50%. The effects of cellular debris and pH on selectivity and recovery of the hydrophobic filtration step are discussed.
Inclined Sedimentation for Selective Retention of Viable Hybridomas in a Continuous Suspension Bioreactor
Authors: Batt Brian C. , Davis Robert H. , Kompala Dhinakar S.
Published: March 2005

The continuous separation of nonviable hybridoma cells from viable hybridoma cells by using a narrow rectangular channel that is inclined from the vertical has been investigated experimentally. The effectiveness of the settler in selectively retaining viable hybridomas in the bioreactor while permitting the removal of nonviable hybridomas has been shown to depend on the flow rate through the settler. Intermediate flow rates through the settler have been found to provide the highest removal of nonviable hybridomas relative to viable hybridoma retention. At high dilution rates through the chemostat, over 95% of the viable cells could be partitioned to the bottom of the settler while over 50% of the nonviable cells are removed through the top of the settler. This successful separation is due to the significantly larger size of the viable hybridomas than the nonviable ones. A continuous perfusion experiment was performed in which an external inclined settler was used to retain virtually all of the viable hybridomas in the culture, while selectively removing from the culture approximately 20% of the nonviable cells that entered the settler. A stable viable cell concentration of 1.0 × 107 cells/mL was achieved, as was an antibody productivity of over 50 μg/(mL‐day). These represent 3‐and 6‐fold increases, respectively, over the values obtained from a chemostat culture without cell retention.
Propagation of Recombinant Vaccinia Virus in HeLa Cells: Adsorption Kinetics and Replication in Batch Cultures
Authors: Chillakuru Rajeev A. , Ryu Dewey D. Y. , Yilma Tilahun
Published: March 2005

The influence of various culture parameters on infection and replication of recombinant vaccinia virus in HeLa cells was examined during various phases of viral replication. A modified form of the model of Valentine and Allison (Biochim. Biophys. Acta 1960, 40, 393–399) model was used to predict successfully the viral adsorption rates in cell suspensions. An experimentally determined aggregation factor, ϵ, was included in the model to account for deviations of the observed adsorption rates from those predicted by the earlier model. It was also shown that the ionic strength, ionic species, and serum proteins present in the medium significantly altered the adsorption kinetics of the virus. The lysosomotropic base chloroquine was found to enhance viral infection more than 2‐fold during the penetration step of viral infection. It was also demonstrated that cells infected during the exponential growth phase gave higher viral yields than those infected during the lag or stationary growth phases and the initial viral MOI did not significantly alter viral yields. Finally, it was demonstrated that viral infection of HeLa cells grown in 4‐L bioreactor batch cultures resulted in increased death and glucose uptake rates and significantly lower growth rates.
Reaction Rates for the Production of Selected Hormones by Solid‐Phase Synthesis
Authors: Wang Shen , Foutch Gary L.
Published: March 2005

Reaction rates have been measured for the production of selected bradykinins, angiotensins, and endorphins by solid‐phase synthesis. Reactor liquid concentrations are monitored by using a UV detector and flow cell. Experimentation has been limited to low excesses of the t‐Boc amino symmetrical anhydride. More than 500 attachments have been monitored. Most data obtained from resins with 1% cross‐linking show second‐order behavior with reaction rate constants between 0.5 and 8L/(mol‐s). The reaction rate is affected by chemical structures of both the attached amino symmetrical anhydride and the anchored amino terminus on the peptide fragment. Increasing reaction temperature and initial solution concentrations promotes reaction rate. The structure of the polymer support affects not only the reaction rate but also the observed reaction kinetics.
Solvent Selection Strategies for Extractive Biocatalysis
Authors: Bruce Lynda J. , Daugulis Andrew J.
Published: March 2005

This report follows the development of systematic solvent screening strategies for the identification of superior pure solvents and introduces techniques for the identification of effective coextractants. Specifically, methods to predict the biocompatibility and extractant capability of solvents are discussed. Biocompatibility is predicted by using heuristic data or the correlations between bioactivity and the logarithm of the partition coefficient of the solvent or the concentration of solvent in the cell membrane. A computer program, known as the extractant screening program or ESP, has been developed to effectively predict the behavior of virtually any product in any solvent/aqueous system. It is demonstrated that a biocompatible yet poor solvent can be mixed with a toxic solvent that has better extractant properties to yield a mixture with improved solvent characteristics that is still biocompatible. The fact that solvents do not mix in an ideal manner is exploited by using ESP to identify solvent mixtures that are still biocompatible at relatively high concentrations of toxic solvent.
Transport and Kinetics in Sandwiched Membrane Bioreactors
Authors: Jeong Yong S. , Vieth W. R. , Matsuura T.
Published: March 2005

A bioreactor in which living yeast cells are sandwiched between an ultrafiltration membrane and a reverse osmosis membrane was constructed, and experiments were performed for the conversion of substrate glucose to product ethanol. A set of equations that include both transport through a series of barrier layers and bioreaction rate were developed to predict the performance of the sandwich bioreactor. The above equations were solved by using numerical values for the transport parameter and the bioreaction rate constant, and the results are compared with the experimental data.
Microscopic Visualization of Insect Cell‐Bubble Interactions. I: Rising Bubbles, Air‐Medium Interface, and the Foam Layer
Authors: Bavarian Farshad , Fan L. S. , Chalmers Jeffrey J.
Published: March 2005

Through the use of microscopic, high‐speed video technology, the interactions of two suspended insect cell lines, Trichoplusia ni (TN‐368) and Spodoptera frugiperda (SF‐9), with air and oxygen bubbles were studied. Events such as cell‐bubble attachment, cell‐bubble collision, cell transport into the foam layer, and trapping of cells in the foam layer are presented and discussed. Based on these observations and those in a companion paper (Chalmers, J. J.; Bavarian, F. Biotechnol. Prog. 1991, following paper in this issue) and the experimental and theoretical work of other researchers, several mechanisms of cell damage as a result of sparging are presented.
Microscopic Visualization of Insect Cell‐Bubble Interactions. II: The Bubble Film and Bubble Rupture
Authors: Chalmers Jeffrey J. , Bavarian Farshad
Published: March 2005

In this paper, the second in the series, the use of a microscopic, high‐speed video system to study the interactions of two suspended insect cells strains, Trichoplusia ni (TN‐368) and Spodoptera frugiperda (SF‐9), with rupturing bubbles is reported. Events such as the adsorption of cells onto the bubble film and the mechanism of bubble rupture were observed. On the basis of these observations and the experimental and theoretical work of other researchers on bubble rupture and cell death as a result of sparging, it is proposed that cells are killed by the rapid acceleration of the bubble film after rupture and the high levels of shear stress in the boundary layer flow associated with bubble jet formation.
Bromodeoxyuridine Labeling and Flow Cytometric Identification of Replicating Saccharomyces cerevisiae Cells: Lengths of Cell Cycle Phases and Population Variability at Specific Cell Cycle Positions
Authors: Dien Bruce S. , Srienc Friedrich
Published: March 2005

An immunofluorescent staining procedure has been developed to identify, with flow cytometry, replicating cells of Saccharomyces cerevisiae after incorporation of bromodeoxyuridine (BrdUrd) into the DNA. Incorporation of BrdUrd is made possible by using yeast strains with a cloned thymidine kinase gene from the herpes simplex virus. An exposure time of 4 min to BrdUrd results in detectable labeling of the DNA. The BrdUrd/DNA double staining procedure has been optimized and the flow cytometry measurements yield histograms comparable to data typically obtained for mammalian cells. On the basis of the accurate assessment of cell fractions in individual cell cycle phases of the asynchronously growing cell population, the average duration of the cell cycle phases has been evaluated. For a population doubling time of 100 min it was found that cells spend in average 41 min in the replicating phase and 24 min in the G2+M cell cycle period. Assuming that mother cells immediately reenter the S phase after cell division, daughter cells spend 65 min in the G1 cell cycle phase. Together with the single cell fluorescence parameters, the forward‐angle light scattering intensity (FALS) has been determined as an indicator of cell size. Comparing different temporal positions within the cell cycle, the determined FALS distributions show the lowest variability at the beginning of the S phase. The developed procedure in combination with multiparameter flow cytometry should be useful for studying the kinetics and regulation of the budding yeast cell cycle.
Regulation of Cellulase Synthesis in Acidothermus cellulolyticus
Authors: Shiang Ming , Linden James C. , Mohagheghi Ali , Grohmann Karel , Himmel Michael E.
Published: March 2005

The regulation of cellulase synthesis by induction and catabolite repression in the thermophilic, aerobic bacterium Acidothermus cellulolyticus was studied in batch fermentations. Various compounds, such as L‐sorbose, cyclic AMP (cAMP), L‐glucose, 2‐deoxyglucose (2‐DG), glucose 1‐phosphate (G‐1‐P), sophorose, salicin, sugar alcohols, and isopropyl thioglucoside (IPTGlu), were added along with Solka Floe to improve extracellular cellulase formation by the culture. When cAMP was added exogenously to A. cellulolyticus cultures in the concentration range of 0.01‐0.2 g/L, cAMP did not affect cell growth; however, cellulase yields were increased with increasing levels of cAMP. The enzyme production rates with the different levels of cAMP addition during Solka‐Floc fermentations were identical. L‐Sorbose, L‐glucose, 2‐DG, G‐1‐P, sophorose, IPTGlu, and sugar alcohols enhanced cellulase activity produced in the medium, but the starting time and the time required to reach the maximum enzyme activity were different in each condition. All these substances may function as moderators of cellulase synthesis. From the experimental results, only cellobiose, xylose, sophorose, and unknown soluble derivatives from cellulose were considered as inducers. In a possible regulatory mechanism of cellulase synthesis, the repressor, inducer, cAMP, and moderator may be all involved in controlling the rate and the yield of enzyme production.
Purification of Glycoproteins by Selective Transport Using Concanavalin‐Mediated Reverse Micellar Extraction
Authors: Paradkar Vikram M. , Dordick Jonathan S.
Published: March 2005

A novel methodology for coupling liquid‐liquid extraction with affinity interaction has been developed to selectively and efficiently purify and separate glycoproteins. The basis for the separation is the selective extraction of glycoproteins from an aqueous solution into a reverse micellar organic phase by using concanavalin A (a sugar‐binding lectin) as a facilitative carrier. Specifically, horseradish peroxidase (a common glycoprotein) can be bound to concanavalin A in an aqueous phase and then extracted into an AOT‐isooctane organic phase with negligible loss in enzyme activity. Virtually no extraction of peroxidase occurs in the absence of concanavalin A. Electron spin resonance studies have shown that the large lectin‐glycoprotein complex (96 000 daltons) resides in a nonaqueous environment within the reverse micelle, perhaps at the surfactant, water‐pool interface; hence, extraction of the large complex is feasible. The facilitative extraction has been extended to selective transport of peroxidase from a mixture of peroxidase and alkaline phosphatase (a nonglycosylated protein). This results in an efficient separation strategy with a separation factor of 16.
Aminotransferase‐Catalyzed Conversion of D‐Amino Acids to L‐Amino Acids
Authors: Fotheringham Ian G. , Kidman Gene E. , Mcarthur Brian S. , Robinson Larry E. , Scollar Mark P.
Published: March 2005

A microbial bioconversion process is described for the conversion of D‐amino acids or the corresponding racemic mixtures to L‐amino acids. The process comprises reacting a D‐amino acid or a D,L mixture of an amino acid with glucose and ammonia in the presence of an aminotransferase‐containing culture of Escherichia coli. The aminotransferase is only moderately stereoselective in converting D‐and L‐amino acids to the keto acids. The aminotransferase‐mediated conversion of α‐keto acid to L‐amino acid exhibits very high stereospecificity, thus rendering the conversion of D‐amino acids irreversible. Conversions of leucine, phenylalanine, and homophenylalanine to the corresponding L isomers are described.
Production of Fusion Protein SpA:: EcoRI in Batch Culture in a 60‐L Airlift Tower Loop Reactor
Authors: Brandes Lutz , Wu Xiaoan , Maschke Hans E. , Jürgens Heike , Reinhardt Birgit , Schügerl Karl
Published: March 2005

Escherichia coli JM103 carrying the expression plasmid pMTC48, repressor plasmid pRK248, and protection plasmid pEcoR4 was grown in a 60‐L working volume airlift tower loop reactor on M9 minimal medium. Production of fusion protein SpA:: EcoRI was induced by a temperature shift from 30 to 38 (optimum), 40, or 42°C. The following parameters were monitored: cell mass concentration (X), total cell counts (TCC), number of colony‐forming units (CFU), concentrations of glucose, acetate, ethanol, pyruvate, lactate, succinate, amino acids, and ammonia, and soluble and total protein content, as well as product concentration (enzyme activity of the fusion protein), dissolved oxygen concentration, oxygen utilization rate (OUR), CO2 production rate (CPR), respiration quotient (RQ), and volumetric mass‐transfer coefficients (kLa). Product formation by temperature shift was only observed if LB concentrate was added to the culture at the same time the aeration rate was increased to avoid oxygen‐transfer limitation. No product accumulation was observed with glucose and ammonia supplementation. During gene expression, X and TCC increased, CFU decreased, acetate increased, and the primary metabolite (ethanol, pyruvate, lactate) concentrations as well as OUR and CPR passed a maximum while RQ changed only slightly. These facts indicate that, during gene expression, the metabolic activity of the cell passes a maximum, and after that it decreases. With increasing aeration rate, the volumetric productivity increased, but the specific productivity with respect to the cell concentration decreased.
Effect of Intraparticle Convection on the Chromatography of Biomacromolecules
Authors: Frey Douglas D. , Schweinheim Eberhard , Horváth Csaba
Published: March 2005

The effect of intraparticle convection in chromatographic columns packed with gigaporous particles (i. e., where dpore/dparticle > 10−2) on the band spreading of unretained biomacromolecules is investigated both experimentally and theoretically. A model is developed for the analysis of mass transfer in spherical particles of bidisperse pore structure when both convection and diffusion take place in the larger pores but only diffusion occurs in the smaller pores. The predictions of the model were experimentally verified. It is demonstrated that gigaporous particles have advantages over conventional porous particles (i. e., where dpore/dparticle < 10−3) for applications that do not require high resolving power, to bring about fast separation. This is because columns packed with gigaporous particles can be operated at high flow velocities without significant loss of efficiency due to the enhancement of mass transfer by intraparticle convection. The results of the model are used to examine the effectiveness of gigaporous column packings for rapid analytical chromatography and for the concentration and recovery of a dilute solute in a saturation‐regeneration cycle utilizing frontal chromatography.
Protein Purification with Novel Porous Sheets Containing Derivatized Cellulose
Authors: Manganaro James L. , Goldberg Bruce S.
Published: March 2005

Novel porous sheets containing commercially available cellulosic ion exchange media of several different functionalities in a PVC matrix have been prepared and evaluated. The advantages of these materials are resistance to alkali, clean in place procedures and low nonspecific binding. These sheets thus provide well‐known and well‐characterized particulate chromatographic media in a porous sheet format. The porous sheet format permits use of short, squat geometries (stacked sheets) as well as tolerance of high pressure gradients. The net result is the ability to handle much higher flow rates (10‐fold or greater) than achievable by comparable cellulosic packed columns of the neat particulate media. Chromatographic separation of natural egg white proteins by a linear salt gradient on a single sheet of 1.3‐mm thickness was demonstrated. In addition, a very significant advantage of the porous sheet format is that it permits the fabrication of devices which are not possible with particulate media. An example of such a device is the Acti‐Mod Spiral Module. In the spiral module a spiral flow channel is formed by wrapping the porous sheet material around a mandrel. Embossed ribbing in the porous sheet provides channel spacing. The small open channel accommodates high flow rates of biological suspensions while the porous sheet walls selectively remove components of the suspension.
Growth of Plant Root Cultures in Liquid‐ and Gas‐Dispersed Reactor Environments
Authors: Mckelvey Scott A. , Gehrig Jason A. , Hollar Kathryn A. , Curtis Wayne R.
Published: March 2005

The growth of Agrobacterium transformed “hairy root” cultures of Hyoscyamus muticus was examined in various liquid‐ and gas‐dispersed bioreactor configurations. Reactor runs were replicated to provide statistical comparisons of nutrient availability on culture performance. Accumulated tissue mass in submerged air‐sparged reactors was 31% of gyratory shake‐flask controls. Experiments demonstrate that poor performance of sparged reactors is not due to bubble shear damage, carbon dioxide stripping, settling, or flotation of roots. Impaired oxygen transfer due to channeling and stagnation of the liquid phase are the apparent causes of poor growth. Roots grown on a medium‐perfused inclined plane grew at 48% of gyratory controls. This demonstrates the ability of cultures to partially compensate for poor liquid distribution through vascular transport of nutrients. A reactor configuration in which the medium is sprayed over the roots and permitted to drain down through the root tissue was able to provide growth rates which are statistically indistinguishable (95% T‐test) from gyratory shake‐flask controls. In this type of spray/trickle‐bed configuration, it is shown that distribution of the roots becomes a key factor in controlling the rate of growth. Implications of these results regarding design and scale‐up of bioreactors to produce fine chemicals from root cultures are discussed.
Measurement of Diffusion Coefficients in Gels Using Holographic Laser Interferometry
Authors: Gustafsson Nils Ove , Westrin Bengt , Axelsson Anders , Zacchi Guido
Published: March 2005

The accuracy and precision of holographic interferometry as a method to measure diffusion coefficients in gels are investigated both experimentally and theoretically. The standard deviations in the experimentally determined diffusion coefficient for ethanol in 4% (w/v) agarose gel were 3.3% for diffusion into the gel and 6.1% for diffusion out of the gel. These are in good agreement with the standard deviations obtained using Monte Carlo simulations. Systematic errors derived from an assumption of constant diffusion coefficients were also investigated.