bluuebunny/crossref_metadata_2025 huggingface dataset:
High‐yield expression of properly folded insulinoma‐associated protein intracellular domain (IA‐2ic) in Escherichia coli
Authors: Sica Mauricio P. , Primo María E. , Ermácora Mario R. , Poskus Edgardo
Published: September 2003

The intracellular domain of insulinoma‐associated protein (IA‐2), IA‐2ic, is a prominent antigen in autoimmune diabetes, and autoantibodies to it are early markers of the disease. The high‐yield expression of properly folded IA‐2ic is needed for basic research and crucial for low‐cost immunoassays aimed at the detection of these autoantibodies in diagnostic and preventive medicine. In previous work, the expression of IA‐2ic fused to glutathione S‐transferase or to a biotinylatable peptide was reported; however, these methods had very poor yield. Here we show that, utilizing a codon‐optimized gene, up to 80 mg of pure and properly folded autoantigen per litre of Escherichia coli culture may be obtained. Furthermore, the addition of a C‐terminal His‐tag greatly facilitates IA‐2ic purification without compromising either its immunoreactivity or its expression yield. To take advantage of the recombinant antigen, an enzyme immunoassay format was developed which proved to be highly specific and sensitive.
Thermal stabilization of trypsin by enzymic modification with β‐cyclodextrin derivatives
Authors: Villalonga Reynaldo , Fernández Michael , Fragoso Alex , Cao Roberto , Mariniello Loredana , Porta Raffaele
Published: September 2003

Streptoverticillum sp. transglutaminase was used as catalyst for the attachment of several β‐cyclodextrin derivatives to the glutamine residues in bovine pancreatic trypsin. The modifying agents used were mono‐6‐ethylenediamino‐6‐deoxy‐β‐cyclodextrin, mono‐6‐propylenediamino‐6‐deoxy‐β‐cyclodextrin, mono‐6‐butylenediamino‐6‐deoxy‐β‐cyclodextrin and mono‐6‐hexylenediamino‐6‐deoxy‐β‐cyclodextrin. The transformed trypsin preparations contained about 3 mol of oligosaccharides/mol of protein. The specific esterolytic activity of trypsin was increased by about 4–21% after conjugation. The Km values for cyclodextrin–trypsin complexes represented about 58–87% of that corresponding to the native enzyme. The optimum temperature for esterolytic activity of trypsin was increased by about 5–10 °C after enzymic modification with the cyclodextrin derivatives. The thermostability was increased by 16 °C for the modified trypsin. Thermal inactivation at different temperatures ranging from 45 to 60 °C was markedly increased for the oligosaccharide–trypsin complexes. This modification also protected the enzyme against autolysis at alkaline pH.
A new method for purification of human plasma retinol‐binding protein and transthyretin
Authors: Raghu Pullakhandam , Ravinder Punjal , Sivakumar Bhattiprolu
Published: September 2003

Retinol is transported in the blood bound to a specific carrier protein called retinol‐binding protein (RBP), which in turn binds to another protein, transthyretin (TTR), a homotetrameric, thyroid‐hormone‐transporting protein. Binding of TTR increases the apparent molecular mass of RBP and thereby prevents glomerular filtration of RBP. Owing to their rapid turnover rates, plasma concentrations of these proteins are sensitive indicators and valuable diagnostic markers of vitamin A nutrition, protein energy malnutrition, infection and renal‐tubule function. Previously RBP and TTR were purified by using different procedures, either from plasma or from pathological urine of humans. In general the procedure for purification of RBP and TTR is laborious, and extensive sample recycling is necessary for purification in appreciable amounts. In the present study, we have purified RBP using a simple method, which involves (NH4)2SO4 fractionation followed by sequential gel filtration under native conditions and 6 M urea. TTR, which was eluted in 60 kDa fractions during urea/Sephadex G‐100 chromatography, was further purified to homogeneity using a combination of two dye‐affinity chromatographic steps on Reactive Yellow and Cibacron Blue coupled to agarose columns. SDS/PAGE and immunoblotting, apart from typical UV absorption and fluorescent properties of RBP, were used for characterizing the purified RBP and TTR. Furthermore, the purified RBP and TTR were found to be functional from mutual binding monitored by fluorescence quenching.
A strategic study using mutant‐strain entrapment in calcium alginate for the production of Saccharomyces cerevisiae cells with high invertase activity
Authors: Rossi‐Alva Juan Carlos , Rocha‐Leão Maria Helena Miguez
Published: September 2003

Entrapped cells and entrapped cells grown inside of a calcium alginate matrix as well as free cells have been investigated using Saccharomyces cerevisiae mutant strains with regard to their pattern of growth and invertase activity. The repression of invertase by glucose and glucose‐consumption ability were considered in the selection process of the mutants. Efficient sucrose bioconversion due to high invertase activity was obtained when entrapped mutant strain Q6R2 cells were grown within calcium alginate gel beads using sucrose plus glucose as the carbon source. Under these conditions, 1 mg (dry weight) of entrapped cells is able to produce 20 μmol of inverted sugar in 3 min (the maximum activity obtained was 20 units·mg−1). The experiments were carried out for 6 months without appreciable loss of either bead integrity or invertase activity. The biocatalyst was also stored at 4 °C for 6 months without appreciable loss of the invertase activity. This work shows that entrapped yeast cells with a weak ability to consume sugar may be used to produce inverted sugar.
A rapid and sensitive fluorometric assay method for the determination of nitrilase activity
Authors: Banerjee Anirban , Sharma Rohit , Banerjee Uttam Chand
Published: September 2003

A rapid, simple and sensitive fluorometric assay method for the determination of nitrilase activity is described. 3‐Cyanopyridine was hydrolysed to nicotinic acid by Rhodococcus rhodochrous and the liberated NH3 was allowed to react with buffered o‐phthaldialdehyde‐2‐mercaptoethanol solution (pH 7.4) to form a fluorochrome. The fluorescence intensity was found to be stable after 20 min incubation at room temperature, and the optimum pH for the reaction was found to be 7.4. The fluorescence intensity was linearly related to enzyme activity with the substrate concentration ranging from 100 to 1000 mM. The activity determined by the proposed method correlates (r=0.9625) well with the established Berthelot method. The proposed method is more sensitive than the existing methods for the determination of nitrilase activity.
Bifunctional constructs of aspirin and ibuprofen (non‐steroidal anti‐inflammatory drugs; NSAIDs) that express antibacterial and alkylation activities
Authors: Bartzatt Ronald , Cirillo Suat L. G. , Cirillo Jeffrey D. , Donigan Laura
Published: September 2003

Ibuprofen and aspirin are two common non‐steroidal anti‐inflammatory drugs (NSAIDs). Both NSAIDs have a carbonyl carbon [‐C(O)‐], which was utilized to attach a nitrogen mustard (N‐mustard) ester group or a tripeptide group. The tripeptide consisted of a l‐Gly‐d‐Ala‐d‐Ala sequence, where d‐Ala‐d‐Ala is the reactive site for antibacterial activity and l‐Gly serves as a linker to the NSAID carrier drug. The aspirin tripeptide and N‐mustard show significant antibacterial activity at 5.0×10−5 M against penicillin‐susceptible or ‐resistant Escherichia coli. The partition coefficients (logKow)logP of aspirin and ibuprofen tripeptide drugs were −1.05 and 2.23, respectively. The NSAIDs served as carrier drugs of the N‐mustard group which expressed alkylation activity directed towards the nucleophilic primary amine of p‐chloroaniline. Hydrolysis of the N‐mustard agents yielded the parent structure of aspirin (or ibuprofen) and an N‐mustard moiety, 2‐[bis(2‐chloroethyl)amino]ethanol. The (logKow)logP for the N‐mustard structures of aspirin and ibuprofen were 2.61 and 5.63, respectively. The (logKow)logP value of 2‐[bis(2‐chloroethyl)amino]ethanol was 0.56. Fluorescamine was utilized to determine unreacted p‐chloroaniline at known time intervals, which permitted calculation of rate constants and rate equations. The aspirin N‐mustard agent expressed strong antibacterial activity against a penicillin‐resistant bacteria and first‐order alkylation kinetics. The ibuprofen N‐mustard and 2‐[bis(2‐chloroethyl)amino]ethanol followed second‐order alkylation kinetics. All N‐mustard and tripeptide compounds showed zero violations of the Rule of 5. Values of TPSA (molecular polar surface area), C logP and molecular dipoles were calculated.
Purification and properties of a transglutaminase produced by a Bacillus circulans strain isolated from the Amazon environment
Authors: De Barros Soares Luís Henrique , Assmann Francine , Záchia Ayub Marco Antônio
Published: September 2003

A new microbial transglutaminase (EC 2.3.2.13) from a Bacillus circulans strain isolated from the aquatic Amazonian environment was purified and characterized. Enzyme purification started with (NH4)2SO4 ‘salting out’ and proceeded with liquid chromatography on Q‐Sepharose FF and octyl‐Sepharose 4 FF. The purification factor was approx. 150‐fold with a yield of 32%. The enzyme's molecular mass was estimated as 45000 Da on SDS/PAGE. The purified transglutaminase had an optimum temperature of 47 °C, the optimum pH of the reaction was 7 and it presented no calcium‐dependent activity.
Purification of soluble human epidermal growth factor (hEGF) from recombinant Escherichia coli culture broth by using expanded‐bed adsorption chromatography
Authors: Lee Yoon Seok , Suh Chang Woo , Park Seung Kook , Lee Eun Kyu
Published: September 2003

Human epidermal growth factor (hEGF) secreted by recombinant Escherichia coli was purified from culture broth by expanded‐bed adsorption (EBA) chromatography, strong anion‐exchange chromatography and finally preparative reversed‐phase HPLC (RP‐HPLC). The EBA chromatography step simultaneously captured the hEGF by cationic exchanger and removed the cellular biomass from the diluted culture broth. This step was carried out at high throughput, and resulted in a high yield (>90%) and a purification factor of approx. 20‐fold to >80% purity. Its process performance was well maintained during a 16‐fold scale‐up. After the successive purification steps of anion‐exchange chromatography and RP‐HPLC, the overall yield was approx. 84% and the purity was satisfactory (>99.5%). It was concluded that the purification process was very efficient and scaleable, warranting its implementation in large‐scale manufacturing.
Effect of Pluronic‐block copolymers on the reduction of serum‐mediated inhibition of gene transfer of polyethyleneimine–DNA complexes
Authors: Kuo Jung‐Hua Steven
Published: September 2003

Serum stability of non‐viral vectors is a crucial factor for successful in vivo gene delivery. Pluronic‐block copolymers consisting of hydrophilic ethylene oxide and hydrophobic propylene oxide blocks were tested to prevent the reduction of serum‐mediated inhibition of gene transfer of polyethyleneimine (PEI)–DNA complexes in NIH/3T3 cells. The order of hydrophilic–lipophilic balance (HLB) of six different types of Pluronics used in this study was F68>F127>P105>P94>L122>L61. Transfection activities of NIH/3T3 cells with PEI–DNA complexes containing Pluronics with higher HLB showed marked improvement of gene‐expression levels in serum media from 10 to 50% fetal bovine serum compared with PEI–DNA complexes alone. Also, higher concentrations (1 and 3%) of Pluronics with higher HLB in the PEI/DNA dispersion provided a stronger steric hindrance in resisting serum components than those obtained in a lower concentration (0.1%). These results suggested that non‐viral vectors incorporated with higher HLB of Pluronics may be used as potential vehicles for in vivo delivery of DNA.
Improvements in spinner‐flask designs for insect‐cell suspension culture
Authors: Annathur Gopinath V. , Pierce Jennifer L. , Combs Rodney G. , Rathore Anurag S. , Banerjee Amit , Steinmeyer David E.
Published: September 2003

Experiments have been conducted to characterize the growth of Spodoptera frugiperda (fall armyworm) (Sf9) cells in standard and modified spinner designs. Results suggest that in standard spinner flasks growth is limited by mixing and oxygen transfer. Modifications to spinner geometry, baffling and agitator aspect ratio provided increases in growth rate and final cell density. Improvements in the performance of spinners, such as those described herein, have the potential to simplify a process and decrease the chance of contamination by decreasing the number of required manipulations and increase process productivity by decreasing inoculum train cycle times.
Optimizing alkaline lysis for DNA plasmid recovery
Authors: Clemson Michael , Kelly William J.
Published: September 2003

Optimization of the alkaline lysis (P2) and neutralization (N3) steps in the recovery of DNA plasmids was pursued. Experiments were conducted at the test‐tube and 5‐litre scales with 3 kb (pUC18) and 20 kb (pQR150) plasmids. The scale and degree of mixing/shear did not affect the optimum yield of supercoiled plasmid during the P2 step, but did effect the time required for the optimum to be achieved. This optimum time for P2 at the large scale was longer (8–9 min), especially when a low‐shear impeller was used. Also, when the yield of supercoiled plasmid reached a maximum during the P2 step, the purity (percentage of plasmids in the supercoiled form) simultaneously reached a minimum. As the duration of the N3 step increased from 1 to 6 min, the yield of the supercoiled plasmids remained fairly constant, provided that a lowshear impeller was used. The neutralized (post‐N3) plasmid solution was shear‐sensitive; however, mixing with a Rushton turbine in a tank (maximum energy dissipation rate in the mixing tank, ɛmax, 12 m2/s3; mixingndash;tank power consumption/volume of mixing tank, 2.0 W/m3) for 5–10 min resulted in a slight decrease in supercoiled plasmid and a notable increase in genomic DNA concentrations. The loss of the larger 20 kb plasmid (20%) was more than for the 3 kb plasmid. Finally, preparing the cells for alkaline lysis with lysozyme or lowndash;pressure homogenization did not increase the plasmid yield. Furthermore, the homogenizer broke up the genomic DNA into fragments that followed through the entire Qiagen prep with the plasmids as impurities.
An apparatus for electrophoretic capture and recovery of circular DNA in thin layers
Authors: Cole Kenneth D.
Published: September 2003

An apparatus was designed for the electrophoretic capture and recovery of circular DNA in thin layers (membranes). Rapid separations were done by the use of a low‐conductivity buffer and high electric field strengths. Two methods that specifically retain circular DNA in the membranes were demonstrated using the supercoiled and open circular forms of two plasmids with sizes of 4.4 and 13 kbp. Electrophoretic trapping (by an impalement mechanism) in agarose‐gel‐filled membranes used electric field strength to immobilize circular DNA in the membranes. The other method of capture utilized the greatly reduced electrophoretic mobility of circular DNA in membranes composed of agarose and the linear polymer hydroxyethylcellulose. The reduction in electrophoretic mobility was not dependent upon the electric field strength, distinguishing it from electrophoretic trapping. Trapping of circular DNA in the membranes followed by size analysis using agarose‐gel electrophoresis could be used as a two‐dimensional separation tool for the analysis of complex mixtures. Captured DNA was recovered by two methods: (i) centrifugation of membranes made with low‐melting‐point agarose resulted in a gel slurry that could be heated to release the DNA; (ii) electroelution of the membranes. Electroelution was done by using an electrode that was isolated from the DNA by a ‘barrier’ membrane. A non‐trapping electric field was used to reverse the DNA out of the membrane into a small volume of buffer above the membrane.
Extraction of cells for single‐stage seeding of vascular‐bypass grafts
Authors: Tiwari A. , Kidane A. , Punshon G. , Hamilton G. , Seifalian A. M.
Published: September 2003

Experimental data are reported for the seeding of prosthetic vascular grafts with either mesothelial or endothelial cells as part of a research strategy in tissue engineering with the aim of improving graft patency and developing new techniques for single‐stage cell extraction and seeding that would give a step reduction in surgery time. New data are reported for two different sources of cells, peritoneal lavage and subcutaneous fat. All experiments were undertaken in patients undergoing abdominal aortic aneurysm repair. Cells extracted from peritoneal lavage were insufficient for a single‐stage seeding process. Subcutaneous fat was processed using either a positive cell‐extraction method using CD31 Dynabeads or by a negative extraction method using CDw90‐coated magnetic beads. Only positive cell extraction gave reliably sufficient numbers of endothelial cells as a source for single‐stage seeding of vascular grafts.
Large‐scale capture and partial purification of plasmid DNA using anion‐exchange membrane capsules
Authors: Zhang Shiying , Krivosheyeva Alla , Nochumson Samuel
Published: September 2003

The demand for larger quantities of high‐purity plasmids continues to increase. Substantial quantities of plasmid DNA are needed to support viral and plasmid‐based gene‐therapy programmes, including drug delivery, preclinical and clinical studies and production of DNA vaccines. Reliance on fermentation, which generates large lysate volumes, for producing the needed quantities of plasmid DNA is becoming more widespread. Development of an efficient capture‐unit operation for very large plasmid DNA molecules from these large lysate volumes can present a considerable challenge. Use of conventional chromatographic beaded media for plasmid capture is problematic, owing to their restrictive pores, which limits access of the large DNA molecules to only those binding sites on the beads' outer surface. Anion‐exchange membranes, on the other hand, have large convective pores that allow the plasmid DNA to readily access all of the membrane's anionic binding sites, even at very high flow rates. The convenience of anion‐exchange membranes, configured in ready‐to‐use capsules, can greatly simplify large‐scale plasmid purification strategies. The effectiveness of membrane‐based technology for the capture of a pCAT reporter plasmid from large volumes of clarified Escherichia coli lysate was demonstrated. The captured and eluted plasmid pool was found to have been concentrated 10‐fold with a reduction in endotoxin of four orders of magnitude.
Scaleable processes for the manufacture of therapeutic quantities of plasmid DNA
Authors: Shamlou Parviz Ayazi
Published: September 2003

The need for scaleable processes to manufacture therapeutic plasmid DNA (pDNA) is easy to overlook when attention is focused primarily on vector design and establishment of early clinical results. pDNA is a large molecule and has properties that are similar to those of the contaminating chromosomal DNA. These, combined with the low initial concentration of plasmids in the host cell, provide unique process challenges that require significant upfront design to establish robust manufacturing processes that can also comply with current Good Manufacturing Practice (‘cGMP’) and produce milligram‐to‐kilogram quantities of pDNA product. This review describes promising scaleable processes that are currently being assessed for production of therapeutic supercoiled pDNA. Fermentation strategies for improving supercoiled plasmid yield and reducing contaminant concentrations are reviewed, and downstream processes are assessed for their ability to efficiently remove cellular contaminants, separate the supercoiled form of the pDNA from its open circular and linear forms, and prepare the purified drug substance for formulation. Current strategies are presented for developing stable delivery systems, and approaches to quality assurance and quality control are discussed.
The influence of physico‐chemical and process conditions on the physical stability of plasmid DNA complexes using response surface methodology
Authors: Mount Clare N. , Lee Li K. , Yasin Ahmad , Scott Andrew , Fearn Tom , Shamlou Parviz Ayazi
Published: September 2003

Research is progressing fast to find safe and effective methods of delivering therapeutic genes to patients afflicted with a range of genetic and acquired diseases that either do not respond at all, or respond poorly, to treatment with small‐molecule drugs or protein‐replacement therapy. A technical barrier that remains relates to the need for scalable operations that can consistently and reproducibly make large quantities of the therapeutic gene vectors under the current Good Manufacturing Practice (‘cGMP’). The present investigation focuses on these issues and introduces a new method of assessing the engineering effects of process and material factors on the colloidal properties of plasmid‐DNA delivery systems based on response surface methodology (RSM) and experimental techniques. Previously, experiments have shown that several factors can reduce the physical stability of non‐viral delivery systems. Specifically, it has been demonstrated that the mean size and charge of plasmid DNA condensed by cationic agents are affected by many factors, including the pH and ionic strength of the buffer, and the method of preparation. For example, the method and intensity of mixing of the DNA with condensing and conjugating agents have been shown to be important. Using RSM to analyse new experimental data in the present paper, we report on the impact of these factors and, more crucially, the effects of interaction between the factors on the colloidal properties of the DNA–vector complexes. Specifically, for plasmid DNA condensed by poly‐l‐lysine, interactions between ionic strength, pH and DNA concentrations play a critical role. Whether poly‐l‐lysine should be used as a condensing agent in the final delivery system remains to be demonstrated. However, the use of RSM combined with the scaleable experimental approach described in this paper may be applied to other delivery systems.
Inhibitory effects in the side reactions occurring during the enzymic synthesis of amoxicillin: p‐hydroxyphenylglycine methyl ester and amoxicillin hydrolysis
Authors: Gonçalves Luciana R. B. , Fernandez‐Lafuente Roberto , Guisan Jose M. , Giordano Raquel L. C. , Giordano Roberto C.
Published: September 2003

Penicillin G acylase immobilized on glyoxyl‐agarose is used to catalyse the reaction between p‐hydroxyphenylglycine methyl ester (POHPGME) and 6‐aminopenicillanic acid (6‐APA). Inhibitory effects affecting the side reactions that occur during the synthesis of amoxicillin have been reported and need to be considered when proposing a kinetic model for the enzymic synthesis. In this work, we present a semi‐empirical kinetic model that successively includes different inhibitory effects in the rate equations. The model performance was always compared with experimental data on amoxicillin synthesis. Enzyme load and stirring rate were chosen to prevent diffusional effects. Our results indicate that POHPGME and amoxicillin were competitive inhibitors of the hydrolysis of amoxicillin and POHPGME, respectively. 6‐APA was a competitive inhibitor of the hydrolysis of amoxicillin. POHPG was a competitive inhibitor and methanol a non‐competitive inhibitor of the hydrolysis of both ester and antibiotic, but the action of methanol was only noticeable at very high concentrations. Adding inhibitory effects to the kinetic model led to a significant increase in the accuracy of the simulations of the overall process of synthesis.
Expression of the Bacillus thuringiensis Cyt2Aa1 toxin in Pichia pastoris using a synthetic gene construct
Authors: Gurkan Cemal , Ellar David J.
Published: September 2003

Bacillus thuringiensis δ‐endotoxins are membrane‐active, pore‐forming proteins with highly specific insecticidal activities. In addition to a well‐established role in the biological control of a wide variety of crop pests and disease vectors, these toxins also have great potential for the development of anti‐tumour agents called immunotoxins (ITs), chimaeric molecules consisting of a cell‐binding ligand coupled to a toxin or its subunits. The ultimate goal of our study was the recombinant production of such ITs based on the Cyt2Aa1 toxin from B. thuringiensis subspecies kyushuensis. We explored the use of Pichia pastoris for recombinant IT production because earlier attempts in our laboratory using the Escherichia coli expression system or various chemical conjugation strategies yielded only low levels of functional product. However, our initial attempts were not successful because the A+T‐rich bacterial cyt2Aa1 gene contained fortuitous polyadenylation sites, causing premature transcription termination in this yeast. Accordingly, we designed and constructed a synthetic cyt2Aa1 gene (syncyt2Aa1) optimized for heterologous expression in P. pastoris. This was achieved by increasing the overall G+C content of the bacterial cyt2Aa1 while changing its codon usage to that preferred by the methylotrophic yeast. Here we describe in detail the design, synthesis and requisite PCR repair of syncyt2Aa1, then present analyses of recombinant Cyt2Aa1 expression in P. pastoris using this synthetic gene. Following the results presented in this paper, the syncyt2Aa1 gene was also successfully used for the recombinant production of a Cyt2Aa1‐based IT in the same expression host [C. Gurkan and D. J. Ellar (2003) Protein Expression Purification 29, 103–116].
Impact of plasmid size on cellular oxygen demand in Escherichia coli
Authors: Kay Alison , O'Kennedy Ronan , Ward John , Keshavarz‐Moore Eli
Published: September 2003

Escherichia coli DH5α harbouring either pBGS18, a 4.4 kb pUC‐based plasmid, or pQR150, a 20 kb derivative of pBGS18, were grown in glucose‐limited chemostat culture to investigate the effects of plasmid size and recombinant protein production on oxygen demand. Under non‐induced conditions, where no recombinant protein was expressed, the cellular oxygen demand of cells harbouring pBGS18 and pQR150 was not significantly different, mean specific oxygen uptake rate (OUR) being 0.75±0.24 and 0.78±0.32 mmol/h per mg of plasmid respectively. Under isopropyl β‐d‐thiogalactoside‐induced conditions, where recombinant protein was expressed, cells harbouring pBGS18 demonstrated a statistically insignificant 37% increase in mean specific OUR while those harbouring pQR150 demonstrated a statistically significant 415% increase. It is concluded that plasmid size does not significantly affect oxygen demand and that increasing oxygen demand observed with increasing plasmid size is due to production of recombinant protein.
Evaluation of an ion‐exchange membrane for the purification of plasmid DNA
Authors: Endres Heather N. , Johnson Julie A. C. , Ross Carl A. , Welp John K. , Etzel Mark R.
Published: September 2003

Separation and purification of large quantities of plasmid DNA (pDNA) is a particularly difficult manufacturing issue because of the relatively low capacity, flow rate and purity observed using traditional bead‐based chromatography. The objective of the present study was to evaluate the performance of anion‐exchange membranes for the purification of pDNA fromEscherichia colilysate solution. The fate of host‐cell protein and endotoxin relative to pDNA was measured and used to calculate recoveries, mass balances, dynamic capacities and purification factors as a function of the flow rate and loading volume of the lysate solution. Breakthrough curves were not sigmoidal and symmetric in shape. They rose sharply at first, and then slowly towards, but never reaching, saturation. Conversely, elution curves were independent of flow rate. pDNA bound tightly to the membranes, whereas protein and endotoxin did not. Dynamic binding capacity for pDNA was 20–25 times greater, and the flow rate was 55–550 times greater, than values observed for beads. However, some pDNA bound irreversibly to the membrane surface and was not removed completely during elution. The intrinsic rate of pDNA adsorption to the membrane was found to be rate‐limiting, whereas effects of liquid‐phase mass transfer and flow non‐idealities were negligible. These results were interpreted using models of adsorption that included steric effects using the ‘car‐parking‐problem’ model, and surface residence time effects using the spreading model. This work demonstrated the advantages of ion‐exchange membranes compared with beads for the purification of large biomolecules such as pDNA.
MINIREVIEW: Perspectives on plasmid‐based gene therapy: challenges for the product and the process
Authors: Kelly William J.
Published: September 2003

Clinical trials for gene‐therapy products began in the 1990s. Currently, most of the 630 completed or on‐going trials are in Phase I and II. Because of the safety issues surrounding the use of viral vectors, approx. 25% of the ongoing clinical trials use DNA plasmids to carry the corrective gene (http://www.wiley.co.uk/genmed). The purpose of this perspective (and this issue) is to highlight some of the unique technical challenges and potential solutions associated with the manufacture and expression of a plasmid‐based gene‐therapy product.
Evaluation of inducible promoters on the secretion of a ZZ‐proinsulin fusion protein in Escherichia coli
Authors: Mergulhão Filipe J. M. , Monteiro Gabriel A. , Larsson Gen , Bostrom Maria , Farewell Anne , Nyström Thomas , Cabral Joaquim M. S. , Taipa M. Ângela
Published: September 2003

Four inducible promoters, uspA, uspB, lacUV5 and malK, were evaluated in the expression of the fusion protein ZZ‐proinsulin by Escherichia coli. The aim was to select for their effects on the most appropriate expression system (promoter and culture medium) for secretion of ZZ‐proinsulin to the periplasmic space and culture medium. All the expression vectors contained the RNase III cleavage site to ensure that the mRNA translation rate remained independent of 5′‐untranslated regions thus making promoter strength comparisons more accurate. The highest ZZ‐proinsulin secretion yields were 6.2 mg/g of dry cell weight in the periplasmic space and 2.6 mg/g of dry cell weight in the culture medium using the malK promoter. It was also demonstrated that the use of M9 minimal medium favours secretion.
Menadione knocks out Vitreoscilla haemoglobin (VHb): the current evidence for the role of VHb in recombinant Escherichia coli
Authors: Aydin Suleyman
Published: September 2003

Genetically engineering the heterologous bacterial host with the gene (vgb) encoding Vitreoscilla haemoglobin (VHb) has been found to provide typical advantages in growth and production, and it has generally been assumed that VHb is responsible for this effect. Here, using matched strains of Escherichia coli that bear a recombinant R‐amylase gene (MK57) or the R‐amylase gene and vgb (MK79), we examined this assumption. Menadione, which is known to oxidize haem proteins, was tested over a range of concentrations for its effects on growth, R‐amylase production, respiration and VHb function in MK57 and MK79. Active VHb accumulated, and VHb was oxidized to the inactive ferric form with the use of menadione at the concentrations of 0.5–10 mM; concentrations that had a much smaller effect on cytochrome oxidase. This decrease in active VHb in strain MK79 was correlated with a reverse in the advantage regarding R‐amylase production of MK79 over MK57 seen at a menadione concentration of 0 mM, thus linking the presence of active VHb with the increase in R‐amylase production. It is concluded that vgb, and not any other Vitreoscilla DNA squences on plasmid pMK79, is the source of the advantages in both the growth and product production of α‐amylase in this system.
Prediction of size distribution of lipid–peptide–DNA vector particles using Monte Carlo simulation techniques
Authors: Sarkar Supti , Zhang Hu , Levy Susana M. , Hart Stephen L. , Hailes Helen C. , Tabor Alethea B. , Shamlou Parviz Ayazi
Published: September 2003

Concerns with insertional mutagenesis for retrovirus and immunogenicity for adenovirus have motivated research into development of non‐viral vectors that can safely deliver desired gene constructs to target cells in tissues and organs. Many non‐viral vectors suffer from unacceptably poor in vivo cell transfection and low transgene expression. Evidence suggests that cell transfection is linked to particle size – vector particles below about 200 nm are considered desirable. Experimental measurements indicate, however, that vector particles are susceptible to significant aggregation under most conditions of pH and ionic strength, including physiological conditions, although there are currently no means of predicting the kinetics of aggregation. The present paper addresses this challenge by presenting a mathematical framework based on the Monte Carlo simulation techniques for modelling the dynamics of aggregation. The approach is used to simulate the evolution of particle‐size distribution for an integrin‐targeting lipid–peptide–DNA vector system in buffers of different pH and ionic strength. The simulations required two input parameters, including the initial‐size distribution of the particles and a fitting parameter (α). Comparison of simulations with experimental data showed that α was closely related to the zeta potential of the particles in the buffer medium, making simulations fully predictive. The modelling approach may be used in other vector systems.
Modulation of neutrophil complement receptor 3 expression by pneumococci
Authors: WILLIAMS James H. , PAHL Madeleine V. , KWONG Donna , ZHANG Jeng , HATAKEYAMA Denise , AHMAD Khawaja , NADERI Majid , KIM Minh , VAZIRI Nosratola
Published: September 2003

Complement receptor 3 (CR3; CD18/CD11b) plays an important role in the recognition and clearance of Streptococcus pneumoniae (pneumococci) by neutrophils. The purpose of the present study was to characterize the modulation of CR3 surface expression on neutrophils exposed to pneumococci and to assess its functional significance. CR3 was detected with fluorescent phytoerythrin-labelled anti-CR3 (CD11b) antibodies, quantified with a fluorescence cell counter (FACS) and localized by confocal fluorescence microscopy. Uptake of fluorescent FITC-labelled pneumococci was quantified by FACS. Whole blood from healthy volunteers was exposed at 37 °C to killed whole type III Streptococcus pneumoniae (KSP; 108/ml) or to a positive control (Escherichia coli lipopolysaccharide) that enhanced CR3 surface expression on neutrophils to a comparable extent. Varying the concentration of KSP between 105 and 108 organisms/ml progressively augmented CR3 surface expression measured at 1 h, whereas the response declined at 109/ml. The diminished response to 109 KSP/ml proved to be time-dependent, with surface CR3 up-regulated maximally within 5 min, and down-regulated thereafter. Labelling of CR3 during exposure demonstrated accelerated receptor sequestration, and confocal fluorescence microscopy demonstrated internalized CR3. Cooling to 16 °C, to inhibit the up-regulation of CR3 surface expression, also inhibited the uptake of FITC-labelled KSP and morphological changes. Accelerated down-regulation of surface CR3 expression by exposure to 109/ml unlabelled KSP diminished the uptake of labelled KSP added subsequently. In contrast, lipopolysaccharide-induced up-regulation of CR3 expression increased the uptake of labelled KSP. Together, these experiments reveal dynamic modulation of CR3 expression on the surface of neutrophils exposed to pneumococci and a functional correlate of this modulation. Thus neutrophil expression of CR3 changes dynamically in response to exposure of neutrophils to progressively higher concentrations of pneumococci, conditions that mimic early neutrophil recruitment to densely infected lung tissue in acute pneumococcal pneumonia.
Diurnal variation in the renal excretion of modified RNA catabolites in humans
Authors: TOPP Heinrich , UNVERZAGT Susanne , RUDLOFF Silvia , SCHÖCH Gerhard , MANZ Friedrich , FUSCH Christoph
Published: September 2003

Renally excreted modified RNA catabolites [pseudouridine (ѱ), N2,N2-dimethylguanosine (m22G) and N6-threoninocarbonyladenosine (t6A)] are markers of whole-body rates of degradation of rRNA and tRNA, and are thought to be potential indicators of the resting metabolic rate. To investigate diurnal variations of these RNA catabolites, the amounts of ѱ, m22G and t6A excreted were determined by HPLC of the urine from eight healthy male adults collected over 47-h periods, which were subdivided into the morning (06.00 or 09.00 to 12.00 hours), the afternoon (12.00 to 18.00 hours), the evening (18.00 to 24.00 hours) and the night (00.00 to 06.00 or 08.00 hours), under two different nutritional regimens with 100 or 50 g of protein/day. Furthermore, rates of degradation of rRNA and tRNA were calculated using values for these RNA catabolites. For comparison, the corresponding excretion of creatinine, which originates from the energy metabolism of muscle, and of 3-methylhistidine (m3His), which is an indicator of muscle protein degradation, was determined. Differences in excretion during the collection periods were tested using the Friedmann test. The excretion of ѱ, creatinine and m3His (μmol·h-1·kg-1) altered significantly (P<0.001) during the day. Medians were: for ѱ, 0.21 (morning), 0.19 (afternoon), 0.19 (evening) and 0.18 (night); for creatinine, 8.8, 8.4, 8.0 and 7.3 respectively; for m3His, 0.13, 0.11, 0.12 and 0.10 respectively. The excretion rates of m22G and t6A (nmol·h-1·kg-1) altered, but not significantly, during the day; corresponding medians were: for m22G, 9.0, 8.4, 8.0 and 8.4 respectively; for t6A, 4.3, 4.1, 3.9 and 3.9 respectively. From these results it can be concluded that, in order to assess the average daily rates of degradation of tRNA and rRNA using modified RNA catabolites, urine collection should be carried out quantitatively over 24 h periods. Likewise, the catabolites creatinine and m3His must be determined using 24 h urine samples when average daily excretion values are required.
Influence of leg position and environmental temperature on segmental volume expansion during venous occlusion plethysmography
Authors: JORFELDT Lennart , VEDUNG Torbjörn , FORSSTRÖM Elisabeth , HENRIKSSON Jan
Published: September 2003

Blood flow determinations by venous occlusion plethysmography applying the strain-gauge technique are frequently used. A problem with the strain-gauge technique is that the relationship between venous volume and transmural pressure is not linear and, furthermore, changes with the sympathetic tone. The present study tests the hypothesis that these factors lead to a redistribution of venous blood, which may impair the accuracy of the technique. The relative volume expansion rates of four leg segments were studied with the leg in different positions and at disparate temperatures, thereby inducing varying venous pressures and sympathetic tone (n=6). With elevated leg and relaxed veins (at 50 °C), the distal thigh showed a relatively low expansion rate (25.8±4.5 ml·min-1·l-1), whereas values in the calf segments were higher (34.5–39.0 ml·min-1·l-1). With lower initial transmural pressure, calf segments can increase their volume much more during occlusion compared with the distal thigh. In a higher transmural pressure region (lowered leg), the difference in compliance between limb segments is less. In this case, compliance and volume expansion rate was higher in the distal thigh (14.2, 13.5 and 22.2 ml·min-1·l-1 at 10, 20 and 50 °C respectively) than in the calf segments (for the distal calf: 6.4, 7.7 and 16.2 ml·min-1·l-1 respectively). There was a significant interaction (P<0.001) between temperature and leg position, indicating a higher degree of sympathetic vasoactivity in the calf. It is concluded that blood flow determination by strain-gauge plethysmography is less accurate, due to a potential redistribution of the venous blood. Therefore possible influences of variations in sympathetic tone and venous pressure must be considered even in intra-individual comparisons, especially in interventional studies.
Expression of Toll-like receptor 4 is associated with enteroviral replication in human myocarditis
Authors: SATOH Mamoru , NAKAMURA Motoyuki , AKATSU Tomonari , IWASAKA Junji , SHIMODA Yudai , SEGAWA Ikuo , HIRAMORI Katsuhiko
Published: September 2003

Previous studies have demonstrated that inflammatory cytokine expression associated with enteroviral (EV) infection may play an important role in human myocarditis. However, the mechanism of the host immune response against viral pathogens has not been fully understood. The aim of the present study was to determine whether Toll-like receptor 4 (TLR4) and EV RNA are present in human myocarditis. Endomyocardial biopsy samples were obtained from 44 patients with myocarditis and five controls. Levels of plus- and minus-strand EV RNAs and TLR4 mRNA were measured by real-time reverse transcriptase–PCR. Immunohistochemical analysis was performed to identify the cellular source of TLR4 and the EV capsid protein VP1. EV RNA was present in 21 patients with myocarditis and these patients were defined as having either active viral replication (n=15) or latent viral persistence (n=6). Neither strand of EV RNA was detected in controls. TLR4 mRNA expression levels were higher in myocarditis patients than in controls (TLR4/glyceraldehyde-3-phosphate dehydrogenase ratio 1.48±0.17 compared with 0.08±0.06, P<0.001). A positive correlation was found between EV RNA and TLR4 levels (plus-strand vs TLR4: r=0.66, P<0.001; minus-strand vs TLR4: r=0.48, P<0.001). TLR4 immunostaining was observed in infiltrating cells and myocytes in patients with myocarditis. The EV capsid protein VP1 was also found in myocytes. The myocarditis group with EV replication and high levels of TLR4 showed significantly lower systolic function. The present study has shown that increased expression of TLR4 is associated with EV replication and that these RNA levels are related to cardiac dysfunction in human myocarditis.
Blood flow and muscle metabolism in chronic fatigue syndrome
Authors: McCULLY Kevin K. , SMITH Sinclair , RAJAEI Sheeva , LEIGH John S. , NATELSON Benjamin H.
Published: September 2003

The purpose of this study was to determine if chronic fatigue syndrome (CFS) is associated with reduced blood flow and oxidative delivery to skeletal muscle. Patients with CFS according to CDC (Center for Disease Control) criteria (n=19) were compared with normal sedentary subjects (n=11). Muscle blood flow was measured with Doppler ultrasound after cuff ischaemia and exercise. Muscle oxygen delivery was measured as the rate of post-exercise and post-ischaemic oxygen-haem resaturation. Oxygen-haem resaturation was measured in the medial gastrocnemius muscle using continuous wavelength near-IR spectroscopy. Muscle metabolism was measured using 31P magnetic resonance spectroscopy. CFS patients and controls were not different in the peak blood flow after cuff ischaemia, the rate of recovery of phosphocreatine after submaximal exercise, and the rate of recovery of oxygen saturation after cuff ischaemia. In conclusion, CFS patients showed no deficit in blood flow or oxidative metabolism. This suggests that CFS symptoms do not require abnormal peripheral function.
Measuring whole-body actin/myosin protein breakdown in mice using a primed constant stable isotope-infusion protocol
Authors: VISSERS Yvonne L. J. , VON MEYENFELDT Maarten F. , BRAULIO Valeria B. , LUIKING Yvette C. , DEUTZ Nicolaas E. P.
Published: September 2003

To measure actin/myosin protein breakdown, the 24 h excretion of Nτ-methylhistidine (3MH) is used. However, in mice, this method is invalid. Therefore we have developed a liquid chromatography-MS technique to measure the tracer/tracee ratio and concentration of 3MH in plasma, enabling an in vivo primed constant infusion protocol with a deuterated stable isotope of 3MH. We tested this model by giving a primed constant infusion of L-[3-methyl-2H3]histidine, L-[phenyl-2H5]phenylalanine and L-[phenyl-2H2]tyrosine to three anaesthetized experimental groups: mice receiving saline intraperitoneally (i.p.) (CON), mice receiving saline i.p. and starved for 9 h (STA), and mice receiving lipopolysaccharide i.p. and starved for 9 h (STA + LPS). The contribution of myofibrillar to total protein breakdown was significantly lower in the STA group than the CON group (30±4% and 54±14% respectively; P<0.05), and was significantly higher in the STA + LPS group than the STA group (52±7% and 30±4% respectively; P<0.05). Whole-body myofibrillar protein breakdown, total protein breakdown, protein synthesis and net protein breakdown were not different between the groups. We conclude that this in vivo primed constant stable isotope-infusion protocol can give valuable information about the role of actin/myosin protein breakdown in mice.
Circulating adhesion molecules are correlated with ultrasonic assessment of carotid plaques
Authors: HASHIMOTO Hiroyuki , KITAGAWA Kazuo , KUWABARA Keisuke , HOUGAKU Hidetaka , OHTSUKI Toshiho , MATSUMOTO Masayasu , HORI Masatsugu
Published: September 2003

The relationship between levels of circulating intercellular cell-adhesion molecule-1 (cICAM-1) or P-selectin (cP-selectin) and the severity of carotid atherosclerosis was examined in 301 outpatients undergoing duplex ultrasonographic examination. Carotid plaque was defined as an intima-media thickness greater than 1.0 mm, and a plaque score (PS) was calculated from the plaque thickness in both carotid arteries. Multivariate analysis demonstrated significant positive associations between cICAM-1 and the number of plaques [β = 0.11; confidence interval (CI), 0.007–0.213], maximum intima-media thickness (β = 0.11; CI, 0.01–0.219), and PS (β = 0.10; CI, 0.001–0.205). In contrast, no significant association was found for cP-selectin. cP-selectin did not increase until atherosclerosis was advanced (PS > 10), showing a marked increase in patients with ≥ 50% stenosis. The circulating levels of both proteins are related to real measurements of plaque formation in the carotid arteries independently of classical risk factors. Marked elevation of cP-selectin occurs in advanced carotid atherosclerosis after gradual elevation of cICAM-1.