bluuebunny/crossref_metadata_2025 huggingface dataset:
Repeated administration of hepatitis C virus core‐encoding plasmid to mice does not necessarily increase the immune response generated against this antigen
Authors: Dueñas‐Carrera Santiago , Alvarez‐Lajonchere Liz , Alvarez Julio Cesar , Ramos Thelvia , Pichardo Dagmara , Morales Juan
Published: November 2003

DNA immunization is a promising approach in generating immune responses to infectious pathogens in many different animal models. In an effort to augment the anti‐[hepatitis C virus (HCV) core] immune response, generated after DNA immunization, the importance of vaccination regimen regarding dose and boosting was investigated in the present study. Balb/c mice were intramuscularly injected with an expression plasmid encoding a truncated variant comprising amino acids 1–176 of the HCV core protein. The highest anti‐core antibody titres (1:3700) were detected in mice inoculated with 50–100 μg of core‐encoding plasmid. Additionally, we demonstrated that antibody levels induced by a single injection of DNA could be further increased by boosting with a second injection of DNA three weeks after primary immunization. However, administration of additional doses or lengthening of the resting period between inoculations resulted in similar or even weaker anti‐core antibody responses. A similar anti‐(HCV core) lymphoproliferative response was also detected in animals that had the highest level of anti‐core antibodies. These results indicate that, in clinical trials, vaccination regimen might be a critical factor in generating optimal anti‐(HCV core) immune responses after genetic immunization.
Characterization of protein glycoforms with N‐linked neutral and phosphorylated oligosaccharides: studies on the glycosylation of endoglucanase 1 (Cel7B) from Trichoderma reesei
Authors: García Rossana , Cremata Jose A. , Quintero Omar , Montesino Raquel , Benkestock Kurt , Ståhlberg Jerry
Published: November 2003

Using anion‐exchange chromatography the catalyticdomain of endoglucanase 1 (Cel7B) from Trichoderma reesei was resolved in multiple fractions with different isoelectric points, presumably related to different glycoforms of the enzyme. The protein fractions were analysed using lectins and electrospray MS. Isolated N‐glycans were analysed by fluorophore‐assisted carbohydrate electrophoresis and amine‐adsorption HPLC. The results show that this particular preparation contained at least 14 different glycoforms. The major isoform contained only one GlcNAc, presumably N‐linked, and one mannose, most probably O‐linked to serine/threonine at a separate site. Except for a small population containing Man5GlcNAc2+1–2 Man, the rest of the protein had negatively charged phosphate‐containing N‐glycans. All glycoforms contained at least one O‐linked mannose residue. The increased negative charge of the protein, introduced by oligosaccharide phosphorylation, is the most probable reason for the different isoelectric points and the occurrence of multiple peaks during purification.
Erythrocyte‐mediated delivery of dexamethasone in patients with chronic obstructive pulmonary disease
Authors: Rossi Luigia , Serafini Sonja , Cenerini Luigi , Picardi Francesco , Bigi Leonardo , Panzani Ivo , Magnani Mauro
Published: November 2003

Human erythrocytes from ten patients with chronic obstructive pulmonary disease (COPD) were loaded with increasing amounts of dexamethasone 21‐phosphate and were re‐infused into the original donors. Drug‐loaded erythrocytes acted as circulating bioreactors, converting the non‐diffusible dexamethasone 21‐phosphate into the diffusible dexamethasone. Pharmacokinetic analyses on these patients showed that a single administration of drug‐loaded erythrocytes was able to maintain detectable dexamethasone concentrations in blood for up to seven days. This continuous release of dexamethasone was paralleled by the suspension of β2‐agonist and oral corticosteroid treatments by all of the patients. Thus dexamethasone 21‐phosphate‐loaded erythrocytes are safe carriers for corticosteroid analogues and are a useful alternative to frequent oral or inhaled drugs in elderly patients with COPD.
Chromosome localization and gene‐copy‐number quantification of three random integrations in Chinese‐hamster ovary cells and their amplified cell lines using fluorescence in situ hybridization
Authors: Davies Julian , Reff Mitchell
Published: November 2003

We have used fluorescence in situ hybridization (FISH) to localize three random Chinese‐hamster ovary (CHO) cell chromosomal integration sites and determine gene copy number in their amplified cell lines. Metaphase FISH showed all three to have integrated into different chromosome positions on different chromosomes. All three Geneticin parent cell lines were found to have single integration sites by Southern‐blot analysis, and these data were confirmed using both metaphase and interphase FISH. Following amplification, metaphase FISH showed that amplification in two of the cell lines occurred by duplication in the chromosome arm where the integration occurred. However, for one cell line, amplification resulted in the translocation of amplified genes from one marker chromosome to another. Interphase FISH showed an expected increase in gene copy number upon amplification with methotrexate.
Activation of diacetyldapsone and a preliminary evaluation of a cyclodextrin–diacetyldapsone complex in cultured lung cells
Authors: Nimbalkar Dipali , Birudaraj Raj , Jones Patrick R. , Smith Timothy J.
Published: November 2003

A diacetyldapsone–2‐hydroxypropyl‐β‐cyclodextrin complex (DADDS–CD) was evaluated with regard to the ability of cultured lung cells to activate DADDS to the active metabolite dapsone. The same system was used to assess the effect of the complex on cell growth. The complex was incubated with cells for periods of 24 to 72 h, followed by extraction of metabolites from the incubation medium and analysis by HPLC. In addition, the Trypan Blue exclusion technique was used to assess cell viability during this time period. Results indicated that lung cells could activate DADDS to dapsone and that, while the complex appeared to delay cell growth in the first 24 h period, no significant difference was seen between cells incubated in the presence and absence of the complex at 72 h. These results indicate that DADDS–CD has significant potential as a drug‐delivery system for DADDS in the lung based upon the ability of the cells to activate DADDS. The mixed effects of the complex on cell growth may have important implications when considering the frequency of administration of the complex to the lung.
Structure and expression of an amylopullulanase gene from Bacillus stearothermophilus TS‐23
Authors: Chen Jen‐Tao , Chen Ming‐Chu , Chen Li‐Lin , Chu Wen‐Shen
Published: November 2003

An amylopullulanase gene (apuTS) from Bacillus stearothermophilus TS‐23 was cloned and characterized. apuTS consisted of an open reading frame of 6054 bp encoding a protein of 2018 amino acids with a calculated Mr of 223811. The deduced amino acid sequence revealed four highly conserved regions that are common among amylolytic enzymes. In the C‐terminal region, a six‐amino‐acid sequence (Pro‐Gly‐Ser‐Gly‐Thr‐Thr) is repeated nine times. It shared the highest degree of homology with the amylopullulanase of Bacillus sp. XAL601. The enzyme also had moderate homology with amylopullulanases from thermophilic anaerobic bacteria. Low levels of homology were observed between the ApuTS of B. stearothermophilus TS‐23 and amylopullulanases of Pyrococcus abyssi Orsay, P. furiosus and Bacillus sp. KSM1378. When the intact coding region of apuTS was expressed in Escherichia coli under the control of the lac promoter, the product was degenerate, as revealed by amylase activity staining after SDS/PAGE. The largest active polypeptide had an Mr of about 220000, while the smallest one had an Mr of about 105000. Upstream of the apuTS gene, a gene orfX was fortuitously cloned. The putative OrfX protein was weakly related to the myosin heavy chain. It was predicted to contain a central, 179‐residue‐long, coiled‐coil domain.
Mammalian cell production of a respiratory syncytial virus (RSV) candidate vaccine recovered using a product‐specific affinity column
Authors: Andersson Christin , Hansson Marianne , Power Ultan , Nygren Per‐åke , Ståhl Stefan
Published: November 2003

The recombinant production of a respiratory syncytial virus (RSV) candidate vaccine BBG2Na in baby hamster kidney cells (BHK‐21 cells) was investigated. BBG2Na consists of a serum‐albumin‐binding region (BB) fused to a 101‐amino‐acid fragment of the RSV G‐protein. Semliki Forest virus‐based expression vectors encoding both intracellular and secreted forms of BBG2Na were constructed and found to be functional. Affinity recovery of BBG2Na employing human serum albumin columns was found to be inefficient due to the abundance of BSA in the applied samples. Instead, a strategy using a tailor‐made affinity ligand based on a combinatorially engineered Staphylococcus aureus protein A domain, showing specific binding to the G‐protein part of the product, was evaluated. In conclusion, a strategy for production and successful recovery of BBG2Na in mammalian cells was created, through the development of a product‐specific affinity column.
Immobilization of native and poly(ethylene glycol)‐treated (‘PEGylated’) bovine serum amine oxidase into a biocompatible hydrogel
Authors: Demers Nicole , Agostinelli Enzo , Averill‐Bates Diana A. , Fortier Guy
Published: November 2003

Native bovine serum amine oxidase (BSAO) and poly(ethylene glycol) (PEG)‐treated (‘PEGylated’) BSAO were immobilized into a hydrogel during its synthesis. The hydrogel was obtained by cross‐linking of BSA with PEG di‐nitrophenyl carbonates with a molecular mass of 10 kDa. Approx. 60% of the amino groups at the surface of BSAO were modified by monomethoxy‐PEG with a molecular mass of 5 kDa when the reaction was carried out for 5 h in borate buffer, pH 9. The number of anchorage points of BSAO in the matrix, which was determined as minimal when PEGylated BSAO was used or maximal when native BSAO was used, did not influence the apparent Km and Vmax values of the different preparations. The apparent Km values of both forms of the enzyme were decreased due to preconcentration of benzylamine substrate by the negatively charged hydrogel. Vmax values were generally lower upon immobilization. We can therefore conclude that the hydrogel swelling has no significant effect on the enzyme's structure. The operational stability, evaluated in the presence of substrate, was generally increased upon enzyme immobilization into the hydrogel. Enzymic hydrogels were very stable during storage in solution at 4 °C, maintaining a high activity even after several weeks. The immobilization of both forms of BSAO did not improve their thermostability at 65 °C. The BSA‐PEG hydrogel is a good matrix for immobilization of enzymes with therapeutic potential such as BSAO.
Adhesion of tear proteins to contact lenses and vials
Authors: Keith Edward O. , Boltz Melissa , Gadh Rundeep , Ghorsriz Roya , Mangatt Donna , Janoff Lester E.
Published: November 2003

Tear‐protein adhesion to contact lenses contributes to lens contamination and deterioration, and depends primarily on the type of contact‐lens material used. Adhesion of tear proteins to contact lenses and glass vials was examined using spectrophotometry, hydrophobic‐interaction HPLC and bicinchoninic acid (BCA) protein assay. Using spectrophotometry at 280 nm, vials containing two proteins (lysozyme and milk lipocalin) at three different concentrations were examined for changes in their protein concentrations over a 3‐week period. Vials containing 5.0 mg/ml lysozyme lost about one‐third of their protein (P<0.005), while vials containing 5.0 mg/ml milk lipocalin lost two‐thirds of their protein (P<0.01). Conversely, vials containing 1.0 mg/ml lysozyme lost two‐thirds of their protein (P<0.005), while vials containing 1.0 mg/ml milk lipocalin lost one‐third of their protein (P>0.05). Subsequently, lysozyme deposition on both glass vials and contact lenses was monitored for 5 days using spectrophotometry, to determine lysozyme content in the vials, and HPLC, to determine lysozyme deposition on contact lenses stored in the same vials. This experiment indicated considerable variation in lysozyme deposition on vials and contact lenses, with vial deposition remaining relatively constant (P>0.05) while lens deposition decreased and then increased (P<0.05). Finally, the same experiment was repeated, monitoring lysozyme deposition using the BCA assay. This experiment yielded the most consistent results, with lysozyme deposition on vials continuing throughout the 5 day experiment (P<0.05), while deposition on lenses again decreased and then increased, although to a much lesser extent than in the previous experiment (P>0.05).
Process characterization for metal‐affinity chromatography of an Fc fusion protein: a design‐of‐experiments approach
Authors: Shukla Abhinav A. , Sorge Laura , Boldman Joshua , Waugh Steve
Published: November 2003

The utility of a design‐of‐experiments approach was investigated for process characterization of a metal‐affinity chromatographic purification process for an Fc fusion protein. This approach gave a better understanding of some of the key process variables as well as robustness for this step in the purification process. Single‐variable experiments were employed to screen some of the potentially important variables in this step. Ranges for these variables were set based on prior experience in clinical manufacturing with similar processes. Following these experiments, a fractional factorial study was employed to further investigate the most important variables and their interactions. Key operational variables that had an impact on step yield and eluate purity were identified. In addition, the study helped identify a worst‐case scenario for the step purity and helped assure that the rest of the process would successfully purify the product. This paper demonstrates the utility of a design‐of‐experiments approach for the characterization and validation of process chromatography steps in downstream processing. In addition, this study emphasizes the utility of robustness studies early in process development and establishes a strategy for future robustness studies.
Two telomerase reverse transcriptases (TERTs) expressed in Candida albicans
Authors: Metz Anneke M. , Love Ruschelle A. , Strobel Gary A. , Long David M.
Published: November 2003

The human pathogenic yeast Candida albicans contains two telomerase reverse transcriptase (TERT) genes. CaTERT1 and CaTERT2 appear either to be two alleles of the same gene or two entirely different genes that encode 867‐residue proteins that differ by five amino acids. Both TERTs have a calculated pI of 9·5 and a Mr of 100·9 kDa and are the smallest TERTs identified to date. Both genes appear to be expressed. Based on sequence similarity between CaTERT1 and the Saccharomyces cerevisiae orthologue Est2p, we suggest a revised alignment for motif E of Est2p. The identification of these TERT genes provides the first opportunity to study telomerase in an important human pathogen.
Impaired l‐arginine uptake in platelets from type‐2 diabetic patients
Authors: Signorello Maria Grazia , Giovine Marco , Pascale Raffaele , Cataldi Luigi , Bordone Caterina , Benatti Umberto , Leoncini Giuliana
Published: November 2003

The l‐arginine uptake of resting platelets from type‐2 diabetic patients and control subjects was measured and the kinetic parameters defined. The effect of platelet stimulation with agonists was also investigated. Kinetic studies showed that the Km value for l‐arginine transport was not different in patients compared with control subjects, while Vmax was significantly decreased in patients compared with controls. Moreover, agonists able to mobilize Ca2+ produced a further decrease in the l‐arginine uptake in controls and in patients, suggesting that Ca2+ intracellular levels down‐regulate l‐arginine transport in platelets from both control subjects and patients. Data suggest that drugs designed to control intracellular Ca2+ might restore platelet function in diabetic patients.
An associated process for the purification of immuno globulin G, catalase, superoxide dismutase and albumin from haemolysed human placenta blood
Authors: Grellet Sheyla , Martins Elizabeth A. L. , Maimoni Gonçalves Viviane , Yague Lopes Alexandre Paulo , Raw Isaías , Cabrera‐Crespo Joaquin
Published: November 2003

The human placenta is a rich raw material for production of many biopharmaceutical products. Here we describe a co‐purification process for the production of four different proteins from haemolysed human placenta blood: IgG, catalase (Cat), superoxide dismutase (Sod) and albumin (Alb). The process can be divided in two parts: the common steps and the specific separation techniques for each protein. The common steps are: extraction, haemoglobin precipitation, concentration/diafiltration and the first Q‐Sepharose chromatography step. At this chromatography step the process is branched: while IgG and Cat were recovered in the flow‐through, Sod and Alb were eluted separately. IgG and Cat were separated in a second Q‐Sepharose chromatography step during which IgG was recovered in the flow‐through, whereas Cat bound to the resin. IgG was purified by S‐Sepharose chromatography, followed by selective precipitation with n‐octanoic acid, yielding about 0.4 g of IgG per kg of placenta. Cat was eluted at the second Q‐Sepharose chromatography step and was purified by Blue Sepharose chromatography. A total of 1.8×106units of Cat were recovered/kg of placenta, with a specific activity of 45000 units/mg of protein. Sod was further purified by S‐Sepharose and Phenyl‐Sepharose chromatography steps and recovered in the non‐adsorbed fractions. The yield of Sod was 2.1×105units/kg of placenta, with a specific activity of 1194 units/mg of protein. Alb purification was followed by a combined process including thermocoagulation and treatment with activated charcoal. The final step was Phenyl‐Sepharose chromatography. The process yielded 3.1 g of Alb/kg of placenta. The described methodology was designed to be easily scaled‐up for industrial production.
Cashew‐tree (Anacardium occidentale L.) exudate gum: a novel bioligand tool
Authors: Da Silveira Nogueira Lima Raquel , Rabelo Lima Jacira , Ribeiro de Salis Celio , De Azevedo Moreira Renato
Published: November 2003

The potential of bioaffinity as a tool for the study of biological‐recognition mechanisms is gaining increasing value. The search continues for alternative products that can be obtained from renewable sources, such as the bark exudate gum from the cashew tree (Anacardium occidentale L.), which grows wild in many tropical and subtropical countries. Its potential use as a chromatographic matrix and/or for bioaffinity ligand for proteins (lectins) has been investigated. The crude gum was cross‐linked in order to obtain a kind of chromatographic matrix (gel). To evaluate the gum's ability to retain glycoproteins (lectins), affinity chromatography was performed and, in addition, the reological behaviour of the gum was characterized.
Enhanced thermal stability of an alkaline protease, AprP, isolated from a Pseudomonas sp. by mutation at an autoproteolysis site, Ser‐331
Authors: Jang Jae Woo , Ko Jung Ho , Kim Eun Kyung , Jang Won Hee , Kang Joo Hyun , Yoo Ook Joon
Published: November 2003

The thermal stability of the alkaline protease extracellular subtilisin‐type serine protease (AprP) from Pseudomonas sp. KFCC 10818 was improved by altering an amino acid residue at an autoproteolytic cleavage site. N‐terminal sequence analysis of the autoproteolytic products of the protein revealed the presence of two cleavage sites, Ser‐307 and Ser‐331. To increase the thermal stability of the enzyme, serine residues of these sites were replaced with aspartate. The S331D mutant enzyme was successfully purified and characterized whereas the S307D mutant was not. The half‐lives of the S331D mutant at 55 °C and 60 °C were 1.5 and 2.4 times longer than that of the wild‐type enzyme, respectively. In addition, the catalytic efficiency was also enhanced.
Transphosphatidylation by immobilized phospholipase D in aqueous media
Authors: Dittrich Nadeshda , Ulbrich‐Hofmann Renate
Published: November 2003

Phospholipase D (PLD) from Streptomyces sp. was immobilized by covalent binding to aminopropyl‐glass activated by glutardialdehyde and to the macroporous synthetic polymer VA‐Epoxy Biosynth (from Riedel‐de Häen, Seelze, Germany) pre‐activated by epoxy groups. The immobilized PLDs were examined for the synthesis of phosphatidylglycerol (PG) from soybean (Glycine max) phosphatidylcholine (PC) in purely aqueous solutions in comparison with commonly used diethyl ether/buffer systems. In contrast with general assumptions, the transphosphatidylation was shown to yield a high percentage of PG, even in pure buffer. With PLD immobilized to VA‐Epoxy Biosynth, the formation of phosphatidic acid (PA) is insignificant, while the yield of PG amounts to 60%. With PLD immobilized to porous glass (average pore diameter 17 nm), higher yields of PG (72%) are reached, but the formation of PA also increases (up to 10%). In comparison with the reaction in the diethyl ether/buffer system, however, the conversion of PC into PG proceeds much more slowly. Detergents such as Triton X‐100 accelerate the reaction.
Immobilization of anl‐aminoacylase‐producing strain ofAspergillus oryzaeinto gelatin pellets and its application in the resolution of D,L‐methionine
Authors: Yuan Ying‐jin , Wang Shu‐hao , Song Zheng‐xiao , Gao Rui‐chang
Published: November 2003

The conditions for immobilization of anl‐aminoacylase‐producing strain ofAspergillus oryzaein gelatin and the enzymic characteristics of the immobilized pellets were studied. The optimal concentrations of gelatin, glutaraldehyde and ethyldiamine and time of immobilization were determined. Scanning electron micrographs reveal the cross‐linked structure differences between the native and immobilized pellets. Optimum pH and temperature of the native and immobilized pellets were determined. Effects of ionic strength and substrate concentration on relative activity of the native and immobilized pellets were investigated in detail. The immobilized pellets were more stable over broader temperature and pH ranges. In addition, the immobilized pellets showed stable activity under operational and storage conditions. The immobilized pellets lost about 20% of their initial activity after five cycles of reuse. The results reported in this paper show the potential for using the immobilizedA.oryzaepellets to resolved,l‐methionine.
Expression, purification, immunological characterization and application of Escherichia coli‐derived hepatitis C virus E2 proteins
Authors: Liu Jing , Zhu Lixin , Zhang Xinxin , Lu Min , Kong Yuying , Wang Yuan , Li Guangdi
Published: November 2003

The envelope glycoprotein E2 of hepatitis C virus (HCV) has been shown to bind human target cells. Anti‐E2 antibodies have been associated with both recovery from natural infection in humans and protection from challenge with homologous HCV in chimpanzees. Therefore E2 has become a major target for the development of anti‐HCV vaccines. Two E2 fragments [amino acids (aa) 450–565 and aa 385–565] derived from a subtype 1b HCV genome were expressed as N‐terminally hexahistidine‐tagged proteins in Escherichia coli and purified to over 85% purity. Both proteins were specifically recognized by homologous hepatitis‐C‐patient's serum on Western blotting, suggesting that these E. coli‐derived E2 proteins displayed E2‐specific antigenicity. E2‐116 (aa 450–565) elicited strong antibody responses in BALB/c mice and rabbits. Rabbit antiserum raised against renatured E2‐116 (RE2–116R) was able to recognize subtype 1b and 1a E2 glycoproteins expressed in mammalian cells on Western blotting. E2‐181 (aa 385–565) reacted with 40% of anti‐HCV+ patients’ sera in ELISA. RE2‐116R and E2‐181 were successfully used in preliminary modified vaccinia virus Ankara‐ and DNA‐based E2 vaccine studies for detecting antigen expression in vitro and assessing induced humoral immune responses in mice. The E2 proteins and rabbit antiserum reported here could find wider applications in the development of effective diagnostic, prophylactic and therapeutic measures against HCV.
Identification of an import signal for, and the nuclear localization of, human lactoferrin
Authors: Penco Silvana , Scarfi Sonia , Giovine Marco , Damonte Gianluca , Millo Enrico , Villaggio Barbara , Passalacqua Mario , Pozzolini Marina , Garrè Cecilia , Benatti Umberto
Published: November 2003

Many different unique functions have been attributed to lactoferrin (Lf), including DNA and RNA binding, and transport into the nucleus, where Lf binds to specific sequences and activates transcription. A pentapeptide, Gly‐Arg‐Arg‐Arg‐Arg, corresponding to a region of the N‐terminal portion of human Lf rich in basic amino acids, was synthesized and its intracellular localization was investigated. Peptide internalization was assayed using the rhodaminated form of the same molecule. This N‐terminal peptide sequence is able to be internalized within less than 10 min at concentration as low as 1 μM, and its intracellular localization is nuclear, mainly nucleolar. Similar behaviour was observed using peptides composed of either all l or d amino acids, the last one being a retro‐inverse peptide. The internalization process does not involve an endocytotic pathway, since no inhibition of the uptake was observed at 4 °C. The kinetics of peptide internalization was also evaluated. The internalization properties of such a short Lf pentapeptide have been assayed for its ability to transport peptide nucleic acids (PNAs) inside cells in order to improve their efficacy. The abundant transmembrane transport and nuclear localization of the proposed peptide, deriving from hLf and, for the first time, identified as a nuclear localization signal, could be used as an alternative strategy to tackle the unsolved problem of intracellular accumulation of antisense and antigene drugs and for the development of new pharmacological tools.
Identification of the ligand‐binding domain of human vascular‐endothelial‐growth‐factor receptor Flt‐1
Authors: Ma Li , Wang Xiaoning , Zhang Zhiqing , Zhou Xiaoming , Chen Aijun , Yao Lihong
Published: November 2003

The vascular‐endothelial‐growth‐factor (VEGF) receptor Flt‐1 has been shown to be involved in vasculogenesis and angiogenesis. The receptor is characterized by seven immunoglobulin‐like loops within the extracellular domain and the first three N‐terminal immunoglobulin‐like loops are involved in high‐affinity binding of VEGF. The minimal extracellular domains of Flt‐1 to achieve VEGF binding were screened using the yeast two‐hybrid system. The result showed that the binding capacity of loop2‐3 was close to that of loop1‐3. The two truncated mutants consisting of loop2‐3 and loop1‐3 were expressed in the methylotrophic yeast Pichia pastoris at high levels (0.3 mg/litre). The corresponding proteins, named soluble (s)Flt‐1(2‐3) and sFlt‐1(1‐3), were purified. An in vitro biological activity assay showed that the binding capacity of sFlt‐1(2‐3) to human VEGF165 and the inhibiting effect of it on human umbilical‐vein endothelial cell proliferation stimulated by human VEGF165 were close to those of sFlt‐1(1‐3). Animal tests showed that sFlt‐1(2‐3) could significantly inhibit the formation of regenerated blood vessels stimulated by hVEGF165.
In vitro phagocytosis of carrier mouse red blood cells is increased by Band 3 cross‐linking or diamide treatment
Authors: Jordán José A. , Alvarez F. Javier , Lotero L. Alfredo , Herráez Angel , Díez José C. , Tejedor M. Cristina
Published: November 2003

Chemical alteration of red blood cells (RBCs) can induce increased phagocytosis of modified cells by macrophages. In this study we have used different chemical treatments for the modification of the mouse red‐blood‐cell membrane surface, namely oxidant compounds, such as ascorbate/Fe+2 and diamide [azodicarboxylic acid bis(dimethylamide)], or Band 3‐cross‐linking reagents. We monitored the phagocytosis of oxidized or Band 3‐cross‐linked mouse red blood cells by peritoneal macrophages. The extent of phagocytosis of RBCs is not affected by oxidation with ascorbate/Fe3+, but it is increased (up to 10%) by oxidation with 2 mM diamide. Furthermore, phagocytosis is greatly increased (up to 40%) as a result of cross‐linking with either of two Band 3 bifunctional reagents [bis(sulphosuccinimidyl) suberate (BS3) and 3,3′‐dithiobis(sulphosuccinimidyl propionate) (DTSSP)]. To evaluate targeting towards macrophages of such modified RBCs for therapeutical purposes, we have determined the phagocytosis of Band 3 carrier RBCs loaded with carbonic anhydrase. In this case phagocytosis is high enough (25%) to deliver the enzyme into macrophages. We have also assayed the influence of serum components and IgG on the efficiency of phagocytosis and discuss the possible phagocytosis mechanisms. In the case of BS3‐cross‐linked carrier RBCs, phagocytosis is markedly enhanced (from 12% up to 25%) by serum components. This opens a way for therapeutic application of these carrier RBCs, with special relevance in short‐term delivery to cells of the mononuclear phagocytic system.
Catalytic activities of intracellular dimeric neopullulanase on cyclodextrin, acarbose and maltose
Authors: Cheong Kyung‐A , Kim Tae‐Jip , Yoon Jong‐Won , Park Cheon‐Seok , Lee Tae‐Soo , Kim Young‐Bae , Park Kwan‐Hwa , Kim Jung‐Wan
Published: November 2003

Multi‐substrate specificity of neopullulanase towards cyclodextrin, acarbose and maltose was investigated using a clone originating from Bacillus stearothermophilus IMA6503. The enzyme purified from Escherichia coli harbouring the corresponding nplA gene hydrolysed β‐cyclodextrin (β‐CD) to maltose and glucose. It exhibited substrate preference for β‐CD, starch and pullulan in the proportions of 10.4:1.2:1. The enzyme not only hydrolysed acarbose, an α‐amylase inhibitor, to a pseudotrisaccharide (PTS) and glucose, but also transferred PTS to glucose, forming isoacarbose. Moreover, it hydrolysed maltose to glucose and transferred the glucose to another maltose molecule to form panose when maltose was present at a low concentration (0.5%) in the reaction solution. The enzyme catalysed condensation between two maltose molecules and subsequent hydrolysis of the resulting 62‐O‐α‐maltosyl‐maltose to glucose and panose, when maltose concentration was increased to 20%. Neopullulanase was likely to be present in monomer–dimer equilibrium with a molar ratio of 1:9 in 50 mM sodium acetate buffer (pH 6.0). The association–dissociation equilibrium of neopullulanase was shifted to monomerization by KCl. When the content of monomer increased in the reaction mixture, the specific activity towards soluble starch increased to 150%, while that towards β‐CD decreased to 80%. Therefore, multi‐substrate specificity of neopullulanase was likely to be modulated by the shift of monomer–dimer association equilibrium.
High‐level expression of human thyroid‐stimulating hormone in Chinese hamster ovary cells by co‐transfection of dicistronic expression vectors followed by a dual‐marker amplification strategy
Authors: Peroni Cibele N. , Soares Carlos R. J. , Gimbo Elizabeth , Morganti Ligia , Ribela Maria Teresa C.P. , Bartolini Paolo
Published: November 2003

The utilization of dicistronic mRNA expression vectors, containing the gene of interest upstream of an amplifiable marker gene, has shown success in rapidly, efficiently and reproducibly obtaining stable cell lines that express high levels of the protein of interest. For this reason, human thyroid‐stimulating hormone (hTSH), a heterodimeric glycoprotein composed of non‐covalently linked α‐ and β‐subunits, was expressed in Chinese hamster ovary (CHO) cells using a system based on dicistronic expression vectors. These contained the genes of interest and the amplifiable gene markers dihydrofolate reductase (DHFR) and adenosine deaminase (ADA), separated by an internal ribosome entry site isolated from the encephalomyocarditis virus. After the cells (CHO‐DHFR−) had been co‐transfected with the expression vectors and submitted to gene amplification in culture medium containing stepwise increments of methotrexate, it was possible to isolate clones that presented a secretion level of up to 7.2±1.3 μg/106 cells per day, the highest ever reported for the expression of this glycoprotein hormone. A second treatment, involving the utilization of deoxycoformycin, directed to amplify the ADA marker gene, provided a clone with an additional 2–3‐fold increase in hTSH secretion, reaching a secretion level of 17.8±7.6 μg/106 cells per day. Cell culture and hTSH production in a hollow‐fibre bioreactor were set up in order to carry out a preliminary physico‐chemical, immunological and biological characterization of this hormone in comparison with pituitary‐extracted hTSH (from the National Institute of Diabetes and Digestive and Kidney Diseases) and the only recombinant hTSH now available (Thyrogen®). The availability of recombinant hTSH is very important in the diagnosis and therapy of thyroid carcinoma, via stimulation of radioiodine uptake.
Oligo‐1,6‐glucosidase from a thermophile, Bacillus thermoglucosidasius KP1006, was efficiently produced by combinatorial expression of GroEL in Escherichia coli
Authors: Watanabe Kunihiko , Fujiwara Hisashi , Inui Kazuyo , Suzuki Yuzuru
Published: November 2003

To improve the production of oligo‐1,6‐glucosidase from the obligately thermophilic Bacillus thermoglucosidasius KP1006 in Escherichia coli, the combined expression of oligo‐1,6‐glucosidase with various chaperone proteins of Hsp (heat‐shock protein) 60 team proteins (GroES and GroEL) or Hsp70 team proteins (GrpE, DnaK and DnaJ) from the same thermophile was examined. This attempt was based on the facts that, (i) among glycosyl hydrolases of Family 13, bacillary oligo‐1,6‐glucosidases share highest homology with yeast α‐glucosidase, and (ii) this yeast enzyme interacts with GroEL. In B. thermoglucosidasius Hsp60 team proteins, in particular, GroEL brought about a remarkable rise in expression of B. thermoglucosidasius oligo‐1,6‐glucosidase, while Hsp70 team proteins had no significant effect. The effect of B. thermoglucosidasius GroEL on oligo‐1,6‐glucosidase expression was supported by the finding that thermally inactivated B. thermoglucosidasius oligo‐1,6‐glucosidase was revived by B. thermoglucosidasius GroEL. Although the molecular mass of B. thermoglucosidasius oligo‐1,6‐glucosidase (66 kDa) exceeds the major range of substrates for GroEL proteins, the GroEL molecules probably recognized the α/β motifs contained in the N‐terminal domain and the subdomain of the oligo‐1,6‐glucosidase. Here we show that (i) the production of B. thermoglucosidasius oligo‐1,6‐glucosidase in E. coli was improved 3.8‐fold by Hsp60 team proteins, (ii) the system can function for the expression of other glycosyl hydrolases of Family 13 that have defects in expression and (iii) the combinatorial expression of thermostable proteins with GroEL from the same thermophile in E. coli can increase the production of thermostable enzymes, preventing problems derived from differences in protein biogenesis.
Immobilization, stability and esterification studies of a lipase from a Bacillus sp.
Authors: Dosanjh Nirpjit S. , Kaur Jagdeep
Published: November 2003

In the present paper a comparative account of the immobilization of a Bacillus lipase on different solid supports with different surface properties and their thermostability is presented. Immobilization enhanced the thermostability of lipase. At higher temperatures, lipase immobilized and cross‐linked on a hydrophobic surface showed the maximum thermostability. The optimum temperature for immobilized lipase was 9 °C higher than for the free enzyme, while the pH optima were the same. The half‐life of soluble lipase at 50 °C was calculated to be 4.5 h, while immobilized lipase did not lose any activity even after 8 h. The temperature stability (for 1 h) of immobilized enzyme was enhanced from 50 to 60 °C in comparison with non‐immobilized enzyme. Applications of immobilized lipase for esterification are also presented.
Optimization of the anti‐(human CD3) immunotoxin DT389–scFv(UCHT1) N‐terminal sequence to yield a homogeneous protein
Authors: Hexham J. Mark , King Vicki , Dudas Debra , Graff Patrick , Mahnke Marion , Wang Y. Karen , Goetschy Jean‐Francois , Plattner David , Zurini Mauro , Bitsch Francis , Lake Phil , Digan Mary Ellen
Published: November 2003

The production and regulatory approval processes for biopharmaceuticals require detailed characterization of potential products. Therapeutic proteins should preferably be homogeneous, although limited, reproducible, heterogeneity may be tolerated. A diphtheria toxin‐based anti‐(human CD3) immunotoxin, DT389–scFv(UCHT1), was expressed in Escherichia coli and purified following refolding [DT389 corresponds to amino acids 1–389 of diphtheria toxin, scFv is single‐chain variable‐region antibody fragment and UCHT1is an anti‐(human CD3) monoclonal antibody]. Biochemical characterization of this molecule by MS and N‐terminal sequencing by Edman degradation revealed that the protein was heterogeneous at the N‐terminus, containing species both with (60%) and without (40%) the initiator methionine residue. In an attempt to generate an N‐terminally homogeneous molecule, a panel of seven N‐terminal variants was designed, based on the published specificity of bacterial methionine aminopeptidase. Following bacterial expression, partial purification and separation on SDS/PAGE, these proteins were subjected to N‐terminal sequencing by Edman degradation. Three of the mutants yielded a 100% homogeneous amino acid sequence. By contrast, the original DT389–scFv(UCHT1) protein and four variant proteins yielded two sequences with varying ratios corresponding to species with and without methionine. The N‐terminal sequences of the three homogeneous clones were MLADD and MLDD, where the methionine was completely retained, and SADD, where the methionine was completely removed. One of the homogeneous mutants (SADD) was expressed, refolded and purified and found to be equipotent with the parent immunotoxin. Thus, using a rational mutagenesis approach, three N‐terminally homogeneous variants of DT389–scFv(UCHT1) have been identified, at least one of which is functionally indistinguishable from the parent immunotoxin. This approach is generally applicable to biopharmaceutical production and immunotoxin development in particular.
Enhancement of enzyme activity and enantioselectivity via cultivation in nitrile metabolism by Rhodococcus sp. CGMCC 0497
Authors: Wu Zhong‐Liu , Li Zu‐Yi
Published: November 2003

Racemic 2‐phenylpropionitrile was resolved enantioselectively by nitrile‐converting enzymes in cells of Rhodococcus sp. CGMCC 0497 to S‐(+)‐2‐phenylpropionic acid and R‐(−)‐2‐phenylpropionamide. By optimization of the culture conditions, great enhancement of enzyme activity and enantioselectivity was achieved. Furthermore, the relationship between cell‐growth periodicity and enzyme accumulation was studied; the addition of inducer was delayed by 1 day and the reaction was further improved. This unusual strategy has almost never been reported with other nitrile‐converting strains. The resulting culture broth, containing methacrylamide as the inducer, beef extract as the nitrogen source and glucose as the carbon source, with methacrylamide added 24 h later, seemed to be most suitable. S‐(+)‐2‐Phenylpropionic acid and R‐(−)‐2‐phenylpropionamide were produced with yields of 48% (enantiomeric excess, 96%) and 42% (enantiomeric excess, 97%) respectively with no nitrile left after 3 h, or with yields of 52% and 39% (enantiomeric excess, 93% and 99%) respectively after 6 h.
A simple fractionation protocol for, and a comprehensive study of the molecular properties of, two major endopolygalacturonases from Aspergillus niger
Authors: Singh Sridevi Annapurna , Appu Rao A. G.
Published: November 2003

A comprehensive study on purification and characterization of the two endopolygalacturonases from Aspergillus niger, PG II and PG IV, accounting for 70% of the total polygalacturonase activity, is reported. These enzymes were purified to homogeneity using ion‐exchange chromatography and gel filtration. The enzymes had specific activities of 982 and 3750 units/mg, and their molecular masses were 61 and 38 kDa, respectively. The pH optimum of PG II was pH 3.8–4.3 and for PG IV it was between pH 3 and 4.6, and the temperature optima also differed for the enzymes. The enzymes preferred pectic acid as a substrate, cleaving it at random, leading to the release of oligogalacturonides as products. The Km values of the two enzymes were found to be 0.12 and 0.72% respectively. The enzymes were rich in hydrophilic amino acids and relatively low in the sulphur‐containing amino acids. Both enzymes were rich in β‐structure and differed in their tertiary folding. The tryptophan residues were in a hydrophobic environment. The enzymes differed in their thermal stability; the midpoint of thermal inactivation, Tm, of the two enzymes was found to be 43 °C for PG II and 46 °C for PG IV.
Modification of a reactive cysteine explains differences between rasburicase and Uricozyme®, a natural Aspergillus flavus uricase
Authors: Bayol Alain , Capdevielle Joël , Malazzi Pascal , Buzy Armelle , Claude Bonnet Marie , Colloc'h Nathalie , Mornon Jean‐Paul , Loyaux Denis , Ferrara Pascual
Published: November 2003

Urate oxidase is used in humans for the control of uric acid in patients receiving chemotherapy. Rasburicase (Fasturtec®/Elitek®), a recombinant urate oxidase expressed in Saccharomyces cerevisiae, was compared with Uricozyme®, the natural enzyme produced by Aspergillus flavus. Rasburicase has a higher purity as demonstrated by SDS/PAGE and chromatographic analysis and a better specific activity. The differences observed for Uricozyme® are likely attributable to the previously used purification process, which modifies the enzyme. The production process of rasburicase, on the other hand, preserves the structure of the molecule. MS analysis shows that Uricozyme® contains a cysteine adduct on Cys103. In the crystal structure, the sulphur atom of the cysteine residue in position 103 is orientated to the external surface of the tetramer, whereas the sulphur atom of two other cysteine residues (Cys35 and Cys290) is orientated to the centre of the canal formed by the tetramer. The same adduct is produced by simple incubation of the rasburicase with cysteine.
Analysis of an ampicillin propyl ester prodrug which inhibits the growth of Escherichia coli
Authors: Bartzatt Ronald , Malesa Cynthia
Published: November 2003

An ampicillin prodrug was synthesized by utilizing the chemical reaction of ampicillin with diazopropane (CH3CH2CHN2) in an organic solvent. The result is esterification of the carboxylic acid functional group. The ampicillin prodrug is a solid that forms yellow crystals which are soluble in water and LB agarose media. The ampicillin prodrug was stable for more than 10 weeks when stored at 0.0 °C. The prodrug has reduced hydrogen‐bonding capability compared with the unmodified structure of ampicillin. Evaluation of the logP parameter (the octanol/water partition coefficient) indicates that the ampicillin prodrug (logP=1.773) has increased lipophilic characteristics relative to the unmodified ampicillin structure (logP=1.06). The lipophilic substituent constant for the esterification of the carboxylic acid is 0.713, a positive value which indicates that the substituent has a lipophilic nature. The ampicillin prodrug was solubilized into LB agarose media at a concentration of 0.228 mg/ml, and was found to induce 100% growth inhibition of an ampicillin‐susceptible and streptomycin‐resistant Escherichia coli strain (designated DH1), and induced greater than 30% growth inhibition of an ampicillin‐resistant E. coli strain (designated PKK). Synthesis of this prodrug utilizing a diazoalkane was highly efficient, with no undesirable by‐products being formed.
Quantitative physical characterization of lipid–polycation–DNA lipopolyplexes
Authors: Tsai Jeannette T. , Furstoss Kevin J. , Michnick Timothy , Sloane David L. , Paul Ralph W.
Published: November 2003

Quantitative assays for the characterization of multi‐component lipopolyplexes and their individual constituents are crucial for determining the consistency of formulation protocols which are ultimately reflected in biological activity. Lipid–polycation–DNA formulations consisting of lipids, polycations and DNA are of interest because they have been demonstrated to be efficient gene‐delivery vehicles when administered systemically. We have developed a panel of analytical techniques to characterize these lipopolyplexes. Complexes were measured for size by dynamic light scattering and surface‐charge characteristics by zeta potential. Interaction between DNA and the polycation, protamine sulphate, was determined using a PicoGreen dye‐exclusion technique. Total DNA in the lipopolyplex was assayed through decomplexation of the formulation by addition of heparin sulphate and subsequent DNA quantification by PicoGreen reagent. Protamine sulphate in the lipopolyplex was determined using a novel Amido Black‐staining protocol which is linearly sensitive in a range of 0.25–3 μg of protein. Lipids were quantified by HPLC after extraction in chloroform/methanol (2:1). In this method elution is conducted over 40 min, with 1,2‐dioleoyl‐3‐trimethylammonium‐propane and cholesterol being resolved by greater than 10 min. Such assays are essential for product characterization and release tests, as well as development of a better understanding of the correlation between physical structure and biological function.
Development of an optimized, simple chemically defined medium for bacterial cellulose production by Acetobacter sp. A9 in shaking cultures
Authors: Heo Moon‐Soo , Son Hong‐Joo
Published: November 2003

The genus Acetobacter can synthesize cellulose when grown in an undefined medium containing glucose. By using the technique of the omission of a single medium component, an optimized and simple chemically defined medium was developed to support cellulose production by Acetobacter sp. A9 in shaking culture. It contained 4.0% (w/v) glucose, 0.2% (w/v) (NH4)2SO4, 0.25% (w/v) KH2PO4, 0.3% (w/v) Na2HPO4·12H2O, 0.05% (w/v) MgSO4·7H2O, 0.0002% (w/v) FeSO4·7H2O, 0.00025% (w/v) H3BO3, 0.00006% (w/v) nicotinamide, 0.00025% (w/v) inositol and 1.4% (v/v) ethanol. A maximum cellulose concentration of around 8 g/l was achieved after 9 days of cultivation at 200 rev./min. The production of cellulose by Acetobacter sp. A9 was greater in simplified synthetic medium than in complex medium (Hestrin and Schramm medium) conventionally used for Acetobacter strains.
Purification and biochemical characterization of the carbamate hydrolase from Pseudomonas sp. 50432
Authors: Chaudhry G. Rasul , Mateen A. , Kaskar B. , Bloda M. , Riazuddin Sheikh
Published: November 2003

A soluble carbamate hydrolase that had a wide specificity was purified 2032‐fold from Pseudomonas sp. 50432. This was achieved using a combination of anion‐exchange, gel‐filtration and hydrophobic‐interaction‐ chromatography techniques. Carbamate hydrolase cleaved the ester linkage of the N‐methylcarbamates. The native enzyme was a monomer with a molecular mass of 88 kDa. The optimum pH and temperature of the enzyme activity were 8.5 and 37 °C respectively. The tested cations or EDTA did not affect the enzyme activity. However, 2‐mercaptoethanol reversibly inhibited the enzyme activity. The enzyme showed the Km values of 16 and 12 μM for carbofuran and carbaryl respectively. The purified enzyme did not hydrolyse o‐nitrophenyl dimethylcarbamate but hydrolysed several N‐methylcarbamates and 1‐naphthyl acetate.