bluuebunny/crossref_metadata_2025 huggingface dataset:
Automated extraction and amplification of DNA from whole blood using a robotic workstation and an integrated thermocycler
Authors: Smit Maarten L. , Giesendorf Belinda A. J. , Heil Sandra G. , Vet Jacqueline A. M. , Trijbels Frans J. M. , Blom Henk J.
Published: November 2003

Growing knowledge of the genetic basis of inheritable diseases has resulted in a rapidly increasing demand for DNA mutation analysis. Current methods are reliable and suitable for low‐throughput mutation analyses, but are unable to cope with the increasing demand for genetic analyses, necessitating the development of new, fully automated and reliable methods. We developed a semi‐automated method for DNA mutation analysis by integrating a thermocycler into a robotic pipetting workstation. DNA was extracted from 84 samples of 10 μl of EDTA‐treated whole blood using magnetic beads within 2 h. Directly after isolation, the DNA was automatically transferred to an integrated thermocycler for amplification. Our semi‐automated method proved to be reliable and robust, showing unambiguously interpretable PCR signals without occurrence of contamination. It is also faster than conventional manual methods. Only a brief manual intervention is required to remove and refit the seal of the PCR plate. This semi‐automated assay is a step forward in the development of fully automated assays for DNA mutation analysis.
High‐level synthesis of human prolactin in Chinese‐hamster ovary cells
Authors: Soares Carlos R. J. , Morganti Ligia , Miloux Brigitte , Lupker Jan H. , Ferrara Pascual , Bartolini Paolo
Published: November 2003

Two eukaryotic human prolactin (hPRL) expression vectors, based on a selectable dihydrofolate reductase (dhfr) marker, were used to transfect dhfr− Chinese‐ hamster ovary (CHO) cells. One vector, p658‐hPRL, contains the hepatitis‐B virus‐X cDNA coding for a viral transactivator and sequences mediating dhfr mRNA degradation. The other, pEDdc‐hPRL, carries the encephalomyocarditis virus leader sequence coupled to hPRL cDNA to provide high‐level protein expression, possibly via a mechanism of internal translation initiation in dicistronic mRNA. Without methotrexate (MTX) amplification, p658‐hPRL‐transfected stable cell lines, secreting up to ≈10 μg of hPRL/106 cells per day, could be rapidly obtained; production by pEDdc‐hPRL‐transfected cells was about 10‐fold lower. However, a three‐step MTX amplification of the latter led to clones secreting up to ≈30 μg of hPRL/106 cells per day. A pilot production using a hollow‐fibre bioreactor indicated that highly concentrated hormone levels in the medium could be obtained, with a production of up to 150 μg of hPRL/ml per day. SDS/PAGE analysis indicated that recombinant hPRL contained ≈10% glycosylated PRL. Chromatographically purified non‐glycosylated and glycosylated recombinant hPRL had bioactivities of 35 and 16 i.u./mg, respectively (Nb2 cell bioassay). This appears to be the first report describing production and purification of recombinant hPRL from CHO cells, secreted at levels higher than reported thus far in eukaryotic systems.
A novel method of isolation of a bone‐morphogenetic‐protein‐like protein from ossein
Authors: Mythili J. , Padmavathy S. , Chandrakasan Gowri
Published: November 2003

A new bone‐morphogenetic‐protein (BMP)‐like protein has been isolated through a new protocol from a novel source, ossein. The BMP‐like protein was hydrophilic and characterized through Fourier‐transform IR studies, SDS/PAGE and coupled with a neutral binder, hydroxypropylmethylcellulose (HPMC) for control release. The IR spectrum of the protein showed peaks in tandem with BMP from bone matrix, and its molecular mass was in the range 18–21 kDa. Sustained release from the surface of HPMC was achieved for a period of 3 days.
Optimization of fermentation conditions for the production of bacterial cellulose by a newly isolated Acetobacter
Authors: Son Hong‐Joo , Heo Moon‐Su , Kim Yong‐Gyun , Lee Sang‐Joon
Published: November 2003

The optimum fermentation conditions for the production of cellulose by a newly isolated Acetobacter sp. A9 were determined by shaken cultures. The strain was able to produce cellulose at 25–30 °C with a maximum at 30 °C. Cellulose production occurred at pH 4.5–7.5 with a maximum at pH 6.5. The improved medium composition was 4% (w/v) glucose, 0.1% (w/v) yeast extract, 0.7% (w/v) polypeptone and 0.8% (w/v) Na2HPO4·12H2O. Under these culture conditions, 3.8 g/l cellulose was produced after 7 days of cultivation, although this strain produced only 2.2 g/l in the standard medium. The addition of ethanol to the improved medium enhanced cellulose production: in an improved medium containing 1.4% (v/v) ethanol, cellulose production was 15.2 g/l, which was about four times higher than that without ethanol. Addition of ethanol was found to eliminate the spontaneous mutation of Acetobacter sp. A9.
A rapid method for detecting conformational changes during differentiation and apoptosis of HL60 cells by Fourier‐transform infrared spectroscopy
Authors: Zhou Jiangbing , Wang Zhao , Sun Suqing , Liu Mingjie , Zhang Hongjun
Published: November 2003

The conformational changes of HL60 cells during differentiation and apoptosis are still not clearly understood. This study uses a new method, Fourier‐transform infrared (FT‐IR) spectroscopy, to analyse the changes of HL60 cells. We detected several differences among normal HL60 cells, differentiated cells induced by all‐trans retinoic acid (ATRA) and PMA, and apoptotic cells induced by vircristine (VCR). The results suggest that the α‐helix content of the membrane protein of differentiated HL60 cells induced by ATRA and PMA increased, and that the β‐sheet content of membrane proteins of apoptotic cells induced by VCR also increased. Also, C–O(H)‐stretching modes of serine, threonine and tyrosine residues of cell proteins were shown to be distinctly different, but no significant differences were detected between cells differentiated along different lineages by ATRA and PMA.
Characterization of EDTA‐soluble polysaccharides from the scape of Musa paradisiaca (banana)
Authors: Raju T. Shantha , Jagadish Ramaswamykanive L. , Anjaneyalu Yernool V.
Published: November 2003

The polysaccharide components present in the scape of Musa paradisiaca (banana) were fractionated into water‐soluble (WSP), EDTA‐soluble (EDTA‐SP), alkali‐soluble (ASP) and alkali‐insoluble (AISP) polysaccharide fractions [Anjaneyalu, Jagadish and Raju (1997) Glycoconj. J. 14, 507–512]. The EDTA‐SP was further fractionated by iso‐amyl alcohol into EDTA‐SP‐A and EDTA‐SP‐B. The homogeneity of these two polysaccharides was established by repeated precipitation with iso‐amyl alcohol, gel‐filtration chromatography and sedimentation analysis. The polysaccharides were characterized by monosaccharide composition analysis, methylation linkage analysis, iodine affinity, ferricyanide number, blue value, hydrolysis with α‐amylase, gold‐electron microscopy and X‐ray diffraction spectroscopy. Data from all of these studies suggest that EDTA‐SP‐A is a branched amylose‐type α‐D‐glucan and that EDTA‐SP‐B is a highly branched amylopectin‐type polymer. The nature of the branching patterns of these polysaccharides suggests that they are unique to M. paradisiaca .
Synthesis and analysis of ethylated tetracycline, an antibiotic derivative that inhibits the growth of tetracycline‐resistant XL1‐Blue bacteria
Authors: Bartzatt Ronald , Koziol Kelly , Benish Theresa , Stoddard Jason
Published: November 2003

Bacterial resistance to antibiotics is a significant problem in medical care facilities, causing increased fatalities due to infection. The present study demonstrates that antibiotic structures can be selectively altered in a manner that revives their ability to inhibit bacterial growth. The antibiotic tetracycline was ethylated at the position of the phenolic hydroxy group with the use of diazoethane, forming an ethyl ether functional group. This derivative was dissolved in Luria–Bertani (LB) agar medium, then placed in tissue culture for screening against a tetracycline‐resistant bacterial strain. The growth of this bacterial strain, designated XL1‐Blue, was inhibited by the ethylated form of tetracycline. The procedure for synthesizing ethylated tetracycline utilizes diazoethane and is presented with the molecular structures and IR spectra. The ethylated form of tetracycline was stable at −20 °C for many weeks, and was soluble in LB agar plate medium. Ethylated tetracycline induced growth inhibition of XL1‐Blue bacteria within the first 24 h of incubation. The level of bacterial growth inhibition was greater than 30%. Calculation of the partition coefficient, log P, was accomplished and indicates that ethylated tetracycline has an increased lipophilic tendency relative to unmodified tetracycline, and therefore has greater solubility in lipid bilayers.
Effect of membrane composition and of co‐encapsulation of immunostimulants in a liposome‐entrapped crotoxin
Authors: Magalhães Tereza , Proietti Viotti Alexandre , Teperino Gomes Roberta , Viana de Freitas Thais
Published: November 2003

Crotoxin isolated from the venom of Crotalus durissus terrificus (South American rattlesnake) was incorporated into liposomes by the dehydration–rehydration vesicle method using different membrane compositions and the co‐encapsulation of immunostimulants. Crotoxin was also encapsulated into liposomes formed from a non‐phospholipid amphiphile, a mixture of polyoxyethylene 2‐cetyl ether, dicetyl phosphate and cholesterol. The preparations were characterized in relation to stability, toxicity and the protection of mice against whole venom after immunization. All liposome preparations were quite stable, retaining more than 75% of the originally encapsulated crotoxin after 1 week of incubation at physiological temperature. Co‐encapsulation with lipopolysaccharide increased the leakage of crotoxin. In contrast, co‐encapsulation of the lipid moiety of lipopolysaccharide did not influence the stability of liposomes. Toxicity of liposomes was dependent on membrane composition. Liposomes made with phospholipids that were resistant to phospholipase A2 activity were less toxic. Mice immunized with three doses of the 1×LD50 of crotoxin encapsulated into liposomes, and with associated immunostimulants, were protected against challenge with 8×subcutaneous LD50 of C. durissus terrificus venom. Using the same immunization schedule, liposomes made from a non‐phospholipid mixture and without immunostimulants achieved 100% protection.
Biochemical and immunological properties of human cardiac troponin I fragments
Authors: Morjana Nihmat , Clark Douglas , Tal Rony
Published: November 2003

Cardiac troponin I (cTnI) is the inhibitory subunit of the troponin complex and is a biochemical marker for myocardial infarction (MI). It is found in human serum within 4–6 h following MI. One of us has shown [Morjana (1998) Biotechnol. Appl. Biochem. 28, 105–111] that MI patient serum TnI is cleaved at the N‐ and C‐terminals and that the TnI fragments exist as a complex with tropinin C (TnC) and troponin T (TnT). In the present study, we have generated C‐terminal truncated TnI fragments and studied their immunological and biochemical properties. Human recombinant TnI (rTnI) expressed in Escherichia coli is cleaved into a major fragment with a molecular mass of 17500 Da using CNBr. The major CNBr fragment contains the first 153 amino acids of human cTnI (TnI153). Cleavage of the rTnI with the endoproteinase Asp‐N generates a smaller TnI fragment (TnI88, residues 6–96). TnI153 has higher immunological activity than that of rTnI and lower activity than that of TnI88, as judged by the Stratus II® TnI Immunoassay. TnI153 exhibits biochemical and immunological properties similar to those of intact TnI. It binds TnC at a molar ratio of 1:1 and forms a ternary complex with TnC and TnT. TnC enhances the immunological activity of TnI153, but has little effect on the activity of TnI88. The TnI153–TnC complex exhibits higher immunological activity than rTnI–TnC and TnI88–TnC, and much higher activity than free rTnI, TnI153 and TnI88. The presence of TnT has no effect on the immunological activity of the TnI153–TnC complex, suggesting that the addition of TnT does not interfere with TnI153 recognition by TnI monoclonal antibodies. Free TnI153 and TnI88 degrade rapidly in human serum. TnC protects TnI153 from proteolytic degradation, but offers less protection for TnI88. The TnI88–TnC complex lost 80% of its immunological activity after incubation for 2 days in human serum at 37 °C. However, there was no loss in the immunological activity of the TnI153–TnC complex under the same conditions. A cTnI fragment (TnI80, residues 1–80), expressed in E. coli as a fusion protein, exhibits immunological activity and stability similar to that of TnI88.
Alginate as a macroaffinity ligand and an additive for enhanced activity and thermostability of lipases
Authors: Sharma Shweta , Gupta Munishwar N.
Published: November 2003

Lipases are being employed increasingly for the synthesis of drug intermediates and pharmaceutically important molecules as well as for the resolution of racemic mixtures for obtaining physiologically active enantiomers. Alginate was used as a macroaffinity ligand to purify lipases from Chromobacterium viscosum, porcine pancreas and wheatgerm by employing the technique of affinity precipitation. In all the cases, the purified preparation showed a single band on SDS/PAGE. The process gave adequate yields of 87, 75 and 62% in the case of Chromobacterium, porcine and wheatgerm lipases respectively. Alginate was also found to activate the enzymes; this effect was most dramatic in the case of wheatgerm lipase, for which a 4–5‐fold activity increase was observed. Furthermore, alginate was also found to protect the enzyme against thermoinactivation.
Purification and conformational properties of a human interferon α2b produced inEscherichia coli
Authors: Beldarraín Alejandro , Cruz Yai , Cruz Oscar , Navarro Monica , Gil Miriela
Published: November 2003

Recombinant human interferon α2b was expressed intracellularly inEscherichia colias insoluble aggregates using a new expression vector, and was purified to homogeneity using essentially two‐step chromatographic procedures, i.e. immobilized metal‐ion‐affinity chromatography and reversed‐phase HPLC. The established purification process is highly reproducible and leads to a total recovery of approx. 12% with a specific biological activity of higher than 1×108i.u./mg of protein, which is comparable with the international requirement for interferon α2b. For purified protein we report conformational stability as a function of pH and temperature using differential scanning calorimetry and CD. Thermal unfolding as a function of pH showed only one endotherm at a temperature higher than 45 °C, and was reversible at pH 2–3.75 and irreversible at pH 4–10. At pH 7.0, the most stable condition, the conformational stability depends on protein concentration and ionic strength. The highly helical secondary structure is very conserved over the whole pH range studied, including at high temperatures.
Preparation of stable, highly active and immobilized glucose oxidase using the anti‐enzyme antibodies and F(ab)′2
Authors: Jan Ulfat , Husain Qayyum , Saleemuddin Mohammad
Published: November 2003

Immobilization of the Aspergillus niger glucose oxidase in high yield was achieved using an immunoaffinity‐based procedure. For this purpose IgGs, isolated from the sera of rabbits immunized with glucose oxidase, were favourably oriented by binding on to cobalt‐charged iminodiacetate–Sepharose. Large amounts of glucose oxidase could be immobilized by incubating the IgG‐bound matrix alternately with the enzyme and either intact IgG or F(ab)′2 derived thereof, leading to the formation of multiple enzyme layers. After three incubation cycles using anti‐(glucose oxidase) IgG, an 8‐fold increase in the amount of enzyme immobilized was observed, while the increase was 11‐fold when the F(ab)′2 replaced intact IgG. The preparations obtained thus were highly active, as also indicated by the high effectiveness factor, η. Immunoaffinity‐layered immobilized preparations were markedly more resistant to inactivation induced by exposure to 60 °C, 4·0 M urea or storage at 4 °C. The preparations also exhibited a remarkable resistance against inactivation induced by the water‐miscible organic solvents tetrahydrofuran, dioxan or acetone. Immobilized glucose oxidase preparations obtained using F(ab)′2 were generally observed to be superior in stability compared with those immobilized with the help of intact IgG.
Purification and characterization of alkaline protease from Alcaligenes faecalis
Authors: Thangam E. Berla , Rajkumar G. Suseela
Published: November 2003

Extracellular alkaline protease from the alkalophilic bacterium Alcaligenes faecalis was purified by a combination of ion‐exchange and size‐exclusion chromatographic methods, and its properties were examined. The purified enzyme had a specific activity of 563.8 μmol of tyrosine/min per mg of protein and gave a single band on native PAGE and SDS/PAGE with a molecular mass of 67 kDa. Gelatin zymogram also revealed one clear zone of proteolytic activity which corresponded to the band obtained with native PAGE and SDS/PAGE. The enzyme had an optimal pH of 9.0 and exhibited its highest activity at 55 °C. The enzyme activity was inhibited by PMSF, suggesting the presence of serine residues at the active site. The enzyme had a Km of 1.66 mg/ml and a Vmax of 526 units/min per mg of protein with casein as the substrate.
Cloning and expression of the external‐glycoprotein gene mutant from HIV‐2 in the methylotrophic yeast Pichia pastoris and identification of the glycoprotein
Authors: Zhang Ying J. , Jin Ning Y. , Jiang Wen Z. , Zhu Xue J. , Shen Jia C.
Published: November 2003

To achieve high‐level expression of HIV‐2ROD external glycoprotein gp105 in Pichia pastoris, the gp105 gene mutant tP1, with the 5′ non‐functional region of the gp105 gene removed, was obtained by PCR amplification and was cloned into secreted expression vector pHILS1. The His+Muts recombinant P. pastoris strain was screened by PCR and induced by methanol. SDS/PAGE and Western‐blot analyses showed that mutation of the low‐usage codon AGG into synonymous codon CGA and the introduction of the optimal codon TTC made P. pastoris overexpress tP1, an 85 kDa heterologous glycoprotein that was secreted into the medium and recognized specifically by HIV‐2 polyclonal antibody. The recombinant strain GS115/tP1 had excellent genetic stability in terms of the properties of growth and expression of gp105, and seven out of 58 recombinant stains with a yield of 29% were selected to be used for further purification of gp105.
Characterization of gelatin‐immobilized pigeonpea urease and preparation of a new urea biosensor
Authors: Srivastava Punit K. , Kayastha Arvind M. , Srinivasan
Published: November 2003

Urease purified from pigeonpea seeds was immobilized on gelatin beads via cross‐linking with glutaraldehyde. The maximum immobilization (75%) was observed at 30 mg/ml gelatin, 0·414 mg of enzyme/bead, 1% (v/v) glutaraldehyde and 4 °C. Beads stored in 50 mM Tris/acetate buffer (pH 7·3) at 4 °C showed a half‐life of 240 days and there was practically no leaching of enzyme (less than 2%) over a period of 30 days. These beads can be reused more than 30 times (with 24 h intervals) without much loss of enzyme activity (i.e. less than 11%). The immobilized urease showed a shift in its optimum pH from 7·3 to 6·5 in Tris/acetate buffer. Optimum temperature also shifted from 47 to 65 °C compared with the soluble enzyme. Gelatin‐immobilized pigeonpea urease had a higher Km (8·3 mM) than that of the soluble enzyme (3·0 mM). The time‐dependent temperature inactivation pattern was also found to change from biphasic to monophasic kinetics. The immobilized beads were used for the preparation of a new urea biosensor with a response time of less than 2 min. At least 14 samples of urea can be measured with this biosensor within an hour. The beads, as well as the biosensor, were used to analyse the urea content in clinical samples from the local clinical pathology laboratories. The results obtained with the biosensor were strikingly similar to those obtained with the various commonly employed biochemical/autoanalyzer® methods used. These immobilization studies also have a potential role in haemodialysis machines that maintain the urea level in kidney patients and in the construction of a portable/wearable kidney. The easy availability of the pigeonpea urease, the ease of its immobilization on gelatin and a significantly lower cost of the urease described in the present study makes it a suitable product for future applications in therapeutics and diagnostics.
Cloning, sequence analysis and functional characterization of DNA polymerase I from the thermophilic eubacterium Rhodothermus marinus
Authors: Blöndal Thórarinn , Thorbjarnardóttir Sigrídur H. , Kieleczawa Jan , Hjörleifsdóttir Sigrídur , Kristjánsson Jakob K. , Einarsson Jón M. , Eggertsson Gudmundur
Published: November 2003

A gene encoding a DNA polymerase I from the thermophilic eubacterium Rhodothermus marinus was identified. The gene was cloned, sequenced and expressed in Escherichia coli. The gene is 2772 bp long and encodes a protein of 924 amino acids with a calculated molecular mass of 104·8 kDa. Sequence analysis showed that a generally conserved Phe residue in the O‐helix is substituted by a Tyr (position 756) in the R. marinus enzyme. A Tyr in this position decreases the discrimination against dideoxynucleotides which is a major advantage in DNA sequencing. The protein was purified, characterized and showed to contain specific DNA‐polymerization activity of 3100 units/mg of protein, 5′→3′ exonuclease activity and a 3′→5′ proofreading activity. Its optimum activity was at 55 °C and it had a half‐life of 2 min at 90 °C. A truncated form of the enzyme lacking the 5′→3′ exonuclease domain was also expressed in E. coli. It had a specific DNA‐polymerization activity of 5000 units/mg of protein and lacked the 5′→3′ exonuclease activity. Its optimum activity was at 65 °C and it had a half‐life of 11 min at 90 °C. It was usable for DNA sequencing. This is the first thermostable DNA polymerase described with the O‐helix Phe→Tyr substitution.
Safety and preliminary immunogenicity of the recombinant outer membrane protein P64k of Neisseria meningitidis in human volunteers
Authors: Pérez Antonio , Dickinson Félix , Cinza Zurina , Ruíz Aroldo , Serrano Teresita , Sosa Jorge , González Sonia , Gutiérrez Yainelis , Nazábal Consuelo , Gutiérrez Oderay , Guzmán Daymi , Díaz Manuel , Delgado Maité , Caballero Evelin , Sardiñas Gretel , Alvarez Anabel , Martín Alejandro , Guillén Gerardo , Silva Ricardo
Published: November 2003

P64k is a meningococcal protein from Neisseria meningitidis that has been obtained by recombinant DNA technology. Recombinant P64k has been extensively characterized by physicochemical and immunological methods. Lately this protein has been found to act as a versatile immunological carrier for weak antigens in mice. In the present work, a Phase I clinical trial was carried out in healthy volunteers who received three inoculations of either placebo or recombinant P64k (20 or 50 μg). No severe adverse events occurred during the trial. Only mild adverse events in ten volunteers were observed. At 1 month after the third dose, 15 out of 18 volunteers (83.3%) who received the recombinant antigen had a P64k‐specific antibody titre 1:100, as detected by ELISA. A fourth dose, given 9 months after the third one, elicited a potent booster immune response in P64k vaccinees. Accordingly, these P64k formulations were considered safe and immunogenic in healthy human volunteers.
Studies on the inactivation of soluble and immobilized papain by the ascorbic acid–Cu2+ system: a model to propose the effect of free radicals on membrane‐bound enzymes in vivo
Authors: Hussain Sazid , Noor Rana , Iqbal Jawaid
Published: November 2003

Free radicals have been suggested to be widely implicated as the species responsible for harmful biological processes, such as aging, carcinogenesis and numerous other diseases. The mechanism of biological damage produced in such processes has been investigated in a wide variety of systems, including studies on proteins, enzymes, nucleic acids and carbohydrates. In the present study we selected an ascorbic acid–transition‐metal ion (ASA–Cu2+) system in order to understand the mechanism of soluble and membrane‐bound enzyme inactivation by generating free radicals. Papain, a thiol protease, was immobilized on an immobilized‐metal‐ion carrier and used as a model to examine the inactivation behaviour of membrane‐bound enzymes. A comparison was made between the inactivation of soluble and immobilized papain by free radicals, and the potential of different radical scavengers to prevent the inactivation of enzyme was examined.
A peroxidase isoenzyme secreted by turnip (Brassica napus) hairy‐root cultures: inactivation by hydrogen peroxide and application in diagnostic kits
Authors: Agostini Elizabeth , Hernández‐Ruiz Josefa , Arnao Marino B. , Milrad Silvia R. , Tigier Horacio A. , Acosta Manuel
Published: November 2003

We have purified various peroxidase isoenzymes from roots and hairy‐root cultures of turnip (Brassica napus) which could potentially be used for commercial applications such as an enzyme immunoassays, diagnostic test kits, wastewater treatment and soil remediation. One of them, a basic peroxidase called HR2, was secreted into the medium of turnip hairy‐root cultures. HR2 had a pI of 9.6, a molecular mass of 39.3 kDa and showed great thermostability. The inactivation of HR2 by H2O2 in the absence of reductant substrates was studied. Under these conditions H2O2 acted as a suicide substrate. The kinetic constants calculated have been compared with those of a basic isoperoxidase from horseradish (Armoracia sp.) roots (HRP‐C), which is commonly used in commercial kits. The results for HR2 indicated that it was more resistant to inactivation because it presented a lower inactivation efficiency and a higher value for the partition ratio (r=1250) than those described for HRP‐C. These results make turnip peroxidase HR2 suitable for use in systems in which high H2O2 concentrations are found. Such an application is demonstrated, namely an enzymic diagnostic kit for determination of uric acid in which HR2 was found to be as efficient as the enzyme originally included in standard kits.
Growth and production of cholesterol oxidase by alginate‐immobilized cells of Rhodococcus equi No. 23
Authors: Chang Yu‐Chih , Chou Cheng‐Chun
Published: November 2003

Rhodococcus equi No. 23 was immobilized in calcium alginate. No detrimental effect on the viability of the test organism was observed during the immobilization procedure. Approx. 98% of the cell population originally present in the alginate solution were immobilized in the gel beads. When the cells of an equal volume of the culture, obtained respectively at exponential phase (12 h preculture), late‐exponential phase (20 h preculture) or stationary phase (36 h preculture) were immobilized, the gel beads prepared with the stationary‐phase culture were found to contain the highest cell population [about 108 colony‐forming units (CFU)/g of beads]. In addition, gel beads, prepared with late‐exponential‐phase culture, exhibited the highest production of cholesterol oxidase (CholOx) after 48 h of incubation. Increasing the bead mass from 3.5 to 14.0 g/100 ml of medium increased CholOx production. However, further increasing the bead mass resulted in a reduction of CholOx production. Furthermore, on the basis of a similar initial cell population, the alginate‐immobilized cells of R. equi No. 23 produced a significantly higher amount of CholOx (P<0·05) than did the free cells.
Sephadex‐based cell‐affinity adsorbents: preparation and performance
Authors: Besselink Geert , De Korte Dirk
Published: November 2003

Sephadex was derivatized consecutively with Staphylococcus Protein A (SpA) and cell‐specific antibodies, and the binding of cells to the resulting material was examined. For comparison, cell binding to commercially obtained SpA–Sepharose was determined. Sephadex G‐10, carboxylated by reaction with glycine and activated subsequently with 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodi‐imide/N‐hydroxysuccinimide (NHS), was allowed to react with SpA. Coupling of SpA to NHS‐activated glycine–Sephadex appeared to be complete (immobilization capacity, ≈300 μg of protein/ml of packed gel) when incubation was carried out at pH 4.0, in buffer of low ionic strength. However, incubation at higher pH values (6.5) led to poor coupling yields. After incubation with rabbit anti‐(human red cell) antiserum, and upon mixing with human red blood cells, SpA–glycine–Sephadex G‐10 could bind up to 5×108 red cells/ml of gel. Cell binding increased when the amount of antiserum, added to SpA–glycine–Sephadex G‐10 for preparing the affinity gel, was increased from 0.5 to 5 μl/ml of gel. Compared with this, SpA–Sepharose CL 4B had to be incubated with much larger amounts of antiserum (100–700 μl/ml of gel) in order to obtain cell‐affinity adsorbent. One obvious advantage of the approach described here is that relatively small amounts of SpA and antisera are needed for preparing cell‐affinity media.
One‐step affinity purification of fetuin from fetal bovine serum
Authors: Cartellieri Simone , Hamer Ole , Helmholz Heike , Niemeyer Bernd
Published: November 2003

Fetuin is a plasma glycoprotein widely distributed in mammals. It has been used as a model protein for structural analyses and investigations into the biological properties of glycoproteins. A convenient one‐step procedure for biospecific isolation of fetuin from fetal bovine serum was developed on the basis of wheatgerm agglutinin (WGA) affinity separation. Two different porous supports, a silica‐based material and a polymer‐based material, were used for the immobilization of WGA. The prepared WGA adsorbents were characterized and process parameters of the affinity separation of fetuin were investigated and optimized. WGA was immobilized on silica and polymer supports with coupling yields of 99.6 and 99.4% respectively and amounts of coupled ligand of 7.9 and 9.2 mg of WGA/ml respectively. It has been shown that the specific capacities for fetuin were 5.1 mg/ml on WGA–silica, 1.8 mg/ml on WGA–polymer and 4.1 mg/ml on WGA–agarose. All three adsorbents proved to be suitable for the biospecific separation of fetuin. The polymer‐based WGA adsorbent was successfully applied in the purification of fetuin from fetal bovine serum in a one‐step separation process. The identity and purity of the isolated product was verified by SDS/PAGE. Under optimized conditions up to 21.6 mg of fetuin could be isolated from 1 ml of serum. The procedure described was designed to be easily scaled‐up for the production of fetuin.
An adventure in biotechnology: the development of haemophilia A therapeutics – from whole‐blood transfusion to recombinant DNA to gene therapy
Authors: Kingdon Henry S. , Lundblad Roger L.
Published: November 2003

The past decade has seen an explosion in the number of therapeutic proteins available for a wide spectrum of diseases. Some of these proteins are obtained from human plasma. Examples of these therapeutic proteins are albumin, intravenous immunoglobulins and prothrombin complex concentrates. The majority of new therapeutic proteins are, however, derived via recombinant DNA technology. There are other examples where the first therapeutic preparation was a crude preparation derived from plasma or tissue and where subsequent development has resulted in a recombinant form of the therapeutic protein. This article focuses on the development of therapeutics for the treatment of haemophilia A (deficiency of Factor VIII activity). The progression from crude plasma fractions to monoclonal‐purified preparations to the more recent development of therapeutic concentrates via recombinant DNA technology is described in some detail. Finally, the current status of gene therapy for haemophilia A is evaluated. Both technical issues as well as market forces are described, as both have had significant impact on the product‐development process.
Production and characterization of a thermostable β‐galactosidase from Bacillus coagulans RCS3
Authors: Batra Navneet , Singh Jagtar , Banerjee Uttam C. , Patnaik Pratap R. , Sobti Ranbir C.
Published: November 2003

A strain of Bacillus coagulans RCS3 isolated from ahot‐water spring produced significant β‐galactosidase activity at 10 days of growth in a flask. While enzyme production was maximum at 50 °C, the highest activity was at 65 °C, where the half‐life was 2 h. A 2 °C decrease in temperature increased the half‐life to 15 h without significantly changing the activity, suggesting that 63 °C is the temperature of preference compared with 65 °C for a combination of good activity and stability. The β‐galactosidase was also stable over pH 5–8, with peak activity at pH 6–7. It was strongly and competitively inhibited by the hydrolysis product galactose. Bivalent cations (Cu2+, Ni2+ and Hg2+) in the concentration range of 0.5–2.0 mM also inhibited enzyme activity. Both lactose solution and whey could be hydrolysed substantially within 36 h at 50 °C. The thermostability and pH‐stability and good hydrolytic capability make this enzyme potentially useful in the dairy industry.
Glycosylation by Chinese hamster ovary cells in dolichol phosphate‐supplemented cultures
Authors: Yuk Inn H. Y. , Wang Daniel I. C.
Published: November 2003

N‐linked glycosylation often imparts important properties to protein therapeutics. An essential step in this intracellular process is the transfer of oligosaccharide from dolichol monophosphate (Dol‐P) to a potential glycosylation site. Variability in the success rate of this reaction affects the extent of protein glycosylation. The critical role of Dol‐P suggests that its availability may influence the extent of glycosylation by limiting the pool of lipid‐linked oligosaccharides (LLOs), the glycosyl donor. To test this hypothesis, the impact of Dol‐P supplementation on protein glycosylation in Chinese hamster ovary (CHO) cells was investigated. Although exogenous Dol‐P was incorporated by CHO cells and processed into LLOs in a dose‐dependent manner, Dol‐P supplementation had no marked effects on LLO or overall cellular glycosylation levels. While concentrations of exogenous Dol‐P exceeding 100 μg/ml were detrimental to CHO cell viability, maximum non‐toxic supplemental doses of Dol‐P had no significant impact on the glycosylation of recombinant interferon‐γ produced by batch cultures of CHO cells. These results show that glycosylation in CHO cells cannot be readily enhanced by Dol‐P feeding under normal culture conditions.
Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli
Authors: Jonasson Per , Liljeqvist Sissela , Nygren Per‐A˚ke , Ståhl Stefan
Published: November 2003

Genetic strategies have been used for more than two decades to improve bacterial bioprocesses and to simplify recovery procedures. Such strategies include the design of efficient expression vectors and the improvement of bacterial production strains in different ways, e.g. by deletion of protease genes or engineering for overexpression of rare‐codon tRNAs, foldases or chaperones. Gene multimerization is another such principle that has proved beneficial to improve production yields. Genetic strategies have furthermore been exploited to facilitate recovery processes by adapting the product for a particular purification principle. In this area, affinity fusions have been commonly used, but other principles, such as modified isoelectric point (pI) or hydrophobic properties have also been successfully investigated. A recent drastic step forward in the use of gene technology to improve recovery processes for recombinant proteins is the introduction of combinatorial protein engineering to generate tailor‐made product‐specific affinity ligands. This strategy, which allows efficient recovery of a recombinant protein in its native form, is likely to be increasingly used also in industrial‐scale bioprocesses, since novel protein ligands have been described that can be sanitized using common industrial cleaning‐in‐place procedures. The examples presented in this review make it evident that genetic strategies will be of utmost importance in the future for facilitating production and recovery of recombinant proteins.
Rapid purification and analysis of α‐synuclein proteins: C‐terminal truncation promotes the conversion of α‐synuclein into a protease‐sensitive form in Escherichia coli
Authors: Choi Ju‐Youn , Sung Young‐Mo , Park Hyo‐Jin , Hur Eun‐Hye , Lee Sun‐Joo , Hahn Chul , Min Byung‐Re , Kim In‐Kyung , Kang Seongman , Rhim Hyangshuk
Published: November 2003

Parkinson's disease (PD) is the most common neurodegenerative movement disorder and is characterized by the loss of dopaminergic neurons and the formation of eosinophilic intracytoplasmic inclusion bodies known as Lewy bodies. Although α‐synuclein is known to be a pivotal factor implicated in the pathogenesis of PD, its function remains to be elucidated. We used the pGEX expression system to develop a simple and rapid method for purifying α‐synuclein proteins in suitable forms for biochemical studies of their functions. The wild‐type α‐synuclein protein was overexpressed in Escherichia coli and purified to approx. 80% purity with relatively high yields. We also used this expression system to investigate the expression pattern of the various domains of α‐synuclein. With the exception of the α‐synuclein protein that was truncated at amino acid residue 95, all domain constructs of α‐synuclein were purified at similar levels with relatively high yields. Unexpectedly, removal of amino acid residues 96–140 in the C‐terminal acidic region of α‐synuclein promotes its conversion to a protease‐sensitive form during expression and purification in E. coli. Our study suggests a method for generating useful reagents to investigate the molecular mechanism by which α‐synuclein regulates the pathogenesis of PD.
The haem‐regulated eukaryotic initiation factor 2α kinase: a molecular indicator of lead‐toxicity anaemia in rabbits
Authors: Anand Sanjay , Pal Jayanta K.
Published: November 2003

The haem‐regulated eukaryotic initiation factor 2α kinase, also called the haem‐regulated inhibitor (HRI), has been shown to increase in the peripheral blood cells as a function of drug‐induced anaemia in rabbits, suggesting that it could be a molecular indicator of drug‐induced anaemia [Anand and Pal (1997) J. Biosci. 22, 287–298]. In the present investigation, we have determined the expression of HRI during lead‐induced anaemia in rabbits. The level of anaemia has been determined by routine procedures such as reticulocyte count, haemoglobin content and packed cell volume. These values were compared with the results obtained for a quantitative Western blot of HRI in the blood cell lysates of drug‐ and lead‐induced anaemic rabbits. These results indicate that HRI could be used as a molecular marker for lead‐induced anaemia since a progressive increase in HRI levels could be detected as a function of the time of lead exposure. In order to understand the role of stress proteins, heat‐shock protein (Hsp) 70 and Hsp90, in inducing anaemia during lead exposure, levels of Hsp70 and Hsp90, and their interaction with HRI, have been determined. Increased levels of these proteins and their intermolecular complexes with HRI suggest their role in regulating protein synthesis during lead‐induced anaemia. These observations further reiterate the use of HRI as a potential indicator for drug‐ and heavy‐metal‐induced anaemia in humans.
Boilysin and thermolysin in dipeptide synthesis: a comparative study
Authors: Kühn Diana , Dürrschmidt Peter , Mansfeld Johanna , Ulbrich‐Hofmann Renate
Published: November 2003

Boilysin (BLN) is an engineered, highly thermostable neutral protease from Bacillus stearothermophilus. Its high resistance is based on the stabilization of a surface loop (amino acid residues 55–69) by eight amino acid exchanges, including the introduction of a disulphide bond. In the present study, BLN was compared with the well‐known and structurally related thermolysin (from B. thermoproteolyticus) with respect to their dipeptide‐ synthetic properties. The synthesis of the aspartame precursor N‐(benzyloxycarbonyl)‐l‐aspartyl‐l‐phenylalanine methyl ester (Z‐Asp‐Phe‐OMe) was used as the model reaction to study its enantioselectivity as well as the influence of neutral salt, temperature and calcium‐ ion concentration on peptide synthesis. The reactions were performed in homogeneous reaction systems containing DMSO or aliphatic alcohols. Furthermore, the substrate specificity in the synthesis of Z‐Asp‐X‐OMe (X=Phe, d‐Phe, Ala, Ile, Leu, Met, Tyr or Val) was examined. The two enzymes showed no difference in time course of reaction by the use of salts as activators or different alcohols as co‐solvents. Both enzymes showed a high enantioselectivity towards the amino component in the reaction. They were strongly activated by NaCl, whereas the final product yields were strongly decreased at high NaCl concentrations. Aliphatic alcohols act as an inhibitor, but allow higher product yields compared with purely aqueous medium. Differences between the two enzymes are found at higher temperatures (60 °C) in the absence or at low concentrations of Ca2+ ions; BLN proved superior under these conditions, because its stability is less dependent on Ca2+ ions. The substrate specificities of BLN and thermolysin at the P′1 position follow the same tendencies, with differences in the initial rates for the conversion of Z‐Asp with Ile‐OMe, Leu‐OMe and Val‐OMe.
Characterization and physicochemical properties of a lipase from Pseudomonas mendocina 3121–1
Authors: Surinenaite Birute , Bendikiene Vida , Juodka Benediktas , Bachmatova Irena , Marcinkevichiene Liucija
Published: November 2003

The lipase from Pseudomonas mendocina 3121–1 was found to be homogeneous with a molecular mass of 30 kDa by SDS/PAGE. It is composed of two identical subunits. A molecular mass of 62 kDa was determined by gel chromatography on a Toyopearl HW‐55F column. Some physicochemical properties of the lipase were investigated using p‐nitrophenyl butyrate (p‐NPB), Tween 80 solution and Sigma olive‐oil emulsion as substrates. The optimum temperature was determined to be 52 °C with p‐NPB, in the range 50–60 °C with Tween 80 and in the range 50–65 °C with olive‐oil emulsion. The optimum pH was determined to be in the pH range 7.2–7.5, both with Tween and the emulsion, but was unusually alkaline (pH 9.5) with p‐NPB. The enzyme was activated for p‐NPB hydrolysis by thermal treatment up to 60 min at 60 °C, pH 7.0–8.2, but was rapidly inactivated at 70–80 °C and at pH 7.0. The lipase was shown to be more thermolabile at 60 °C with respect to other two substrates. Using the emulsified substrate, no activity was obtained after preincubating the enzyme for 30 min at 70 °C. The enzyme was found to be pH‐tolerant when stored at 20 °C, pH 6.3–10.3 (100 mM Briton–Robson buffer) as the half‐life (t1/2) was more than 240 h when p‐NPB was used as the substrate. By contrast, the pH‐stability range was more narrow (pH 8.0–10.5) with olive‐oil emulsion. The effect of various metal ions and EDTA depended on the nature of the substrate.
Chlorpromazine oxidation by hydroperoxidase activity of covalent immobilized lipoxygenase
Authors: Santano Esperanza , Pinto María del Carmen , Macías Pedro
Published: November 2003

The application of the hydroperoxidase activity of immobilized lipoxygenase to xenobiotic metabolization is reported in this work. Soya bean lipoxygenase has been immobilized by covalent coupling to oxirane acrylic beads. This immobilized system has been applied to the oxidation of chlorpromazine (CPZ), a phenothiazine of wide pharmacological use which in some cases presents toxicity. Immobilized lipoxygenase produces the oxidation of CPZ in the presence of hydrogen peroxide at acidic pH, maintaining a high level of activity and stability. In comparison with free lipoxygenase, the immobilized enzyme system shows higher catalytic efficiency and protection against enzymic inactivation produced by the presence of H2O2. When the system of immobilized enzyme was loaded into a bioreactor operating in continuous mode, the level of CPZ oxidation was higher than obtained using a discontinuous procedure or the free enzyme. The results obtained in this work suggest that a system of covalent immobilized lipoxygenase operating in continuous mode may constitute a valuable tool for xenobiotic detoxification or metabolization studies.
Quantifying Phytophthora medicaginis in Susceptible and Resistant Alfalfa with a Real‐Time Fluorescent PCR Assay
Authors: Vandemark G. J. , Barker B. M.
Published: November 2003

A real‐time fluorescent PCR assay using a set of specific primers and a fluorochrome‐labelled probe (TaqMan) was developed to quantify the amount of Phytophthora medicaginis DNA in alfalfa plants that were classified as either resistant or susceptible to the pathogen based on visual assessment of disease response. The assay clearly discriminated among three standard check alfalfa populations with different levels of resistance based on the analysis of DNA extracted from the roots of bulked plant samples. In two independent experiments, the Spearman rank correlation between pathogen DNA content and the number of susceptible plants in a bulked sample was greater than 0.89 and highly significant (P<0.0001). Significantly less pathogen DNA was detected in bulked samples of a highly resistant check population than in bulked samples from more susceptible check populations. Analysis of individual plants indicated that significantly less pathogen DNA was detected in resistant plants than in susceptible plants. Applications of the assay are considered for breeding programs and the study of microbial population dynamics in plants simultaneously infected with different pathogens.
Occurrence, Symptom Expression and Characterization of Phytoplasma Associated with Pear Decline Disease in Catalonia (Spain)
Authors: Garcia‐Chapa M. , Laviña A. , Sanchez I. , Medina V. , Batlle A.
Published: November 2003

A total area of 1500 ha of commercial plots was surveyed to study the extent of pear decline disease and its relative importance in northeastern Spain. A preliminary evaluation indicated that around 7% of the plots had symptoms of the disease. At the same time, pear decline incidence was evaluated in 45 plots, by visual inspection of 500 trees in each plot. In September, the incidence of trees with symptoms ranged from 8 to 59% depending on the cultivar selected. The presence of pear decline (PD) phytoplasma in these plots was confirmed by polymerase chain reaction (PCR) amplification of phytoplasma DNA with universal or group‐specific primers. Restriction fragment length polymorphism analyses also showed the presence of a unique phytoplasma strain.

The symptom expression of PD disease in different cultivars was evaluated throughout the year. The relationship between the presence of symptoms and detection of PD by PCR in these cultivars was also studied. Results showed that the nested‐PCR, using specific primers to detect the DNA from PD phytoplasma, is the most accurate method to identify the total percentage of affected trees.