bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interplay of SOS induction, recombinant gene expression, and multimerization of plasmid vectors in Escherichia coli
Authors: Lee Jeewon , Kim Hyung‐Cheol , Kim Sung‐Woo , Kim Seung Wook , Hong Suk In , Park Young‐Hoon 
Published: September 2002

AbstractUsing pBR322‐ and pUC‐derived plasmid vectors, a homologous (Escherichia coli native esterase) and three heterologous proteins (human interleukin‐2, human interleukin‐6, and Zymomonas levansucrase) were synthesized in E. coli IC2015(recA::lacZ) and GY4786 (sfiA::lacZ) strains. Via time‐course measurement of β‐galactosidase activity in each recombinant culture, the SOS induction was estimated in detail and the results were systematically compared. In recombinant E. coli, the SOS response did not happen either with the recombinant insert‐negative plasmid backbone alone or the expression vectors containing the homologous gene. Irrespective of gene expression level and toxic activity of synthesized foreign proteins, the SOS response was induced only when the heterologous genes were expressed using a particular plasmid vector, indicating strong dependence on the recombinant gene clone and the selection of a plasmid vector system. It is suggested that in recombinant E. coli the SOS response (i.e., activation of recA expression and initial sfiA expression) may be related neither to metabolic burden nor toxic cellular event(s) by synthesized heterologous protein, but may be provoked by foreign gene‐specific interaction between a foreign gene and a plasmid vector. Unlike in E. coli XL1‐blue(recA−) strains used, all expression vectors encoding each of the three heterologous proteins were multimerized in E. coli IC2015 strains in the course of cultivation, whereas the expression vectors containing the homologous gene never formed the plasmid multimers. The extent of multimerization was also dependent on a foreign gene insert in the expression vector. As a dominant effect of the SOS induction, recombinant plasmid vectors used for heterologous protein expression appear to significantly form various multimers in the recA+ E. coli host. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 84–92, 2002.
Biodegradation of gasoline by gellan gum‐encapsulated bacterial cells
Authors: Moslemy Peyman , Neufeld Ronald J. , Guiot Serge R. 
Published: September 2002

AbstractEncapsulated cell bioaugmentation is a novel alternative solution to in situ bioremediation of contaminated aquifers. This study was conducted to evaluate the feasibility of such a remediation strategy based on the performance of encapsulated cells in the biodegradation of gasoline, a major groundwater contaminant. An enriched bacterial consortium, isolated from a gasoline‐polluted site, was encapsulated in gellan gum microbeads (16–53 μm diameter). The capacity of the encapsulated cells to degrade gasoline under aerobic conditions was evaluated in comparison with free (non‐encapsulated) cells. Encapsulated cells (2.6 mgcells g−1 bead) degraded over 90% gasoline hydrocarbons (initial concentration 50–600 mg L−1) within 5–10 days at 10°C. Equivalent levels of free cells removed comparable amounts of gasoline (initial concentration 50–400 mg L−1) within the same period but required up to 30 days to degrade the highest level of gasoline tested (600 mg L−1). Free cells exhibited a lag phase in biodegradation, which increased from 1 to 5 days with an increase in gasoline concentration (200–600 mg L−1). Encapsulation provided cells with a protective barrier against toxic hydrocarbons, eliminating the adaptation period required by free cells. The reduction of encapsulated cell mass loading from 2.6 to 1.0 mgcells g−1 bead caused a substantial decrease in the extent of biodegradation within a 30‐day incubation period. Encapsulated cells dispersed within the porous soil matrix of saturated soil microcosms demonstrated a reduced performance in the removal of gasoline (initial concentrations of 400 and 600 mg L−1), removing 30–50% gasoline hydrocarbons compared to 40–60% by free cells within 21 days of incubation. The results of this study suggest that gellan gum‐encapsulated bacterial cells have the potential to be used for biodegradation of gasoline hydrocarbons in aqueous systems. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 175–184, 2002.
Glucose‐to‐fructose conversion at high temperatures with xylose (glucose) isomerases from Streptomyces murinus and two hyperthermophilic Thermotoga species
Authors: Bandlish Rockey K. , Michael Hess J. , Epting Kevin L. , Vieille Claire , Kelly Robert M. 
Published: September 2002

AbstractThe conversion of glucose to fructose at elevated temperatures, as catalyzed by soluble and immobilized xylose (glucose) isomerases from the hyperthermophiles Thermotoga maritima (TMGI) and Thermotoga neapolitana 5068 (TNGI) and from the mesophile Streptomyces murinus (SMGI), was examined. At pH 7.0 in the presence of Mg2+, the temperature optima for the three soluble enzymes were 85°C (SMGI), 95° to 100°C (TNGI), and >100°C (TMGI). Under certain conditions, soluble forms of the three enzymes exhibited an unusual, multiphasic inactivation behavior in which the decay rate slowed considerably after an initial rapid decline. However, the inactivation of the enzymes covalently immobilized to glass beads, monophasic in most cases, was characterized by a first‐order decay rate intermediate between those of the initial rapid and slower phases for the soluble enzymes. Enzyme productivities for the three immobilized GIs were determined experimentally in the presence of Mg2+. The highest productivities measured were 750 and 760 kg fructose per kilogram SMGI at 60°C and 70°C, respectively. The highest productivity for both TMGI and TNGI in the presence of Mg2+ occurred at 70°C, pH 7.0, with approximately 230 and 200 kg fructose per kilogram enzyme for TNGI and TMGI, respectively. At 80°C and in the presence of Mg2+, productivities for the three enzymes ranged from 31 to 273. A simple mathematical model, which accounted for thermal effects on kinetics, glucose–fructose equilibrium, and enzyme inactivation, was used to examine the potential for high‐fructose corn syrup (HFCS) production at 80°C and above using TNGI and SMGI under optimal conditions, which included the presence of both Co2+ and Mg2+. In the presence of both cations, these enzymes showed the potential to catalyze glucose‐to‐fructose conversion at 80°C with estimated lifetime productivities on the order of 2000 kg fructose per kilogram enzyme, a value competitive with enzymes currently used at 55° to 65°C, but with the additional advantage of higher fructose concentrations. At 90°C, the estimated productivity for SMGI dropped to 200, whereas, for TNGI, lifetime productivities on the order of 1000 were estimated. Assuming that the most favorable biocatalytic and thermostability features of these enzymes can be captured in immobilized form and the chemical lability of substrates and products can be minimized, HFCS production at high temperatures could be used to achieve higher fructose concentrations as well as create alternative processing strategies. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 185–194, 2002.
Optical waveguide lightmode spectroscopy (OWLS) to monitor cell proliferation quantitatively
Authors: Hug T. S. , Prenosil J. E. , Maier P. , Morbidelli M. 
Published: September 2002

AbstractThe use of microscopic observations used for in situ monitoring of cell proliferation in the production of epidermal autografts is not satisfactory. In particular, the identification of the projected cell area from microscopic pictures by image analysis (IA) depends on intensity edges and level of contrasts and is thus limited to subconfluent cultures. Some of these problems can be solved by using optical waveguide lightmode spectroscopy (OWLS), which measures the effective refractive index of a thin layer above an Si(Ti)O2 waveguide surface. In this study the use of OWLS to monitor cell adhesion, spreading, and growth was studied. The sensitivity of the method was investigated by using three different cell lines, two fibroblasts and one hepatoma cell line. Cell proliferation of two strains of fibroblasts and hepatoma cells was monitored up to 2 days with the OWLS. In parallel, cell density was determined at different time points microscopically using an additional window in the measuring chamber. The cell density of fully spread cells (∼4 h after attachment) was found to be proportional to the OWLS signal. In long‐term cultures the influence of the cell density from single cells to confluent cell cultures upon the OWLS signal was investigated. The exponentially growing number of hepatoma resulted in a linear increase of the sensor signal. Due to this and to the fact that the proliferating cells exhibit contact inhibition, it was concluded that the cell contact area must decrease exponentially. The results show the strength of OWLS for monitoring the adhesion and proliferation of anchorage‐dependent cells in applications where an on‐line indicator of the total biomass is needed. Additionally, OWLS provides metabolic information through detection of the cell mass in close contact with the waveguide. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 213–221, 2002.
Macroaffinity ligand‐facilitated three‐phase partitioning (MLFTPP) for purification of xylanase
Authors: Sharma Aparna , Gupta M. N. 
Published: September 2002

AbstractIt is shown that eudragit S‐100, a copolymer of methylacrylic acid and methylmethacrylate, undergoes three‐phase partitioning. It was found that 95% eudragit S‐100 could be recovered as the interfacial precipitate by using 30% (w/v) ammonium sulfate, 1:1 ratio of t‐butanol to polymer solution at 40°C. Three‐phase partitioning of proteins uses simultaneous addition of ammonium sulfate and t‐butanol to precipitate proteins in an interfacial layer separating the aqueous phase and organic solvent. Exploiting the affinity of xylanases towards eudragit S‐100, it was possible to purify xylanase from Aspergillus niger; 60% recovery of activity with 95‐fold purification could be obtained by this process. The purified enzyme showed A single band on SDS‐PAGE. The technique shows promise to develop into a general method that could be termed “macroaffinity ligand‐facilitated three‐phase partitioning (MLFTPP).” © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 228–232, 2002.
The heat shock response of Mycobacterium tuberculosis: linking gene expression, immunology and pathogenesis
Authors: Stewart Graham R. , Wernisch Lorenz , Stabler Richard , Mangan Joseph A. , Hinds Jason , Laing Ken G. , Butcher Philip D. , Young Douglas B. 
Published: September 2002

AbstractThe regulation of heat shock protein (HSP) expression is critically important to pathogens such as Mycobacterium tuberculosis and dysregulation of the heat shock response results in increased immune recognition of the bacterium and reduced survival during chronic infection. In this study we use a whole genome spotted microarray to characterize the heat shock response of M. tuberculosis. We also begin a dissection of this important stress response by generating deletion mutants that lack specific transcriptional regulators and examining their transcriptional profiles under different stresses. Understanding the stimuli and mechanisms that govern heat shock in mycobacteria will allow us to relate observed in vivo expression patterns of HSPs to particular stresses and physiological conditions. The mechanisms controlling HSP expression also make attractive drug targets as part of a strategy designed to enhance immune recognition of the bacterium. Copyright © 2002 John Wiley & Sons, Ltd.
Microarray analysis of bacterial gene expression: towards the regulome
Authors: Kendall Sharon L. , Movahedzadeh Farahnaz , Wietzorrek Andreas , Stoker Neil G. 
Published: September 2002

AbstractMicroarray technology allows co‐regulated genes to be identified. In order to identify genes that are controlled by specific regulators, gene expression can be compared in mutant and wild‐type bacteria. However, there are a number of pitfalls with this approach; in particular, the regulator may not be active under the conditions in which the wild‐type strain is cultured. Once co‐regulated genes have been identified, protein‐binding motifs can be identified. By combining these data with a map of promoters, or operons (the operome), the regulatory networks in the cell (the regulome) can start to be built up. Copyright © 2002 John Wiley & Sons, Ltd.
BµG@Sbase—a microarray database and analysis tool
Authors: Witney Adam A. , Hinds Jason 
Published: September 2002

AbstractThe manufacture and use of a whole‐genome microarray is a complex process and it is essential that all data surrounding the process is stored, is accessible and can be easily associated with the data generated following hybridization and scanning. As part of a program funded by the Wellcome Trust, the Bacterial Microarray Group at St. George's Hospital Medical School (BµG@S) will generate whole‐genome microarrays for 12 bacterial pathogens for use in collaboration with specialist research groups. BµG@S will collaborate with these groups at all levels, including the experimental design, methodology and analysis. In addition, we will provide informatic support in the form of a database system (BµG@Sbase). BµG@Sbase will provide access through a web interface to the microarray design data and will allow individual users to store their data in a searchable, secure manner. Tools developed by BµG@S in collaboration with specific research groups investigating analysis methodology will also be made available to those groups using the arrays and submitting data to BµG@Sbase. Copyright © 2002 John Wiley & Sons, Ltd.
Harvesting the mouse genome
Authors: Botcherby Marc 
Published: September 2002

AbstractThe sequencing of the black 6 mouse (strain C57Bl/6) has reached an important juncture. The BAC fingerprint map is almost complete, the BACs have been end‐sequenced and a seven‐fold coverage whole‐genome shotgun has been assembled. Now the BAC‐by‐BAC sequencing phase is under way and in‐depth comparative analysis can be carried out on regions that have been the subject of targeted sequencing. This paper reviews the progress so far and looks forward to the promises of finished sequence. Copyright © 2002 John Wiley & Sons, Ltd.
Heterologous array analysis in Pinaceae: hybridization of Pinus taeda cDNA arrays with cDNA from needles and embryogenic cultures of P. taeda, P. sylvestris or Picea abies
Authors: van Zyl Leonel , von Arnold Sara , Bozhkov Peter , Chen Yongzhong , Egertsdotter Ulrika , MacKay John , Sederoff Ronald R. , Shen Jing , Zelena Lyubov , Clapham David H. 
Published: September 2002

AbstractHybridization of labelled cDNA from various cell types with high‐density arrays of expressed sequence tags is a powerful technique for investigating gene expression. Few conifer cDNA libraries have been sequenced. Because of the high level of sequence conservation between Pinus and Picea we have investigated the use of arrays from one genus for studies of gene expression in the other. The partial cDNAs from 384 identifiable genes expressed in differentiating xylem of Pinus taeda were printed on nylon membranes in randomized replicates. These were hybridized with labelled cDNA from needles or embryogenic cultures of Pinus taeda, P. sylvestris and Picea abies, and with labelled cDNA from leaves of Nicotiana tabacum. The Spearman correlation of gene expression for pairs of conifer species was high for needles (r2 = 0.78 − 0.86), and somewhat lower for embryogenic cultures (r2 = 0.68 − 0.83). The correlation of gene expression for tobacco leaves and needles of each of the three conifer species was lower but sufficiently high (r2 = 0.52 − 0.63) to suggest that many partial gene sequences are conserved in angiosperms and gymnosperms. Heterologous probing was further used to identify tissue‐specific gene expression over species boundaries. To evaluate the significance of differences in gene expression, conventional parametric tests were compared with permutation tests after four methods of normalization. Permutation tests after Z‐normalization provide the highest degree of discrimination but may enhance the probability of type I errors. It is concluded that arrays of cDNA from loblolly pine are useful for studies of gene expression in other pines or spruces. Copyright © 2002 John Wiley & Sons, Ltd.
Amorphin is phosphorylase; Phosphorylase is an alpha‐actinin‐binding protein
Authors: Chowrashi Prokash , Mittal Balraj , Sanger Jean M. , Sanger Joseph W. 
Published: September 2002

AbstractIn a study of myofibrillar proteins, Chowrashi and Pepe [1982: J. Cell Biol. 94:565–573] reported the isolation of a new, 85‐kD Z‐band protein that they named amorphin. We report that partial sequences of purified amorphin protein indicate that amorphin is identical to phosphorylase, an enzyme important in the metabolism of glycogen. Anti‐amorphin antibodies also reacted with purified chicken and rabbit phosphorylase. To explore the basis for phosphorylase's (amorphin's) localization in the Z‐bands of skeletal muscles, we reacted biotinylated alpha‐actinin with purified amorphin and with purified phosphorylase and found that alpha‐actinin bound to each. Radioimmune assays also indicated that phosphorylase (amorphin) bound to alpha‐actinin, and, with lower affinity, to F‐actin. Negative staining of actin filaments demonstrated that alpha‐actinin mediates the binding of phosphorylase to actin filaments. There are several glycolytic enzymes that bind actin (e.g., aldolase, phosphofructokinase, and pyruvate kinase), but phosphorylase is the first one demonstrated to bind alpha‐actinin. Localization of phosphorylase in live cells was assessed by transfecting cultures of quail embryonic myotubes with plasmids expressing phosphorylase fused to Green Fluorescent Protein (GFP). This resulted in targeting of the fusion protein to Z‐bands accompanied by a diffuse pattern in the cytoplasm. Cell Motil. Cytoskeleton 53:125–135, 2002. © 2002 Wiley‐Liss, Inc.
Localised depletion of polymerised actin at the front of Walker carcinosarcoma cells increases the speed of locomotion
Authors: Keller Hansuli , Zadeh Alireza Dehghani , Eggli Peter 
Published: September 2002

AbstractSpontaneously migrating Walker carcinosarcoma cells usually form lamellipodia at the front. Combined treatment with 10‐5M colchicine and 10‐7M latrunculin A produces large defects in the cortical F‐actin layer at the leading front and suppresses lamellipodia. However, the cortical actin layer at the rear is intact and shows myosin IIA accumulation. These cells, showing no or little detectable cortical F‐actin at the front and no morphologically recognisable protrusions, migrate faster than control cells with lamellipodia and an intact cortical actin layer. This documents that the cortical actin layer or actin‐powered force generation at the front is redundant for locomotion. Colchicine and latrunculin A have synergistic effects in compromising the cortical layer at the front and in increasing the speed of locomotion, but antagonistic effects on the relative amount of F‐actin per cell. Colchicine but not latrunculin A, can increase the proportion of polarised and locomoting cells under appropriate conditions. Locomotion and polarity of cells treated with latrunculin A and colchicine is inhibited at latrunculin A concentrations >10‐7M, by the myosin inhibitor BDM or the ROCK inhibitor Y‐27632. Colchicine and Y‐27632 have antagonistic effects on polarity and the speed of locomoting cells. The data show that locomotion of metazoan cells, which normally form lamellipodia, can be driven by actomyosin contraction behind the front (cell body, uropod). They are best compatible with a cortical contraction/frontal expansion model, but they are not compatible with models implying that actin polymerisation or actomyosin contraction at the front drive locomotion of the cells studied. Cell Motil. Cytoskeleton 53:189–202, 2002. © 2002 Wiley‐Liss, Inc.
Pseudopodium dynamics and rapid cell movement in Dictyostelium Ras pathway mutants
Authors: Chubb Jonathan R. , Wilkins Andrew , Wessels Deborah J. , Soll David R. , Insall Robert H. 
Published: September 2002

AbstractLoss of either of the Ras pathway members RasS or GefB causes growing Dictyostelium cells to move aberrantly rapidly. In this study, we describe the changes in motility that underlie these phenotypes using computer‐assisted 3D dynamic image analysis. Unexpectedly, the two mutants use different mechanisms to achieve rapid migration. The rasS− cells' motility is characterised by highly dynamic cell morphology, with rapidly extending and retracting pseudopodia. The gefB− cells do not have an unusually dynamic morphology, and achieve their efficient translocation by the continual remodelling of an existing dominant anterior pseudopodium. In spite of these dramatic changes in pseudopodium behaviour, the underlying motility cycle of both mutants remains normal. The levels of F‐actin in both mutant cell lines are significantly elevated with respect to the wild‐type parental cells, suggesting a possible biochemical basis for these emphatic phenotypes. Cell Motil. Cytoskeleton 53:150–162, 2002. © 2002 Wiley‐Liss, Inc.
A talin fragment as an actin trap visualizing actin flow in chemotaxis, endocytosis, and cytokinesis
Authors: Weber Igor , Niewöhner Jens , Du Aiping , Röhrig Ursula , Gerisch Günther 
Published: September 2002

AbstractA C‐terminal 63‐kDa fragment of talin A from Dictyostelium discoideum forms a slowly dissociating complex with F‐actin in vitro. This talin fragment (TalC63) has been tagged with GFP and used as a trap for actin filaments in chemotactic cell movement, endocytosis, and mitotic cell division. TalC63 efficiently sequesters actin filaments in vivo. Its translocation reflects the direction and efficiency of an actin flow. Along the body of a migrating Dictyostelium cell, this flow is directed from the front to the tail. If during chemotaxis one or two new fronts are induced, the flow is always directed away from these fronts. The flow thus reflects the re‐programming of cell polarity in response to changing gradients of chemoattractant. In endocytosis, the fluorescent complexes are translocated to the base of a phagocytic or macropinocytic cup. During mitosis, the complexes of F‐actin with TalC63 accumulate within the midzone of anaphase cells. If TalC63 is strongly expressed, the entire cleavage furrow is filled out by sequestered actin filaments, and cytokinesis is severely impaired. These cells are considered to mimic the phenotype of mutants deficient in the shredding of actin filaments that normally occurs in the mid‐zone of a dividing cell. Cell Motil. Cytoskeleton 53:136–149, 2002. © 2002 Wiley‐Liss, Inc.