bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of low culture temperature on specific productivity, transcription level, and heterogeneity of erythropoietin in Chinese hamster ovary cells
Authors: Yoon Sung Kwan , Song Ji Yong , Lee Gyun Min
Published: February 2003

To determine the effect of low culture temperature on erythropoietin (EPO) production in recombinant Chinese hamster ovary (rCHO) cells, rCHO cells producing EPO (LGE10‐9‐27) were cultivated at 30, 33, and 37°C. At a culture temperature lower than 37°C cell growth was suppressed, but cell viability remained high for a longer culture period. When the culture temperature was lowered from 37°C to 33°C, more than a 2.5‐fold increase in the maximum EPO concentration was achieved. This enhanced EPO production at 33°C was not just because of the extended culture longevity with the decreased release of proteolytic enzymes from dead cells, but mainly because of enhanced qEPO. The qEPO at 33°C was 0.35 ± 0.08 μg/106 cells/h, which was approximately 4‐fold higher than that at 37°C. Although the highest qEPO of 0.49 ± 0.14 μg/106 cells/h was obtained at 30°C, the maximum EPO concentration was lowest because the detrimental effect of lowering culture temperature on cell growth outweighed its beneficial effect on qEPO. Like qEPO, the relative EPO mRNA content increased by lowering culture temperature, indicating that the increased transcription level of EPO was responsible in part for the enhanced qEPO at low culture temperature. The quality of EPO produced at 33°C in regard to isoform pattern, sialic acid content, and in vivo biological activity was comparable to or even better than that produced at 37°C. Taken together, the results obtained demonstrate the potential of the application of low culture temperature to the commercial EPO production in rCHO cells. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 289–298, 2003.
Characterization of an oxygen‐dependent inducible promoter, the Escherichia coli nar promoter, in gram‐negative host strains
Authors: Lee Kil Ho , Cho Moo Hwan , Chung Taeowan , Chang Ho Nam , Lim Sun Ha , Lee Jongwon
Published: February 2003

The Escherichia coli nar promoter is maximally induced under anaerobic conditions in the presence of nitrate ion or under anaerobic only conditions, depending on the genotype of the E. coli nar promoter. Previously, we found that the E. coli nar promoter has some desirable characteristics as an inducible promoter in the E. coli host strains. In this study, the E. coli nar promoter with lacZ gene at the downstream was cloned onto a broad‐host‐range Gram‐negative vector, pBBR122. It was then induced in some other Gram‐negative host strains, such as Agrobacterium, Pseudomonas, and Rhizobium, to determine whether the E. coli nar promoter could be used as an inducible promoter in these strains. From shake‐flask experiments it was found that the wild‐type E. coli nar promoter cloned onto pBBR122, pNW61, was suppressed under aerobic conditions in an Agrobacterium host strain, was partially induced under microaerobic only conditions, and was maximally induced under microaerobic conditions in the presence of nitrate ion. Whereas the mutant‐type E. coli nar promoter cloned onto pBBR122, pNW618, was suppressed under aerobic conditions and was maximally induced under microaerobic conditions, regardless of the presence of nitrate ion. This kind of induction pattern observed for the E. coli nar promoters in the Agrobacterium host strain was similar to that observed for the E. coli nar promoters in the E. coli host strain. On the other hand, it was found that both of the E. coli nar promoters, pNW61 and pNW618, in a Pseudomonas host strain were partially induced under aerobic conditions and were maximally induced under microaerobic conditions, regardless of the presence of nitrate. Finally, it was found that both of the E. coli nar promoters in a Rhizobium host strain were minimally induced, regardless of the presence of oxygen or nitrate ion. Similar induction patterns for the three strains were also observed from fermentor experiments in which the dissolved oxygen (DO) level was tightly controlled. From an evolutionary point of view, the results from the three Gram‐negative host strains indicate that the E. coli nar promoter system, including the promoter and regulatory proteins, was best conserved in the Agrobacterium host strain and the least conserved in the Rhizobium host strain. From an industrial point of view, the results indicate that the E. coli nar promoter system can be used as an oxygen‐dependent inducible promoter in both Agrobacterium and Pseudomonas host strains. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 271–277, 2003.
Rheology, oxygen transfer, and molecular weight characteristics of poly(glutamic acid) fermentation by Bacillus subtilis
Authors: Richard Andrew , Margaritis Argyrios
Published: February 2003

Poly(glutamic acid) (PGA) is a water‐soluble, biodegradable biopolymer that is produced by microbial fermentation. Recent research has shown that PGA can be used in drug delivery applications for the controlled release of paclitaxel (Taxol) in cancer treatment. A fundamental understanding of the key fermentation parameters is necessary to optimize the production and molecular weight characteristics of poly(glutamic acid) by Bacillus subtilis for paclitaxel and other applications of pharmaceuticals for controlled release. Because of its high molecular weight, PGA fermentation broths exhibit non‐Newtonian rheology. In this article we present experimental results on the batch fermentation kinetics of PGA production, mass transfer of oxygen, specific oxygen uptake rate, broth rheology, and molecular weight characterization of the PGA biopolymer.© 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 299–305, 2003.
Formation of steady‐state oxygen gradients in vitro: Application to liver zonation
Authors: Allen Jared W. , Bhatia Sangeeta N.
Published: February 2003

We have developed a perfusion bioreactor system that allows the formation of steady state oxygen gradients in cell culture. In this study, gradients were formed in cultures of rat hepatocytes to study the role of oxygen in modulating cellular functions. A model of oxygen transport in our flat‐plate reactor was developed to estimate oxygen distribution at the cell surface. Experimental measurements of outlet oxygen concentration from various flow conditions were used to validate model predictions. We showed that cell viability was maintained over a 24‐h period when operating with a physiologic oxygen gradient at the cell surface from 76 to 5 mmHg O2 at the outlet. Oxygen gradients have been implicated in the maintenance of regional compartmentalized metabolic and detoxification functions in the liver, termed zonation. In this system, physiologic oxygen gradients in reactor cultures contributed to a heterogeneous distribution of phosphoenolpyruvate carboxykinase (predominantly localized upstream) and cytochrome P450 2B (predominantly localized downstream) that correlates with the distribution of these enzymes in vivo. The oxygen gradient chamber provides a means of probing the oxygen effects in vitro over a continuous range of O2 tensions. In addition, this system serves as an in vitro model of zonation that could be further extended to study the role of gradients in ischemia–reperfusion injury, toxicity, and bioartificial liver design. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 253–262, 2003.
Effect of specific oxygen uptake rate on Enterobacter aerogenes energetics: Carbon and reduction degree balances in batch cultivations
Authors: Converti Attilio , Perego Patrizia , Del Borghi Marco
Published: February 2003

The effect of oxygen availability on the metabolism of Enterobacter aerogenes NCIMB 10102 was studied through batch fermentations of glucose performed increasing the specific oxygen uptake rate up to 72.7 mmolO2 C‐molDW−1 h−1. The final concentrations of fermentation products of this biosystem (2,3‐butanediol, hydrogen, acetoin, formate, acetate, carbon dioxide, ethanol, lactate, succinate, and biomass) were utilized to check the use of simple carbon mass and reduction degree balances for the study of microbial energetics even in batch cultivations. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 370–377, 2003.
Computer simulation of the delivery of etanidazole to brain tumor from PLGA wafers: Comparison between linear and double burst release systems
Authors: Tan Wilson Hor Keong , Wang Fangjing , Lee Timothy , Wang Chi‐Hwa
Published: February 2003

This paper presents the computer simulation results on the delivery of Etanidazole (radiosensitizer) to the brain tumor and examines several factors affecting the delivery. The simulation consists of a 3D model of tumor with poly(lactide‐co‐glycolide) (PLGA) wafers with 1% Etanidazole loading implanted in the resected cavity. A zero‐order release device will produce a concentration profile in the tumor which increases with time until the drug in the carrier is depleted. This causes toxicity complications during the later stages of drug treatment. However, for wafers of similar loading, such release results in a higher drug penetration depth and therapeutic index as compared to the double drug burst profile. The numerical accuracy of the model was verified by the similar results obtained in the two‐dimensional and three‐dimensional models. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 278–288, 2003.
Aerobic biological treatment of synthetic municipal wastewater in membrane‐coupled bioreactors
Authors: Klatt Christian G. , LaPara Timothy M.
Published: February 2003

Membrane‐coupled bioreactors (MBRs) offer many benefits compared to conventional biological wastewater treatment systems; however, their performance characteristics are poorly understood. Laboratory‐scale MBRs were used to study bacterial adaptations in physiology and community structure. MBRs were fed a mixture of starch, gelatin, and polyoxyethylene‐sorbitan monooleate to simulate the polysaccharide, protein, and lipid components of municipal wastewater. Physiological adaptations were detected by measuring ectoenzyme activity while structural dynamics were studied by denaturing gradient gel electrophoresis of PCR‐amplified 16S rRNA gene fragments. As cell biomass accumulated in the MBRs, pollutant removal efficiency initially improved and then stabilized with respect to effluent concentrations of chemical oxygen demand, protein, and carbohydrate. Comparison of the MBR effluent to filtered reactor fluid indicated that a portion of the observed pollutant removal was due to filtration by the membrane rather than microbial activity. The rates of ectoenzyme‐mediated polysaccharide (α‐glucosidase) and protein (leucine aminopeptidase) hydrolysis became relatively constant once pollutant removal efficiency stabilized. However, the maximum rate of lipid hydrolysis (heptanoate esterase) concomitantly increased more than 10‐fold. Similarly, α‐glucosidase and leucine aminopeptidase ectoenzyme affinities were relatively constant, while the heptanoate esterase affinity increased more than 30‐fold. Community analysis revealed that a substantial community shift occurred within the first 7 days of operation. A Flavobacterium‐like bacterial population dominated the community (>50% of total band intensity) and continued to do so for the remainder of the experiment. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 313–320, 2003.
Effects of intermittent addition of cellulase for production of L‐lactic acid from wastewater sludge by simultaneous saccharification and fermentation
Authors: Nakasaki Kiyohiko , Adachi Tomohiko
Published: February 2003

An attempt was made to create L‐lactic acid, a precursor of poly‐lactic acid, which is a biodegradable plastic, from wastewater sludge from the paper‐manufacturing industry. The sludge contained a high percentage of cellulose and needed to be hydrolyzed to glucose by the action of the cellulase before being treating with lactic acid bacteria. Therefore, a method involving simultaneous saccharification and fermentation (SSF) was carried out. The optimum pH of the SSF for production of the lactic acid by the newly isolated lactic acid bacterium with a high selectively of L‐lactic acid was found out to be around pH = 5.0, and the optimum temperature to be approximately 40°C. On the basis of the measurement of the cell density changes in the lactic acid bacteria, it was ascertained that the bacterial activity could continue at a high level for a relatively long period of time, and that the L‐lactic acid productivity was diminished by the rapid deactivation of the cellulase. With the intermittent addition of cellulase once daily for the sake of compensating for the cellulase deactivation, the L‐lactic acid attained a maximum concentration of 16.9 g/L, i.e., a 72.2% yield based on the potential glucose contained in the sludge under optimum pH and temperature conditions.© 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 263–270, 2003.
Bracketed generic inactivation of rodent retroviruses by low pH treatment for monoclonal antibodies and recombinant proteins
Authors: Brorson Kurt , Krejci Sherrie , Lee Kitty , Hamilton Elizabeth , Stein Kathryn , Xu Yuan
Published: February 2003

Viral safety is a predominant concern for monoclonal antibodies (mAbs) and other recombinant proteins (RPs) with pharmaceutical applications. Certain commercial purification modules, such as nanofiltration and low‐pH inactivation, have been observed to reliably clear greater than 4 log10 of large enveloped viruses, including endogenous retrovirus. The concept of “bracketed generic clearance” has been proposed for these steps if it could be prospectively demonstrated that viral log10 reduction value (LRV) is not impacted by operating parameters that can vary, within a reasonable range, between commercial processes. In the case of low‐pH inactivation, a common step in mAb purification processes employed after protein A affinity chromatography, these parameters would include pH, time and temperature of incubation, the content of salts, protein concentration, aggregates, impurities, model protein pI, and buffer composition. In this report, we define bracketed generic clearance conditions, using a prospectively defined bracket/matrix approach, where low‐pH inactivation consistently achieves ≥4.6 log10 clearance of xenotropic murine leukemia virus (X‐MLV), a model for rodent endogenous retrovirus. The mechanism of retrovirus inactivation by low‐pH treatment was also investigated. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 321–329, 2003.
Quantification of toxic and inhibitory impact of copper and zinc on mixed cultures of sulfate‐reducing bacteria
Authors: Utgikar Vivek P. , Tabak Henry H. , Haines John R. , Govind Rakesh
Published: February 2003

The adverse effects of copper and zinc on an acetate‐utilizing mixed cultures of sulfate‐reducing bacteria (SRB) at concentrations below the toxic concentration (minimum metal concentration at which no sulfate reduction is observed) are reported in this paper. Mathematical models were developed to incorporate the toxic and inhibitory effects (defined as the reduction in bacterial population upon exposure to the metal and the decrease in the metabolic rate of sulfate reduction by the SRB, respectively) into the sulfate‐reduction biokinetics. The characteristic toxicity and inhibition constants were obtained from the measurements of bacterial populations and dissolved metal concentrations in serum bottle studies conducted at 35°C and pH 6.6. Both copper and zinc had toxic and inhibitory effects on SRB. The toxicity constants for copper and zinc were 10.6 and 2.9 mM−1, respectively, indicating that exposure to copper resulted in a higher mortality of SRB than did exposure to zinc. The values of the inhibition constants were found to be 17.9 ± 2.5 and 25.2 ± 1.0 mM−1 for copper and zinc, respectively. This implies that dissolved zinc was slightly more inhibitory to SRB than copper. The models presented in the paper can be used to predict the response of a sulfate‐reduction bioreactor to heavy metals during acid mine drainage treatment. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 306–312, 2003.
Engineering the aveC gene to enhance the ratio of doramectin to its CHC‐B2 analogue produced in Streptomyces avermitilis
Authors: Stutzman‐Engwall Kim , Conlon Steve , Fedechko Ronald , Kaczmarek Frank , McArthur Hamish , Krebber Anke , Chen Yan , Minshull Jeremy , Raillard Sun Ai , Gustafsson Claes
Published: February 2003

Avermectin and its analogues are produced by the actinomycete Streptomyces avermitilis and are major commercial products for parasite control in the fields of animal health, agriculture, and human infections. Historically, the avermectin analogue doramectin (CHC‐B1), which is sold commercially as Dectomax™, is co‐produced during fermentation with the undesired analogue CHC‐B2 at a CHC‐B2:CHC‐B1 ratio of 1.6:1. Although the identification of the avermectin gene cluster has allowed for characterization of most of the biosynthetic pathway, the mechanism for determining the avermectin B2:B1 ratio remains unclear. The aveC gene, which has an essential role in avermectin biosynthesis, was inactivated by insertional inactivation and mutated by site‐specific mutagenesis and error‐prone PCR. Several unrelated mutations were identified that resulted in improved ratios of the desirable avermectin analogue CHC‐B1, produced relative to the undesired CHC‐B2 fermentation component. High‐throughput (HTP) screening of cultures grown on solid‐phase fermentation plates and analysis using electrospray mass spectrometry was implemented to significantly increase screening capability. An aveC gene with mutations that result in a 4‐fold improvement in the ratio of doramectin to CHC‐B2 was identified. Subsequent integration of the enhanced aveC gene into the chromosome of the S. avermitilis production strain demonstrates the successful engineering of a specific biosynthetic pathway gene to significantly improve fermentation productivity of a commercially important product. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 359–369, 2003.
Ester synthesis from trimethylammonium alcohols in dry organic media catalyzed by immobilized Candida antarctica lipase B
Authors: Lozano Pedro , Daz Mirta , De Diego Teresa , Iborra José L.
Published: February 2003

Twenty‐one different organic solvents were assayed as possible reaction media for the synthesis of butyryl esters from trimethylammonium alcohols in dry conditions catalyzed by immobilized Candida antarctica lipase B. The reactions were carried out following a transesterification kinetic approach, using choline and L‐carnitine as primary and secondary trimethylammonium alcohols, respectively, and vinyl butyrate as acyl donor. The synthetic activity of the enzyme was strictly dependent on the water content, the position of the hydroxyl group in the trimethylammonium molecule, and the Log P parameter of the assayed solvent. Anhydrous conditions and a high excess of vinyl butyrate over L‐carnitine were necessary to synthesize butyryl‐L‐carnitine. The synthetic reaction rates of butyryl choline were practically 100‐fold those of butyryl‐L‐carnitine with all the assayed solvents. In both cases, the synthetic activity of the enzyme was dependent on the hydrophobicity of the solvent, with the optimal reaction media showing a Log P parameter of between −0.5 and 0.5. In all cases, 2‐methyl‐2‐propanol and 2‐methyl‐2‐butanol were shown to be the best solvents for both their high synthetic activity and negligible loss of enzyme activity after 6 days. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 352–358, 2003.
Characterization of antibody binding to three cancer‐related antigens using flow cytometry and cell tracking velocimetry
Authors: Chosy E. Julia , Nakamura Masayuki , Melnik Kristie , Comella Kristin , Lasky Larry C. , Zborowski Maciej , Chalmers Jeffrey J.
Published: February 2003

Proper antibody labeling is a fundamental step in the positive selection/isolation of rare cancer cells using immunomagnetic cell separation technology. Using either a two‐step or single‐step labeling protocol, we examined a combination of six different antibodies specific for three different antigens (epithelial specific antigen, epithelial membrane antigen, and HER‐2/Neu) on two different breast cancer cell lines (HCC1954 and MCF‐7). When a two‐step labeling protocol was used (i.e., anti‐surface marker–fluoroscein‐isothiocyanate [FITC] [primary Ab], anti‐FITC magnetic colloid [secondary Ab]) saturation of the primary antibody was determined using fluorescence intensity measurements from flow cytometry (FCM). The saturation of the secondary antibody (or saturation of a single‐step labeling) was determined using magnetophoretic mobility measurements from cell tracking velocimetry (CTV). When the maximum magnetophoretic mobility was the primary objective, our results demonstrate that the quantities necessary for antibody saturation with respect to fluorescence intensity were generally higher than those recommended by the manufacturer. The results demonstrate that magnetophoretic mobility varies depending on the types of cell lines, primary antibodies, and concentration of secondary magnetic colloid‐conjugated antibody. It is concluded that saturation studies are a vital preparatory step in any separation method involving antibody labeling, especially those that require the specificity of rare cell detection. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 340–351, 2003.
Application of hydrophobic interaction displacement chromatography for an industrial protein purification
Authors: Sunasara Khurram M. , Xia Fang , Gronke Robert S. , Cramer Steven M.
Published: February 2003

Recently it has been established that low molecular weight displacers can be successfully employed for the purification of proteins in hydrophobic interaction chromatography (HIC) systems. This work investigates the utility of this technique for the purification of an industrial protein mixture. The study involved the separation of a mixture of three protein forms, that differed in the C‐terminus, from their aggregate impurities while maintaining the same relative ratio of the three protein forms as in the feed. A batch high‐throughput screening (HTS) technique was employed in concert with fluorescence spectroscopy for displacer screening in these HIC systems. This methodology was demonstrated to be an effective tool for identifying lead displacer candidates for a particular protein/stationary‐phase system. In addition, these results indicate that surfactants can be employed at concentrations above their CMCs as effective displacers. Displacement of the recombinant proteins with PEG‐3400 and the surfactant Big Chap was shown to increase the productivity as compared to the existing step‐gradient elution process. © 2003 Wiley Periodicals, Inc. Biotechol Bioeng 82: 330–339, 2003.
Products of phosphoinositide specific phospholipase C can trigger dephosphorylation of cofilin in chemoattractant stimulated neutrophils
Authors: Zhan Qian , Bamburg James R. , Badwey John A.
Published: February 2003

The signal transduction pathways that trigger dephosphorylation of cofilin in neutrophils stimulated with the chemoattractant fMet‐Leu‐Phe (fMLP) were investigated with a phospho‐specific antibody that recognized cofilin only when this protein was phosphorylated on ser‐3. Unlike earlier studies that monitored changes in 32P‐labeled cofilin, this Ab allowed us to monitor changes in the total mass of phosphorylated cofilin during neutrophil stimulation. Neutrophils stimulated with fMLP (1.0 μM) for 1.0 min exhibited a massive loss (> 85%) of phosphate from cofilin, which was blocked by an antagonist of phosphoinositide‐specific phospholipase C (PI‐PLC) (1.0 μM U73122). Products of PI‐PLC, sn‐1,2‐diglyceride and inositol (1,4,5)‐trisphosphate, are known to activate protein kinase C (PKC) and increase intracellular Ca2+, respectively. Treatment of neutrophils with agents that selectively activate PKC [4β‐phorbol 12‐myristate 13‐acetate (PMA) ] or cellular Ca2+ (ionophore A23187) also triggered dephosphorylation of cofilin. Both a nonspecific (100 nM staurosporine) and a highly selective antagonist of PKC (200 nM bisindolylmaleimide I) blocked dephosphorylation of cofilin in neutrophils stimulated with PMA but not with fMLP or ionophore A23187. The calmodulin (CaM) antagonists trifluoperazine (15 μM) and W‐7 (50 μM) blocked dephosphorylation of cofilin in stimulated neutrophils whereas inactive/less‐active analogs of these inhibitors (15 μM promethazine, 50 μM W‐5) were substantially less effective. Calyculin A (40 nM), an antagonist of type 1 and 2A protein phosphatases, also triggered a massive dephosphorylation of cofilin in unstimulated neutrophils through a pathway that was insensitive to inhibitors of type 2B phosphatases. These data suggest that both PKC‐dependent and independent pathways can trigger dephosphorylation of cofilin in neutrophils with the latter pathway predominating in fMLP‐stimulated cells. These pathways may also contain CaM and a type 2C and/or novel phosphatase (e.g., slingshot). Cell Motil. Cytoskeleton 54:1–15, 2003. © 2003 Wiley‐Liss, Inc.
Dynamic localization of myosin‐I to endocytic structures in Acanthamoeba
Authors: Ostap E. Michael , Maupin Pamela , Doberstein Steven K. , Baines Ivan C. , Korn Edward D. , Pollard Thomas D.
Published: February 2003

We used fluorescence microscopy of live Acanthamoeba to follow the time course of the concentration of myosin‐I next to the plasma membrane at sites of macropinocytosis and phagocytosis. We marked myosin‐I with a fluorescently labeled monoclonal antibody (Cy3‐M1.7) introduced into the cytoplasm by syringe loading. M1.7 binds myosin‐IA and ‐IC without affecting their activities, but does not bind myosin‐IB. Cy3‐M1.7 concentrates at two different macropinocytic structures: large circular membrane ruffles that fuse to create macropinosomes, and smaller endocytic structures that occur at the end of stalk‐like pseudopodia. These dynamic structures enclose macropinosomes every 30–60 s. Cy3‐M1.7 accumulates rapidly as these endocytic structures form and dissipate rapidly after they internalize. Double labeling fixed cells with Cy3‐M1.7 and polyclonal antibodies specific for myosin‐IA, ‐IB, or ‐IC revealed that all three myosin‐I isoforms associate with macropinocytic structures, but individual structures vary in their myosin‐I isoform composition. Myosin‐I and actin also concentrate transiently at sites where amoebae ingest yeast or the pseudopodia of neighboring cells (heterophagy) by the process of phagocytosis. Within 3 min of yeast attachment to the amoeba, myosin‐I concentrates around the phagocytic cup, yeast are internalized, and myosin‐I de‐localizes. Despite known differences in the regulation of macropinocytosis and phagocytosis, the morphology, protein composition, and dynamics of phagocytosis and macropinocytosis are similar, indicating that they share common structural properties and contractile mechanisms. Cell Motil. Cytoskeleton 54:29–40, 2003. © 2003 Wiley‐Liss, Inc.
Erythroid spectrin in miceller detergents
Authors: Ray Sibnath , Chakrabarti Abhijit
Published: February 2003

We have studied the interaction of spectrin, the major protein of the erythrocyte cytoskeleton, with four commonly used detergents at concentrations above their critical miceller concentrations (cmc). Fluorescence spectroscopic studies on the emission intensity, steady state polarization, quenching with acrylamide, and time‐resolved fluorescence measurements were done with spectrin in anionic detergents, e.g., SDS, deoxycholate, and nonionic detergents, e.g., Triton‐X‐100 and octylglucoside at concentrations double their respective cmc's. The spectrin‐detergent complexes in all four systems have been characterized by far‐UV CD and measurements on tryptophan fluorescence in combination with fluorescence of the extrinsic probe, pyrene. Tryptophan fluorescence studies revealed quaternary structural changes due to unzipping of the spectrin subunits in Triton‐X‐100 without complete dissociation. Both Triton‐X‐100 and SDS were found to partially denature spectrin indicated by the far‐UV CD. Octylglucoside and deoxycholate are shown to have the least structural perturbations on the cytoskeletal protein, rationalizing the use of octylglucoside, in particular and also deoxycholate to be the most effective in preparing cytoskeletal fractions from erythrocytes rather than the Triton‐X‐100 that has long been used for preparing the Triton shells. Cell Motil. Cytoskeleton 54:16–28, 2003. © 2003 Wiley‐Liss, Inc.
Actin‐based endosome and phagosome rocketing in macrophages: activation by the secretagogue antagonists lanthanum and zinc
Authors: Southwick Frederick S. , Li Wei , Zhang Fangliang , Zeile William L. , Purich Daniel L.
Published: February 2003

Although motile endocytic vesicles form actin‐rich rocket tails [Merrifield et al., 1999: Nature Cell Biol 1:72–74], the mechanism of intracellular organelle locomotion remains poorly understood. We now demonstrate that bone marrow macrophages treated with lanthanum and zinc ions, well‐known secretagogue antagonists, reliably exhibit vesicle motility. This treatment results in accentuated membrane ruffling and the formation of phagosomes and early endosomes that move rapidly through the cytoplasm by assembling actin filament rocket tails. Protein‐specific immunolocalization demonstrated the presence of Arp2/3 complex in the polymerization zone and throughout the actin‐rich tail, whereas N‐WASP was most abundant in the polymerization zone. Although Arp2/3 and N‐WASP play essential roles in nucleating filament assembly, other processes (i.e., elongation and filament cross‐linking) are required to produce forces needed for motility. Efficient elongation was found to require zyxin, VASP, and profilin, proteins that interact by means of their ABM‐1 and ABM‐2 proline‐rich motifs. The functional significance of these motifs was demonstrated by inhibition of vesicle motility by the motif‐specific ABM‐1 and ABM‐2 analogues. Furthermore, lanthanum/zinc treatment also facilitated the early onset of actin‐based vaccinia motility, a process that also utilizes Arp2/3 and N‐WASP for nucleation and the zyxin‐VASP‐profilin complex for efficient elongation. Although earlier studies using cell extracts clouded the role of oligoproline sequences in activating the polymerization zone, our studies emphasize the importance of evaluating motility in living cells. Cell Motil. Cytoskeleton 54:41–55, 2003. © 2003 Wiley‐Liss, Inc.
How spermatozoa come to be confined to surfaces
Authors: Cosson J. , Huitorel P. , Gagnon C.
Published: February 2003

In most detailed studies, sea urchin sperm movement has been analyzed mainly from observations of spermatozoa swimming at the interface between two media: water/air or water/glass. When spermatozoa are placed on a microscope slide, they rapidly appear to swim near those interfaces. The aim of this article is to determine how they become confined to the vicinity of surfaces. High‐speed observations of moving spermatozoa reveal blurred portions in the flagellum images that propagate from base to tip, suggesting that flagellar waves contain an out‐of‐plane component. The model we have developed depicts how this tri‐dimensional component tends to keep spermatozoa close to interfaces and, as a consequence, increases the time of contact between the egg surface and spermatozoa. Cell Motil. Cytoskeleton 54:56–63, 2003. © 2003 Wiley‐Liss, Inc.
Keratinocyte motility induced by TGF‐β1 is accompanied by dramatic changes in cellular interactions with laminin 5
Authors: Décline Françoise , Okamoto Osamu , Mallein‐Gerin Frédéric , Helbert Bruno , Bernaud Janine , Rigal Dominique , Rousselle Patricia
Published: February 2003

Transforming growth factor‐β1 (TGF‐β1) has the ability to induce epithelial cell migration while stopping proliferation. In this study, we show that, concomitant to promoting migration of normal human keratinocytes in vitro, TGF‐β1 induced a marked decrease in their adhesion capacity to processed α3‐containing laminin 5‐coated surfaces. Indeed, the expression levels of α3 and α6 integrin subunit mRNA and protein, as well as the cell surface α3β1 and α6β4 integrins, were down‐regulated. Recent studies showed that keratinocytes over express and deposit laminin 5 during migration and we have shown that laminin 5 found in the matrix of TGF‐β1 induced migrating keratinocytes is present in its unprocessed form [Décline and Rousselle, 2001: J. Cell Sci. 114:811–823]. We show here that TGF‐β1 treatment of the cells promoted a significant increase in their adhesion to the α3 chain carboxy‐terminal LG4/5 subdomain and that this interaction is likely to be mediated by a heparan sulfate proteoglycan type of receptor. Our results indicate that α6β4 and α3β1 integrin interactions with laminin 5 are diminished during migration while a specific interaction occurs between an additional cellular receptor and the α3 LG4/5 module present on unprocessed laminin 5. Cell Motil. Cytoskeleton 54:64–80, 2003. © 2003 Wiley‐Liss, Inc.
Identification and characterisation of a novel human isoform of Arp2/3 complex subunit p16‐ARC/ARPC5
Authors: Millard Thomas H. , Behrendt Barbara , Launay Sophie , Fütterer Klaus , Machesky Laura M.
Published: February 2003

The Arp2/3 complex is an actin filament nucleator that activates regulated actin assembly in response to extracellular signals. The mammalian complex is composed of seven subunits, the smallest of which is known as ARPC5 or p16‐Arc. We have identified a human cDNA sequence with homology to ARPC5 and here provide evidence that this encodes a novel ARPC5 isoform. Specific antibodies were generated against the novel protein, which we have termed ARPC5B, as well as the previously characterised ARPC5 isoform, henceforth ARPC5A. The presence of both ARPC5 isoforms was detected in Arp2/3 complex affinity purified from human neutrophil extract. The tissue distribution of ARPC5A and B was analysed using the isoform‐specific antibodies and it was found that the two isoforms exhibited significant differences; ARPC5A was found to be highly enriched in spleen and thymus, while ARPC5B exhibits a more regular expression, with levels in the brain being highest. Myc‐tagged ARPC5A and B co‐localised with the Arp2/3 complex when expressed in C2C12 cells and the cellular distribution of the two isoforms could not be distinguished. Our data show for the first time that mammalian cells contain multiple forms of the Arp2/3 complex. Cell Motil. Cytoskeleton 54:81–90, 2003. © 2003 Wiley‐Liss, Inc.
Calyculin A–induced actin phosphorylation and depolymerization in renal epithelial cells
Authors: Gu Luo , Zhang Hui , Chen Qi , Chen Jing
Published: February 2003

This study reports actin phosphorylation and coincident actin cytoskeleton alterations in renal epithelial cell line, LLC‐PK1. Serine phosphorylation of actin was first observed in vitro after the cell lysate was incubated with phosphatase inhibitors and ATP. Both the phosphorylated actin and actin kinase activities were found in the cytoskeletal fraction. Actin phosphorylation was later detected in living LLC‐PK1 cells after incubation with the phosphatase inhibitor calyculin A. Calyculin A–induced actin phosphorylation was associated with reorganization of the actin cytoskeleton, including net actin depolymerization, loss of cell‐cell junction and stress fiber F‐actin filaments, and redistribution of F‐actin filaments in the periphery of the rounded cells. Actin phosphorylation was abolished by 3‐h ATP depletion but not by the non‐specific kinase inhibitor staurosporine. These results demonstrate that renal epithelial cells contain kinase/phosphatase activities and actin can be phosphorylated in LLC‐PK1 cells. Actin phosphorylation may play an important role in regulating the organization of the actin cytoskeleton in renal epithelium. Cell Motil. Cytoskeleton 54:286–295, 2003. © 2003 Wiley‐Liss, Inc.
Entropy and information in flagellar axoneme cybernetics: A radial spokes integrative function
Authors: Cibert Christian
Published: February 2003

Radial spokes and the consequences of their relationships with the central apparatus seem to play a very important role in the regulation of axonemal activity. We modeled their behavior and observed that it appears to differ in the cilium and the flagellum with respect to the development of bending as a function of time. Specifically, our calculation raises the question of the real function of the radial spokes in the regulation of the axoneme, because a given curvature of the flagellar axoneme may correspond to two opposite of their tilts. The stable nil/low amplitude shear points that we had characterized along the flagellum allowed us to describe their axoneme as a series of modules [Cibert, 2002: Cell Motil. Cytoskeleton 51:89–111]. We observed that a nil/low shearing point moves along each module during beating when a new bend is created at the base of the flagellum [Cibert, 2001: Cell Motil. Cytoskeleton 49:161–175]. We propose that the structural gradients of isoforms of tubulin could be basic verniers that act as structural references for the axonemal machinery during the beating. This allowed us to interpret the axonemal organization as a segmented structure, that could be analyzed according to the complexion1 theory and Shannon's information theory, which associate entropy and probability in the creation of information. The important consequence of this interpretation is that regulation of the axonemal machinery appears to be due to the upstream and downstream cross‐talk between the axonemal segments that do not involve any dedicated integrative structure but depend on the energy level of the entire length of each module. Cell Motil. Cytoskeleton 54:296–316, 2003. © 2003 Wiley‐Liss, Inc.

1Complexion was defined by Boltzman in 1877 and used by Planck in 1900 to calculate the energy distribution of a normal spectrum [Morowitz, 1970].
Expression of WASP and Scar1/WAVE1 actin‐associated proteins is differentially modulated during differentiation of HL‐60 cells
Authors: Launay Sophie , Brown Geoffrey , Machesky Laura M.
Published: February 2003

The Wiskott‐Aldrich Syndrome (WAS) is a disease associated with mutations in the WAS gene and characterised by developmental defects in haematopoietic cells such as myeloid cells. The Wiskott‐Aldrich Syndrome protein (WASP)‐family includes Scar1 and WASP, which are key regulators of actin reorganization in motile cells. To understand the roles of Scar1 and WASP in myeloid cells and their cytoskeletal control in haematopoietic tissues, we have explored their expression during differentiation of the promyeloid cell line HL‐60. Undifferentiated HL‐60 cells expressed Scar1 and WASP, and differentiation to neutrophils, induced by retinoic acid or non‐retinoid agent treatments, led to a decrease in the level of expression of Scar1, whereas WASP expression was unaffected. Differentiation to monocytes/macrophages, induced by phorbol ester treatment, resulted in a decreased expression of both proteins in the adherent mature cells. Vitamin D3 treatment or cytochalasin D in combination with PMA treatment did not affect WASP expression suggesting that adhesion and cytoskeletal integrity were both essential to regulate WASP expression. Scar1 expression was regulated by differentiation, adhesion, and cytoskeletal integrity. Recently, WASP was found to colocalize with actin in the podosomes. In contrast, we show here that Scar1 did not localize with the podosomes in mature monocytes/macrophages. These observations show for the first time that modulation of Scar1 and WASP expression is a component of the differentiation program of myeloid precursors and indicate that WASP and Scar1 have different roles in mature myeloid cells. Cell Motil. Cytoskeleton 54:274–285, 2003. © 2003 Wiley‐Liss, Inc.
Muscle‐specific microtubule‐associated protein 4 is expressed early in myogenesis and is not sufficient to induce microtubule reorganization
Authors: Casey Liam M. , Lyon Hiram D. , Olmsted Joanna B.
Published: February 2003

The expression of a muscle‐specific variant of microtubule‐associated protein 4 (mMAP4) has been analyzed during myogenesis of C2C12 cells using an isoform‐specific antibody. MMAP4 localizes to microtubules (MTs) and is expressed prior to a very early morphogenetic event, the formation of mononucleate spindle‐shaped cells. MMAP4 protein appears at about the same time as titin and coincident with Golgi reorganization, but antedates myosin expression. Misexpression of EGFP‐mMAP4 in non‐muscle and proliferating C2C12 cells does not induce dramatic changes in MT organization or stability, nor in Golgi organization. Expression of full‐length mMAP4 or of a truncated form lacking the MT‐binding domain does not disrupt myotube formation or myofibrillogenesis. While previous antisense studies indicated that mMAP4 is necessary for normal myotube formation [Mangan and Olmsted, 1996: Development 122:771–781], these data indicate mMAP4 is not sufficient to induce the reorganization of MTs or the Golgi into patterns typical of muscle cells. Thus, with respect to MT organizing properties, this tissue‐specific variant differs from related neuronal MAPs, MAP2, and tau, which induce neural‐like changes in MT organization. Cell Motil. Cytoskeleton 54:317–336, 2003. © 2003 Wiley‐Liss, Inc.
Beg, borrow, or steal. finding ideas for new NMR experiments
Authors: Freeman Ray
Published: February 2003

Inspiration for new NMR experiments can sometimes come from other disciplines, far removed from magnetic resonance. Five disparate examples are presented: optimum weighing strategies, radar technology, Darwin's theory of evolution, quantum computers, and simulation of brain function. The applications range from multiple selective irradiation experiments that retain the multiplex advantage, radiofrequency oscillators that “follow” the magnetic field, a solvent suppression scheme, a pulse sequence for the “universal swap” operation, and “designer” radiofrequency pulses. © 2003 Wiley Periodicals, Inc. Concepts Magn Reson Part A 17A: 71–85, 2003.
A primer for nuclear magnetic relaxation in liquids
Authors: Murali Nagarajan , Krishnan V.V.
Published: February 2003

This article is a primer that is intended to serve as a concise source of information for researchers interested in learning relaxation aspects in NMR. Explicit expressions have been derived for the equation of motion of nuclear spins in the solution state. General expressions valid for all motional regimes, including anisotropy of spin interactions and molecular motions, are presented. Intricate details that impact the validity of various expressions have been elaborated and efforts have been made to maintain consistent notation. The suitable examples given here should help in the understanding of the concepts developed in the theory. © 2003 Wiley Periodicals, Inc. Concepts Magn Reson Part A 17A: 86–116, 2003.
Computer simulations in solid‐state NMR. I. Spin dynamics theory
Authors: Edén Mattias
Published: February 2003

This article is the first in a series of publications that discuss the basics of the writing of computer programs for simulating solid‐state NMR experiments with static and rotating samples. The present article gives an account of the relevant NMR theory needed for writing NMR simulation computer codes. The concept of irreducible spherical tensors is reviewed, as is how it may be used to construct Hamiltonians that represent the various NMR interactions. The spin dynamics that links the Hamiltonian to the time evolution of the nuclear spins and the detection of the NMR time‐domain signal is discussed for static and rotating solids, as well as the relationship between the form of the Hamiltonian and the resulting NMR spectrum. © 2003 Wiley Periodicals, Inc. Concepts Magn Reson Part A 17A: 117–154, 2003.
Nitric oxide suppression triggers apoptosis through the FKHRL1 (FOXO3A)/ROCK kinase pathway in human breast carcinoma cells
Authors: Radisavljevic Zivotije
Published: February 2003

BACKGROUND

The winged helix/forkhead transcriptional factor FKHRL1 (FOXO3a) triggers apoptosis, but its mode of action is not well understood. ROCK kinase is an effector molecule in human breast carcinoma cell apoptosis, but its relation to FKHRL1 is unknown. Because the human breast carcinoma T47D cell line releases a great amount of nitric oxide (NO), I investigated signaling of FKHRL1 to ROCK kinase during NO suppression.

METHODS

Expression of phosphorylated FKHRL1 in T47D cells was analyzed using Western blotting. Apoptosis was evaluated by flow cytometry. Transfection of FKHRL1‐HA wild‐type and mutant FKHRL1‐HA T32A constructs were performed by lipofectamine plus reagent. Measurement of NO generation was performed by Griess reaction.

RESULTS

Nitric oxide suppression promotes FKHRL1 thr‐32‐enhanced phosphorylation, which was significantly (P < 0.005) sensitive to Y‐27632, a specific inhibitor of the ROCK kinase, but not to capase‐3 inhibitor or wortnannin, a specific inhibitor of phosphoinositol‐3‐OH kinase (PI3K). Nitric oxide suppression by N‐G‐monomethyl‐L‐arginine, an inhibitor of NO synthase, causes a significant (P < 0.001) increase in the apoptosis of T47D cells. However, a significant decrease (P < 0.01) in NO generation and a significant (P < 0.01) increase in apoptosis were observed when FKHRL1‐HA wild‐type cells were transfected, which caused increased FKHRL1 thr‐32 phosphorylation.

CONCLUSIONS

This novel unknown phenomenon of breast carcinoma cell apoptosis was triggered by NO suppression, which promotes FKHRL1 thr‐32‐enhanced phosphorylation and initiates signaling of FKHRL1 to ROCK kinase as an effector molecule. This apoptotic signalling process is caspase‐3 as well as PI3K/Akt independent. Cancer 2003;97:1358–63. © 2003 American Cancer Society.

DOI 10.1002/cncr.10081
Socioeconomic status and breast carcinoma survival in four racial/ethnic groups
Authors: O'Malley Cynthia D. , Le Gem M. , Glaser Sally L. , Shema Sarah J. , West Dee W.
Published: February 2003

BACKGROUND

Although overall survival for invasive breast carcinoma remains high, black women experience poorer survival than whites. Less is known about the survival of Hispanics and Asians, who may share clinical and socioeconomic risk factors similar to blacks. To better understand racial/ethnic survival patterns, we investigated the effect of socioeconomic status (SES) and disease stage on racial/ethnic differences in breast carcinoma survival in a large population‐based cohort.

METHODS

Using data from the Surveillance, Epidemiology, and End Results program (SEER), we identified 10,414 white, 940 black, 1100 Hispanic, and 1180 Asian females diagnosed with breast carcinoma in the Greater San Francisco Bay Area between 1988 and 1992. We used the Kaplan–Meier method to generate survival rates and Cox proportional hazards regression to estimate the risk of death by race/ethnicity, after adjustment for clinical, demographic, and census‐derived SES variables.

RESULTS

The 10‐year unadjusted survival rates were 81% for whites, 69% for blacks, 75% for Hispanics, and 79% for Asians. Adjusting for stage decreased the relative risk of mortality for blacks from 1.81 to 1.29; the stage‐adjusted relative risk for Hispanics (1.11) and Asians (1.02) did not differ significantly from whites. Additional adjustment for age, tumor characteristics, and treatment factors did little to alter the relative risk in blacks; adding blue‐collar status to the model further decreased the relative risks for blacks to 1.22. Residing in a blue‐collar neighborhood was independently associated with a 1.16 increase in risk of death.

CONCLUSIONS

After adjustment for multiple factors, blacks continue to have slight but significantly poorer survival after breast carcinoma compared with whites, whereas the survival of Hispanics and Asians did not differ from whites. Cancer 2003;97:1303–11. © 2003 American Cancer Society.

DOI 10.1002/cncr.11160