bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractThe 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

AbstractAvermectin 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

AbstractTwenty‐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

AbstractProper 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

AbstractRecently 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

AbstractThe 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

AbstractWe 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

AbstractWe 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

AbstractAlthough 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

AbstractIn 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

AbstractTransforming 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

AbstractThe 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

AbstractThis 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

AbstractRadial 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].