bluuebunny/crossref_metadata_2025 huggingface dataset:
Microbial Hydrogen Production with Immobilized Sewage Sludge
Authors: Wu Shu‐Yii , Lin Chi‐Num , Chang Jo‐Shu , Lee Kuo‐Shing , Lin Ping‐Jei
Published: October 2002

Municipal sewage sludge was immobilized to produce hydrogen gas under anaerobic conditions. Cell immobilization was essentially achieved by gel entrapment approaches, which were physically or chemically modified by addition of activated carbon (AC), polyurethane (PU), and acrylic latex plus silicone (ALSC). The performance of hydrogen fermentation with a variety of immobilized‐cell systems was assessed to identify the optimal type of immobilized cells for practical uses. With sucrose as the limiting carbon source, hydrogen production was more efficient with the immobilized‐cell system than with the suspended‐cell system, and in both cases the predominant soluble metabolites were butyric acid and acetic acid. Addition of activated carbon into alginate gel (denoted as CA/AC cells) enhanced the hydrogen production rate ( vH2) and substrate‐based yield ( YH2/sucrose) by 70% and 52%, respectively, over the conventional alginate‐immobilized cells. Further supplementation of polyurethane or acrylic latex/silicone increased the mechanical strength and operation stability of the immobilized cells but caused a decrease in the hydrogen production rate. Kinetic studies show that the dependence of specific hydrogen production rates on the concentration of limiting substrate (sucrose) can be described by Michaelis‐Menten model with good agreement. The kinetic analysis suggests that CA/AC cells may contain higher concentration of active biocatalysts for hydrogen production, while PU and ALSC cells had better affinity to the substrate. Acclimation of the immobilized cells led to a remarkable enhancement in vH2 with a 25‐fold increase for CA/AC and ca. 10‐ to 15‐fold increases for PU and ALSC cells. However, the ALSC cells were found to have better durability than PU and CA/AC cells as they allowed stable hydrogen production for over 24 repeated runs.
Cell Population Modeling and Parameter Estimation for Continuous Cultures of Saccharomyces cerevisiae
Authors: Mhaskar Prashant , Hjortsø Martin A. , Henson Michael A.
Published: October 2002

Saccharomyces cerevisiae is known to exhibit sustained oscillations in chemostats operated under aerobic and glucose‐limited growth conditions. The oscillations are reflected both in intracellular and extracellular measurements. Our recent work has shown that unstructured cell population balance models are capable of generating sustained oscillations over an experimentally meaningful range of dilution rates. A disadvantage of such unstructured models is that they lack variables that can be compared directly to easily measured extracellular variables. Thus far, most of our work in model development has been aimed at achieving qualitative agreement with experimental data. In this paper, a segregated model with a simple structured description of the extracellular environment is developed and evaluated. The model accounts for the three most important metabolic pathways involved in cell growth with glucose substrate. As compared to completely unstructured models, the major advantage of the proposed model is that predictions of extracellular variables can be compared directly to experimental data. Consequently, the model structure is well suited for the application of estimation techniques aimed at determining unknown model parameters from available extracellular measurements. A steady‐state parameter selection method developed in our group is extended to oscillatory dynamics to determine the parameters that can be estimated most reliably. The chosen parameters are estimated by solving a nonlinear programming problem formulated to minimize the difference between predictions and measurements of the extracellular variables. The efficiency of the parameter estimation scheme is demonstrated using simulated and experimental data.
Computational Fluid Dynamics Modeling of Steady‐State Momentum and Mass Transport in a Bioreactor for Cartilage Tissue Engineering
Authors: Williams Kenneth A. , Saini Sunil , Wick Timothy M.
Published: October 2002

Computational fluid dynamics (CFD) models to quantify momentum and mass transport under conditions of tissue growth will aid bioreactor design for development of tissue‐engineered cartilage constructs. Fluent CFD models are used to calculate flow fields, shear stresses, and oxygen profiles around nonporous constructs simulating cartilage development in our concentric cylinder bioreactor. The shear stress distribution ranges from 1.5 to 12 dyn/cm2 across the construct surfaces exposed to fluid flow and varies little with the relative number or placement of constructs in the bioreactor. Approximately 80% of the construct surface exposed to flow experiences shear stresses between 1.5 and 4 dyn/cm2, validating the assumption that the concentric cylinder bioreactor provides a relatively homogeneous hydrodynamic environment for construct growth. Species mass transport modeling for oxygen demonstrates that fluid‐phase oxygen transport to constructs is uniform. Some O2 depletion near the down stream edge of constructs is noted with minimum pO2 values near the constructs of 35 mmHg (23% O2 saturation). These values are above oxygen concentrations in cartilage in vivo, suggesting that bioreactor oxygen concentrations likely do not affect chondrocyte growth. Scale‐up studies demonstrate the utility and flexibility of CFD models to design and characterize bioreactors for growth of tissue‐engineered cartilage.
Reactor Operation and Scale‐Up of Whole Cell Baeyer‐Villiger Catalyzed Lactone Synthesis
Authors: Doig Steven D. , Avenell Philip J. , Bird Paul A. , Gallati Patrick , Lander Katie S. , Lye Gary J. , Wohlgemuth Roland , Woodley John M.
Published: October 2002

The recombinant whole cell biocatalyst Escherichia coli TOP10 [pQR239], expressing cyclohexanone monooxygenase from Acinetobacter calcoaceticus NCIMB 9871, was used in 1.5‐ and 55‐L fed‐batch processes to oxidize bicyclo[3.2.0]hept‐2‐en‐6‐one to its corresponding regioisomeric lactones, (–)‐(1 S,5 R)‐2‐oxabicyclo[3.3.0]oct‐6‐en‐3‐one and (–)‐(1 R,5 S)‐3‐oxabicyclo[3.3.0]oct‐6‐en‐2‐one. By employing a bicyclo[3.2.0]hept‐2‐en‐6‐one feed rate below that of the theoretical volumetric biocatalyst activity (275 μmol·min−1·L−1), the reactant concentration in the bioreactor was successfully maintained below the inhibitory concentration of 0.2–0.4 g·L−1. In this way approximately 3.5 g·L−1 of the combined regioisomeric lactones was produced with a yield of product on reactant of 85–90%. The key limitation to the process was shown to be product inhibition. This process was scaled up to 55 L, producing over 200 g of combined lactone product. Using a simple downstream process (centrifugation, adsorption to activated charcoal, 5‐fold concentration with ethyl acetate elution, and silica gel chromatography), we have shown that the two regioisomeric lactone products could be isolated and purified at this scale.
Multiple Substrate Growth Kinetics of Leptothrix discophoraSP‐6
Authors: Yurt Nurdan , Sears John , Lewandowski Zbigniew
Published: October 2002

The growth parameters of Leptothrix discophora SP‐6 were quantified on the basis of the steady‐state concentrations and utilization rates of pyruvate, dissolved oxygen, and concentration of microorganisms in a chemostat operated at 25 °C, pH 7.2, and an agitation rate of 350 rpm. The results showed that the microbial growth was limited by both pyruvate and dissolved oxygen. A combined growth kinetics model using Monod growth kinetics for pyruvate and Tessier growth kinetics for oxygen showed the best correlation with the experimental data when analyzed using an interactive multiple substrate model. The growth kinetics parameters and the respective confidence limits, estimated using the Monte Carlo simulation, were μmax = 0.576 ± 0.021 h−1, KsMp = 38.81 ± 4.24 mg L−1, KsTo = 0.39 ± 0.04 mg L−1, YX/p = 0.150 (mg microorganism mg−1 pyruvate), YX/o = 1.24 (mg microorganism mg−1 oxygen), the maintenance factors for pyruvate and oxygen were mp = 0.129 (mg pyruvate consumed mg−1 microorganism h−1) and mo = 0.076 (mg oxygen consumed mg−1 microorganism h−1), respectively.
Decreased Protein Expression and Intermittent Recoveries in BiP Levels Result from Cellular Stress during Heterologous Protein Expression in Saccharomyces cerevisiae
Authors: Kauffman Kenneth J. , Pridgen Eric M. , Doyle Francis J. , Dhurjati Prasad S. , Robinson Anne Skaja
Published: October 2002

Cells are inherently robust to environmental perturbations and have evolved to recover readily from short‐term exposure to heat, pH changes, and nutrient deprivation during times of stress. The stress of unfolded protein accumulation has been implicated previously in low protein yields during heterologous protein expression. Here we describe the dynamics of the response to this stress, termed the unfolded protein response (UPR), during the expression of the single chain antibody 4–4–20 (scFv) in Saccharomyces cerevisiae. Expression of scFv decreased the growth rate of yeast cells whether the scFv was expressed from single‐copy plasmids or integrated into the chromosome. However, the growth rates recovered at longer expression times, and surprisingly, the recovery occurred more quickly in the high‐copy integration strains. The presence of a functional UPR pathway was necessary for a recovery of normal growth rates. During the growth inhibition, the UPR pathway appeared to be activated, resulting in decreased intracellular scFv levels and intermittent recovery of the chaperone BiP within the endoplasmic reticulum. Intracellular scFv was observed primarily in the endoplasmic reticulum, consistent with activation of the UPR pathway. Although the intracellular scFv levels dropped over the course of the expression, this was not a result of scFv secretion. A functional UPR pathway was necessary for the drop in intracellular scFv, suggesting that the decrease was a direct response of UPR activation. Taken together, these results suggest that control of heterologous gene expression to avoid UPR activation will result in higher production levels.
Protein Purification via Aqueous Two‐Phase Extraction (ATPE) and Immobilized Metal Affinity Chromatography. Effectiveness of Salt Addition To Enhance Selectivity and Yield of GFPuv
Authors: Li Yi , Beitle Robert R.
Published: October 2002

This study illustrates the compatibility and complementary nature of aqueous two‐phase extraction (ATPE) and immobilized metal affinity chromatography (IMAC) in a general recovery scheme. The purification of green fluorescent protein (GFPuv) from extracts of Eschericia coli was investigated using a combination of these two techniques. High molarity of sodium chloride was found effective in increasing selectivity, with the promotion of hydrophobic interaction the probable mechanism that drove the target protein to a particular phase in ATPE, as well as that which enhanced GFPuv adsorption in IMAC. Moreover, the similar salt condition allows the direct application of the GFPuv‐containing phase to the IMAC column without additional adjustment step. A simple screen of conditions was therefore performed to generate a favorable two‐step purification scheme for GFP leading to an overall high purity.
Isoprene Formation in Bacillus subtilis: A Barometer of Central Carbon Assimilation in a Bioreactor?
Authors: Shirk Megan C. , Wagner William P. , Fall Ray
Published: October 2002

Isoprene (2‐methyl‐1,3‐butadiene) is a volatile hydrocarbon of uncertain function in Bacillus subtilis, and we hypothesized that it is an overflow metabolite produced during excess carbon utilization. Here we tested this idea for phase 2 of isoprene release, a phase that occurs during extracellular acetoin accumulation and its reassimilation. Phase 2 isoprene formation could be disrupted in three different ways, all related to acetoin metabolism. Disruption of a gene essential for acetoin biosynthesis (acetolactic acid synthase, alsS) blocked acetoin formation and led to cessation of phase 2 isoprene formation as well as a variety of pleiotropic effects related to loss of pH control. Growth of the alsS mutant with external pH control reversed most of these effects. Disruption of acetoin catabolism (acetoin dehydrogenase, acoA), also eliminated phase 2 isoprene formation and caused cells to transition directly from phase 1 to phase 3; the latter is attributed to amino acid catabolism. A third alteration of acetoin metabolism was detected in the widely used strain 168 ( trpC2) but not in strain MS175, a trpC mutant constructed in the Marburg strain genetic background. Strain 168 exhibited slow acetoin assimilation compared to that of MS175 or the parental strain, with little or no isoprene formation during this growth phase. These findings support the idea that isoprene release occurs primarily when the rate of carbon catabolism exceeds anabolism and that this volatile hydrocarbon is a product of overflow metabolism when precursors are not required for higher isoprenoid biosynthesis. It is suggested that isoprene release might serve as a useful barometer of the rise and fall of central carbon fluxes during the growth of Bacillus strains in industrial bioreactors.
Exquisite Regioselectivity and Increased Transesterification Activity of an Immobilized Bacillus subtilis Protease
Authors: Ferreira Lino , Ramos M. A. , Gil Maria Helena , Dordick Jonathan S.
Published: October 2002

Commercially available proteases and lipases were screened for their ability to acylate regioselectively sucrose with divinyladipate either in pyridine or dimethylformamide (DMF). The protease (EC 3.4.21.62) from Bacillus subtilis (Proleather FG‐F) exhibited the highest conversion (100% in 24 h of reaction in DMF) yielding sucrose 2‐ O‐vinyladipate as main product. The enzyme preference for a secondary hydroxyl group is a distinct feature of this biocatalyst compared to others described in the literature. Two sets of chemically distinct silica supports were used for Proleather immobilization presenting terminal amino (SAPTES) or hydroxyl groups (STESPM‐pHEMA). The percentage of immobilized enzyme was smaller in SAPTES (7–17%) than in STESPM‐pHEMA (52–56%), yet Proleather immobilized into SAPTES supports presented higher total and specific hydrolytic activity. The highest total and specific activities were obtained with STESPM‐pHEMA and SAPTES, respectively. Silicas with large pore (bimodal distribution of pores, 130/1200 Å, denoted as S1000) presented higher specific activities relative to those with smaller pore sizes. Furthermore, the synthetic specific activity of S1000SAPTES immobilized protease was ca. 10‐fold higher than that of the free enzyme. In addition to sucrose, the immobilized protease was used to acylate methyl α‐d‐glucopyranoside, trehalose, and maltose in nearly anhydrous DMF. Finally, immobilized Proleather was reasonably stable, retaining ca. 55% activity after six reaction cycles.
Continuous Selective Extraction of Secondary Metabolites from Catharanthus roseus Hairy Roots with Silicon Oil in a Two‐Liquid‐Phase Bioreactor
Authors: Tikhomiroff Cyril , Allais Ségolène , Klvana Maya , Hisiger Steve , Jolicoeur Mario
Published: October 2002

A two‐liquid‐phase bioreactor was designed to extract indole alkaloids from Catharanthus roseus hairy roots with silicon oil. Partition studies between silicon oil and culture medium showed that the silicon oil did not alter the availability of nutrients. The affinity of tabersonine and löchnericine for silicon oil is nine times higher than for the aqueous phase. Cultures were elicited with 25 mg/L of jasmonic acid. The growth of the hairy roots was not significantly modified by the presence of silicon oil. The overall specific yields of tabersonine and löchnericine were increased by 100–400 % and 14–200 %, respectively, with the use of silicon oil in nonelicited control cultures. In elicited cultures, these values were 10–55 % for tabersonine and 20–65 % for löchnericine. Serpentine was never found in the silicon oil. All measured alkaloids' specific yields were higher using silicon oil and elicitation, suggesting that the silicon oil, while acting as a metabolic sink for tabersonine and löchnericine, was efficient in increasing metabolic fluxes of the secondary metabolism pathways.
Dual Excitation Ratiometric Fluorescent pH Sensor for Noninvasive Bioprocess Monitoring: Development and Application
Authors: Kermis Haley R. , Kostov Yordan , Harms Peter , Rao Govind
Published: October 2002

The development and application of a fluorescent excitation‐ratiometric, noninvasive pH sensor for continuous on‐line fermentation monitoring is presented. The ratiometric approach is robust and insensitive to factors such as source intensity, photobleaching, or orientation of the patch, and since measurements can be made with external instrumentation and without direct contact with the patch, detection is completely noninvasive. The fluorescent dye 8‐hydroxy‐1,3,6‐pyrene trisulfonic acid was immobilized onto Dowex strongly basic anion‐exchange resin, which was subsequently entrapped into a proton‐permeable hydrogel layer. The sensor layer was polymerized directly onto a white microfiltration membrane backing that provided an optical barrier to the fluorescence and scatter of the fermentation medium. The ratio of emission intensity at 515 nm excited at 468 nm to that excited at 408 nm correlated well with the pH of clear buffers, over the pH range of 6–9. The sensor responded rapidly (<9 min) and reversibly to changes in the solution pH with high precision. The sterilizable HPTS sensor was used for on‐line pH monitoring of an E. coli fermentation. The output from the indwelling sensor patch was always in good agreement with the pH recorded off‐line with an ISFET probe, with a maximum discrepancy of 0.05 pH units. The sensor is easily adaptable to closed‐loop feedback control systems.
Model‐Independent Relationships between Hematocrit, Blood Viscosity, and Yield Stress Derived from Couette Viscometry Data
Authors: Yeow Y. Leong , Wickramasinghe S. Ranil , Leong Yee‐Kwong , Han Binbing
Published: October 2002

This paper describes a procedure, based on Tikhonov regularization, for obtaining the shear rate function or equivalently the viscosity function of blood from Couette viscometry data. For data sets that include points where the sample in the annulus is partially sheared the yield stress of blood will also be obtained. For data sets that do not contain partially sheared points, provided the shear stress is sufficiently low, a different method of estimating the yield stress is proposed. Both the shear rate function and yield stress obtained in this investigation are independent of any rheological model of blood. This procedure is applied to a large set of Couette viscometer data taken from the literature. Results in the form of shear rate and viscosity functions and yield stress are presented for a wide range of hematocrits and are compared against those reported by the originators of the data and against independently measured shear properties of blood.
The Application of a Generic Feedstock from Wheat for Microbial Fermentations
Authors: Wang Ruohang , Domínguez‐Espinosa Rosa M. , Leonard Karen , Koutinas Apostolis , Webb Colin
Published: October 2002

The feasibility of a generic fermentation feedstock produced from wheat flour has been confirmed in several fermentations of yeasts, bacterium, and filamentous fungus for the production of commodity chemicals. Saccharomyces cerevisiae was incubated for the observation of yeast growth and ethanol production, Pichia farinosa for glycerol production, Monascus purpureus for fungal growth and pigment production, and Lactobacillus bulgaricus for bacteria growth and lactic acid production. The results confirmed that the feedstock contained no inhibitory components to the strains tested. Similar or higher metabolite yields were obtained in comparison with other studies carried in commonly used media.
Verbs of placement in Swedish children with SLI
Authors: Hansson Kristina , Bruce Barbro
Published: October 2002

This study investigated lexical aspects of verb knowledge in Swedish children with specific language impairment (SLI). The type of verbs studied were those of placement, a semantic area with a language‐specific differentiation in Swedish. It was hypothesized that owing to their cross‐linguistic uncommonness, these verbs cause difficulties for children with SLI, that these problems are more marked in production than in comprehension, and that children with SLI show less change over time than controls. Ten children with SLI and 10 language‐matched controls participated in an acting‐out task testing the comprehension and production of four different verbs of placement. The children were tested on two occasions, with a 6‐month interval. The groups did not differ in their performance on the comprehension task at either testing. In production, the children with SLI had significantly more difficulties in choosing the correct verb than the controls at the second testing. The controls performed significantly better on production at the second testing than at the first, whereas the children with SLI did not enhance their performance between testings. This indicates that children with SLI have lexical problems and that these problems do not diminish with time. The results are related to the results from studies of second‐language learners of Swedish who often have difficulties in the usage of these verbs. The results are also related to the results from studies of the corresponding semantic field in English‐ and Dutch‐speaking children with SLI. The comparisons show some interesting differences, depending on the type of learner and the language learned. The approach adopted seems promising. Studies of lexical specificity in children with SLI would give interesting information, useful both for the development of clinical assessment instruments and for our understanding of lexical acquisition in these children.
Cultural variations in attention regulation: A comparative analysis of British and Chinese‐immigrant populations
Authors: Vigil Debra C.
Published: October 2002

Recent intervention for young children has included parent training. The premise for this training is based in research on parent‐child interaction. However, these studies have focused on white, middle‐class families. More recent research suggests that not all cultures follow the same pattern. This study explored the relationship between attention regulation, pragmatic input, object manipulation and later language competence in Chinese‐immigrant and British caregiver‐infant dyads. Microanalytical techniques were used to explore observational data derived from a longitudinal study of 10 caregivers and their infants at 9 and 12 months of age. Relations among selected mother and infant activities, notably caregiver control of and responsiveness to attentional focus in infants and object/infant manipulation, were evaluated. Two styles of attention regulation were found across groups. British caretakers responded to their infant's behaviours by following their lead, whereas Chinese caretakers directed their infant's attention. These styles were supported by non‐verbal behaviours such as object manipulation on the part of the caretaker. No differences between the two groups in expressive vocabulary size at age 18 months were found, suggesting that attention regulation styles of directing a child's attention or following a child's lead need further investigation to determine the relationship with vocabulary acquisition. Results show that parents begin to use a particular style of interaction with their children at a very young age. Further research is required to determine if this style continues through the language‐learning years. Furthermore, this study suggests that speech and language therapists must be aware that the communication style of ethnic minorities on their caseloads maybe different from mainstream culture; and with this knowledge service providers will be able to serve those communities better and avoid potential cultural bias.
A protein disulfide isomerase expressed in the embryonic midline is required for left/right asymmetries
Authors: Hoshijima Kazuyuki , Metherall James E. , Grunwald David Jonah
Published: October 2002

Although the vertebrate embryonic midline plays a critical role in determining the left/right asymmetric development of multiple organs, few genes expressed in the midline are known to function specifically in establishing laterality patterning. Here we show that a gene encoding protein disulfide isomerase P5 (PDI-P5) is expressed at high levels in the organizer and axial mesoderm and is required for establishing left/right asymmetries in the zebrafish embryo.pdi-p5 was discovered in a screen to detect genes down-regulated in the zebrafish midline mutant one-eyed pinheadand expressed predominantly in midline tissues of wild-type embryos. Depletion of the pdi-p5 product with morpholino antisense oligonucleotides results in loss of the asymmetric development of the heart, liver, pancreas, and gut. In addition, PDI-P5 depletion results in bilateral expression of all genes known to be expressed asymmetrically in the lateral plate mesoderm and the brain during embryogenesis. The laterality defects caused by pdi-p5antisense treatment arise solely due to loss of the PDI-P5 protein, as they are reversed when treated embryos are supplied with an exogenous source of the PDI-P5 protein. Thus the spectrum of laterality defects resulting from depletion of the PDI-P5 protein fully recapitulates that resulting from loss of the midline. As loss of PDI-P5 does not appear to interfere with other aspects of midline development or function, we propose that PDI-P5 is specifically involved in the production of midline-derived signals required to establish left/right asymmetry.
A widely conserved bacterial cell division protein that promotes assembly of the tubulin-like protein FtsZ
Authors: Gueiros-Filho Frederico J. , Losick Richard
Published: October 2002

Cell division in bacteria is mediated by the tubulin-like protein FtsZ, which assembles into a structure known as the Z ring at the future site of cytokinesis. We report the discovery of a Z-ring-associated protein in Bacillus subtilis called ZapA. ZapA was found to colocalize with the Z ring in vivo and was capable of binding to FtsZ and stimulating the formation of higher-order assemblies of the cytokinetic protein in vitro. The absence of ZapA alone did not impair cell viability, but the absence of ZapA in combination with the absence of a second, dispensable division protein EzrA caused a severe block in cytokinesis. The absence of ZapA also caused lethality in cells producing lower than normal levels of FtsZ or lacking the division-site-selection protein DivIVA. Conversely, overproduction of ZapA reversed the toxicity of excess levels of the division inhibitor MinD. In toto, the evidence indicates that ZapA is part of the cytokinetic machinery of the cell and acts by promoting Z-ring formation. Finally, ZapA is widely conserved among bacteria with apparent orthologs in many species, including Escherichia coli, in which the orthologous protein exhibited a strikingly similar pattern of subcellular localization to that of ZapA. Members of the ZapA family of proteins are likely to be a common feature of the cytokinetic machinery in bacteria.
A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins
Authors: Ishizuka Akira , Siomi Mikiko C. , Siomi Haruhiko
Published: October 2002

Fragile X syndrome is a common form of inherited mental retardation caused by the loss of FMR1 expression. The FMR1 gene encodes an RNA-binding protein that associates with translating ribosomes and acts as a negative translational regulator. InDrosophila, the fly homolog of the FMR1 protein (dFMR1) binds to and represses the translation of an mRNA encoding of the microtuble-associated protein Futsch. We have isolated a dFMR1-associated complex that includes two ribosomal proteins, L5 and L11, along with 5S RNA. The dFMR1 complex also contains Argonaute2 (AGO2) and a Drosophila homolog of p68 RNA helicase (Dmp68). AGO2 is an essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease complex that mediates RNA interference (RNAi) in Drosophila. We show that Dmp68 is also required for efficient RNAi. We further show that dFMR1 is associated with Dicer, another essential component of the RNAi pathway, and microRNAs (miRNAs) in vivo, suggesting that dFMR1 is part of the RNAi-related apparatus. Our findings suggest a model in which the RNAi and dFMR1-mediated translational control pathways intersect inDrosophila. Our findings also raise the possibility that defects in an RNAi-related machinery may cause human disease.
c-Myc is essential for vasculogenesis and angiogenesis during development and tumor progression
Authors: Baudino Troy A. , McKay Catriona , Pendeville-Samain Helene , Nilsson Jonas A. , Maclean Kirsteen H. , White Elsie L. , Davis Ann C. , Ihle James N. , Cleveland John L.
Published: October 2002

c-Myc promotes cell growth and transformation by ill-defined mechanisms. c-myc−/− mice die by embryonic day 10.5 (E10.5) with defects in growth and in cardiac and neural development. Here we report that the lethality of c-myc−/−embryos is also associated with profound defects in vasculogenesis and primitive erythropoiesis. Furthermore, c-myc−/−embryonic stem (ES) and yolk sac cells are compromised in their differentiative and growth potential. These defects are intrinsic to c-Myc, and are in part associated with a requirement for c-Myc for the expression of vascular endothelial growth factor (VEGF), as VEGF can partially rescue these defects. However, c-Myc is also required for the proper expression of other angiogenic factors in ES and yolk sac cells, including angiopoietin-2, and the angiogenic inhibitors thrombospondin-1 and angiopoietin-1. Finally, c-myc−/− ES cells are dramatically impaired in their ability to form tumors in immune-compromised mice, and the small tumors that sometimes develop are poorly vascularized. Therefore, c-Myc function is also necessary for the angiogenic switch that is indispensable for the progression and metastasis of tumors. These findings support the model wherein c-Myc promotes cell growth and transformation, as well as vascular and hematopoietic development, by functioning as a master regulator of angiogenic factors.
Fragile X-related protein and VIG associate with the RNA interference machinery
Authors: Caudy Amy A. , Myers Mike , Hannon Gregory J. , Hammond Scott M.
Published: October 2002

RNA interference (RNAi) is a flexible gene silencing mechanism that responds to double-stranded RNA by suppressing homologous genes. Here, we report the characterization of RNAi effector complexes (RISCs) that contain small interfering RNAs and microRNAs (miRNAs). We identify two putative RNA-binding proteins, the Drosophilahomolog of the fragile X mental retardation protein (FMRP), dFXR, and VIG (Vasa intronic gene), through their association with RISC. FMRP, the product of the human fragile X locus, regulates the expression of numerous mRNAs via an unknown mechanism. The possibility that dFXR, and potentially FMRP, use, at least in part, an RNAi-related mechanism for target recognition suggests a potentially important link between RNAi and human disease.
Ku complex controls the replication time of DNA in telomere regions
Authors: Cosgrove Andrew J. , Nieduszynski Conrad A. , Donaldson Anne D.
Published: October 2002

We have investigated whether the Ku complex is involved in regulating DNA replication in the yeast Saccharomyces cerevisiae. We find that Ku proteins control the replication time of telomeric regions; replication origins located close to telomeres or within subtelomeric repeat sequences normally initiate late, but are activated much earlier in mutants lacking Ku function. In contrast, origins distant from telomeres initiate replication at the normal time. Ku is one of the first components identified as important for replication timing, and specification of the replication time of chromosome ends by Ku is consistent with its role in maintaining telomere localization.
Single-molecule analysis reveals clustering and epigenetic regulation of replication origins at the yeast rDNA locus
Authors: Pasero Philippe , Bensimon Aaron , Schwob Etienne
Published: October 2002

How eukaryotes specify their replication origins is an important unanswered question. Here, we analyze the replicative organization of yeast rDNA, which consists of ∼150 identical repeats, each containing a potential origin. Using DNA combing and single-molecule imaging, we show that functional rDNA origins are clustered and interspersed with large domains where initiation is silenced. This repression is largely mediated by the Sir2p histone-deacetylase. Increased origin firing insir2Δ mutants leads to the accumulation of circular rDNA species, a major determinant of yeast aging. We conclude that rDNA replication is regulated epigenetically and that Sir2p may promote genome stability and longevity by suppressing replication-dependent rDNA recombination.
Requirement for two copies of RNA polymerase α subunit C-terminal domain for synergistic transcription activation at complex bacterial promoters
Authors: Lloyd Georgina S. , Niu Wei , Tebbutt John , Ebright Richard H. , Busby Stephen J.W.
Published: October 2002

Transcription activation by the Escherichia coli cyclic AMP receptor protein (CRP) at different promoters has been studied using RNA polymerase holoenzyme derivatives containing two full-length α subunits, or containing one full-length α subunit and one truncated α subunit lacking the α C-terminal domain (αCTD). At a promoter having a single DNA site for CRP, activation requires only one full-length α subunit. Likewise, at a promoter having a single DNA site for CRP and one adjacent UP-element subsite (high-affinity DNA site for αCTD), activation requires only one full-length α subunit. In contrast, at promoters having two DNA sites for CRP, or one DNA site for CRP and two UP-element subsites, activation requires two full-length α subunits. We conclude that a single copy of αCTD is sufficient to interact with one CRP molecule and one adjacent UP-element subsite, but two copies of αCTD are required to interact with two CRP molecules or with one CRP molecule and two UP-element subsites.
Bicoid associates with the 5′-cap-bound complex of caudal mRNA and represses translation
Authors: Niessing Dierk , Blanke Stephen , Jäckle Herbert
Published: October 2002

Translational control plays a key role in many biological processes including pattern formation during early Drosophilaembryogenesis. In this process, the anterior determinant Bicoid (BCD) acts not only as a transcriptional activator of segmentation genes but also causes specific translational repression of ubiquitously distributed caudal (cad) mRNA in the anterior region of the embryo. We show that translational repression of cad mRNA is dependent on a functional eIF4E-binding motif. The results suggest a novel mode of translational repression, which combines the strategy of target-specific binding to 3′-untranslated sequences and interference with 5′-cap-dependent translation initiation in one protein.
Identification of a polymorphic, neuron-specific chromatin remodeling complex
Authors: Olave Ivan , Wang Weidong , Xue Yutong , Kuo Ann , Crabtree Gerald R.
Published: October 2002

A variety of chromatin remodeling complexes are thought to assist sequence-specific transcription factors. The complexes described to date are expressed ubiquitously, suggesting that they have general transcriptional functions. We show that vertebrate neurons have a specialized chromatin remodeling complex, bBAF, specifically containing the actin-related protein, BAF53b, which is first expressed in postmitotic neurons at about murine embryonic day 12.5 (E12.5). BAF53b is combinatorially assembled into polymorphic complexes with ubiquitous subunits including the two ATPases BRG1 and BRM. We speculate that bBAF complexes create neuronal-specific patterns of chromatin accessibility, thereby imparting new regulatory characteristics to ubiquitous sequence-specific transcription factors in neurons.
Mass Spectrometric Identification of Isoforms of PR Proteins in Xylem Sap of Fungus-Infected Tomato
Authors: Rep Martijn , Dekker Henk L. , Vossen Jack H. , de Boer Albert D. , Houterman Petra M. , Speijer Dave , Back Jaap W. , de Koster Chris G. , Cornelissen Ben J.C.
Published: October 2002

The protein content of tomato (Lycopersicon esculentum) xylem sap was found to change dramatically upon infection with the vascular wilt fungus Fusarium oxysporum. Peptide mass fingerprinting and mass spectrometric sequencing were used to identify the most abundant proteins appearing during compatible or incompatible interactions. A new member of the PR-5 family was identified that accumulated early in both types of interaction. Other pathogenesis-related proteins appeared in compatible interactions only, concomitantly with disease development. This study demonstrates the feasibility of using proteomics for the identification of known and novel proteins in xylem sap, and provides insights into plant-pathogen interactions in vascular wilt diseases.
From bricks to clicks: understanding the e‐consumer
Authors: Dennis Charles , Harris isa , Sandhu Balraj
Published: October 2002

It is well known that online shopping is growing, but recent reports have indicated that e‐retailers are failing to deliver. In this paper, the authors consider aspects of shopping and shopping styles, comparing e‐shopping with bricks and mortar. First, a small exploratory pilot study comparing Internet vs an exemplar shopping centre, and comparing the centre with an “ideal” centre is reported. In this initial stage, the respondents were selected as the “shoppers of tomorrow” – sixth‐form students – more Web‐literate than older age groups. Second, the results of a further small exploratory pilot study are reported with slightly more mature shoppers – university students. The qualitative findings from this stage of the research form the basis for our conclusions. Finally, we speculate on the possible future of shopping.
Assessments of the “new economy” scenario
Authors: Fuentelsaz Lucio , Pablo Maicas‐López Juan , Polo Yolanda
Published: October 2002

This paper is devoted to a consideration of the present‐day situation faced by the digital economy. The appearance of this phenomenon as one of the basic pillars of modern economies has provoked increasing interest in academic circles which, for the moment, is not fully reflected in the economic literature. Against this background, the central aim of this study is to synthesise the current state of the question, accepting the limitations imposed on such an exercise that is supposed by the scarcity of sources alluded to earlier. Qualitative research in this area has concentrated on the strategies that generate competitive advantages in a digital economy scenario. We follow this line by focusing on this question, trying to determine whether these studies converge in terms of proposals that can be used as a reference point for firms operating in virtual markets. Establishing the foundations of this new scenario is essential in order to consolidate the digital economy as an independent discipline.
The Joy of Stargazing
Authors: Catchpole Robin M.
Published: October 2002

Seeing in the Dark
How Backyard Stargazers Are Probing Deep Space and Guarding Earth from Interplanetary Peril. Timothy Ferris. Simon and Schuster, New York, 2002. 399 pp. $26, C$39.50. ISBN 0-684-86579-3.

In this account for a lay audience, Ferris weaves together his personal experiences in stargazing, an overview of the fairly sophisticated level of current amateur astronomy, and descriptions of some targets to tempt readers into taking up their own studies of the night sky.
Transient Induction of Cyclin T1 during Human Macrophage Differentiation Regulates Human Immunodeficiency Virus Type 1 Tat Transactivation Function
Authors: Liou Li-Ying , Herrmann Christine H. , Rice Andrew P.
Published: October 2002

The human immunodeficiency virus type 1 (HIV-1) Tat protein is essential for viral replication and stimulates transcription of the integrated provirus by recruiting the kinase complex TAK/P-TEFb, composed of cyclin T1 (CycT1) and Cdk9, to the viral TAR RNA element. TAK/P-TEFb phosphorylates the RNA polymerase II complex and stimulates transcriptional elongation. In this report, we investigated the regulation of TAK/P-TEFb in primary human macrophages, a major target cell of HIV infection. While Cdk9 levels remained constant, CycT1 protein expression in freshly isolated monocytes was very low, increased early during macrophage differentiation, and, unexpectedly, decreased to very low levels after about 1 week in culture. The kinase activity of TAK/P-TEFb paralleled the changes in CycT1 protein expression. RNA analysis indicated that the transient induction of CycT1 protein expression involves a posttranscriptional mechanism. In transient transfection assays, the ability of Tat to transactivate the HIV long terminal repeat (LTR) in the late differentiated macrophages was greatly diminished relative to its ability to transactivate the HIV LTR in early differentiated cells, strongly suggesting that CycT1 is limiting for Tat function in late differentiated macrophages. Interestingly, lipopolysaccharide, a component of the cell wall of gram-negative bacteria, reinduced CycT1 expression late in macrophage differentiation. These results raise the possibility that regulation of CycT1 expression may be involved in establishing latent infection in macrophages and that opportunistic infection may reactivate the virus by inducing CycT1 expression.
Equine Infectious Anemia Virus Envelope Evolution In Vivo during Persistent Infection Progressively Increases Resistance to In Vitro Serum Antibody Neutralization as a Dominant Phenotype
Authors: Howe Laryssa , Leroux Caroline , Issel Charles J. , Montelaro Ronald C.
Published: October 2002

Equine infectious anemia virus (EIAV) infection of horses is characterized by well-defined waves of viremia associated with the sequential evolution of distinct viral populations displaying extensive envelope gp90 variation; however, a correlation of in vivo envelope evolution with in vitro serum neutralization phenotype remains undefined. Therefore, the goal of the present study was to utilize a previously defined panel of natural variant EIAV envelope isolates from sequential febrile episodes to characterize the effects of envelope variation during persistent infection on viral neutralization phenotypes and to define the determinants of EIAV envelope neutralization specificity. To assess the neutralization phenotypes of the sequential EIAV envelope variants, we determined the sensitivity of five variant envelopes to neutralization by a longitudinal panel of immune serum from the source infected pony. The results indicated that the evolution of the EIAV envelope sequences observed during sequential febrile episodes produced an increasingly neutralization-resistant phenotype. To further define the envelope determinants of EIAV neutralization specificity, we examined the neutralization properties of a panel of chimeric envelope constructs derived from reciprocal envelope domain exchanges between selected neutralization-sensitive and neutralization-resistant envelope variants. These results indicated that the EIAV gp90 V3 and V4 domains individually conferred serum neutralization resistance while other envelope segments in addition to V3 and V4 were evidently required for conferring total serum neutralization sensitivity. These data clearly demonstrate for the first time the influence of sequential gp90 variation during persistent infection in increasing envelope neutralization resistance, identify the gp90 V3 and V4 domains as the principal determinants of antibody neutralization resistance, and indicate distinct complex cooperative envelope domain interactions in defining sensitivity to serum antibody neutralization.
Poliovirus Internal Ribosome Entry Segment Structure Alterations That Specifically Affect Function in Neuronal Cells: Molecular Genetic Analysis
Authors: Malnou Cécile E. , Pöyry Tuija A. A. , Jackson Richard J. , Kean Katherine M.
Published: October 2002

Translation of poliovirus RNA is driven by an internal ribosome entry segment (IRES) present in the 5′ noncoding region of the genomic RNA. This IRES is structured into several domains, including domain V, which contains a large lateral bulge-loop whose predicted secondary structure is unclear. The primary sequence of this bulge-loop is strongly conserved within enteroviruses and rhinoviruses: it encompasses two GNAA motifs which could participate in intrabulge base pairing or (in one case) could be presented as a GNRA tetraloop. We have begun to address the question of the significance of the sequence conservation observed among enterovirus reference strains and field isolates by using a comprehensive site-directed mutagenesis program targeted to these two GNAA motifs. Mutants were analyzed functionally in terms of (i) viability and growth kinetics in both HeLa and neuronal cell lines, (ii) structural analyses by biochemical probing of the RNA, and (iii) translation initiation efficiencies in vitro in rabbit reticulocyte lysates supplemented with HeLa or neuronal cell extracts. Phenotypic analyses showed that only viruses with both GNAA motifs destroyed were significantly affected in their growth capacities, which correlated with in vitro translation defects. The phenotypic defects were strongly exacerbated in neuronal cells, where a temperature-sensitive phenotype could be revealed at between 37 and 39.5°C. Biochemical probing of mutated domain V, compared to the wild type, demonstrated that such mutations lead to significant structural perturbations. Interestingly, revertant viruses possessed compensatory mutations which were distant from the primary mutations in terms of sequence and secondary structure, suggesting that intradomain tertiary interactions could exist within domain V of the IRES.
Engineered CD4- and CXCR4-Using Simian Immunodeficiency Virus from African Green Monkeys Is Neutralization Sensitive and Replicates in Nonstimulated Lymphocytes
Authors: König Renate R. , Flory Egbert , Steidl Stefanie , Neumann Jeanette , Coulibaly Cheick , Holznagel Edgar , Holzammer Silke , Norley Stephen , Cichutek Klaus
Published: October 2002

During human immunodeficiency virus type 1 (HIV-1) infection, disease progression correlates with the occurrence of variants using the coreceptor CXCR4 for cell entry. In contrast, apathogenic simian immunodeficiency virus (SIV) from African green monkeys (SIVagm), specifically the molecular virus clone SIVagm3mc, uses CCR5, Bob, and Bonzo as coreceptors throughout the course of infection. The influence of an altered coreceptor usage on SIVagm3mc replication was studied in vitro and in vivo. The putative coreceptor binding domain, the V3 region of the surface envelope (SU) glycoprotein, was replaced by the V3 loop of a CD4- and CXCR4-tropic HIV-1 strain. The resulting virus, termed SIVagm3-X4mc, exclusively used CD4 and CXCR4 for cell entry. Consequently, its in vitro replication was inhibited by SDF-1, the natural ligand of CXCR4. Surprisingly, SIVagm3-X4mc was able to replicate in vitro not only in interleukin-2- and phytohemagglutinin-stimulated but also in nonstimulated peripheral blood mononuclear cells (PBMCs) from nonhuman primates. After experimental infection of two pig-tailed macaques with either SIVagm3-X4mc or SIVagm3mc, the coreceptor usage was maintained during in vivo replication. Cell-associated and plasma viral loads, as well as viral DNA copy numbers, were found to be comparable between SIVagm3mc and SIVagm 3-X4mc infections, and no pathological changes were observed up to 14 months postinfection. Interestingly, the V3 loop exchange rendered SIVagm3-X4mc susceptible to neutralizing antibodies present in the sera of SIVagm3-X4mc- and SIVagm3mc-infected pig-tailed macaques. Our study describes for the first time a successful exchange of a V3 loop in nonpathogenic SIVagm resulting in CD4 and CXCR4 usage and modulation of virus replication in nonstimulated PBMCs as well as sensitivity toward neutralization.