bluuebunny/crossref_metadata_2025 huggingface dataset:
Knicknachweis von Mikropfählen in geschichteten Böden
Authors: Ofner Robert , Wimmer Heinz
Published: December 2007

Mikropfähle werden als Gründungs‐ oder Nachgründungselement eingesetzt. Diese Pfähle mit Durchmesser kleiner als 0,3 m sind in der EN14199 geregelt. Aufgrund der hohen Schlankheit sind druckbeanspruchte Mikropfähle, bei einer nicht ausreichenden seitlichen Bettung durch den Boden, knickgefährdet. In bestehenden Normen werden Knicknachweise nur für Mikropfähle in Böden mit undränierten Scherfestigkeiten unter 10 bis 15 kN/m2 gefordert. In einer kürzlich erschienenen Veröffentlichung wurde ein Knicknachweis für Mikropfähle mit konstanter Normalkraft vorgestellt und gezeigt, dass auch bei höheren Scherfestigkeiten ein Knickversagen nicht ausgeschlossen werden kann. In der vorliegenden Veröffentlichung wird der Knicknachweis für Mikropfähle erweitert, die geschichtete Böden durchörtern und die Belastung über die Mantelreibung in den Boden einleiten.
Modellversuche zum Erd‐ und Auflastdruck auf Winkelstützwände
Authors: Arnold Michael , Franke Dietrich
Published: December 2007

Der Nachweis der Bauteilquerschnitte von Winkelstützwänden erfordert die Kenntnis der direkt an der Wand angreifenden Einwirkungen, insbesondere des Erddrucks am Vertikalschenkel und des Auflastdrucks am Horizontalschenkel. Es werden großmaßstäbliche Modellversuche vorgestellt, mit denen die Ausbildung dieser Kontaktdrücke infolge Eigenlast der Hinterfüllung sowie infolge gleichmäßiger Oberflächenlasten untersucht wird. Errichtung und Hinterfüllung der Wand werden unter Variation der Wandgeometrie und der Nachgiebigkeit der Gründungssohle simuliert. Aus den Versuchsergebnissen werden grundlegende Erkenntnisse zum Erd‐ und Auflastdruck abgeleitet.
Knowledge‐making distinctions in synthetic biology
Authors: A. O'Malley Maureen , Powell Alexander , Davies Jonathan F. , Calvert Jane
Published: December 2007

Synthetic biology is an increasingly high‐profile area of research that can be understood as encompassing three broad approaches towards the synthesis of living systems: DNA‐based device construction, genome‐driven cell engineering and protocell creation. Each approach is characterized by different aims, methods and constructs, in addition to a range of positions on intellectual property and regulatory regimes. We identify subtle but important differences between the schools in relation to their treatments of genetic determinism, cellular context and complexity. These distinctions tie into two broader issues that define synthetic biology: the relationships between biology and engineering, and between synthesis and analysis. These themes also illuminate synthetic biology's connections to genetic and other forms of biological engineering, as well as to systems biology. We suggest that all these knowledge‐making distinctions in synthetic biology raise fundamental questions about the nature of biological investigation and its relationship to the construction of biological components and systems. BioEssays 30:57–65, 2008. © 2007 Wiley Periodicals, Inc.
Light resonance energy transfer‐based methods in the study of G protein‐coupled receptor oligomerization
Authors: Gandía Jorge , Lluís Carme , Ferré Sergi , Franco Rafael , Ciruela Francisco
Published: December 2007

Since most of the functions in cells are mediated by multimeric protein complexes, the determination of protein–protein interactions is an important step in the study of cellular mechanisms. Traditionally, after screening for possible target interactors by means of a yeast two‐hybrid screen, several methods are used to validate the initial result before carrying out functional experiments. Nowadays, non‐invasive fluorescence‐based methods like Bioluminescence Resonance Energy Transfer (BRET) and Fluorescence Resonance Energy Transfer (FRET) are widely used in the study of protein–protein interactions in living cells. In the present review, we address the individual strengths and weaknesses of both RET approaches, providing information on their possible future use in the study of G protein‐coupled receptor oligomerization. BioEssays 30:82–89, 2008. © 2007 Wiley Periodicals, Inc.
Asymmetric nuclear reprogramming in somatic cell nuclear transfer?
Authors: Loi Pasqualino , Beaujean Nathalie , Khochbin Saadi , Fulka Josef , Ptak Grazyna
Published: December 2007

Despite the progress achieved over the last decade after the birth of the first cloned mammal, the efficiency of reproductive cloning remains invariably low. However, research aiming at the use of nuclear transfer for the production of patient‐tailored stem cells for cell/tissue therapy is progressing rapidly. Yet, reproductive cloning has many potential implications for animal breeding, transgenic research and the conservation of endangered species. In this article we suggest that the changes in the epi‐/genotype observed in cloned embryos arise from unbalanced nuclear reprogramming between parental chromosomes. It is probable that the oocyte reprogramming machinery, devised for resident chromosomes, cannot target the paternal alleles of somatic cells. We, therefore, suggest that a reasonable approach to balance this asymmetry in nuclear reprogramming might involve the transient expression in donor cells of chromatin remodelling proteins, which are physiologically expressed during spermatogenesis, in order to induce a male‐specific chromatin organisation in the somatic cells before nuclear transfer. BioEssays 30:66–74, 2008. © 2007 Wiley Periodicals, Inc.
Damage‐induced reactivation of cohesin in postreplicative DNA repair
Authors: Ball Alexander R. , Yokomori Kyoko
Published: December 2007

Cohesin establishes sister‐chromatid cohesion during S phase to ensure proper chromosome segregation in mitosis. It also facilitates postreplicative homologous recombination repair of DNA double‐strand breaks by promoting local pairing of damaged and intact sister chromatids. In G2 phase, cohesin that is not bound to chromatin is inactivated, but its reactivation can occur in response to DNA damage. Recent papers by Koshland's and Sjögren's groups describe the critical role of the known cohesin cofactor Eco1 (Ctf7) and ATR checkpoint kinase in damage‐induced reactivation of cohesin, revealing an intricate mechanism that regulates sister‐chromatid pairing to maintain genome integrity.1,2 BioEssays 30:5–9, 2008. © 2007 Wiley Periodicals, Inc.
Alternative splicing: current perspectives
Authors: Kim Eddo , Goren Amir , Ast Gil
Published: December 2007

Alternative splicing is a well‐characterized mechanism by which multiple transcripts are generated from a single mRNA precursor. By allowing production of several protein isoforms from one pre‐mRNA, alternative splicing contributes to proteomic diversity. But what do we know about the origin of this mechanism? Do the same evolutionary forces apply to alternatively and constitutively splice exons? Do similar forces act on all types of alternative splicing? Are the products generated by alternative splicing functional? Why is “improper” recognition of exons and introns allowed by the splicing machinery? In this review, we summarize the current knowledge regarding these issues from an evolutionary perspective. BioEssays 30:38–47, 2008. © 2007 Wiley Periodicals, Inc.
From priming to plasticity: the changing fate of rhizodermic cells
Authors: Savage Natasha Saint , Schmidt Wolfgang
Published: December 2007

The fate of root epidermal cells is controlled by a complex interplay of transcriptional regulators, generating a genetically determined, position‐biased arrangement of root hair cells. This pattern is altered during postembryonic development and in response to environmental signals to confer developmental plasticity that acclimates the plant to the prevailing conditions. Based on the hypothesis that events downstream of this initial mechanism can modulate the pattern installed during embryogenesis, we have developed a reaction diffusion model that reproduces the root hair patterning previously observed experimentally. Under all growth conditions, an almost equal spacing between root hair forming cells was observed both in vitro and in silico, indicating that long‐range intercellular communication is crucial for the trichoblasts' decision to form a root hair. We assume that a hair growth promoter (HGP) is upregulated in root‐hair‐forming cells by a trichoblast‐specific component. Once established, HGP production is self‐enhancing. The autocatalytic regulation of HGP is antagonized by an HGP‐produced hair growth inhibitor (HGI). HGI is exported from trichoblasts and diffuses to neighboring cells, where it inhibits further HGP production and promotes the non‐hair cell fate. Under conditions of phosphate deficiency, we hypothesise that HGP production is increased and HGI diffusion rate is reduced, leading to a position‐independent formation of extra root hairs. BioEssays 30:75–81, 2008. © 2007 Wiley Periodicals, Inc.
Right or left turn? RecA family protein filaments promote homologous recombination through clockwise axial rotation
Authors: Wang Ting‐Fang , Chen Li‐Tzu , Wang Andrew H.‐J.
Published: December 2007

The RecA family proteins mediate homologous recombination, a ubiquitous mechanism for repairing DNA double‐strand breaks (DSBs) and stalled replication forks. Members of this family include bacterial RecA, archaeal RadA and Rad51, and eukaryotic Rad51 and Dmc1. These proteins bind to single‐stranded DNA at a DSB site to form a presynaptic nucleoprotein filament, align this presynaptic filament with homologous sequences in another double‐stranded DNA segment, promote DNA strand exchange and then dissociate. It was generally accepted that RecA family proteins function throughout their catalytic cycles as right‐handed helical filaments with six protomers per helical turn. However, we recently reported that archaeal RadA proteins can also form an extended right‐handed filament with three monomers per helical turn and a left‐handed protein filament with four monomers per helical turn. Subsequent structural and functional analyses suggest that RecA family protein filaments, similar to the F1‐ATPase rotary motor, perform ATP‐dependent clockwise axial rotation during their catalytic cycles. This new hypothesis has opened a new avenue for understanding the molecular mechanism of RecA family proteins in homologous recombination. BioEssays 30:48–56, 2008. © 2007 Wiley Periodicals, Inc.
“JIP”ing along the axon: the complex roles of JIPs in axonal transport
Authors: Koushika Sandhya P.
Published: December 2007

JIPs are JNK interacting proteins and bind to JNK cascade kinases. JIP1 and JIP3 were known to be adaptors linking cargo to Kinesin‐I, a major molecular motor for axonal transport. Recent research sheds further light on JIPs' complex roles in axonal transport, namely in activation of Kinesin‐I and in cargo release. In Drosophila, APLIP1/JIP1 allows the Kinesin‐I complex to enable cargo release through activation of JNK signaling.1 In mammalian cell culture, JIP1 is necessary and, together with UNC‐76/FEZ1, sufficient for activating Kinesin‐I.2 I discuss and compare the many roles played by JIP1 and JIP3 through interactions with several distinct players, in retrograde as well as anterograde transport. BioEssays 30:10–14, 2008. © 2007 Wiley Periodicals, Inc.
Live cells‐based cytotoxic sensorchip fabricated in a microfluidic system
Authors: Wada Ken‐Ichi , Taniguchi Akiyoshi , Kobayashi Jun , Yamato Masayuki , Okano Teruo
Published: December 2007

We report here a live cells‐based sensorchip fabricated in microfluidic channels in which several laminar flows were achieved. In addition, we established a cytotoxic sensor cell line, which was transfected with a green fluorescence protein (GFP) plasmid derived from the heat shock protein 70B′ (HSP70B′) promoter, which is induced by cytotoxic reagents. The fluorescence in the sensor cells increased in a CdCl2 dose‐dependent manner in the microfluidic channels. In this system, cytotoxic reagents can be quantitatively detected in a quick, sensitive and high‐throughput manner. The combination of sensor cells and microfluidic systems will provide an important basis for the development of micro‐total analysis systems (µ‐TAS) technology, and can be applied to toxicology, environmental assessment and drug screening. Biotechnol. Bioeng. 2008;99: 1513–1517. © 2007 Wiley Periodicals, Inc.
Kinetic and stoichiometric parameters estimation in a nitrifying bubble column through “in‐situ” pulse respirometry
Authors: Ordaz Alberto , Oliveira Catarina S. , Aguilar Ricardo , Carrión Manuel , Ferreira Eugénio C. , Alves Madalena , Thalasso Frédéric
Published: December 2007

This article proposes a simple “in‐situ” pulse respirometric method for the estimation of four important kinetic and stoichiometric parameters. The method is validated in a suspended biomass nitrifying reactor for the determination of (i) maximum oxygen uptake rate (OURexmax), (ii) oxidation yield (fE), (iii) biomass growth yield (fS), and (iv) affinity constant (KS). OURexmax and fE were directly obtained from respirograms. In the presented case study, a minimum substrate pulse of 10 mgNH‐N L−1 was necessary to determine OURexmax which was 61.15 ± 4.09 mgO2 L−1 h−1 (5 repetitions). A linear correlation (r2 = 0.93) obtained between OURexmax and the biomass concentration in the reactor suggests that biomass concentration can be estimated from respirometric experiments. The substrate oxidation yield, fE, was determined along 60 days of continuous operation with an average error of 5.6%. The biomass growth yield was indirectly estimated from the substrate oxidation yield fE. The average obtained value (0.10 ± 0.04 mgCOD mg−1COD) was in accordance with the fS estimation by the traditional COD mass balance method under steady‐state conditions (0.09 ± 0.01). The affinity constant KS was indirectly estimated after fitting the ascending part of the respirogram to a theoretical model. An average value of 0.48 ± 0.08 mgNH‐N L−1 was obtained, which is in the range of affinity constants reported in the literature for the nitrification process (0.16–2 mgNH‐N L−1). Biotechnol. Bioeng. 2008;100: 94–102. © 2007 Wiley Periodicals, Inc.
Plasmid DNA transfection using magnetite cationic liposomes for construction of multilayered gene‐engineered cell sheet
Authors: Ino Kosuke , Kawasumi Tamayo , Ito Akira , Honda Hiroyuki
Published: December 2007

Modification of cellular functions by overexpression of genes is being increasingly practiced for tissue engineering. In the present study, we investigated whether transfection efficiency could be enhanced by magnetofection that involves the use of plasmid DNA (pDNA)/magnetite cationic liposomes (MCLs) complexes (pDNA/MCL) and magnetic force. The transfection efficiencies of the magnetofection technique by pDNA/MCL in fibroblasts and keratinocytes using reporter genes were 36‐ and 10‐fold higher, respectively, than those of a lipofection technique by cationic liposomes. Moreover, in vitro construction of three‐dimensional (3D) tissues is an important challenge. We recently proposed a novel technique termed “magnetic force‐based tissue engineering” (Mag‐TE) to produce 3D tissues. Since the fibroblasts after magnetofection incorporated both magnetite nanoparticles and pDNA, we investigated whether multilayered heterotypic cell sheets expressing transgene could be fabricated by Mag‐TE. First, the fibroblasts were seeded onto an ultra‐low attachment culture plate. When a magnet was placed under the plate, the cells accumulated at the bottom of the culture plate. After 24 h of culture, the transgene‐expressing cells formed a multilayered cell sheet‐like structure. These results indicated that MCLs are a potent biomanipulation tool for both gene transfer and 3D tissue construction, suggesting that these techniques are useful for tissue engineering. Biotechnol. Bioeng. 2008;100: 168–176. © 2007 Wiley Periodicals, Inc.
Functionalization of a poly(D,L)lactic acid surface with galactose to improve human keratinocyte behavior for artificial epidermis
Authors: Renò F. , Traina V. , Gatti S. , Battistella E. , Cannas M.
Published: December 2007

The production of artificial epidermis using reabsorbable polymeric matrices is one of possible goals; one of most used strategies in this field is the polymer substrate functionalitation using specific growth factors, in order to accelerate and improve keratinocyte adhesion and proliferation. In this study films of poly(D,L)lactide (P(D,L)LA), have been functionalized with various concentrations of galactose (GAL, 1–5–10%, w/v) conjugated with poly‐L‐lysine (PLL) using 1‐etil‐3‐(3‐diaminopropil) carbodiimide (EDC) as a coupling agent. GAL is a disaccharide present in the extracellular matrix (ECM) and it is bind by Galectines, a family of cell receptors whose activation regulate the cell–matrix interaction and cell growth and apoptosis. One of these receptors, Galectin‐7 (Gal‐7), is selectively expressed by human keratinocytes. Spontaneously immortalized human keratinocytes (HaCaT) that express high level of Gal‐7 were allowed to adhere for 4 h in serum free condition on control P(D,L)LA (PLA), and on PLA‐GAL and cell proliferation; the production of matrix metalloproteinases (MMP‐2, MMP‐9, and MMP‐28), involved in cellular migration and tissue homeostasis have been analyzed after 24 h. The presence of GAL onto the polymer surface increased both cell adhesion, spreading and proliferation along with MMP‐9 and MMP‐28 production, suggesting that polymer functionalization using GAL could be an useful tool for the production of an artificial epidermis. Biotechnol. Bioeng. 2008;100: 195–202. © 2007 Wiley Periodicals, Inc.
Biosilicification of dual‐fusion enzyme immobilized on magnetic nanoparticle
Authors: Chien Liang‐Jung , Lee Cheng‐Kang
Published: December 2007

Rapid recovery, immobilization, and silica encapsulation of a dual‐fusion enzyme was achieved by using iminodiacetic acid (IDA) modified magnetic nanoparticle as a carrier. D‐amino acid oxidase (DAAO) of Rhodosporidium toruloides was used as a model enzyme in which a silica‐precipitating peptide R5 and a metal ion complexing peptide (His)6 were fused to its N‐ and C‐terminal, respectively. After charging the magnetic particle with Cu2+, the dual‐fusion DAAO of 0.43 g could be directly recovered from the recombinant E. coli crude extract and immobilized on 1 g of the magnetic particle. Once in contact with hydrolyzed tetramethoxysilane (TMOS), the homogeneously dispersed immobilized dual‐fusion DAAO was biosilicificated to form aggregates with size about 50 µm. The silica‐encapsulated immobilized DAAO demonstrated a pyruvic acid production rate comparable with that of the naked immobilized DAAO in five repeated batch reactions when D‐alanine was used as substrate. Furthermore, 85% of its activity remained after incubation at 60°C for 1 h while the naked immobilized DAAO lost all its activity. This process provides the advantages that recombinant fusion enzyme can be directly recovered from crude extract, silica encapsulation protects the enzyme from leakage and denaturation, and the enzyme activity can be easily retrieved by applying a magnetic field. Biotechnol. Bioeng. 2008;100: 223–230. © 2007 Wiley Periodicals, Inc.
Spectrophotometric ferric ion biosensor from Pseudomonas fluorescens culture
Authors: Gupta Varun , Saharan Krishna , Kumar Lalit , Gupta Roohi , Sahai Vikram , Mittal Aditya
Published: December 2007

Pseudomonas fluorescens cultures produce fluorescent siderophores. By utilizing optimal conditions for maximizing siderophore production in shake flask cultures of P. fluorescens, we report successful characterization of the culture broth supernatant as a robust ferric ions biosensor. For characterizing the ferric ions biosensor, we tested the effects of pH, buffers, different ferric salts and possible interference by ferrous ions under different solution conditions. We find that the biosensor is very specific to ferric ions only with sensitivity to concentrations as low as 10 µM. Further, the response time of the biosensor is the shortest (∼5 min or smaller) for citrate as the accompanying anion with ferric ions. While the response time is longer than that expected of normal biosensors, it is well compensated by the simplicity and economics of the biosensor production. Extremely low standard deviations in several experimental repeats also highlight the robustness of the ferric ions biosensor. Most importantly, the biosensor is extremely easy to use due to its straightforward spectrophotometric applications. We also show the utility of the biosensor with the high resolution technique of fluorescence microscopy. Finally, we report a novel mechanistic finding that siderophores present in the culture broth supernatants have two distinct optically active sites on them, which can be monitored independently in presence or absence of ferric ions. Biotechnol. Bioeng. 2008;100: 284–296. © 2007 Wiley Periodicals, Inc.
Serum‐free transient protein production system based on adenoviral vector and PER.C6 technology: High yield and preserved bioactivity
Authors: Havenga M.J.E. , Holterman L. , Melis I. , Smits S. , Kaspers J. , Heemskerk E. , Vlugt R. van der , Koldijk M. , Schouten G.J. , Hateboer G. , Brouwer K. , Vogels R. , Goudsmit J.
Published: December 2007

Stable E1 transformed cells, like PER.C6, are able to grow at scale and to high cell densities. E1‐deleted adenoviruses replicate to high titer in PER.C6 cells whereas subsequent deletion of E2A from the vector results in absence of replication in PER.C6 cells and drastically lowers the expression of adenovirus proteins in such cells. We therefore considered the use of an ΔE1/ΔE2 type 5 vector (Ad5) to deliver genes to PER.C6 cells growing in suspension with the aim to achieve high protein yield. To evaluate the utility of this system we constructed ΔE1/ΔE2 vector carrying different classes of protein, that is, the gene coding for spike protein derived from the Coronavirus causing severe acute respiratory syndrome (SARS‐CoV), a gene coding for the SARS‐CoV receptor or the genes coding for an antibody shown to bind and neutralize SARS‐CoV (SARS‐AB). The ΔE1/ΔE2A‐vector backbones were rescued on a PER.C6 cell line engineered to constitutively over express the Ad5 E2A protein. Exposure of PER.C6 cells to low amounts (30 vp/cell) of ΔE1/ΔE2 vectors resulted in highly efficient (>80%) transduction of PER.C6 cells growing in suspension. The efficient cell transduction resulted in high protein yield (up to 60 picogram/cell/day) in a 4 day batch production protocol. FACS and ELISA assays demonstrated the biological activity of the transiently produced proteins. We therefore conclude that ΔE1/ΔE2 vectors in combination with the PER.C6 technology may provide a viable answer to the increasing demand for high quality, high yield recombinant protein. Biotechnol. Bioeng. 2008;100: 273–283. © 2007 Wiley Periodicals, Inc.
Case study and application of process analytical technology (PAT) towards bioprocessing: Use of on‐line high‐performance liquid chromatography (HPLC) for making real‐time pooling decisions for process chromatography
Authors: Rathore Anurag S. , Yu Marcella , Yeboah Samuel , Sharma Ashutosh
Published: December 2007

Process analytical technology (PAT) has been gaining a lot of momentum in the biopharmaceutical community due to the potential for continuous real time quality assurance resulting in improved operational control and compliance. This paper presents a PAT application for one of the most commonly used unit operation in bioprocessing, namely liquid chromatography. Feasibility of using a commercially available online‐high performance liquid chromatography (HPLC) system for real‐time pooling of process chromatography column is examined. Further, experimental data from the feasibility studies are modeled and predictions of the model are compared to actual experimental data. It is found that indeed for the application under consideration, the online‐HPLC offers a feasible approach for analysis that can facilitate real‐time decisions for column pooling based on product quality attributes. It is shown that implementing this analytical scheme allows us to meet two of the key goals that have been outlined for PAT, that is, “variability is managed by the process” and “product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions.” Finally, the implications of implementing such a PAT application in a manufacturing environment are discussed. The application presented here can be extended to other modes of process chromatography and/or HPLC analysis. Biotechnol. Bioeng. 2008;100: 306–316. © 2007 Wiley Periodicals, Inc.
Effect of electric currents on bacterial detachment and inactivation
Authors: Hong Seok Hoon , Jeong Joonseon , Shim Soojin , Kang Heekyoung , Kwon Sunghoon , Ahn Kyung Hyun , Yoon Jeyong
Published: December 2007

Since biofilms show strong resistance to conventional disinfectants and antimicrobials, control of initial bacterial adhesion is generally accepted as one of the most effective strategies for preventing biofilm formation. Although electrical methods have been widely studied, the specific properties of cathodic, anodic, and block currents that influence the bacterial detachment and inactivation remained largely unclear. This study investigated the specific role of electric currents in the detachment and inactivation of bacteria adhered to an electrode surface. A real‐time bacterial adhesion observation and control system was employed that consisted of Pseudomonas aeruginosa PAO1 (PAO1) with green fluorescent protein as the indicator microorganism and a flow cell reactor mounted on a fluorescent microscope. The results suggest that the bacteria that remained on the electrode surface after application of a cathodic current were alive, although the extent of detachment was significant. In contrast, when an anodic current was applied, the bacteria that remained on the surface became inactive with time, although bacterial detachment was not significant. Further, under these conditions, active bacterial motions were observed, which weakened the binding between the electrode surface and bacteria. This phenomenon of bacterial motion on the surface can be used to maximize bacterial detachment by manipulation of the shear rate. These findings specific for each application of a cathodic or anodic electric current could successfully explain the effectiveness of block current application in controlling bacterial adhesion. Biotechnol. Bioeng. 2008;100: 379–386. © 2007 Wiley Periodicals, Inc.
Determination of diffusion coefficients and diffusion characteristics for chlorferon and diethylthiophosphate in Ca‐Alginate gel beads
Authors: Ha Jiyeon , Engler Cady R. , Lee Seung Jae
Published: December 2007

Diffusion characteristics of chlorferon and diethylthiophosphate (DETP) in Ca‐alginate gel beads were studied to assist in designing and operating bioreactor systems. Diffusion coefficients for chlorferon and DETP in Ca‐alginate gel beads determined at conditions suitable for biodegradation studies were 2.70 × 10−11 m2/s and 4.28 × 10−11 m2/s, respectively. Diffusivities of chlorferon and DETP were influenced by several factors, including viscosity of the bulk solution, agitation speed, and the concentrations of diffusing substrate and immobilized cells. Diffusion coefficients increased with increasing agitation speed, probably due to poor mixing at low speed and some attrition of beads at high speeds. Diffusion coefficients also increased with decreasing substrate concentration. Increased cell concentration in the gel beads caused lower diffusivity. Theoretical models to predict diffusivities as a function of cell weight fraction overestimated the effective diffusivities for both chlorferon and DETP, but linear relations between effective diffusivity and cell weight fraction were derived from experimental data. Calcium‐alginate gel beads with radii of 1.65–1.70 mm used in this study were not subject to diffusional limitations: external mass transfer resistances were negligible based on Biot number calculations and effectiveness factors indicated that internal mass transfer resistance was negligible. Therefore, the degradation rates of chlorferon and DETP inside Ca‐alginate gel beads were reaction‐limited. Biotechnol. Bioeng. 2008;100: 698–706. © 2007 Wiley Periodicals, Inc.
Hypoxic fed‐batch cultivation of Pichia pastoris increases specific and volumetric productivity of recombinant proteins
Authors: Baumann Kristin , Maurer Michael , Dragosits Martin , Cos Oriol , Ferrer Pau , Mattanovich Diethard
Published: December 2007

High cell density cultivation of Pichia pastoris has to cope with several technical limitations, most importantly the transfer of oxygen. By applying hypoxic conditions to chemostat cultivations of P. pastoris expressing an antibody Fab fragment under the GAP promoter, a 2.5‐fold increase of the specific productivity qP at low oxygen supply was observed. At the same time the biomass decreased and ethanol was produced, indicating a shift from oxidative to oxidofermentative conditions. Based on these results we designed a feedback control for enhanced productivity in fed batch processes, where the concentration of ethanol in the culture was kept constant at approximately 1.0% (vv−1) by a regulated addition of feed medium. This strategy was tested successfully with three different protein producing strains, leading to a three‐ to sixfold increase of the qP and threefold reduced fed batch times. Taken together the volumetric productivity QP increased 2.3‐fold. Biotechnol. Bioeng. 2008;100: 177–183. © 2007 Wiley Periodicals, Inc.
Creating a spectrum of fibrocartilages through different cell sources and biochemical stimuli
Authors: Hoben Gwendolyn M. , Athanasiou Kyriacos A.
Published: December 2007

In this study a scaffoldless approach was employed with two different cell sources and biochemical stimuli to engineer a spectrum of fibrocartilages representative of the different regions of the knee meniscus. Constructs composed of 100% fibrochondrocytes (FC) or a 50:50 co‐culture of fibrochondrocytes and chondrocytes (CC) were cultured in 10% fetal bovine serum medium or serum‐free “chondrogenic” medium, each ±10 ng/mL TGF‐β1 (+T). Constructs from these two cell groups and four culture conditions were cultured for 6 weeks. By varying the cell type and presence of the growth factor, GAG per dry weight of the constructs spanned that of native tissue, ranging 16–45% and 1–7% in the CC and FC groups, respectively. Collagen density was most dependent on cell type and was significantly lower than tissue values. The collagen I/II ratio could be manipulated by cell type and serum presence to span the native range, from 3.5 in the serum‐free CC group to over 1,000 in the FC groups treated with serum‐containing medium. Using the CC cell group in the presence of serum‐free medium dramatically increased the compressive stiffness to 128 ± 34 kPa, similar to native tissue. Similarly, serum‐free medium or TGF‐β1 treatment enhanced the tensile modulus by an order of magnitude, up to 3,000 kPa. Using two cell sources and manipulating biochemical stimuli, a range of fibrocartilaginous neotissues was engineered. Fibrocartilages such as the knee meniscus are characterized by heterogeneity in matrix and functional properties, and this work demonstrates a strategy for recreating these heterogeneous tissues. Biotechnol. Bioeng. 2008;100: 587–598. © 2007 Wiley Periodicals, Inc.
Synthesis and Antifungal Activity of Aromatic Bis‐γ‐lactones Analogous to Avenaciolide
Authors: Castelo‐Branco Pedro A. , Rubinger Mayura M. M. , Alves Leandro de C. , de Barros Pauline M. , Pereira Sinval G. , de Melo Vanessa J. , Pilo‐Veloso Dorila , Zambolim Laércio
Published: December 2007

Avenaciolide is a bis‐γ‐lactone isolated from Aspergillus avenaceus and possesses antifungal activity. Here, we describe the total syntheses and characterization by elemental analyses, and IR and NMR spectroscopy of three new bis‐γ‐lactones analogous to avenaciolide, where the octyl group of the natural product was replaced by aromatic groups. The effects of the avenaciolide, the novel compounds, and their synthetic precursors on the mycelia development and conidia germination of Colletotrichum gloeosporioides were evaluated in vitro. The new compounds were as active as avenaciolide in the tested conditions, while the synthetic precursors were inactive. The preparation and characterization of 15 new synthetic intermediates are also described.
Acute Toxicity of the Water‐Soluble Fraction of Spent Lubricating Oil on the African Catfish Clarias gariepinus
Authors: Ogali Regina E. , Osuji Leo C. , Ayodele Olufemi
Published: December 2007

Static tests were employed to assess the acute toxicity of the water‐soluble fraction (WSF) of spent automotive lubricating oil (of mixed SAE grades) on Clarias gariepinus, a freshwater fish commonly cultured in Nigeria. Median lethal concentrations (LC50) of the WSF were found to decrease as a function of exposure time from 690±21 (after 24 h) to 513±58 mg/l (after 96 h). The characteristics of the WSF such as mean acidity (pH 6.6), turbidity (40 NTU), total dissolved solids (TDS; 40 mg/l) and significantly reduced (P<0.05) dissolved‐oxygen (DO) values (1.44 mg/l) were not compliant with existing standards set for discharged effluents. The solubility of the detected straight‐chain aliphatics ranked as C14>C16>C32>C18>C28; that of the simple aromatics was ortho‐xylene>para‐xylene; and that of the polyaromatic hydrocarbons (PAHs) was acenaphthylene>9H‐fluorene>naphthalene>anthracene>phenanthrene>chrysene>benzo[k]fluoranthene>benzo[a]pyrene>benzo[b]fluoranthene, most of which being serious carcinogens. These oil constituents and the overall physico‐chemical properties of the WSF are expected to act synergistically on the test organism (C. gariepinus), eliciting the quantal responses observed. The toxicity of the WSF points to the base constituent, oxidative degradation, and mechano‐chemical reactions associated with aged crankcase oils. These oils, therefore, should definitely no longer be disposed into water streams or landscape, not even at sub‐lethal concentrations, because of the inherent toxicity of their soluble fractions and the associated danger of bioaccumulation.
Evolution of ‘Ligand‐Diffusion Chreodes’ on Protein‐Surface Models: A Genetic‐Algorithm Study
Authors: Marashi Sayed‐Amir , Kargar Mehdi , Katanforoush Ali , Abolhassani Hassan , Sadeghi Mehdi
Published: December 2007

Lattice models have been previously used to model ligand diffusion on protein surfaces. Using such models, it has been shown that the presence of pathways (or ‘chreodes’) of consecutive residues with certain properties can decrease the number of steps required for the arrival of a ligand at the active site. In this work, we show that, based on a genetic algorithm, ligand‐diffusion pathways can evolve on a protein surface, when this surface is selected for shortening the travel length toward the active site. Biological implications of these results are discussed.
Two New Antibacterial Sesquiterpenoids from Centipeda minima
Authors: Liang Heng‐Xing , Bao Fu‐Kai , Dong Xiao‐Ping , Zhu Hua‐Jie , Lu Xiao‐Jie , Shi Ming , Lu Qing , Cheng Yong‐Xian
Published: December 2007

Two new guaiane‐type sesquiterpene lactones, compounds 1 and 2, along with three known guaianolide‐ or pseudoguaianolides, were isolated from Centipeda minima (whole plant). Their structures were identified by spectroscopic and mass‐spectrometric analyses. The configuration at C(5) of the guaiane framework of 1 was rationalized by quantum‐mechanical calculations (Table 2). All compounds were found to be active against eight different microbial pathogens (Table 3), with MIC values in the range of 6.25–100 μg/ml.
Qualitative and Quantitative HPLC/MS Determination of Proanthocyanidins in Areca Nut (Areca catechu)
Authors: Wu Qingli , Yang Yonghong , Simon James E.
Published: December 2007

Proanthocyanidins (PACs) in areca nut (Areca catechu L.) were analyzed by high‐performance liquid chromatography (HPLC) combined with electrospray‐ionization mass spectrometry (ESI‐MS) and compared to grape seed extract. Under optimized conditions, the separated PACs were individually analyzed and identified on the basis of their [M+H]+ peaks. The PAC distribution in areca nut was found to be very similar to that in grape seed, but lacking any gallate conjugates. Based on reverse‐phase HPLC separation, the PAC monomers (+)‐catechin (CA, 1) and (−)‐epicatechin (EC; 2) were successfully quantified by ESI‐MS in the selected‐ion‐monitoring (SIM) mode, (−)‐epigallocatechin (EGC; 3) being used as internal standard. Detailed quality and validation assays showed that the accuracy and repeatability (n=8) were within 10% for each analyte.
Solid‐Phase Total Synthesis and Antimicrobial Activities of Loloatins A–D
Authors: Ding Yan , Qin Chuanguang , Guo Zhihong , Niu Weining , Zhang Ruijie , Li Yang
Published: December 2007

The first total synthesis of the decapeptide antibiotics loloatins A–D (1–4), originally isolated from the marine bacterial isolate MK‐PNG‐276A, possibly in the genus Bacillus, was accomplished by solid‐phase peptide synthesis (SPPS), followed by ‘head‐to‐tail’ cyclization of the activated linear precursors, without protection of nucleophilic side‐chain functions, on a safety‐catch resin. The synthetic peptides were equally active as the natural products isolated from the bacterial source and found to possess similar bacterial selectivity as other members in the family of amphipathic antimicrobial cyclic decapeptides.
Studies on the Cytotoxicity of Miscellaneous Compounds from Eupatorium betonicaeforme (D.C.) Baker (Asteraceae)
Authors: Rocha Danilo D. , Dantas Ivana N. F. , Albuquerque Maria Rose Jane R. , Montenegro Raquel C. , Pessoa Cláudia , de Moraes Manoel Odorico , Pessoa Otília Deusdênia L. , Silveira Edilberto R. , Costa‐Lotufo Letícia V.
Published: December 2007

A detailed study on the cytotoxic effects of five known constituents isolated from the flowers and roots of Eupatorium betonicaeforme is reported, including 2,2‐dimethyl‐6‐vinylchroman‐4‐one (1), 2‐senecioyl‐4‐vinylphenol (2), 6‐acetyl‐2,2‐dimethylchroman‐4‐one (3), (4E)‐8β‐angeloyloxy‐9β,10β‐dihydroxy‐1‐oxogermacra‐4,11(13)‐dien‐12,6α‐olide (4), and 3β‐hydroxyicosan‐1,5β‐olide (5). The sesquiterpene lactone 4 exhibited the highest cytotoxicity, with IC50 values ranging from 3.9 to 9.9 μM, showing some degree of cell selectivity. The antiproliferative activity of 4 was examined towards HL‐60 cells, and found to diminish cell viability in a dose‐dependent manner. Moreover, at all concentrations tested, there was a decrease in the number of cells capable of incorporating 5‐bromo‐2′‐deoxyuridine (BrdU), indicating disruption of DNA synthesis. The morphological changes induced by 4 were compatible with apoptotic cell death. This work, thus, corroborates the anticancer potential of Eupatorium secondary metabolites.
High Diversity of Ligularia dictyoneura in Chemical Composition and DNA Sequence
Authors: Nagano Hajime , Iwazaki Yukiko , Matsushima Mika , Sato Masahiko , Gong Xun , Shen Yuemao , Hirota Hiroshi , Kuroda Chiaki , Hanai Ryo
Published: December 2007

The chemical composition of root extracts of the title species collected at 20 different places in the Hengduan Mountains of China was examined. From these samples, a total of 17 eremophilane derivatives were isolated, three of which were new franoeremophilane derivatives: 3β‐acetoxy‐6β‐(angeloyloxy)furanoeremophilan‐10β‐ol, 1α‐acetoxyfuranoeremophilan‐15,6α‐olide, and 6β‐[2‐(hydroxymethyl)prop‐2‐enoyloxy]furanoeremophil‐1(10)‐ene. Based on the chemical composition, the samples could be classified into as many as seven types: one type containing non‐furanoeremophilane derivatives and the other six containing furanoeremophilane derivatives with different oxidation levels. Results of DNA sequencing of the atpB‐rbcL region and the internal transcribed spacers of the ribosomal RNA gene also indicated a high diversity in the species.
Microcalorimetric and Spectroscopic Investigation of the Antibacterial Properties of Cationic Ytterbium(III)–Porphyrin Complexes Lacking Charged Peripheral Groups
Authors: Hou An‐Xin , Xue Zhi , Liu Yi , Qu Song‐Sheng , Wong Wai‐Kwok
Published: December 2007

The antibacterial activities towards Escherichia coli of two cationic YbIII–monoporphyrin complexes, [YbIII(TMP)(H2O)3]Cl (1) and [YbIII(TTP)(H2O)3]Cl (2), were investigated at the cellular and sub‐cellular levels. The biological effects of the complexes on the growth of E. coli were evaluated by microcalorimetry and by analysis of the resulting metabolic thermogenic curves, from which IC50 values and metabolic parameters such as growth rate and generation time were derived. At the subcellular level, DNA‐binding experiments were performed by means of UV/VIS‐ and fluorescence‐titration experiments, as well as by near‐infrared (NIR) emission, which revealed that 1 and 2 strongly bind to herring‐sperm DNA (HS‐DNA), though by different binding modes.
Three Novel, Structurally Unique Spirocyclic Alkaloids from the Halotolerant B‐17 Fungal Strain of Aspergillus variecolor
Authors: Wang Wen‐Liang , Zhu Tian‐Jiao , Tao Hong‐Wen , Lu Zhen‐Yu , Fang Yu‐Chun , Gu Qian‐Qun , Zhu Wei‐Ming
Published: December 2007

During our search for novel antitumor lead compounds from microorganisms living under extreme conditions, three novel alkaloids, variecolortides A–C (1–3), were isolated from the mycelia of the halotolerant fungal strain Aspergillus variecolor B‐17. The new compounds were found to share an unprecedented ‘spiro‐anthronopyranoid diketopiperazine’ structure, with a stable hemiaminal function, as corroborated by in‐depth NMR‐spectroscopic and mass‐spectrometric analyses, as well as by a single‐crystal X‐ray analysis of 1. All compounds were shown to exhibit weak cytotoxic and antioxidant activities.
4′′′‐Acetylvitexin‐2″‐O‐rhamnoside, Isoorientin, Orientin, and 8‐Methoxykaempferol‐3‐O‐glucoside as Markers for the Differentiation of Crataegus monogyna and Crataegus pentagyna from Crataegus laevigata (Rosaceae)
Authors: Prinz Sonja , Ringl Alexandra , Huefner Antje , Pemp Enne , Kopp Brigitte
Published: December 2007

In our chemotaxonomic investigation of pharmaceutically relevant Crataegus species, the qualitative and quantitative flavonoid fingerprint of Crataegus monogyna and C. pentagyna is presented. Six flavonoids were identified as vitexin‐2″‐O‐rhamnoside (1), vitexin (2), isovitexin (3), rutin (4), hyperoside (5), and isoquercitrin (6). Besides the verification of the main compounds isoorientin (7) and orientin (8) in C. pentagyna, further four flavonoids were isolated and identified as isoorientin‐2″‐O‐rhamnoside (9), orientin‐2″‐O‐rhamnoside (10), isovitexin‐2″‐O‐rhamnoside (11), and 8‐methoxykaempferol‐3‐O‐glucoside (12) by means of 1D‐ and 2D‐NMR, MS, and UV analyses. Compound 12 was isolated for the first time from C. pentagyna. In contrast to C. pentagyna, C. monogyna samples were predominated by 4′′′‐acetylvitexin‐2″‐O‐rhamnoside (13), which was missing in C. pentagyna. Hence, 13 represents an interesting compound for chemotaxonomy of C. monogyna, whereas the main flavonoids 7, 8, and 12 could be proposed as markers for C. pentagyna. The absence of 7, 8, 12, and 13 in C. laevigata offers an appropriate tool for additional differentiation from C. monogyna and C. pentagyna, and for sample identification and quality control of the three main Crataegus species used in European phytotherapy.
Intra‐ and Intermolecular Interactions Influence the Reactivity of RNA Oligonucleotides
Authors: Linjalahti Heidi , Mikkola Satu
Published: December 2007

The transesterification of RNA oligonucleotides was studied over a wide pH range. The rate constants obtained indicate that, under neutral conditions, oligonucleotides with an adenosine moiety as the 5′‐linked nucleoside can be up to 1000‐fold more reactive than the reference oligonucleotide, a 13‐mer oligo‐U (1). Experiments with the modified oligonucleotide with N6,N6‐dimethyladenosine (9) as the 5′‐linked nucleoside moiety suggest that the exocyclic amino group is involved in the reaction, influencing the reactivity of the neighboring phosphodiester bond. In addition to such intramolecular interactions, weak intermolecular interactions most probably contribute to the reactivity.
Two New Alkaloids from Brachystemma calycinum and Their Inhibitory Effects on Lymphocyte Proliferation
Authors: Lu Qing , Zhang Li , He Guo‐Rong , Liang Heng‐Xing , Du Guan‐Hua , Cheng Yong‐Xian
Published: December 2007

Two new alkaloids, brachystemidines F (1) and G (2), were isolated from the roots of Brachystemma calycinum. Their structures were established on the basis of detailed spectroscopic analyses, including extensive NMR and HR‐MS techniques. Compound 2, which exhibits an unusual N‐hydroxydiazenyl (HONN) moiety, is a potent immunosuppressive agent, as demonstrated by inhibition of mouse T‐ and B‐lymphocyte proliferation, with IC50 values of 6.33 and 5.60 μg/ml, resp.
New Chiral Lanthanide Amide Ate Complexes for the Catalysed Synthesis of Scalemic Nitrogen‐Containing Heterocycles
Authors: Aillaud Isabelle , Collin Jacqueline , Duhayon Carine , Guillot Régis , Lyubov Dmitrii , Schulz Emmanuelle , Trifonov Alexander
Published: December 2007

New chiral binaphthylamido yttrium and ytterbium ate complexes with lithium and potassium counterions have been synthesised and characterised. X‐ray structures have been obtained for [Li(thf)4][Ln{(R)‐C20H12(NC5H9)2}2] (Ln=Yb, Y) and [K(thf)5][Yb{(R)‐C20H12(NCH2CMe3)2}2] as isostructural complexes. The efficiency of these complexes for the enantioselective intramolecular hydroamination was examined. [Li(thf)4][Yb{(R)‐C20H12(NC5H9)2}2] afforded the highest enantiomeric excess (up to 87 %) for the synthesis of a spiropyrrolidine, while [Li(thf)4][Y{(R)‐C20H12(NC5H9)2}2] proved to be slightly more active. The role of the counter cation in the active catalytic species was evidenced by the comparison between lithium and potassium ate complexes. The most active catalyst of this series, [Li(thf)4][Yb{(R)‐C20H12(NCH2CMe3)2}2], was successfully used for the cyclisation of aminopentenes with internal double bonds.
Antiaromaticity and Reactivity of a Planar Cyclooctatetraene Fully Annelated with Bicyclo[2.1.1]hexane Units
Authors: Nishinaga Tohru , Uto Takayuki , Inoue Ryota , Matsuura Akira , Treitel Noach , Rabinovitz Mordecai , Komatsu Koichi
Published: December 2007

A detailed investigation has been made into the antiaromaticity and chemical reactivity of a planar cyclooctatetraene (COT) molecule fully annelated with bicyclo[2.1.1]hexane units 2. In spite of its planar 8π‐electronic structure, theoretical calculations have indicated that the antiaromaticity of COT 2 is considerably decreased in comparison with a planar COT 16 with D4h symmetry. This behavior appears to be related to the wider HOMO–LUMO gap of 2 relative to 16, which is caused by the raised LUMO level as a result of the effective σ–π* orbital interaction between the strained bicyclic framework and the COT π system. The two‐electron reduction of 2 required the use of potassium mirror or a combination of lithium/corannulene in highly dried [D8]THF at −78 °C under vacuum. In contrast, the [4+2] cycloaddition of 2 with tetracyanoethylene (TCNE) proceeded quite smoothly owing to the high‐lying HOMO. Reaction of 2 with meta‐chloroperbenzoic acid gave all‐trans tetraepoxide 23 in the same way as the corresponding benzene derivative 3. While the Simons–Smith‐type cyclopropanation of benzene 3 gave tricyclopropanated derivative 21, the reaction of 2 only afforded isomers of dicyclopropanated derivatives 25 and 26. Yet, the reactivity of 2 is higher than the parent COT, which does not show any reactivity under the same conditions. On the basis of homodesmic reactions, it was concluded that release of strain is also an important factor for such relatively high reactivity in the epoxidation and cyclopropanation of bicycloannelated COT 2 as well as benzene 3.