bluuebunny/crossref_metadata_2025_split huggingface dataset:
Determination of Compound Binding to Plasma Proteins
Authors: Dow Natasha 
Published: September 2006

AbstractThe pharmacokinetic and pharmacodynamic properties of a compound are profoundly affected by the extent of its binding to plasma proteins. Consequently, the determination of the plasma protein binding properties of a compound is essential during drug development and is increasingly required during lead prioritization. The protocols described in this unit detail the techniques of ultrafiltration and equilibrium dialysis for determination of plasma protein binding. Equilibrium dialysis is the more robust of the two techniques, whereas ultrafiltration is the more rapid. Two versions of the equilibrium dialysis technique are provided: the traditional approach and a higher‐throughput 96‐well plate version.
In Vitro Species Comparisons and Metabolite Identification
Authors: Dow Paul 
Published: September 2006

AbstractThe metabolism and elimination of a drug has a direct bearing on its potential to cause toxicity in humans as well as to influence its clinical activity. The confidence with which data from preclinical safety studies, particularly toxicology studies, can be extrapolated to humans depends upon knowing whether humans are exposed to the same chemical entities (i.e., a parent drug and its metabolites) as the laboratory animals used to study toxicity and pharmacological responses. To address this issue, the FDA Guidance for Industry (1997) recommends the use of in vitro interspecies metabolic comparisons at an early stage in the drug development process. Knowledge about the metabolism of a compound is also important from a drug discovery perspective. In vitro studies provide a convenient and cost‐effective way to obtain this information. This unit presents protocols for comparing metabolite profiles across several species, as well as methods for identifying metabolites.
Using Biacore to Measure the Binding Kinetics of an Antibody‐Antigen Interaction
Authors: Murphy Michael , Jason‐Moller Laure , Bruno JoAnne 
Published: September 2006

AbstractThe optical phenomenon of surface plasmon resonance (SPR) used by Biacore systems enables the detection and measurement of protein‐protein interactions in real time, without the use of labels. In this unit, the application of Biacore technology to measure a protein‐protein interaction is described using an antibody and its antigen as an example. The affinity of the antibody for its antigen is determined by measuring the binding kinetics of the interaction. The protocols are divided into three major steps that are required for measuring binding kinetics using Biacore: (1) surface preparation, (2) assay development, and (3) kinetic analysis.
Generation, Isolation, and Maintenance of Rodent Mast Cells and Mast Cell Lines
Authors: Jensen Bettina M. , Swindle Emily J. , Iwaki Shoko , Gilfillan Alasdair M. 
Published: September 2006

AbstractAntigen‐mediated mast cell activation, with subsequent mediator release, is a major initiator of the inflammatory allergic response associated with such conditions as asthma. A comprehensive understanding of the principles involved in this process therefore is key to the development of novel therapies for the treatment of these disease states. In vitro models of mast cell function have allowed significant progress to be made in the recognition of the fundamental principles of mast cell activation via the high‐affinity IgE receptor (FcɛRI) and, more recently, other receptors expressed on mast cells. In addition to human mast cells, the major cell culture systems employed to investigate these responses are rat and mouse peritoneal mast cells, mouse bone‐marrow‐derived mast cells, the rat basophilic leukemia cell line RBL‐2H3, and the mouse MC/9 mast cell line. In this unit, we describe the protocols used for the isolation and/or culture of these cells and discuss the relative merits of their use.
Safe Use of Radioisotopes
Authors: Meisenhelder Jill , Semba Kentaro 
Published: September 2006

AbstractThe use of radioisotopes to label specific molecules in a defined way has greatly furthered the discovery and dissection of biochemical pathways. The development of methods to synthesize such tagged biological compounds inexpensively on an industrial scale has enabled them to be used routinely in laboratory protocols, including many detailed in this manual. Although most of these protocols involve the use of only microcurie amounts of radioactivity, some (particularly those describing the metabolic labeling of proteins or nucleic acids within cells) require amounts on the order of millicuries. In all cases where radioisotopes are used, depending on the quantity and nature of the isotope, certain precautions must be taken to ensure the safety of the scientist. It is essential to use good safety practices and proper protection to handle radioactive substances. This unit discusses handling, storage, and disposal of the isotopes most frequently used in biological research.
Measuring Retrograde Transport to the Trans‐Golgi Network
Authors: Amessou Mohamed , Popoff Vincent , Yelamos Belèn , Saint‐Pol Agnès , Johannes Ludger 
Published: September 2006

AbstractThe recently described retrograde transport route is a highly selective pathway that allows some internalized molecules to reach the trans‐Golgi network from early/recycling endosomes, bypassing the recycling route to the plasma membrane and the late endocytic pathway. The non‐toxic receptor‐binding B‐subunit of bacterial Shiga toxin has played an important role in the discovery and molecular dissection of membrane trafficking at the early/recycling endosomes–TGN interface. This unit describes several recent methods for quantitative biochemical and morphological analysis of retrograde transport. The sulfation assay permits the detection and quantification of cargo protein transport from endosomes to the TGN, describing how sulfation‐site peptides can be chemically coupled to cargo proteins. Furthermore, a variant of the sulfation assay on permeabilized cells is presented. The chemical crosslinking theme is extended to horseradish peroxidase for the ultrastructural study of the Shiga toxin–containing early/recycling endosomes by whole mount analysis. Finally, an endocytosis assay describes concomitant analysis of cellular uptake of Shiga toxin and transferrin.
Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells
Authors: Snapp Erik L. , Hegde Ramanujan S. 
Published: September 2006

AbstractFluorescence resonance energy transfer (FRET) refers to the nonradiative transfer of energy from one fluorescent molecule (the donor) to another fluorescent molecule (the acceptor). Measurement of FRET between two fluorophore‐labeled proteins can be used to infer the subnanometer spatial and temporal characteristics of protein interactions in their native cellular environment. Multiple experimental methods exist for measuring FRET. The method that can be most widely and simply implemented, quantified, and interpreted is the acceptor‐photobleaching FRET technique. In this method, the presence of FRET between a donor and acceptor is revealed upon destruction (by photobleaching) of the acceptor. Acceptor photobleaching can be exploited to detect changes in the composition and organization of subunit proteins within a multiprotein complex and to even gain insight into relative stoichiometries of proteins within the complex. In this unit, strategies, tools, and background for designing and interpreting acceptor‐photobleaching FRET experiments in cells are described.
Comparative Protein Structure Modeling Using Modeller
Authors: Eswar Narayanan , Webb Ben , Marti‐Renom Marc A. , Madhusudhan M.S. , Eramian David , Shen Min‐yi , Pieper Ursula , Sali Andrej 
Published: September 2006

AbstractFunctional characterization of a protein sequence is one of the most frequent problems in biology. This task is usually facilitated by accurate three‐dimensional (3‐D) structure of the studied protein. In the absence of an experimentally determined structure, comparative or homology modeling can sometimes provide a useful 3‐D model for a protein that is related to at least one known protein structure. Comparative modeling predicts the 3‐D structure of a given protein sequence (target) based primarily on its alignment to one or more proteins of known structure (templates). The prediction process consists of fold assignment, target‐template alignment, model building, and model evaluation. This unit describes how to calculate comparative models using the program MODELLER and discusses all four steps of comparative modeling, frequently observed errors, and some applications. Modeling lactate dehydrogenase from
Trichomonas vaginalis
(TvLDH) is described as an example. The download and installation of the MODELLER software is also described.
Detection, Isolation, and Identification of Vibrio cholerae from the Environment
Authors: Huq Anwar , Grim Christopher , Colwell Rita R. , Nair G. Balakrish 
Published: September 2006

AbstractMicrobiological techniques for sampling the aquatic realm have become increasingly sophisticated, especially with advances in molecular biology. These techniques have been used to detect microorganisms that cannot be cultured by conventional bacteriological methods. This has resulted in a deeper and a clearer understanding of the ecology and epidemiology of microorganisms. Important advances have been made in isolation, detection, and identification of Vibrio cholerae over the past decade. The understanding that V. cholerae, like several other pathogenic bacteria, can enter into a state known as “viable but nonculturable” (VBNC) provided important clues on the epidemiology of the pathogen and its ability to cause sudden explosive epidemics at multiple places almost simultaneously. The advances in techniques have also allowed investigators to discern the intricate aspects of the ecology of this pathogen in the aquatic world. In this unit, we present the most accepted methods for the isolation and detection of V. cholerae.
Hamster Model of Leptospirosis
Authors: Haake David A. 
Published: September 2006

AbstractThis unit describes the techniques involved in the hamster model of leptospirosis. Hamsters are exquisitely susceptible to infection with pathogenic Leptospira species. The LD50 of virulent Leptospiral strains by intraperitoneal inoculation of 3‐ to 4‐week‐old hamsters is as low as a single organism. The pathogenesis of severe leptospirosis in the hamster is a model of accidental infections observed in nature in many mammals, including humans. Animal husbandry issues and the reproducibility of the hamster model make it the animal model of choice for Leptospiral vaccine and antibiotic treatment studies.
Human Immunodeficiency Viruses: Propagation, Quantification, and Storage
Authors: Richards Kathryn H. , Clapham Paul R. 
Published: September 2006

AbstractDescribed in this unit are basic protocols frequently used in the research of human immunodeficiency viruses (HIVs). Provided are methods for propagating and quantifying HIV, as well as recommendations for long‐term storage. Background information about these methods is also provided and includes their advantages, disadvantages, and troubleshooting.
Diffusion and reaction in a porous catalyst slab: Perturbation solutions
Authors: Cardoso Silvana S. S. , Rodrigues Alírio E. 
Published: September 2006

AbstractThe problem of diffusion and reaction in a porous catalyst slab is revisited. A combination of perturbation and integral mathematical techniques are used to derive approximate analytical solutions for the concentration and temperature profiles, as well as the effectiveness factor, for a first order, non‐isothermal reaction. The development of thermal and concentration boundary layers near the surface of the particle is characterized. The internal particle resistances to mass and heat transfer are also calculated. Our analytical solutions are validated by comparison with previous numerical results. © 2006 American Institute of Chemical Engineers AIChE J, 2006
Role of neurofilament aggregation in motor neuron disease
Authors: Lin Hong , Schlaepfer William W. 
Published: September 2006

AbstractA major question in the pathogenesis of motor neuron disease is why motor neurons are selectively susceptible to mutations in widely expressed gene products. Reexamination of motor neuron degeneration due to alterations of neurofilament (NF) expression suggests that disruption of assembly with aggregation of the light neurofilament (NFL) protein may be an upstream event and contributing factor leading to the preferential degeneration of motor neurons. The implications of these findings are that aggregation of NFL is not only a triggering mechanism to account for the hallmark aggregates of NF protein in sporadic and familial forms of amyotrophic lateral sclerosis, but that aggregates of NFL may also promote aggregation of wildly expressed proteins that are destabilized by missense mutations, such as by mutations in superoxide dismutase‐1 protein. This review examines the potential role of NFs in determining and promoting the preferential degeneration of motor neurons in motor neuron disease. The underlying premise is that motor neurons are selectively susceptible to alterations in NF expression, that alterations in NF expression lead to NF aggregates in motor neurons, and that elevated levels of NF aggregates provide a favorable microenvironment for the formation of neurotoxic aggregation and degeneration of motor neurons. Ann Neurol 2006
Cleavage of cystatin C is not associated with multiple sclerosis
Authors: Del Boccio Piero , Pieragostino Damiana , Lugaresi Alessandra , Di Ioia Maria , Pavone Barbara , Travaglini Daniela , D'Aguanno Simona , Bernardini Sergio , Sacchetta Paolo , Federici Giorgio , Di Ilio Carmine , Gambi Domenico , Urbani Andrea 
Published: September 2006

AbstractRecently, Irani and colleagues proposed a C‐terminal cleaved isoform cystatin C (12.5kDa) in cerebrospinal fluid as a marker of multiple sclerosis. In this study, we demonstrate that the 12.5kDa product of cystatin C is formed by degradation of the first eight N‐terminal residues. Moreover, such a degradation is not specific in the cerebrospinal fluid of multiple sclerosis, but rather is given by an inappropriate sample storage at −20°C. We conclude that the use of the 12.5kDa product of cystatin C in cerebrospinal fluid might lead to a fallacious diagnosis of multiple sclerosis. Preanalytical validation procedure is mandatory for proteomics investigations. Ann Neurol 2007
Nanocrystalline Ag from Supramolecular Stabilization of Metals in 4‐tert‐Butylcalix[4]arene Lattices
Authors: Brown Philip O. , Enright Gary D. , Ripmeester John A. 
Published: September 2006

AbstractCompetition between intermolecular forces is a prime method for directing the formation of self‐assembled structures without resorting to the use of covalently modified host molecules. In this study, we demonstrate how the competition between hydrogen bonding, coordinate bonding, and van der Waals interactions guides the formation of a complex crystalline clathrate based on a Ag–ethylenediamine coordination complex and 4‐tert‐butylcalix[4]arene. Owing to the coordinative flexibility of Ag and the stabilizing interactions with the calixarene framework, a four‐coordinate Ag complex is included in the structure. Subsequent removal of the amine by heating the crystalline inclusion compound results in reduction of the Ag+ ion such that metallic Ag nanocrystals are formed, thus indicating that this self‐assembly approach is a viable method for obtaining simple calixarene frameworks loaded with silver. In the light of the experimental evidence, we comment on how this process relates to the reduction of silver in other systems.
Endoplasmic reticulum‐retention C‐terminal sequence enhances production of an 11S seed globulin fromAmaranthus hypochondriacusinPichia pastoris
Authors: Medina‐Godoy Sergio , Valdez‐Ortiz Angel , Valverde María Elena , Paredes‐López Octavio 
Published: September 2006

AbstractThe methylotrophic yeastPichia pastoriswas used to express an 11S seed globulin fromAmaranthus hypochondriacus.Three different plasmids were tested for expression of amarantin. One of them, which included the untranslated regions (UTR) of the full cDNA, failed to express the amarantin under tested conditions, whereas the other plasmids, one without UTR and the other similar but including the endoplasmic reticulum‐retention signal KDEL, were able to express the proamarantin inP. pastoris. After 48 h of induction, KDEL‐proamarantin had accumulated quite significantly compared to unmodified proamarantin. Different solubilization patterns were also obtained from both proamarantin versions; only soluble protein was obtained from the system that included the KDEL retrieval signal. Protein fractionation was carried out by differential precipitation with ammonium sulfate, and proamarantin purification was performed using an HPLC ion exchange column. The endoplasmic reticulum‐retention C‐terminal sequence (KDEL retrieval signal), not commonly employed in this heterologous expression system, can therefore be used to enhance accumulation of recalcitrant protein inP. pastoris.The results obtained here also suggest that this expression system is suitable for expression and evaluation of engineered seed globulin proteins.
Plant growth promoting potential of free‐living diazotrophs and other rhizobacteria isolated from Northern Indian soil
Authors: Ahmad Farah , Ahmad Iqbal , Aqil Farrukh , Ahmed Wani Aijaz , Sousche Yogesh S. 
Published: September 2006

AbstractThe viable count of free‐living diazotrophic bacteria in different crop rhizospheres varied from 1.11 × 104 to 8.5 × 105 CFU/g of soil. The majority of the diazotrophs phenotypically belong to either Azotobacter chroococcum, non‐A. chroococcum type and to a heterogenous group tentatively named putative nitrogen‐fixing (PNF) bacteria. In this study, 25 isolates of the PNF group were screened for their multiple plant growth‐promoting (PGP) traits and grouped into 5 PGP types. An isolate, PNF11 showed promising PGP potential in vitro and was characterized as a species of Achromobacter by 16S rRNA analysis. The isolate PNF11 along with three other previously isolated PGP bacteria, Azotobacter sp. (AZS3), fluorescent pseudomonas (Ps5), Bacillus sp. (Bc1) were selected for crop inoculation response in green house experiment on Vigna radiata var.T44. Plants from inoculated and control pots were sampled and analyzed at 30, 45 and 60 days after sowing for various vegetative, nodule‐related data and yield parameters. The findings indicated that selected isolate of PNF bacteria, and other PGP isolates with multiple activities significantly improve the plant growth parameters, yield parameters of Vigna radiata T44 over control and also show good compatibility with Bradyrhizobium inoculation.
Genetic analysis and epigenetic silencing ofAt4CL1andAt4CL2expression in transgenicArabidopsis
Authors: Soltani Bahram M. , Ehlting Jürgen , Douglas Carl J. 
Published: September 2006

Abstract4‐coumarate::CoA ligase (4CL) gene family members are involved in channeling carbon flow into branch pathways of phenylpropanoid metabolism. TransgenicArabidopsisplants containing theAt4CL1orAt4CL2promoter fused to the beta‐glucuronidase (GUS) reporter gene show developmentally regulated GUS expression in the xylem tissues of the root and shoot. To identify regulatory genes involved in the developmental regulation ofAt4CLand other phenylpropanoid‐specific genes, we generated ethyl methyl sulfate mutagenized populations ofAt4CL1::GUSandAt4CL2::GUStransgenic lines and screened ∼16 000 progeny for reduced or altered GUS expression. Several lines with reproducible patterns of reduced GUS expression were identified. However, the GUS‐expression phenotype segregated in a non‐Mendelian manner in all of the identified lines. Also, GUS expression was restored by 5‐azacytidine (aza) treatment, suggesting inhibitory DNA methylation of the transgene. Southern analysis confirmed DNA methylation of the proximal promoter sequences of the transgene only in the mutant lines. In addition, retransformation ofAt4CL::GUSlines with furtherAt4CLpromoter constructs enhanced the GUS‐silencing phenotype. Taken together, these results suggest that the isolated mutants are epimutants. Apparently, two different modes of silencing were engaged in theAt4CL1::GUSandAt4CL2::GUSsilenced lines. While silencing in the seedlings of theAt4CL1::GUSlines was root specific in seedlings, it affected all organs in theAt4CL2::GUSlines. Also,At4CL1::GUStransgene silencing was confined to the transgene butAt4CL2::GUSsilencing extended to the endogenousAt4CL2gene. Organ‐specific silencing of theAt4CL1::GUStransgene cannot be explained by current models in the literature.
Two new disposable bioreactors for plant cell culture: The wave and undertow bioreactor and the slug bubble bioreactor
Authors: Terrier Bénédicte , Courtois Didier , Hénault Nicolas , Cuvier Arnaud , Bastin Maryse , Aknin Aziz , Dubreuil Julien , Pétiard Vincent 
Published: September 2006

AbstractThe present article describes two novel flexible plastic‐based disposable bioreactors. The first one, the WU bioreactor, is based on the principle of a wave and undertow mechanism that provides agitation while offering convenient mixing and aeration to the plant cell culture contained within the bioreactor. The second one is a high aspect ratio bubble column bioreactor, where agitation and aeration are achieved through the intermittent generation of large diameter bubbles, “Taylor‐like” or “slug bubbles” (SB bioreactor). It allows an easy volume increase from a few liters to larger volumes up to several hundred liters with the use of multiple units. The cultivation of tobacco and soya cells producing isoflavones is described up to 70 and 100 L working volume for the SB bioreactor and WU bioreactor, respectively. The bioreactors being disposable and pre‐sterilized before use, cleaning, sterilization, and maintenance operations are strongly reduced or eliminated. Both bioreactors represent efficient and low cost cell culture systems, applicable to various cell cultures at small and medium scale, complementary to traditional stainless‐steel bioreactors. Biotechnol. Bioeng. 2007;96:914–923. © 2006 Wiley Periodicals, Inc.
Expansion of mouse embryonic stem cells on microcarriers
Authors: Abranches Elsa , Bekman Evguenia , Henrique Domingos , Cabral Joaquim M.S. 
Published: September 2006

AbstractEmbryonic stem (ES) cells have been shown to differentiate in vitro into a wide variety of cell types having significant potential for tissue regeneration. Therefore, the operational conditions for the ex vivo expansion and differentiation should be optimized for large‐scale cultures. The expansion of mouse ES cells has been evaluated in static culture. However, in this system, culture parameters are difficult to monitor and scaling‐up becomes time consuming. The use of stirred bioreactors facilitates the expansion of cells under controlled conditions but, for anchorage‐dependent cells, a proper support is necessary. Cytodex‐3, a microporous microcarrier made up of a dextran matrix with a collagen layer at the surface, was tested for its ability to support the expansion of the mouse S25 ES cell line in spinner flasks. The effect of inocula and microcarrier concentration on cell growth and metabolism were analyzed. Typically, after seeding, the cells exhibited a growth curve consisting of a short death or lag phase followed by an exponential phase leading to the maximum cell density of 2.5–3.9 × 106 cells/mL. Improved expansion was achieved using an inoculum of 5 × 104 cells/mL and a microcarrier concentration of 0.5 mg/mL. Medium replacement allowed the supply of the nutrients and the removal of waste products inhibiting cell growth, leading to the maintenance of the cultures in steady state for several days. These conditions favored the preservation of the S25 cells pluripotent state, as assessed by quantitative real‐time PCR and immunostaining analysis. Biotechnol. Bioeng. 2007;96:1211–1221. © 2006 Wiley Periodicals, Inc.