bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differential metabolism of benzo[α]pyrene in vitro by human placental tissues exposed to active maternal cigarette smoke
Authors: Sanyal Mrinal K. , Li You Lan 
Published: February 2007

AbstractOBJECTIVES: Generation of different metabolites and DNA‐adduct(s) of metabolites of benzo[α]pyrene (B[α]P) in vitro by placental tissues (microsomes) of mothers who actively smoked cigarettes (tobacco) and those who did not smoke were analyzed to determine the variability in metabolism of the B[α]P substrate among individual placental samples. METHODS: Overall B[α]P metabolism was assayed by alkaline aqueous extraction of metabolites, and reactive metabolites by DNA adducts of B[α]P‐metabolites produced by salmon sperm DNA added to the incubation mixtures of the substrate and microsomes of exposed‐ and unexposed‐placentas to maternal cigarette smoke. Array of B[α]P‐metabolites produced by the same incubations were identified by high pressure liquid chromatography of the aqueous extracts. RESULTS: Subsets of smoke‐exposed placentas assessed by cluster analysis had augmented metabolic activity, others did not respond to smoke exposure. CYP1A1 expression in trophoblast cells analyzed by immunohistochemistry did not correlate with smoke exposure. The DNA‐adducts generated was variable, regardless of verbally reported levels of maternal exposure. The amounts of different B[α]P‐metabolites produced by individual samples matched for similar levels of exposure during pregnancy by self‐reported smoking (1 pack/day) were also not comparable. Metabolism of B[α]P into different metabolites, and production of toxic DNA adducts from metabolites in vitro by human placenta were variable and unrelated to the extent of smoke exposure. CONCLUSIONS: The metabolic characteristic of human placenta for xenobiotic exposure substrates is based on the expression and function of diverse enzymes, and such metabolism exhibited inter‐individual variation for toxic metabolite production or detoxification of the substrates in response to maternal smoke exposure. Birth Defects Res (Part B) 80:49–56, 2007. © 2007 Wiley‐Liss, Inc.
An optimized ATP/PPi‐exchange assay in 96‐well format for screening of adenylation domains for applications in combinatorial biosynthesis
Authors: Otten Linda G. , Schaffer Michelle L. , Villiers Benoit R.M. , Stachelhaus Torsten , Hollfelder Florian 
Published: February 2007

AbstractWe report a new format for measuring ATP/[32P]pyrophosphate exchange in a higher throughput assay of adenylation domains (A‐domains) of non‐ribosomal peptide synthetases. These enzymes are key specificity determinants in the assembly line biosynthesis of non‐ribosomal peptides, an important class of natural products with an activity spectrum ranging from antibiotic to antitumor activities. Our assay in 96‐well format allows the rapid measurement of approximately 1000 data points per week as a basis for precise assessment of the kinetics of A‐domains. The assay also allows quantitative high‐throughput screening of the substrate specificity of A‐domains identifying alternative, promiscuous substrates. We show that our assay is able to give high quality data for the T278A mutant of the A‐domain of the tyrocidine synthetase module TycA with a 330‐fold lower kcat/KM. The large dynamic range of this assay will be useful for the screening of libraries of mutant A‐domains. Finally we describe and evaluate a procedure for the high‐throughput purification of A‐domains in 96‐well format for the latter purpose. Our approach will be of utility for mechanistic analysis, substrate profiling and directed evolution of the A‐domains, to ultimately enable the combinatorial biosynthesis of non‐natural analogues of non‐ribosomal peptides that may have potential as alternative drug candidates.
Updating the metagenomics toolbox
Authors: Gabor Esther , Liebeton Klaus , Niehaus Frank , Eck Juergen , Lorenz Patrick 
Published: February 2007

AbstractMetagenomics – the application of the genomics suit of technologies to uncultivated microorganisms – is coming of age. Sophisticated technologies are being developed and adapted to this promising genetic resource to make increasing use of the seemingly boundless molecular and functional diversity. Particular progress has been made in the areas of randomly proliferating limited‐source DNA, massively parallel sequencing without cloning, isolating specific target sequences from highly complex template mixtures, high‐throughput assay systems targeting metabolic pathways, artificial transcriptional regulators activating reporter genes to indicate enzymatic substrate conversion and cDNA cloning from extracted mRNA to directly clone actively expressed genes from a microbial consortium. However, challenges still lie ahead. Most prominently, the efficient heterologous expression of a plethora of potentially interesting enzymes from unknown source organisms is not readily achieved.
The most proficient enzyme as the central theme in an integrated, research‐based biochemistry laboratory course
Authors: Smiley Jeffrey A. 
Published: February 2007

AbstractThe enzyme orotidine‐5′‐monophosphate decarboxylase is an attractive choice for the central theme of an integrated, research‐based biochemistry laboratory course. A series of laboratory exercises common to most instructional laboratories, including enzyme assays, protein purification, enzymatic characterization, elementary kinetics, and recombinant DNA methods, has been devised. The series of exercises requires biochemical instrumentation common to most laboratories and some specialized materials available from the author's institution to laboratory instructors.
Nitrophorins: Lipocalin‐based heme proteins transporting nitric oxide
Authors: Ascenzi Paolo , Nardini Marco , Bolognesi Martino , Montfort William R. 
Published: February 2007

AbstractNitrophorins 1–4 (NPs; NP1 to NP4) are salivary heme proteins secreted from the blood‐sucking insect Rhodnius prolixus. NPs transfer nitric oxide (NO) to the victim, resulting in vasodilatation, sequester histamine, reducing host inflammation and immune response, and inhibit blood coagulation. The NP fold is based on an eight‐stranded antiparallel β‐barrel, typically observed in the lipocalin homology family, which hosts a heme group. Although NPs use the ferric heme to bind NO and histamine, they are unrelated to the six/eight α‐helix (non)vertebrate hemoglobins, which bind NO preferentially to the ferrous heme, but do not bind histamine. Therefore, NPs and hemoglobins can be considered as an evolutionary experiment, where the heme group has been adapted to structurally different protein families to achieve similar function(s).
Detection of legume protease inhibitors by the Gel‐X‐ray film contact print technique
Authors: Mulimani Veerappa H. , Sudheendra Kulkarni , Giri Ashok P. 
Published: February 2007

AbstractRedgram (Cajanus cajan L.) extracts have been analyzed for the protease inhibitors using a new, sensitive, simple, and rapid method for detection of electrophoretically separated protease inhibitors. The detection involves equilibrating the gel successively in the protease assay buffer and protease solution, rinsing the gel in assay buffer, and later exposing it to an unprocessed x‐ray film. Gelatin on the film in places corresponding to the inhibitor bands remains unhydrolyzed. A simple and inexpensive laboratory experiment to detect protease inhibitors of legume seeds by the gel‐x‐r ay film contact print technique is described.
Introducing students to DNA: Identifying nutritional plants in a simple tried and tested laboratory experiment
Authors: Streicher Hansjörg , Brodte Annette 
Published: February 2007

AbstractIn this paper we report on a practical experiment, containing isolation, purification, and characterization of plant DNA, which is designed to give students unexperienced in DNA handling and molecular biology a glimpse of the field. Based on well established procedures, genomic DNA of a nutritional crop is isolated, and a non‐coding region of chloroplast DNA is amplified by use of PCR with universal primers. The origin of the sample is revealed by the size of the amplified fragment and in case of indistinguishable fragment sizes of wheat and barley by selective digestion with a suitable restriction endonuclease.
Vitamin requirements: Relationship to basal metabolic need and functions
Authors: Rucker Robert B. , Steinberg Francene M. 
Published: February 2007

AbstractThe dietary requirements for most water‐soluble vitamins in homeothermic animals, particularly vitamins utilized in energy‐related pathways, are related directly to metabolic rate. As a consequence, vitamin requirements are similar when expressed relative to empirical functions of metabolic body size, e.g. (Wtkg)3/4 or body surface area. The vitamin requirements for a range of animals are expressed relative to their corresponding rates of basal metabolism. Data for the rates of ascorbic acid production and turnover in animals that produce ascorbic acid are also compared with the ascorbic acid requirements and turnover in humans and guinea pigs, species that require ascorbic acid as a dietary essential. Factors that are most important in dictating the relative need for a given vitamin from a chemical perspective include chemical stability, the relative number of catalytic events that are involved in the process, the nature of the interactions with associated enzymes, and the presence or absence of pathways for partial synthesis or regeneration of the given vitamin. Vitamins that are required daily in millimolar amounts are usually less stable chemically, are involved in numerous reactions, and often exist in tissues as dissociable cofactors. In contrast, vitamins that are required daily in micromolar amounts are involved in fewer reactions and are often covalently bound to the proteins or enzymes for which they serve as cofactors. Development of the preceding concepts allows linkages of relative vitamin requirements to energy utilization and related biochemical and oxidative processes, which can aid in developing a better understanding of the integrative nature of nutritional and biochemical relationships.
Nitrate reductase: A model system for the investigation of enzyme induction in eukaryotes
Authors: Pike Carl S. , Cohen William S. , Monroe Jonathan D. 
Published: February 2007

AbstractPlant nitrate reductase is an easily studied enzyme whose induction by various signals can be explored in inquiry‐based undergraduate laboratories. Barley, mustard, and radish seedlings are grown for 3–7 days with different light conditions and nitrogen nutrition. The enzyme activity of crude extracts is assayed in a colorimetric procedure. The protocols can be used in student independent research projects.
Liposomes as tools to study drug diffusion and toxin‐induced leaks
Authors: Levillain Florence , Lanéelle Gilbert 
Published: February 2007

AbstractMultilayered vesicles made of egg‐phosphatidylcholine and of phosphatidic acid were used to teach in a 4‐h session of practical work with a low cost spectrophotometer how to determine osmolarity inside multilayered vesicles and to show, by using two anti‐tuberculous drugs (isonicotinic acid hydrazide, p‐aminosalicylate), that a small and non‐ionized molecule diffused freely through phospholipid vesicles, whereas a charged one did not. In addition, the permeabilizing effect of melittin, a membrane‐targeted bee‐venom toxin, was tested.
Assessment of the purification of a protein by ion exchange and gel permeation chromatography
Authors: Pugh Michael Eugene , Schultz Emeric 
Published: February 2007

AbstractA 3‐week group laboratory project experiment that involves the partial purification of myoglobin (Mb) from bovine hamburger is described. The experiment compares alternate purification methods (gel filtration and ion exchange chromatography) as to both overall yield and enhancement in relative purity. The purity and molecular weight of purified Mb is established by SDS‐PAGE. Group reports are directed toward having students put together all the information that is collected in the experiment. We discuss what we have learned and continue to learn from experiments done in the cooperative learning mode.
Dealing with the challenges of teaching molecular biophysics to biochemistry majors through an heuristics‐based approach
Authors: Castanho Miguel A. R. B. 
Published: February 2007

AbstractThe main distinction between the overlapping fields of molecular biophysics and biochemistry resides in their different approaches to the same problems. Molecular biophysics makes more use of physical techniques and focuses on quantitative data. This difference encounters two difficult pedagogical challenges when teaching molecular biophysics to biochemistry majors; an aversion to math, “Mathematophobia,” combined with little appreciation for the importance of models in scientific progress. To overcome these problems, I have used an heuristic approach to student learning. Although inspired by problem‐based learning, it has a lesser demand for material resources and can be managed by a single teacher in small classes. This approach is especially suited for elective courses in the final years of a program of study. It consists of teaching/touching the art of discovery by turning laboratories into original research projects and lectures into seminars.
Protein tryptophan accessibility studied by fluorescence quenching
Authors: Möller Matías , Denicola Ana 
Published: February 2007

AbstractA laboratory class is described to introduce the biochemistry major students to the basic concepts and various applications of fluorescence spectroscopy. Through simple and inexpensive experiments the students learn how to record excitation and emission spectra and measure intrinsic protein fluorescence and its quenching to elucidate the local tryptophan environment. Free tryptophan, ovalbumin, and bovine serum albumin are used for the experiments. The structural information that can be retrieved from emission spectra, as well as from quenching data, is discussed.
The adaptive response to ambient pH in Neurospora crassa: Contribution of a model organism to the elucidation of gene expression in eukaryotes
Authors: Nozawa Sérgio R. , Thedei Geraldo , Ciancaglini Pietro , Rossi Antonio 
Published: February 2007

AbstractThe main objective of this class experiment is to show that the extracellular levels of inorganic phosphate and the pH of the growth medium affect the synthesis and secretion of alkaline phosphatase in the filamentous mold Neurospora crassa. This type of study integrates knowledge of biochemistry and genetics through the regulation of gene expression and shows that the secretion of alkaline phosphatase is responsive to extracellular Pi levels and ambient pH. However, because N. crassa is a eukaryotic organism, other factors have to be considered, like the post‐translational modifications necessary for the movement of secreted enzymes through the secretory pathway, which may alter their catalytic properties. Thus, the enzymatic activity data, although useful, should not be analyzed alone but should be considered as just one of the tools needed to understand the biological phenomena involved in the gene expression of adaptivity in eukaryotic organisms.
Using computers to teach biochemistry
Authors: Whitehead Joyce P. , Pence Harry E. 
Published: February 2007

AbstractThis paper describes a symposium entitled “Using Computers to Teach Biochemistry,” which was presented at the fall, 2001 National Meeting of the American Chemical Society in Chicago, IL. The use of computers has increased in all areas of chemical education; however, the visualization capabilities needed to convey some aspects of biochemistry are quite complex, so the upward trend in the use of computers for this purpose has been especially significant. The various speakers in this symposium discussed the use of a wide range of instructional technologies, including presentation software, Chime, online data exercises, interactive visualization tools, self‐designed tutorial programs, and bioinformatics.
Strategies for introducing computer technologies into a biology laboratory program
Authors: Tillotson Joanne Kivela 
Published: February 2007

AbstractComputers have been installed in the General Biology laboratory at Purchase College and incorporated into the laboratory curriculum for all biology majors at the introductory level. The goal is to ensure that all students become familiar with general computer applications in the biological sciences and are comfortable enough to use them regularly. Thus, students will be prepared for later courses involving use of computer applications in bioinformatics or other technologically advanced subjects.
Incorporation of bioinformatics exercises into the undergraduate biochemistry curriculum
Authors: Feig Andrew L. , Jabri Evelyn 
Published: February 2007

AbstractThe field of bioinformatics is developing faster than most biochemistry textbooks can adapt. Supplementing the undergraduate biochemistry curriculum with data‐mining exercises is an ideal way to expose the students to the common databases and tools that take advantage of this vast repository of biochemical information. An integrated collection of exercises based on pet proteins has been assembled. The exercises described are applicable to either a lecture or laboratory format and require only basic desktop computers, an Internet connection, a current web browser, and the free Chime plug‐in module. In an open‐ended, inquiry‐based format, the assignments ask students to explore concepts such as the relative information content of the different biopolymers, the relationship between primary sequence and tertiary structure, and how sequence conservation can be used to find an enzyme active site.
Study of protein‐ligand binding by fluorescence
Authors: Möller Matías , Denicola Ana 
Published: February 2007

AbstractA practical class experiment is described to show the students an application of fluorescence spectroscopy in the study of protein‐ligand binding. This class is part of an undergraduate physical biochemistry course for life science students. The major aim is to introduce the students to the basic concepts of fluorescence emission (intrinsic and extrinsic), the effect of environment on fluorescence parameters, and how this could be applied to the study of ligand binding to proteins. In particular, binding of the fluorescent probe 1‐anilinonaphthalene‐8‐sulfonate to albumin (native and oxidized) is described. From the obtained results, the stoichiometry of complex formation, the binding equilibrium constants, and alterations of the environment of the binding sites are determined and discussed with the students.
Insulin signaling
Authors: Szeberényi József 
Published: February 2007

AbstractTerms to be familiar with before you start to solve the test: Signal transduction, insulin, insulin receptor, insulin receptor substrate 1 (IRS‐1), adaptor proteins, Western blotting, immunoprecipitation, tyrosine protein kinases, tyrosine phosphorylation, SH2 and SH3 domains.
Hemopexin: The primary specific carrier of plasma heme*
Authors: Mattu Marco , Fasano Mauro , Spallarossa Andrea , Bolognesi Martino , Ascenzi Paolo 
Published: February 2007

AbstractHemopexin (HPX) is the primary specific carrier of plasma heme and participates in its clearance by transport to the liver. After delivering the heme intracellularly, HPX is released intact into the bloodstream. HPX is formed by two four‐bladed β‐propeller domains, resembling two thick disks that lock together at a 90° angle; the face of the N‐terminal β‐propeller domain packs against one edge of the C‐terminal domain. Each propeller blade comprises a four‐stranded antiparallel β‐sheet, with the first and the fourth blades tied together by disulfide bridges. The heme ligand is bound between the two four‐bladed β‐propeller domains in a pocket formed by the interdomain linker peptide. Residues His‐213 and His‐266 coordinate the heme iron atom giving a stable bis‐histidyl Fe(III) complex. Heme release results from opening of the heme binding pocket, through movement of the two β‐propeller domains and/or the interdomain linker peptide.
Building a foundation for bioenergetics
Authors: Hamori Eugene 
Published: February 2007

AbstractTo give students a lasting comprehension of bioenergetics, first such basics as heat, work, equilibrium, entropy, free energy, closed versus open systems, steady state, and reversibility should be explained to them in a meticulous manner, albeit with a minimal use of mathematical formulae. The unique feature of thermodynamics, that it does not require any structural, mechanistic, and kinetic assumptions and models about the microscopic nature of the matter, should be exploited. Its laws could be considered as given, reflecting simply empirical observations that science has found unassailable so far. The familiarization of the students with the basic concepts should rely more on problem solving exercises than on the molecular mechanistic explanations. Only at a later phase should structural molecular inferences be made and (if justified) more rigorous mathematical derivations given. When the students are later confronted with the seemingly complex energetics of biological processes the idea of a steady‐state reaction should be stressed. Even without nonequilibrium thermodynamics a wealth of useful information can be deduced from this simple model of metabolic reactions. Because biological processes are typically homeostatic and progress at de facto constant temperature and pressure, using the steady‐state assumption, their thermodynamic treatment can be surprisingly straight forward. Thus the conceptual role of the steady‐state model in bioenergetics might be viewed as similar in pedagogical significance to the ideal gas model of basic chemistry.
Lactose operon mutants
Authors: Szeberényi József 
Published: February 2007

AbstractTerms to be familiar with before you start to solve the test: Lactose operon, operator, electrophoretic mobility shift assay, repressor, catabolite activator protein, RNA polymerase, σ‐factor, gel filtration, exclusion limit, void volume (V0), elution volume (Ve), expression plasmid.
Using the human genome: A case study in education*
Authors: Boyle John A. 
Published: February 2007

AbstractThe working drafts of the human genome, announced in February 2001, have clearly provided a breakthrough in biochemistry and molecular biology research. The scientific data also provide an opportunity to vary a typical approach to teaching. Advanced graduate students at our university can elect to take a course in molecular genetics. The human genome drafts and the initial publications generated by the drafts were used as the framework for a variation of this course in the fall of 2001. Instead of a traditional, linear lecture‐driven course, this approach provided a more methods‐driven focus to the topics considered. It also required the introduction of some recent historical facts to put the material into proper context. The bulk of the topics normally taught remained unchanged; however, their order of appearance was greatly modified. Nevertheless, the material remained coherent because of the constant tie‐in with the genome data. Evaluation of student performance was challenging because of the variety of material presented. Web‐based assignments proved useful as mechanisms to probe the level of student understanding as well as providing another means of imparting information.