Showing papers in "Methods in Enzymology in 1999"

Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent

Abstract: Publisher Summary This chapter discusses the analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Analyses of the Folin-Ciocalteu (FC) type are convenient, simple, and require only common equipment and have produced a large body of comparable data. Under proper conditions, the assay is inclusive of monophenols and gives predictable reactions with the types of phenols found in nature. Because different phenols react to different degrees, expression of the results as a single number—such as milligrams per liter gallic acid equivalence—is necessarily arbitrary. Because the reaction is independent, quantitative, and predictable, analysis of a mixture of phenols can be recalculated in terms of any other standard. The assay measures all compounds readily oxidizable under the reaction conditions and its very inclusiveness allows certain substances to also react that are either not phenols or seldom thought of as phenols (e.g., proteins). Judicious use of the assay—with consideration of potential interferences in particular samples and prior study if necessary—can lead to very informative results. Aggregate analysis of this type is an important supplement to and often more informative than reems of data difficult to summarize from various techniques, such as high-performance liquid chromatography (HPLC) that separate a large number of individual compounds .The predictable reaction of components in a mixture makes it possible to determine a single reactant by other means and to calculate its contribution to the total FC phenol content. Relative insensitivity of the FC analysis to many adsorbents and precipitants makes differential assay—before and after several different treatments—informative.

Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration

Abstract: Publisher Summary This chapter discusses ferric reducing/antioxidant power (FRAP) assay. The ferric reducing/antioxidant power (FRAP) assay is a recently developed, direct test of “total antioxidant power.” The FRAP assay is robust, sensitive, simple, and speedy and facilitates experimental and clinical studies investigating the relationship among antioxidant status, dietary habits, and risk of disease. Measurement of the total antioxidant power of fresh biological fluids—such as blood plasma—can be measured directly; the antioxidant content of various dietary agents can be measured objectively and reproducibly and their potential for improving the antioxidant status of the body investigated and compared. The FRAP assay is also sensitive and analytically precise enough to be used in assessing the bioavailability of antioxidants in dietary agents to help monitor longitudinal changes in antioxidant status associated with an increased intake of dietary antioxidants and to investigate the effects of disease on antioxidant status.

Quantification of beta -sheet amyloid fibril structures with thioflavin t

Abstract: Publisher Summary Despite the presence of a significant amount of carbohydrate in these fibrils, the staining reaction was eventually shown to be because of the protein component. The histologic benzothiazole dyes thioflavin S (ThS) and thioflavin T (ThT) under the appropriate conditions selectively stain amyloid structures in a number of pathological settings, as does the diazobenzidine sulfonate dye, Congo red, which is also birefringent when bound to fibrils. Phorwhite BBU, Sirius Red, and several other fluorescent and nonfluorescent aromatic molecules also show this property. Investigation of the amyloid fibril formation process requires not only the ability to distinguish the characteristic amyloid B-sheet structure from amorphous aggregates of the monomer or nonamyloid fibril forms of the precursor protein, but quantitation of the amyloid form as well. Congo red and thioflavin T undergo characteristic spectral alterations on binding to a variety of amyloid fibrils that do not occur on binding to the precursor polypeptides, monomers, or amorphous aggregates of peptide. Both dyes have been adapted to in vitro measurements of amyloid fibril formation.

DNA arrays for analysis of gene expression.

Abstract: Publisher Summary This chapter describes one of the currently used microarray technologies commonly called “spotting” or “printing” because DNAs are physically spotted on a solid substrate in which short oligonucleotides is synthesized directly on a solid support. In standard spotting applications, large collections of DNA samples are assembled in 96- or 384-well plates. DNA microarrays are used for a variety of purposes; essentially any property of a DNA sequence that can be made experimentally to result in differential recovery of that sequence can be assayed for thousands of sequences at once by DNA microarray hybridization. The chapter focuses on the application of DNA microarrays to gene expression studies and discusses general principles of whole genome expression monitoring as well as detailing the specific process of making and using spotted DNA microarrays.

Genetic approaches to study of biofilms

Abstract: Interest in the study of microbial biofilms has increased greatly in recent years due in large part to the profound impact biofilms have in clinical, industrial, and natural settings. Traditionally, the study of biofilms has been approached from an ecological or engineering perspective, using a combination of classical microbiology and advanced microscopy. We and others have begun to use genetic approaches to understand the development of these complex communities. To begin we must answer the question: What is a biofilm? This definition, by necessity, may be quite broad because it is clear that many organisms can attach to a variety of surfaces under diverse environmental conditions. Therefore, in the context of this article we will operationally define a biofilm as bacteria that are attached to a surface in sufficient numbers to be detected macroscopically.

Preparation of nucleosome core particle from recombinant histones.

Abstract: Publisher Summary This chapter describes the preparation method of nucleosome core particles (NCPs) from recombinant histones. The ability to make defined NCPs, or arrays of nucleosomes, from histone proteins expressed in bacteria has several advantages over previously used methods using histones isolated from natural sources. The chapter discusses protocols for the overexpression and purification of histones H2A, H2B, H3, and H4, both as full-length proteins and as corresponding trypsin-resistant globular domains. The chapter presents a method for the refolding and purification of a histone octamer from denatured recombinant histone proteins together with a protocol for the assembly and purification of a nucleosome core particle using 146 base pairs of DNA. The purity and homogeneity of the final core-particle preparation are assessed by a high-resolution gel shift assay. The chapter presents a flow chart that describes the procedures involved in the preparation of synthetic nucleosomes.

[34] Screening of dietary carotenoids and carotenoid-rich fruit extracts for antioxidant activities applying 2,2′-azinobis(3-ethylenebenzothiazoline-6-sulfonic acid radical cation decolorization assay

Abstract: Publisher Summary This chapter discusses the screening of dietary carotenoids and carotenoid-rich fruit extracts for antioxidant activities applying 2, 2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation decolorization assay. The decolorization of the ABTS radical cation is an efficient, accurate assay for screening the antioxidant activities of lipophilic substances and food extracts. The inhibitory response of the radical cation is proportional to the antioxidant concentration, and the time point selected (2.5 min) for the analysis explains that this is an ideal measuring point when the reaction is complete. Results for the carotenoids are of the same order as previously reported data using alternative systems for generating the ABTS.+ radical cation. It is essential to examine the relative bioavailability, absorption, and hypoactivity of dietary carotenoids and flavonoids/phenolics to understand the importance of these diverse nutritional components in the maintenance of health and disease prevention. The chapter concludes with a discussion of trolox reference standard for relative antioxidant activities.

Analysis of protein aggregation kinetics.

Abstract: Given a set of kinetic data, then, the preceding discussions suggest the following approach to its analysis. 1. 1. For purposes of establishing the reaction, ignore the final stages and concentrate on the initial 10–20% of the reaction at first. A globally optimized model may be based on a faulty assumption for the initial steps. Thus, although the whole data set may look reasonably well fit, the reaction could be misrepresented, and thus the fit unhelpful if accuracy at the later stages has come at the expense of the initial phase of the reaction. 2. 2. What is the time course of the initial reaction? (A) Is the reaction exponential? Exponential growth gives dramatic lag times (see Fig. 3), whereas nonexponential “lag times” have a visible signal from time 0 (i.e., Fig. 2). If the data set shows the abrupt appearance of signals after a period of quiescence, the chances are excellent that the time course is exponential. High sensitivity measurement of the signal at times during the lag phase should be used to confirm the exponential nature quantitatively. Exponential reactions mean a secondary pathway is operative. (a) A cascade ( t n ) can look similar to an exponential, but may proceed from a multistep single-path reaction. Thus the exponential needs to be ascertained with some accuracy. (b) It is possible that some or all of the lag results from a stochastic process, i.e., formation of a single nucleus being observed. This, however, is likely to be accompanied by a secondary process, as few techniques are sensitive enough to detect a single polymer at a time, and having one nucleus form many polymers is a hallmark of a secondary process. Thus, the reproducibility of the kinetics must be established to rule out stochastics. If data show wide variation, stochastic methods as described earlier may be employed. (c) Given a secondary process, one must separate the primary nucleation process from the secondary process (by stochastic means or by use of the product B 2 A , as described earlier). (B) If the reaction does not begin with an exponential, is it parabolic? If so, it falls in the general class of linear polymerizations. 3. 3. What is the concentration dependence of the reaction(s)? This will separate nucleation processes from growth, and so on. 4. 4. If the initial reaction is neither exponential nor parabolic, a reaction mechanism needs to be proposed and evaluated. Solving the resulting equations is best done by linearization, which has the best chance of giving equations whose solutions and their sensitivity to parameters are readily understood. If this proves fruitful, full numeric solutions may be useful. 5. 5. At this point, the full reaction may be considered to completion. 6. 6. The physical basis of the description (sizes of parameters and their dependencies) needs to be finally considered to ensure that the mathematical success of the description rests on tenable physical grounds.

Measurement of oxygen radical absorbance capacity in biological samples

Abstract: Publisher Summary Several methods have been developed to assess the total antioxidant capacities of various biological samples, particularly complex matrices such as plasma, serum, wine, fruits, vegetables, and animal tissues. This chapter presents a method called “oxygen radical absorbance capacity” (ORAC) assay based largely on the work reported by Glazer's laboratory, which depends on the unique properties of phycoerythrin (PE). The ORAC assay is the only method that takes reactive species (RS) reaction to completion and uses an “area under the curve” (AUC) technique for quantitation, thus combining both inhibition time and inhibition percentage of the RS action by antioxidants into a single quantity. The chapter discusses the general principles of ORAC assay for assessing antioxidant capacity against peroxyl radicals. By integrating inhibition percentages over the whole inhibition time period, the ORAC assay successfully overcomes all related problems in quantitation of the antioxidant capacity of a biological sample. Either B- or R-phycoerythrin (B-PE or R-PE) can be used in the ORAC assay. The sensitivity of B- or R-PE to hydroxyl radical damage may be different even for the same PE with different lot numbers. The concentrations of Cu 2+ and standard (Tro lox) can be adjusted, when it is necessary. The aforementioned procedures are based on using B- or R-PE that loses more than 90% of its fluorescence within 30 rains. The chapter concludes with a discussion of ORAC assay for assessing antioxidant capacity against transition metals.

Mass spectrometric quantification of F2-isoprostanes in biological fluids and tissues as measure of oxidant stress.

Abstract: This chapter has outlined methods to assess lipid peroxidation associated with oxidant injury in vivo by quantifying concentrations of free F2-IsoPs in biological fluids and levels of F2-IsoPs esterified in tissue lipids. The mass spectrometric assay described herein is highly precise and accurate. A potential shortcoming with this approach is that it requires expensive instrumentation, i.e., a mass spectrometer. However, several immunoassays for an F2-IsoP, 8-iso-PGF2α, have become available from commercial sources. At this time, the accuracy and reliability of these assay for quantifying F2-IsoPs in biological fluids has not been fully validated by mass spectrometry. If these immunoassays prove to be a reliable measure of F2-IsoPs, however, this should greatly expand the use of F2-IsoPs to assess oxidant stress. In conclusion, studies carried out over the past several years have shown that measurement of F2-IsoPs has overcome many of the limitations associated with other methods to assess oxidant status, especially when applied to the measurement of oxidant stress in vivo in humans. Therefore, the quantification of F2-IsoPs represents an important advance in our ability to assess the role of oxidant stress and lipid peroxidation in human disease.

Suppression subtractive hybridization: a versatile method for identifying differentially expressed genes.

Abstract: A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries It is based primarily on a technique called suppression PCR, and combines normalization and subtraction in a single procedure The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations As a result only one round of subtractive hybridization is needed and the subtracted library is normalized in terms of abundance of different cDNAs It dramatically increases the probability of obtaining low-abundance differentially expressed cDNA and simplifies analysis of the subtracted library The SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes This chapter provides detailed protocols for the generation of subtracted cDNA and differential screening of subtracted cDNA libraries As a representative example we demonstrate the usefulness of the method by constructing a testis-specific cDNA library as well as using the subtracted cDNA mixture as a hybridization probe Finally, we discuss the characteristics of subtracted libraries, the nature and level of background nondifferentially expressed clones in the libraries, as well as a procedure for the rapid identification of truly differentially expressed cDNA clones

Quantifying amyloid by congo red spectral shift assay.

Abstract: Publisher Summary The specific protocols discussed in this chapter refer to the use of the CR spectral shift assay to quantify A β aggregation. However, this method has the potential to be very general and the strategy for optimizing the method for other amyloid proteins is later discussed in detail. The CR spectral shift assay has several advantages over other approaches. First, this method requires only a simple spectrophotometer, making it broadly applicable. Second, it requires no radioisotopes or other expensive reagents. It is rapid and technically simple because there is no need to separate amyloid-bound from free indicator. Perhaps the greatest advantage of this technique is its ability to quantitate dye–amyloid interactions in absolute terms, allowing the direct comparison of results from day to day and from one laboratory to another. Initial discussion of the mathematical derivation of the equations used in this method incurs the risk of obscuring the relative ease and straightforward nature of this technique. To avoid this, this chapter first presents a basic protocol that allows rapid and direct application of the assay to the quantitation of A β fibril test samples prior to discussion of the theoretical basis of the method.

Preparation and assay of mammalian thioredoxin and thioredoxin reductase.

Abstract: Publisher Summary This chapter describes the preparation and assay of mammalian thioredoxin and thioredoxin reductase (TrxR). The amino acid sequences of mammalian TrxR revealed a strikingly high homology to glutathione reductase. 14,19 The conserved features of all the structural components of glutathione reductase are preserved in mammalian TrxR, including a redox active disulfide motif in the N-terminal FAD region, the NADPH binding region, and the carboxyterminal interface region that governs the association of the two subunits in the homodimeric holoenzyme. Mammalian thioredoxin reductase has gained increased interest due to its wide reductive capacity, the discovery of selenium in the enzyme, and its lipid hydroperoxide reductase activity. As thioredoxin shows a growing number of new roles in redox regulation of cellular processes and as an extracellular cytokine, the interest in TrxR in these contexts naturally follows. Future studies of mammalian thioredoxin systems should be an exciting area of research, yielding results required for a deeper understanding of thiol redox control and mechanisms protecting against oxidative stress.

Kinetic analysis of amyloid fibril formation.

Abstract: Publisher Summary A nucleation-dependent polymerization model has been proposed to explain the mechanisms of amyloid fibril formation in vitro. This model consists of two phases—the nucleation and extension phases. Nucleus formation requires a series of association steps of monomers that are thermodynamically unfavorable, representing the rate-limiting step in amyloid fibril formation. Once the nucleus ( n -mer) has been formed, the further addition of monomers becomes thermodynamically favorable, resulting in a rapid extension of amyloid fibrils. This chapter developes a first-order kinetic model of amyloid fibril extension in vitro and confirmed that the extension of amyloid fibrils proceeds via the consecutive association of monomeric precursor proteins onto the ends of existing fibrils. A characteristic sigmoidal time-course curve of amyloid fibril formation from monomeric precursor proteins at a physiological pH is widely believed to represent the essence of a nucleation-dependent polymerization model—that is, an initial lag phase represents the thermodynamically unfavorable nucleus formation. However, no convincing kinetic models to explain the sigmoidality of the curve have been reported. This chapter describes the paradigm to analyze the kinetic aspect of amyloid fibril formation in vitro. During the investigation of murine senile amyloidosis observed in the senescence-accelerated mouse (SAM), it develops a novel fluorometric method to determine amyloid fibrils in vitro based on the unique characteristics of thioflavin T (ThT). In recent years, this method has been applied to the analysis of several human amyloidoses, including gelsolin-derived amyloidosis, dialysis-related amyloidosis, and Alzheimer's β -amyloidosis. With this fluorometric method, this chapter successfully measures the polymerization velocity of three types of amyloid fibrils—that is, murine senile amyloid fibrils (fAApoAII), Alzheimer's β -amyloid fibrils (fA β ), and dialysis-related amyloid fibrils (fA β 2 -m), and performs kinetic analysis of amyloid fibril polymerization in vitro.

Inhibition of aggregation side reactions during in vitro protein folding.

Abstract: Publisher Summary When synthetic or natural genes or cDNAs are overexpressed in the cytosol of microbial host cells such as Escherichia coli, recombinant proteins can be produced in large amounts. Although high-level expression is usually achieved using standard recombinant DNA techniques, the polypeptides are often sequestered in the form of insoluble, inactive inclusion bodies. These large, dense particles often span the whole diameter of the host cell. After proper isolation they consist primarily of the recombinant protein. Active protein can be recovered from the inclusion bodies by solubilization in chaotrophic buffer systems and subsequent in vitro folding. However, unproductive side reactions (predominantly aggregation) often compete with correct folding during in vitro folding. Various techniques, some of which are summarized in this chapter, have been developed to inhibit aggregation side reactions and to ensure efficient in vitro protein folding. These methods, which can now be considered as standard laboratory techniques, allow the refolding of many inclusion body proteins on the laboratory scale or even in industrial production processes.

Staining methods for identification of amyloid in tissue.

Abstract: Publisher Summary The staining reaction given by amyloid after treatment with iodine was often used in the earlier studies of amyloidosis, and amyloid is still identified by its characteristic histological staining reactions. Despite the enormous amount of knowledge now known regarding the molecular nature of amyloid, histological staining methods are crucial for the diagnosis of amyloidosis and are also used commonly in amyloid research. Also, the introduction of modern immunohistochemical techniques has made it possible to identify normal and abnormal components in tissue. Immunohistochemistry (often used interchangeably with immunocytochemistry) has become an important tool in amyloid research. Amyloid was first recognized by its tinctorial properties, which were elicited when amyloid-laden tissues were treated with iodine at the autopsy table. This reaction is now known to depend on the presence of minor carbohydrate components in the amyloid deposits. Iodine reacts with the amyloid, giving it a mahogany-like color that changes to blue when sulfuric acid is subsequently added. The staining properties of amyloid with rosaniline dyes (e.g., methyl violet and cresyl violet), which were the main staining methods for amyloid before Congo red staining was introduced in the 1920s, are also based on the presence of these same carbohydrate components. Because of their low sensitivity and lack of specificity, these methods are not commonly used any longer. Most, if not all, dyes used for the identification of amyloid are compounds developed for use by the textile industry. This includes the dye Congo red, which was introduced as the first direct cotton dye in 1884. Much of the background knowledge regarding the properties of these amyloid-associated dyes comes from textile staining.

Molecular tools for study of biofilm physiology.

Abstract: Publisher Summary This chapter describes methods for the handling and analysis of microbial behavior of organisms in biofilm communities at both microscopic and macroscopic levels. Only methods and reporter systems that can be applied without disturbing the spatial organization of the organisms in the biofilm are presented. The in situ methods described in this chapter can be used for more than just identifying or tracing cells or genes in biofilms. By combining promoters that respond to specific environmental signals with appropriate marker genes, it may be possible to tag specific organisms and use these as monitor systems to estimate local chemical composition directly in the biofilms. Changes in environmental conditions will also have significant effects on the physiological state of the organisms. Such shifting conditions may result in several responses, such as altered growth rates, stress response, starvation, or even cell death. Most of these responses can be visualized directly using specific promoter–reporter fusions. The ribosome number is a reliable indicator of growth rate in bacteria growing in balanced growth and has been used as a standard for growth rates in biofilm-embedded bacteria as well.

High-efficiency full-length cDNA cloning.

Abstract: Publisher Summary A full-length cDNA library is advantageous in that it allows cloning of a complete sequence in a single step. However, the representation of full-length cDNA clones has been low in cDNA libraries prepared using standard techniques. In the preparation of full-length cDNA libraries, two problems have arisen. The first is the difficulty in reaching the cap site in the first-strand synthesis with the reverse transcriptase (RT), the first enzyme involved in the preparation of a cDNA library. The other problem in library preparation is that there have not been effective methods for selection of full-length cDNAs from incompletely extended cDNAs. This chapter describes protocols using the powerful “thermostabilized” RT that make it possible to prepare efficiently full-length cDNAs longer than 10 kb, combined with the selection of cDNA by biotinylated cap trapper to remove residual non-full-length cDNAs. Using these protocols, full-length cDNA libraries can be prepared at high yield without using polymerase chain reaction (PCR) that introduces sequence bias and causes overrepresentation of short clones in a library.

Membrane filter assay for detection of amyloid-like polyglutamine-containing protein aggregates.

Abstract: Publisher Summary The accumulation of polyglutamine-containing protein aggregates in neuronal intranuclear inclusions (NIIs) has been demonstrated for several progressive neurodegenerative diseases such as Huntington's disease (HD), dentatorubral pallidoluysian atrophy (DRPLA), and spinocerebellar ataxia (SCA) types 1, 3, and 7. Furthermore, it has been shown in vitro that the proteolytic cleavage of fusion proteins of glutathione S-transferase (GST) and the polyglutamine-containing huntingtin peptide coded for by the first exon of the HD gene s leads to the formation of insoluble high molecular weight protein aggregates with a fibrillar or ribbonlike morphology reminiscent of β -amyloid fibrils in Alzheimer's disease and scrapie prion rods. This chapter demonstrates that the cellulose acetate filter retardation assay can be a useful tool for the identification, structural characterization, and quantification of SDS-insoluble polyglutamine-containing protein aggregates formed in vitro and in vivo. In addition to the histochemical identification of amyloids, it useful in detecting insoluble protein aggregates in all types of human and animal amyloidoses, including the polyglutamine diseases, and also in screening compound libraries for potential aggregation inhibitors. Currently, attempts to develop a microtiter plate-based high-throughput filter retardation assay to identify chemical compounds that slow down the rate of formation of polyglutamine-containing fibrils in vitro are in progress. The amyloid-binding agents arising from this screen then will be tested in a HD cell culture model system and in the HD animal model for their therapeutic potential.

Isoforms of mammalian peroxiredoxin that reduce peroxides in presence of thioredoxin

Abstract: Publisher Summary All three mammalian Prx proteins (Prx I, II, and III) were shown to exhibit peroxidase activity toward H202 in the presence of the thioredoxin system (thioredoxin, thioredoxin reductase, and NADPH) or nonphysiological hydrogen donor DTT. This chapter describes two different assay procedures for the detection of Prx enzymes and purification procedures of mammalian Prx I, II, and III. The first assay procedure is based on the fact that glutamine synthetase is inactivated by oxygen radicals generated from the thiol oxidation system and that Prx protein can prevent this oxidative inactivation by removing H202, the precursor of radical species. The second assay is based on the fact that the reduction of H2O2 by a Prx is coupled to the oxidation of NADPH via thioredoxin and thioredoxin reductase. The chapter also describes the purification procedure of all the three mammalian Prx.

Ferrous ion oxidation in presence of xylenol orange for detection of lipid hydroperoxides in plasma.

Abstract: Publisher Summary This chapter discusses ferrous ion oxidation in presence of xylenol orange for detection of lipid hydroperoxides (ROOHs) in plasma The ferrous oxidation in xylenol orange 2 (FOX2) assay in conjugation with triphenylphosphine (TPP) has been implemented for the measurement of plasma ROOHs TPP reduces ROOHs to their corresponding alcohols while itself being converted to triphenylphosphine oxide This maneuver was also necessary to generate a proper control, since plasma contains interfering components, mainly ferric ions that are detected by xylenol orange There are other advantages of the FOX2 assay over existing techniques: (a) the kinetics of the reaction are independent of the chemical structure of ROOHs, (b) no extraction step is normally needed for analysis of liposomes and lipoprotein suspensions because of the use of 90% methanol/25 mM H2SO4 that denatures proteins sufficiently for access of the ferrous ions to available ROOHs

8-Hydroxydeoxyguanosine and 8-hydroxyguanine as biomarkers of oxidative DNA damage.

Abstract: Publisher Summary Modifications and alternatives to the original HPLC-EC (high-performance liquid chromatography- electrochemical) methods have produced a burgeoning literature on oxidative DNA damage. At the same time, they have resulted in discordant results and disagreements about the most appropriate techniques. Because of the apparent importance of DNA damage, and the interest in 8-Oxo-2'-deoxyguanosine (oxo 8 dG) and oxo 8 Gua as biomarkers for this damage, it is important to identify the best available assay methods. This chapter reviews the HPLC-EC methods that find to be the most reliable, indicate the common pitfalls associated with these techniques, and uncertainties of interpretation. Methods for DNA isolation continue to improve, with minor modification of the chaotropic NaI technique producing the lowest oxo 8 dG values reported to date using HPLC-EC. Although the use of urinary oxo 8 Gua adducts to monitor DNA oxidation in vivo remains attractive, the results can be difficult to interpret.

Methodological and chemical factors affecting amyloid beta peptide amyloidogenicity.

Abstract: Publisher Summary The immense difficulties and unexpected questions presented by hundreds of amyloid toxicity studies beg for a careful study of A β assembly processes, evaluating the kinetics and structures of different aggregation pathways that convert the soluble monomer into the higher order A β assemblies that harbor neurotoxic properties. A large array of sophisticated instrumental techniques, including analytical ultracentrifugation, atomic force microscopy, solid-state and solution NMR, and fluorescence tracer techniques, will help elucidate the bioactive A β structure(s), whereas specific cellular and biochemical techniques will elucidate the specific steps involved in the aftermath of A β cellular interactions. Nonetheless, all these potentially useful high technology methods are ineffective unless proper care regarding A β sample preparation is taken into account. This chapter briefly summarizes potential problems involved with A β sample preparation and has also described methods to deal with these issues. The most critical complications are related to the starting structures and aggregation states of the A β .

Ratio of reduced to oxidized glutathione as indicator of oxidative stress status and DNA damage

Abstract: Publisher Summary This chapter discusses the ratio of reduced to oxidized glutathione (GSH) as an indicator of oxidative stress status and DNA damage. Several methods have been proposed for the determination of GSH status in biological samples. Accurate determination of this status is largely dependent on the prevention of GSH autoxidation during sample processing. As the disulfide form (GSSG) is present only in minimal amounts with respect to the reduced form, a small GSH autoxidation during sample processing can give erroneously high GSSG level. The chapter describes high-performance liquid chromatography (HPLC) method for determining GSSG. It also presents a method for glutathione determination and explores the possible relationship between glutathione oxidation and apoptosis in fibroblasts. The resulting glutathione redox ratio (GSSG/GSH) accurately reflects the redox ratio of cells or of subcellular organelles and relationships can be found between glutathione oxidation and other metabolic parameters, such as lactate levels in exercise, oxidized DNA bases in aging, or apoptosis. The methods described in the chapter may be used to determine the glutathione redox ratio in various physiological and pathophysiological situations.

Mapping DNA interaction sites of chromosomal proteins using immunoprecipitation and polymerase chain reaction.

Abstract: Publisher Summary Chromosomal proteins that affect the maintenance, propagation, and expression of the genome often interact with DNA only indirectly through other DNA-binding factors. This chapter describes a method that helps determine the position at which such factors interact directly or indirectly with DNA in the genome. The yeast Saccharomyces cerevisiae is used for this purpose because of its wholly sequenced genome and the ease by which it is possible to introduce targeted mutations in specific genes. The initial step is the cross-linking of live cells. Formaldehyde (FA) is a reagent particularly useful for this purpose; it has long been used in studies of histone organization in the nucleosome or protein–DNA interactions. Thus, cross-linking can be done with intact cells, which reduces the risk of redistribution or reassociation of chromosomal proteins during the preparation of cellular or nuclear extracts. Finally, the polymerase chain reaction (PCR) products are analyzed by polyacrylamide gel electrophoresis. The chapter also discusses immunoprecipitation and DNA isolation.

Markers of oxidative damage to DNA: Antioxidants and molecular damage

Abstract: Publisher Summary More than 100 different oxidative modifications have been observed in DNA. However, so far only a few of the base modifications have been used as biomarkers, and of these, the oxidative C-8 adduct of guanine is by far the most studied as either the nucleoside or base. In principle, the level in DNA from target or surrogate tissues or cells or the excretion of repair products into the urine can be measured. Under the usual steady-state conditions, the latter will reflect the rate of damage, whereas the former will reflect the balance between damage and repair. The chapter discusses the involved assays, the interpretation of the methods, and some results from antioxidant intervention studies, particularly in humans. The assays for the urinary DNA repair products include high-performance liquid chromatography (HPLC) with electrochemical detection (EC) for 8-oxodG and 8-oxoGua and with ultra violet (UV) absorbance detection for thymidine glycol (dTg) and thymine glycol (Tg), whereas all the repair products can potentially be measured by gas chromatography-mass spectrometry (GC/MS). The major problem with all these assays involves separation of the very small amounts of analyte from urine, which is a very complicated matrix.

Candida biofilms and their susceptibility to antifungal agents.

Abstract: Publisher Summary This chapter discusses the formation of Candida biofilm on catheter disks, on cylindrical cellulose filters, and on perfused biofilm fermenter. Growth of the biofilms is monitored quantitatively using dry weight, colorimetric, or radioisotope assays and can be visualized readily by scanning electron microscopy. Different types of catheters, made from various plastics, can be used for biofilm formation. These include central venous catheters (composed of polyvinyl chloride (PVC) or polyurethane) and urinary Foley catheters. A much simpler model system is the formation of biofilm within small, cylindrical, cellulose filters that are perfused with culture medium. The perfused biofilm fermenter represents a model system in which the growth rate of biofilms can be controlled accurately. The chapter discusses the susceptibility of Candida biofilms to antifungal agents. Like bacterial biofilms, Candida biofilms are resistant to a range of antimicrobial agents. Drug susceptibility of biofilms can be assayed using any of these three model systems.

Antioxidative homeostasis: characterization by means of chemiluminescent technique.

Abstract: Publisher Summary This chapter discusses the characterization of antioxidative homeostasis by means of chemiluminescent technique. The state of antioxidative homeostasis can be made based on the measurement of antioxidative reserves in a biological substrate and the extent of its oxidative damage. For oxidative damage to occur, a local decrease in antioxidative factors under a critical limit is the cause for subsequent pathological events. The centrally determined antioxidative capacity of water (ACW) reflects the reaction of the organism toward these local processes. Isolated ACW measurements yield information on the actual antioxidant concentration but do not demonstrate whether their concentration is sufficient under the given circumstances. An approach based on correcting the concentration of antioxidative vitamins that deviate from normal levels appears somewhat promising. Clinically relevant approaches involve quality, dosage, and timing of an adequate effect on antioxidative homeostasis. These can be determined by systematic investigation of the antioxidative capacity of blood plasma with particular emphasis on vitamin C as one of the biologically most relevant components of the water-soluble portion of ACW or vitamin E as the respective lipid-soluble antioxidant, together with hydrogen peroxidestimulated chemiluminescence.

Preparation of cDNA from single cells and subcellular regions.

Abstract: Phenotypic characterization of cells in conjunction with single-cell mRNA analysis, which yields information regarding expression of multiple genes in individual neurons, facilitates a detailed and comprehensive view of neuronal cell biology. More specifically, the aRNA amplification method has provided an approach to analyze mRNA levels in single cells that have been phenotypically characterized on the basis of electrophysiology, morphology, and/or protein expression. In this way, relative mRNA abundances can be directly assayed from a well-defined population of neurons. The concept of expression profiling led to the development of robotics methods for arraying thousands of cDNAs on microarrays. These cDNA arrays can be screened with labeled aRNA or cDNA to generate a molecular fingerprint of a specific cell type, disease state, or therapeutic efficacy. A broad view of how gene expression is altered in single neurons affected by a particular disease process may provide clues to pathogenetic disease mechanisms or avenues for therapeutic interventions. The use of mRNA profiles to produce diagnostics and therapeutics is called transcript-aided drug design (TADD). When coupled with single-cell resolution, TADD promises to be an important tool in diagnosis of disease states, as well as provide a blueprint on which to develop therapeutic strategies. For example, mRNA abundances in an individual diseased cell may increase, decrease, or remain constant, and thus it is possible that a pharmaceutical alone or in combination with other drugs may be specifically designed to restore mRNA abundances to a normal state. Alternatively, if functional protein levels parallel the mRNA level changes, then drugs targeting the function of the proteins translated from these altered mRNAs may prove to be therapeutic. One promise of such an approach is that information about mRNA abundances that are altered in a diseased cell may provide new therapeutic indications for existing drugs. For example, if the abundance of mRNA for the beta-adrenergic receptor is altered as shown by the microarrays for a particular disease, already available adrenergic receptor agonists or antagonists that had not previously been used in this particular disease paradigm may prove to be therapeutically efficacious. The expression profile of a given cell is a measure of the potential for protein expression. Proteins are generally the functional entities within cells and differences in protein function often result in disease. The ability to monitor the coordinate changes in gene expression, in single phenotypically identified cells, that correlate with disease will provide unique insight into the expressed genetic variability of cells and will likely furnish unforeseen insight into the underlying cellular mechanisms that produce disease etiology.