Replace Toxic Products in your home

Everyday Roots

This book includes home remedies, natural beauty recipes and Diy household product tutorials. Discover over 215 suprising natural home remedies using common ingredients like onion, lemons and apple cider vinegar. EveryDay Roots will help you to make healthy changes in your life. Learn how to treat coughs, headaches and other health conditions with common ingredients like honey and watermelon. When you buy the book you get a 328 page Pdf with a clickable table of contents. Read more...

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Choice of Detergents for Structural Studies

Integral membrane proteins like GPCRs are adapted to function within lipid bilayers, making them difficult to manipulate, and are generally considered to be the most difficult type of protein to crystallize. For successful crystallization of a membrane protein to occur, a number of issues have to be addressed to generate and maintain the crystal lattice. To date, most of the membrane protein structures that have been solved by X-ray crystallography have involved the use of nonionic and zwitterionic detergents. Of the structures that have been solved, the vast majority have involved the use of alkyl maltosides, glucosides, dimethyl N-oxides (e.g., LDAO), and polyoxyethylene glycols (e.g., C12E8) 145 . Furthermore, the majority of the detergents tend to have an alkyl chain length of between C7 and C12, which are thought to mimic the approximate thickness of the hydrophobic portion of the cell membrane 160 . A number of considerations have to be made when assessing which detergent to use...

Application of Biosurfactant as a Substitute of Synthetic Chemical Surfactant in Commercial Laundry Detergents

Almost all surfactants, an important component used in modern day commercial laundry detergents, are chemically synthesized and exert toxicity to fresh water living organisms. Furthermore, these components often produce undesirable effects. Therefore, growing public disquiet about the environmental hazards and risks associated with chemical surfactants has stimulated the search for ecofriendly, natural substitutes of chemical surfactants in laundry detergents. A recent study from our laboratory has shown that cyclic lipopeptide (CLP) biosurfactants produced by B. subtilis strains were stable over a pH range of 7.0-12.0 and heating them at 80 C for 60 min did not result in any loss of their surface-active property.69 Crude CLP biosurfactants showed good emulsion formation capability with vegetable oils and demonstrated excellent compatibility and stability with commercial laundry detergents favoring their inclusion in laundry detergents formulations.69

Approaches To Clone Opioid Receptors

One of the main approaches to clone opioid receptors copied techniques used to identify beta-adrenergic receptor 15 and opioid peptide precursor transcripts 16 . This well-established strategy involved isolation and purification of the protein and determination of partial amino acid sequence by Edman degradation. Protein sequence information could then be used to design DNA probes, screen cDNA or genomic libraries, and identify nucleic acid stretches encoding the protein. The major issue was that the opioid receptor is membrane bound and required solubilization prior to isolation. Unfortunately, removing the receptor from its membrane environment either compromised or completely eliminated binding, an important characteristic required for purification. A review by Simon and Hiller in 1988 17 revealed many issues which thwarted first attempts to purify opioid receptors, including the lack of a rich source of opioid receptors, the sensitivity of opioid binding to detergents, and the...

Cyclo Oxygenase Biochemistry

However, our main concern here is with the cyclo-oxygenase enzyme itself. It had been recognised since the 1960s that tissue homogenates generated prostaglandins and that the enzyme activity seemed to be located in the high-speed membrane fraction, but it wasn't until the mid 1970s that convincing data were obtained about the intracellular location of the cyclo-oxygenase. Experiments such as those performed by Bohman and Larsson in 1975 (11) on the rabbit kidney suggested that the enzyme was associated with the endoplasmic reticulum. This membrane-bound location of the cyclo-oxygenase complex frustrated many early efforts to characterise this haem-containing enzyme. The subsequent discovery that it could be solubilized using detergents was important in providing a more convenient source of enzyme that could be subjected to further analysis. Useful data were gathered by Roth et al. (12) who ingeniously adapted Vane's finding (see below) to assess the molecular weight of the enzyme by...

Oligonucleotide Antisense Agents

Although considerable efforts have been devoted to taking advantage of the boranophosphate linkage, numerous limitations are inherent in the use of the BH3 moiety, especially in chemical syntheses. The highly reducing nature of this group can cause base degradation. It has been reported that the borane group is incompatible with some commonly used protecting groups in modified oligo-nucleotide synthesis.29 Likewise, the BH3 moiety has severe toxicity implications in that borohydrides, boranocarbonates and amineboranes typically have LD50 values in the tens of mg kg by ip injection in mice. The syntheses and properties of biologically important molecules that contain substituted boranes of the form BH2X (X COOR, C(O)NHR, CN, etc.) have been investigated extensively.1,79 The use of these substituted boranes in oligonucleotide modification may ameliorate some of the problems encountered with the unsubsti-tuted boranonucleic acids. Since the boron analogue of glycine, H3NBH2COOH, was...

Is Dimerization Required for Class A Gpcr Activation

Experimental proof of class A dimers through mutagenesis is challenging because the monomers interact within highly conserved hydrophobic TM domains, even for the glycoprotein GPCRs with large N-termini 114 . Cross-linking experiments indicate an interface between helices IV and V of opposing monomers in opsin, 5HT, and dopamine GPCRs 115-117 , with additional formation of higher-order oligomers (e.g., through helix I 115, 117 ). Targeting the interface by mutagenesis, or isolated expression of the relevant isolated helices, inhibits dimer formation and associated GPCR maturation and signaling 96, 111, 118-121 , but side effects on appropriate folding of the individual proteins cannot be excluded. Complementation of coexpressed nonbinding and noncoupling GPCRs, akin to the GABAB receptor, can restore function through dimerization 114, 122, 123 , but swapping individual domains in these receptors might also achieve a signaling monomeric unit. Importantly, these approaches (in common...

Expression and Purification of Membrane Proteins

After expression, the protein is solubilized and separated from the lipid components by the use of detergents. This process very often requires an intensive screening process, since different detergents have to be used for different trans-membrane proteins 10 . After solubilization, the recombinant protein is often purified by affinity chromography methods, after insertion of histidine tags into the N- or C-terminal of the protein.

Structure of Plasma Membrane Bound Nitrate Reductase

The cNR is composed of several redox centers with individual domains containing the co-factors FAD, heme and molybdopterin, which can independently catalyze partial reactions. As components of PM-NR these domains may also participate in plasma membrane redox reactions. In plasma membrane vesicles purified from roots or leaves, each of the known partial reactions of cNR was demonstrated, indicating that cNR and PM-NR share a similar composition (Stohr et al., 1993 Kunze et al., 1997 Wienkoop et al., 1999). However, the PM-NR in roots additionally catalyzes electron transfer from succinate to nitrate, resulting in formation of fumarate and nitrite (Stohr and Ullrich, 1997). Since this property has been observed neither for the cNR in roots or leaves nor for the PM-NR in leaves, there must be structural differences between these enzymes and root PM-NR. Comparison with the mitochondrial membrane-bound succinate dehydrogenase suggests that this electron transfer is likely to be mediated by...

Determination of the Three Dimensional Structure of the Chemokine Receptors

One of the disadvantages of chemokine receptors and other GPCRs compared with rhodopsin is the relatively low-level expression of these GPCRs. The structural determination of rhodopsin was facilitated by the extremely high levels of expression found in native mammalian sources. Thus, a key feature of many of the initial studies in chemokine receptor purification and structural analysis is that they have involved efforts at increasing expression levels. This has been achieved with CCR5 and CXCR4 by codon optimization using codons typically found in the highly expressed opsin family of GPCRs (85,86). With CCR5, this increased expression two- to fivefold compared with the wild-type receptor. In addition, expression of the receptor in heterologously transfected mammalian cells can be increased by treating cells with the histone-deacetylase inhibitor sodium butyrate. With CCR5, this increases expression by up to threefold. Both CCR5 and CXCR4 have now been purified to homogeneity in an...

Molecular Size Of Native Aspnat

Whether or not the large molecular size of native Asp-NAT was an artifact of the enzyme preparation methods was tested by using different homogenization and solubilization conditions. A buffer that is commonly used in mitochondrial protein purification (Tris-sucrose medium Tris-HCl, 50 mM 1 mM EDTA 0.32 M sucrose 1 mM DTT protease inhibitor cock-tail added according to the quantity of processed tissue pH adjusted to 7.4) was employed to obtain a crude mitochondrial pellet from rat brains. Various detergents were tested to solubilize the pellet deoxycholate (DC) (negatively charged and one of the most commonly used detergents in mitochondrial studies), hexadecyl trimethyl ammonium bromide (positively charged, commonly called CTAB), Triton X-100 (non-ionic), laurylmaltoside (LM) (non-ionic, commonly used in mitochondrial protein reconstitution experiments) and CHAPS. Crude mitochondrial pellets were obtained by homogenizing frozen rat brain tissue in the Tris-sucrose medium using a...

Solubilization Of Substrates

The majority of the techniques that are currently used for the determination of lipase activities, have in common the use of detergents as solubilizers of the hydrophobic substrates in water. However, the resulting micellar systems can rarely be prepared in a reproducible manner and this creates one of the main problems that has to be overcome in lipase analytics.

G Detecting Orderof Addition Effects

Samples containing solubilizing agents, like Triton and Tween detergents, or polyethylene glycol are especially likely to cause order-of-addition artifacts, as are those samples requiring agents, such as dimethylsulfoxide, to dissolve sparingly soluble reactants and or inhibitors.

The Phospholipase D Signaling Pathway

Phospholipase D catalyzes the phosphodiesteratic hydrolysis of membrane phospholipids (primarily phosphatidylcholine) producing phosphatidic acid and the polar head group (41,45,46). Multiple isoforms of phospholipase D activity apparently exist that differ in subcellular distribution, in effects of ions, detergents, and fatty acids on activity, and in overall mode of regulation. Many agonists for GPCR, including peptide, monoamine, nucleotide, and lipid agents, activate phospholipase D in a broad range of cell types. Although the physiological significance of PLD activation is unclear, phosphatidic acid generation has been implicated in regulation of inflammation, secretion, cell growth, superoxide generation, and the activity of a variety of enzymes including specific isoforms of protein kinase C (47). Phosphatidic acid inhibits the activity of adenylyl cyclase through a mechanism involving Gi, activates other Gi-promoted signaling responses, and has been shown to disrupt protein...

Homodimerization of 5HT2C Receptors Biochemical and Biophysical Properties

Fig. 7.1 5-HT2C receptors in choroid plexus. (a) Freshly isolated, intact choroid plexus tissue from an E19 rat pup, stained with 5-HT2C antibody (Santa Cruz, C-20) and Alexa 568. Scale bar 20 mm. (b) Western blot Choroid plexus tissue was briefly sonicated in Laemmli sample buffer and run under nonreducing conditions. Mature 5-HT2C receptor 55 kDa. (c) Detergent and cross-linker sensitivity of 5-HT2C receptor homodimers. Cell membranes from a 5-HT2C YFP HEK 293 stable cell line were solubilized with different detergents as indicated (+), run on 10 PAGE, and probed with GFP antibody. Lanes 1-4 5-HT2C YFP monomers (80-90 kDa) and dim-ers (160-180 kDa). Lane 5 Cells were pretreated with BS3 cross-linker prior to solubilization in CHAPS (Reproduced from Herrick-Davis et al. 2004. With permission from American Chemical Society ACS ) Fig. 7.1 5-HT2C receptors in choroid plexus. (a) Freshly isolated, intact choroid plexus tissue from an E19 rat pup, stained with 5-HT2C antibody (Santa Cruz,...

Solubility Modulation

Solubilizers (e.g., organic solvents, detergents, and Pluronics) are often used to solubilize drugs in aqueous solution without considering their effects on biological systems, such as lipid membranes and multidrug resistance (MDR) efflux transporters (e.g., P-glycoprotein or MDR1). Liposomal solubilization is an effective approach for the delivery of potent, insoluble drug candidates. An alternative to other methods developed is the production of drug nanoparticles by high-pressure homogenization either as pearl milling or as the continuous high-pressure homogenization. Of importance is the consideration of metallic contamination during fast speed milling processes to keep it less than 1 ppm. Drug nanoparticles are produced by the dispersion of drug powder in an aqueous surfactant solution the obtained presuspension is passed through a high-pressure piston-gap homog-enizer, for example, 5-20 homogenization cycles at typically 1000-1500 bars and works on the principle that cavitation...

Plausibility of Adverse Effects of EDCs Mechanisms of Gonadal and Accessory Reproductive Organ Differentiation

Developmental exposure to low doses of estrogenic chemicals has been related to a permanent decrease in testicular function in rats and mice. Sharpe et al. 145 fed octylphenol (an alkylphenol surfactant used in detergents, plastics, and many other products) to pregnant and lactating rats and reported a decrease in daily sperm production in the adult male offspring. Specifically, developmental exposure to a dose of octylphenol estimated to be within the range of 100400 g kg day, while not influencing body weight of adult males, significantly reduced the ratio of testes body weight, as well as weight of kidneys and ventral prostate, relative to untreated males. In addition, daily sperm production was significantly reduced. Moreover, administration of only 20 g kg day dose of octylphenol to pregnant mice during days 11-17 resulted in a small (15 ),but significant, decrease in daily sperm production in adult male offspring 146 .

Additional Methodological Notes

The use of the anionic detergent sarkosyl to solubilize the overexpressed CTP from the inclusion body fraction is particularly critical. We have tested many nonanionic detergents that proved ineffective at solubilizing the CTP. Fiermonte et al. (1993) first demonstrated the effectiveness of sarkosyl in solubilizing mitochondrial transporters. We subsequently showed that 20 different mitochondrial transporters, which differ substantially in amino acid sequence, are effectively solubilized by this detergent (Kaplan et al., 1995 Mayor et al., 1997). It should be pointed out that, during the solubilization step, patience is required, as the complete extraction of transporter from inclusion bodies can take > 1 h of careful, repetitive pipetting and scraping. In addition, we have shown based on CD analysis that the detergent solubilized Cys-less CTP contains a substantial amount of a-helical content, which approaches that observed with transporter that has been reconstituted into...

Potential Applications of Biosurfactant

Biosurfactants are becoming important biotechnology products for industrial and medical applications due to their specific modes of action, low toxicity, relative ease of preparation and widespread applicability.63-65 Biosurfactants also exhibit natural physiological roles in increasing bioavailability ofhydrophobic molecules and can complex with heavy metals, promoting improved degradation of chemical contaminants.66 They can be used as emulsifiers, de-emulsifiers, wetting and foaming agents, functional food ingredients and as detergents in petroleum, petrochemicals, environmental management, agrochemicals, foods and beverages, cosmetics and pharmaceuticals, commercial laundry detergents and in the mining and metallurgical industries.67-71

Technical Biological and Economical Limits for Assay Miniaturization in High Density Plates

Liquid handling in high-density plates tends to generate randomly appearing air bubbles in some wells of the plate. This effect is more pronounced in small wells and when the solutions contain proteins or detergents. The disturbing bubbles can be easily removed by a short (1 min) centrifugation of the plate. Therefore, high-density assay plates are often centrifuged before optical readout of the analytical result. A centrifuge should be integrated into an HTS system that processes high-density plates. Centrifugation also generates exceedingly uniform menisci over the entire plate and thereby yields assay data with higher precision, compared to readout without centrifugation. This difference can be attributed to the inevitable partial reflection of the light used for optical readout at the menisci.

Biosurfactant in Oil Clean Up of Storage Tanks

Due to excellent emulsifying properties of biosurfactants, they are used as detergents in cleaning up hydrocarbon crude oil storage tank. Banat et al75 reported the ability of biosurfactants produced by a bacterial strain (Pet 1006) for cleaning up oil storage tanks and to recover hydrocarbons from emulsified sludge. In a test for cleaning up of oil storage tank, about 91 crude oil could be recovered from the total sludge. Such clean up process is highly desirable as it is economically rewarding and environmentally friendly.76

MS Binding Assays Quantifying the Bound Marker

The liberation of the marker from the target-marker complex after separation should be complete and reproducible and include both the specifically and non-specifically bound marker. To achieve this, common methods for protein denatu-ration should be suitable (e.g. change in pH value, addition of organic solvents, chaotropic salts, detergents or increase in temperature) 76, 77 . However, it has to be kept in mind that the denaturation method should not interfere with the subsequent quantitation. When using ESI-MS for quantitation for example, high salt concentrations can lead to signal suppression and impair the LLOQ of the method 78, 79 . Therefore, denaturation with organic solvents seems to be more advantageous when using ESI-MS for quantitation.

Evidence For Endocrine Disruption

Endocrine disruption in wildlife was first reported in the 1950s, though the observations were not immediately associated with disturbance of the endocrine system. The deleterious effects of organochlorine pesticides, in particular DDT and its metabolites, became apparent when breeding failure in raptors in the USA was observed, leading to dramatic falls in populations in exposed areas. The subsequent restrictions in use of such pesticides in Western countries has been associated with a gradual recovery in most affected areas. Nonetheless, owing to the marked environmental stability of these compounds, residues and metabolites are still detected globally. Other chemicals suspected of causing disruption have been identified, including the polyaromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), dioxins (PCDD) and furans (PCDFs), non-ionic surfactants (used in detergents) and phthalate esters (used as plastisers), together with a number of agrochemicals (such as lindane and...

Biofilm Formation and Detachment

Initial attachments of microbes at the start of biofilm formation is due to weak van der Waals forces. Later the biofilm is strengthened by microbial polymeric substances including pili.50,51 Biofilms exhibit co-operative metabolic capabilities among the member organisms and they also are characterized by having increased resistence to detergents and antibiotics.53 It has been suggested that the greater antibiotic resistence of some biofilm surface bacteria, described as 'persisters', is due to their low level of metabolic activity.53

Key Problems Limiting Production Of 3d Gpcr Structures

The natural environment for GPCRs is the lipid bilayer of the plasma membrane. Detergents are used to solubilize receptors and remove them from the lipid bilayer. During the purification of GPCRs, it is often necessary to include the presence of lipids to ensure that the protein remains in its functional conformation 6 . The selection of detergents, in particular, those that are most compatible with crystallization, is fraught with difficulty and often results in a loss of function. Selection of the right detergent and advances in the use of suitable detergent lipid combinations have been one of the critical steps in obtaining crystals of GPCRs. The choice of detergent and the lipid requirement varies considerably from one receptor to another and must be determined experimentally for each receptor protein 7, 8 . Many membrane proteins have specific lipid binding sites, which may be required to preserve correct function. In addition, some receptors also have cholesterol binding sites,...

Lipid to Protein Ratio

The reconstitution of membrane proteins into bilayers is achieved by mixing lipids and protein, both solubilized in detergents, and decreasing the detergent concentration. Figure 1 shows an example where the concentration of octyl-polyoxyethylene (8-POE) was decreased by dilution, and the region of detergent concentration where lipid bilayer and mixed micellar structures coexist. The micelle-bilayer transition region (Stage II) was found to be the key to reconstitution and, by implication, to 2D crystallization (7,11). Cryo-electron microscopy (9) has shown for several lipid detergent systems that this transition involves the formation of worm-like extended lipid micelles, probably capped by detergents that must convert to vesicles on detergent removal. Such rod-like structures are therefore thought to be important intermediates in the formation of 2D crystals.

Classification Of Surfactants

The most commonly encountered anionic surfactants have carboxylate, sulfate, sulfonate, and phosphate polar groups in combination with counterions such as sodium and potassium (for water solubility) or calcium and magnesium (for oil solubility). Sodium and potassium salts of carboxylic acids derived from animal fats or vegetable oils are generically referred to as soaps and constitute the largest single type of surfactant. Linear alkylbenzene sulfonates are commonly used in household detergents and a variety of industrial applications. A commonly used surfactant for pharmaceutical application is sodium lauryl sulfate, which is a mixture of sodium alkyl sulfates, the chief of which is sodium dodecyl sulfate (SDS), C12H25SOzT Na+. It is very soluble in water and is used pharmaceutically as a preoperative skin cleaner, having bacteriostatic action against gram-positive bacteria, and also in medicated shampoos. It is a component of emulsifying wax. An alkyl sulfonate useful for the...

Rhodopsin as a Model for Other GPCRs

The years of research that have culminated in the various structures of rho-dopsin described here have not only benefited research into the mechanism and disorders of vision. In the absence of other GPCR structures, they have also provided an invaluable model and template for the entire GPCR family of receptors. Rhodopsin continues to be the model receptor, leading the way in our understanding of GPCR function at the molecule level. However, despite sharing many common features, seven transmembrane domains, conserved sequence motifs, and the ability to couple to G proteins, rhodopsin is less than 25 homologous to most other Family A GPCRs and has no homol-ogy with the other GPCR families, such as the secretin (Family B) and metabo-tropic (Family C) receptors. Nevertheless, pioneering work in the development of methods for obtaining crystal structures of rhodopsin, including methods for purification, selection of detergents, and conditions for crystallization, as well as the...

Crystallization in Native Membranes

Some membrane proteins have a natural propensity to form regular arrays within the native membrane. Since the membrane protein does not dissociate from the lipid bilayer, its native orientation is maintained. Additionally, the proteins are not exposed to harsh detergents and, therefore, are kept under stabilizing conditions. One disad

Protein Purification And Solubilization

One of the factors that has aided the study of membrane proteins is the large and diverse range of detergents that are available. Detergents can be classified into three main classes nonionic, zwitterionic, and ionic. Nonionic are characterized by their uncharged, hydrophilic head group and include mild detergents such as digitonin, DDM, and Triton X- 100 (Sigma-Aldrich, St. Louis, MO). Zwitterionic detergents contain both a positive and a negative charge in their hydrophilic head group and include detergents such as Fos-choline 12 (Anatrace, Maumee, OH), CHAPS, and LDAO. Anionic detergents are characterized by their charged hydrophilic head groups and include detergents such as SDS, sodium cholate, and decyltrimethylammonium chloride (HDTCI). When considering the ideal detergent for use in structural studies, a number of points have to be considered. The most important requirement is that the detergent has to be able to solubilize the receptor without affecting the structural...

Multiprotein Form Of Dna Polymerase Mediates Papovavirus Dna Replication In Vitro

Treatment with detergents, salt, RNase or DNase, suggesting that the association of the proteins with one another was independent of nonspecific interactions with other cellular macromolecular components. It was proposed that the isolated mouse cell multiprotein form of DNA polymerase represented a mammalian Multiprotein DNA Replication Complex (MRC). We introduced a model to represent the mouse cell MRC based on the fractionation and chromatographic profiles of the individual proteins found to co-purify with the complex.76 The proposed model for the MRC was recently extended to include the human cell multiprotein form of DNA polymerase.

Morphological Aspects And Protein Components Of Rosenthal Fibers

The parallels between RFs of Alexander's disease and the intracellular inclusions of common neurodegenerative diseases (e.g., neurofibrillary tangles, Lewy bodies) are striking. Common to these inclusions are extreme insolubility of accumulating protein, resistance to detergents and proteases, ubiquitination, high lysine content, absence of mutation, and accumulation with disease in a region-specific manner that correlates with clinical deficits (7,11,12,21). In neurodegenerative diseases such as Alzheimer's disease, increasing data indicate that oxidative post-translational modifications confer these characteristics (22-24). Thus, it may also be that similar posttranslational protein modifications in RFs confer the biochemical properties necessary for their insolubility and, therefore, accumulation, and that such accumulation is intimately related to disease expression and pathogenesis.

The Distribution of G Proteins in the Plasma Membrane

Measurements of the absolute levels of expression of GPCRs and G proteins invariably conclude that the G proteins are in considerable molar excess with regard to any specific GPCR 17-18 . This poses certain problems in pharmacology, because models of GPCR-G protein function that are both robust and predictive in nature derive from the premise that GPCR > G protein . Furthermore, the distribution of GPCRs and G proteins in the plasma membrane is non-random. Although the literature on the distribution of GPCRs is both large and complex, the equivalent literature for G proteins is relatively straightforward. Rather than being equally and randomly distributed, a substantial proportion of cellular G protein is targeted to, and located in, lipid 'rafts'. These are specialized structures enriched in cholesterol and sphingolipids that are both relatively resistant to dissolution by treatment with detergents and have low buoyant density 19 . This has allowed enrichment of such fractions on...

Industrial Applications

Other industries have also utilized non-aqueous conditions for their biotechno-logical application, and this literature base can be applied to pharmaceutical applications. For example, industrial enzymes have been used for detergents, textiles, food production, dairy, animal feed, leather, bioremediation, paper, cosmetics and diagnostics (Matsuura et al., 1993 Dai and Klibanov, 1999 Laboret and Perraud, 1999 Stevenson, 1999 Loughlan and Hawkes, 2000 Gonzales-Navarro et al., 2001 Schmid et al., 2001 Secundo and Carrea, 2003 Kuntz et al., 2003 Sardessai and Bhosle, 2003 Gupta et al., 2004 Houde et al., 2004 Sardessai and Bhosle, 2004 Spreti et al., 2004).

Biological Samples foR proteomic Analysis 15221 Protein Isolation

Proteomic studies generally entail isolation of a protein or proteins of interest prior to analysis by MS. Cellular proteins must be extracted from material containing other biological molecules including carbohydrates, lipids, and nucleic acids. Thus protein extraction protocols involve the homogenization of cells and tissues with subsequent application of detergents such as 3-(dimeth-ylammonio )-1 propane sulfate (CHAPS).1 Tween and sodium dodecyl sulfate (SDS) facilitate solubilization of the proteins and separate them from the lipid components, reducing agents such as dithiothreitol (DTT), denaturants such as urea that disrupt the bonds leading to formation of secondary and tertiary conformational structures, and enzymes that degrade nucleic acids such as DNAses and RNAses.

Mechanism Of Action On Biofilms In Chronic Infection

One benefit of this environment is increased resistance to detergents and antibiotics, as the dense extracellular matrix and the outer layer of cells protect the interior of the community. The biofilm environment provides physical protection to bacteria from a potentially hostile external environment and also a habitat where bacteria can communicate with each other (quorum sensing), which may lead to increase in virulence and propensity to cause infection (Kievit and Iglewski, 2000). Theoretically, chronic wounds offer ideal conditions for biofilm production because proteins (collagen, fibronectin) and damaged tissues are present, which can allow attachment. The biofilm, in turn, becomes a primary impediment to the healing of chronic wounds (Wolcott and Rhoads, 1996). Bacteria within a biofilm live in microcolonies that are encapsulated in a matrix composed of an extracellular polymeric substance separated by open water channels that act as a pseudocirculatory system for the delivery...

Receptors in Brain Gonad and Liver Hormone Action

Most research in the field of endocrine disruption has focused on xenobi-otics that mimic estrogens, although more recently some research has been carried out on environmental androgens, antiandrogens, and antiestrogens. Environmental xenobiotic compounds such as pesticides, surfactants from detergents, industrial waste, sewage treatment plant effluent, paper mill effluent, heavy metals, and phytosterols have been shown to be estrogenic 71 . Certain fungicides, such as the metabolites of vinclozolin, have been identified as an-tiandrogens in rats. Interestingly, metabolites of other compounds can have very different receptor-binding affinity from the parent compound. For example, o,p'-DDT is a known estrogen agonist, and one of its metabolites p,p'-DDE is antiandrogenic in rodents 72 .

Specified Variations To The General Method

Variations to the general method required for the analysis of specific substances may be specified in detail in individual monographs. Variations may include the addition of urea in the running gel (a 3 M concentration is often satisfactory to keep the protein in solution, but up to 8 M can be used). Some proteins precipitate at their isoelectric point. In this case, urea is included in the gel formulation to keep the protein in solution. If urea is used, only fresh solutions should be used to prevent carbamylation of the protein. Other variations include the use of alternative staining methods and the use of gel additives such as nonionic detergents (e.g., octylglucoside) or zwitterionic detergents (e.g., CHAPS or CHAPSO) to prevent proteins from aggregating or precipitating.

Incidence Of Acute Poisoning

The true incidence of poisoning in the U.S. is not known, but in 2003, nearly 2.4 million cases were voluntarily reported to the American Association of Poison Control Centers. The number of actual poisonings almost certainly exceeds by far the number reported. Deaths in the U.S. owing to poisoning number more than 1100per year. The incidence of poisoning in children younger than 5 years of age has decreased dramatically over the past four decades (e.g., there were no reported childhood deaths from aspirin in 2003, compared with 140 deaths year in the early 1960s). This favorable trend probably is due to safety packaging of drugs, drain cleaners, turpentine, and other household chemicals improved medical training and care and increased public awareness of potential poisons.

Oxidative Damage to Macromolecules

Free radicals are important because they can damage cells. This capacity lies in their ability to alter biological molecules. The types of modifications caused by such radicals pose a risk to cellular homeostasis and could provoke the death of an individual cell and eventually an organism. All molecules are more or less sensitive to oxidation however, the effect is different depending on the type of molecule affected, the role ofsuch a molecule in cellular physiology, the existence ofrepair mechanisms and the possibility of a removal of toxic products.

Critical Issues in TB Drug Development 51 Cell Penetration

Historically, the intrinsic resistance of mycobacteria to antibiotics has been attributed to the low permeability of the cell wall. For example, perturbations in the cell envelope caused by detergents, mutations, or through inhibition of cell wall polymer biosynthesis increases the susceptibility of mycobacteria to various classes of antibiotics, including aminoglycosides, EMB, and RIF 332-335 . Moreover, data indicate that the outer membrane of Mycobacterium chelonei is 1,000-fold less

Localization and Some Physiological Roles of Esterases

Tetrachloroethylene Degradation Pathway

The physiological functions of carboxylesterases are still partly obscure but these enzymes are probably essential, since their genetic codes have been preserved throughout evolution 84 96 . There is some evidence that micro-somal carboxylesterases play an important role in lipid metabolism in the en-doplasmic reticulum. Indeed, they are able to hydrolyze acylcarnitines, pal-mitoyl-CoA, and mono- and diacylglycerols 74a 77 97 . It has been speculated that these hydrolytic activities may facilitate the transfer of fatty acids across the endoplasmic reticulum and or prevent the accumulation of mem-branolytic natural detergents such as carnitine esters and lysophospholipids. Plasma esterases are possibly also involved in fat absorption. In the rat, an increase in dietary fats was associated with a pronounced increase in the activity of ES1. In the mouse, the infusion of lipids into the duodenum decreased ES1 levels in both lymph and serum, whereas an increase in ES2 levels was observed....

Design Of Novel Antidyslipidemic Agents

In atheromatosis the following pathologic events take place hypercholesterol-emia, hypertriglyceridemia, oxidation of LDL to oxLDL, (a form not recognized by the LDL receptor), a free radical attack to the vascular endothelium, and the accumulation of macrophages that release superoxide anion radicals in atheromatic plaques .4 Further injury is caused via nitric monoxide, peroxynitrite, hydrogen peroxide, and hydrolytic enzymes . Macrophages induce platelet-stimulating factors, generation of potentially toxic peroxidation products, which in turn aggravate the initial endothelial damage. 5 Oxidized LDL is a potent chemoattractive factor for human blood monocytes in atheromatic lesion and further induces eicosanoid production Monocyte-derived macrophages are sources of growth factors derived of platelets, as well as smooth muscle cell growth factors . 6 Oxidized lipids induce production of interleukine-1 . Lysophosphatidyl choline, a product of LDL oxidation, is chemoattractive for...

Propylene Glycol Diabetic Insulin

Stoichiometric ratios of anionic detergents, such as sodium dodecyl sulfate (SDS), have been used to complex peptides and increase their partition coefficient into non-polar solvents by two- to four-fold (Powers et al., 1993). For example, insulin was complexed with SDS and resulted in a ten-fold increase in solubility in 1-octanol. The insulin remained in its native conformation with

O Cationic Surfactants

How Surfactants Work

Surfactants present several difficulties. Soaps and other anionic detergents inactivate them. All traces of soap must be removed from skin and other surfaces before they are applied. Tissue debris, blood, serum, and pus reduce the effectiveness of the surfactants. Cationic surfactants are also adsorbed on glass, talc, and kaolin to reduce or prevent their action. The bactericidal action of cationic surfactants is slower than that of iodine. Solutions of cationic surfactants intended for disinfecting surgical instruments, gloves, etc. should never be reused because they can harbor infectious microorganisms, especially Pseudomonas and Enterobacter spp. The antimicrobial properties of the biguanides were discovered as a result of earlier testing of these compounds as possible antimalarial agents (Chapter 7). Although the biguanides are technically not bisquaternary ammonium compounds and, therefore, should probably be classified separately, they share many physical, chemical, and...

ABCA1interacting proteins and lipid membrane microdomains

DRMs associated with the ABCA1 pathway using different detergents and density gradient separations were characterized by our laboratory. Various cell types including macrophages and fibroblasts from controls and monogenetic human diseases (ABCA1-, NPC1 deficiency) were analyzed. ABCA1 copurifies only with Lubrol- but not with Triton-or Brij-DRMs. Lubrol-DRM association of ABCA1 is modulated by cholesterol loading and apoA-I stimulated lipid efflux upon apoA-I interaction. ABCA1 may act as a raft disrupter, and this function is disturbed in ABCA1-deficient cells as assessed in patient macrophages and fibroblasts. To analyze cell type-dependent dynamic differences of the lipid species composition in DRMs and shedded membrane vesicles from filipodia upon HDL3 apoA-I mediated ABCA1 activation, quantitative tandem mass spectrometry methods with stable isotope labeling for the analysis of various lipid species from total and fractionated cellular extracts were developed (Binder et al. 2006...

Identification and Characterization of FET3

Although the above studies showed that FET3 was a multicopper oxidase, the specific role of the oxidase remained unclear. It is possible that Fet3p oxidizes a protein, rather than iron. The suggestion that Fet3p acted as a ferroxidase required the isolation of Fet3p and a direct measurement of ferroxidase activity. This was first accomplished by deSilva et al., who demonstrated that purified Fet3p could oxidize iron and that it could also load iron onto transferrin (18). Examination of a number of potential substrates for multicopper oxidases showed iron to be the preferred substrate. The Km for iron oxidation, 0.5 uM was very close to the Km for iron transport. Purified Fet3p could iron load transferrin, although the rate of iron loading was low relative to that of ceruloplasmin. Reasons for the low rate may be that detergent was use to isolate Fet3p and as isolated Fet3p is heavily glycosylated. The presence of both detergent and carbohydrate may reduce the rate of transferrin iron...

Physicochemical drug interactions and incompatibilities

Chemical, as well as physical, instability may result from changes in pH, buffering capacity, salt formation or complexation. Chemical instability may give rise to the formation of inactive or toxic products. Although infusion times are generally not greater than 2 h, chemical changes following a change in pH may occur rapidly. pH changes often follow from the addition of a drug substance or solution to an infusion fluid, as shown in Table 10.1. This increase or decrease in pH may then produce physical or chemical changes in the system.

Diseased Compromised Skin and Solvent Selection

Erupted skin surface will allow increased water loss from the body. Psoriasis is a chronic recurring non-infectious scaling skin condition characterised by erythematous plaques covered with silvery scales. For topical therapy the loss of skin barrier integrity has been shown to be valuable for targeting drugs to the required site of action while minimising side effects (Anigbogu et al., 1996). Lichenoid eruptions are characterised by intensely itchy flat-topped papules while eczema is a further non-infectious eruptive condition, in which blistering occurs. Contact dermatitis can result from a direct irritant action of a substance on the skin (irritant contact dermatitis) or further exposure, following previous sensitisa-tion of the skin, from a contact allergen (allergic contact dermatitis). Irritant dermatitis is the more common of the two manifestations, and can be caused by many chemicals, solvents and detergents sodium lauryl sulphate was used to induce irritant dermatitis before...

Stabilization of XRay Structures

An X-ray structure of a protein that is closely related to p2AR, the turkey p1 receptor complexed with the antagonist cyanopindolol, was published recently I 28 , Employing a strategy to identify a combination of mutations in p1AR that result in thermal stability, the authors found a set of six point mutations that sufficiently stabilized the complex without the introduction of a companion (Fab5 or T4L) protein. It should be noted that residues in ICL3 and the C-terminus were also deleted. The mutated protein was stable in many detergents used for crystallization and remained in an antagonist conformation even when the ligand was no longer present. Interestingly, the

Uridine Diphosphate Glucuronosyltransferase UDPGT

Glucuronosyltransferase

Latency and Membrane Disruption by Detergents The active site of the UDPGT lies on the lumenal side of the endoplasmic reticulum (ER), which restricts the access of substrates and UDPGA from cytosol. It has been suggested that UDPGA transport from cytosol onto the lumenal side of the ER across the ER membranes may be the rate-determining step for glucuroni-dation in the intact microsomes. Owing to this membrane barrier, UDPGTs are latent enzymes and their activities in freshly isolated microsomes cannot be fully revealed without disrupting the membranes to a certain degree. For instance, more than 95 of UDPGT activity can be latent in liver microsomes prepared in 0.25 M sucrose with 5 mM HEPES, pH 7.4. Disruption of microsomal membranes with detergents such as Lubrol Px can remove UDPGT latency and increase enzyme activity by up to 10- to 20-fold under the optimal conditions. Often, the ratio of protein and detergent (0.01-0.5 mg detergent mg microsomal protein) has to be...

ALIS An Affinity Selection Mass Spectrometry System based on Continuous SEC

Mass Spectrometry Pharmacology

In the affinity selection step, a protein target is incubated with one or more compounds in a physiologically relevant buffer containing any necessary cofac-tors, salts, metal ions, and detergents. The ALIS system is generic with respect to target class, and the binding reaction can be performed using proteins of virtually any variety, including both soluble targets and membrane-associated proteins enzymes such as proteases, kinases, and phosphatases nuclear hormone receptors and G protein-coupled receptors (GPCRs). Genomic targets from bacterial or viral pathogens, especially novel target proteins that are derived from lethal gene product deletions but are of otherwise unknown function, can be readily studied to yield potential anti-infective compounds. Since the binding reaction is solution based, potential ligands can query all protein surfaces and not just the ''active site,'' enabling the discovery of ligands that act through allosteric binding and other mechanisms. The use of...

Applications of Hemicelluloses

In industrial applications, hemicelluloses are used to control water content and the rheology of aqueous phases. Thus they may be used as food additives, thickeners, emulsifiers, gelling agents, adhesives and adsorbents. Thus, derivatives of xylans (acetate, butyrate and benzoate) are used as extruders for fatty acids. Carboxymethyl xylans are used as detergents, flocculants and adhesives in coating paper. Due to their antitumoral and hypocholesterolemic activities, xylan sulfate derivatives have applications in medicine. Arabinoxylans are used as emulsifiers, thickeners or

The Implications of Endocrine Dysfunction for Fish

Alkylphenols, ammonia, asbestos, chlorinated paraffins, 4-chloroaniline, cyanide, detergents, di-n-butyl phthalate, polyaromatic hydrocarbons (PAHs e.g. anthracene, benzopyrene, methylcholanthrene, B-naphthoflavone), nitrate, nitrite, petroleum oil, phenol, pentachlorophenol, 4-nitrophenol, dinitro-o-cresol, polychlorinated biphenyls (PCBs especially coplanar), polychlorinated dioxins, polybrominated naphthalenes, -sitosterol, sulfide, thiourea, urea, acid water, coal dust

Tetrazolium Reduction Assays

Mtt Assay Cell Precipitate

The formazan product resulting from reduction of MTT is deposited as a precipitate both inside and outside of cells. The precipitate must be solubilized before recording 570 nm absorbance that requires addition of a second reagent to generate a uniformly colored solution within the assay well. A variety of different combinations of organic solvents and detergents were developed as reagents to solubilize the formazan product, stabilize the color, avoid evaporation, and reduce interference by phenol red often present in culture medium (Tada et al. 1986 Hanson, Nielsen, and Berg 1989 Denizot and Lang 1986).

Biological Reactivity Formation And Metabolism Of Epoxides

Hydration Epoxide

Many organisms have detoxification enzymes that catalyze the transformation of epoxides to less toxic products, specifically, glutathione S-transferases, which form glutathione conjugates of epoxides (Figure 1a) (9), and epoxide hydrolases, which hydrate epoxides to the corresponding dihydrodiols (Figure 1 b) (10). Epoxide hydrolases initiate epoxide catabolism in certain bacteria that grow using epoxypropane or epichlorohydrin (3-chloroepoxypropane) as carbon sources (11, 12). In the case of bacteria that grow using isoprene (2-methyl-1,3-butadiene) and styrene (phenylethene) as carbon sources, the epoxide products are further metabolized by a conventional strategy (glutathione conjugation in the case of isoprene monoxide metabolism) (13, 14) or a nonconventional one (isomerization to phenylacetaldehyde in the case of phenylepoxyethane metab

Growth Of 2d Crystals

Molecular Packing

Not only excess lipid but also other contaminating proteins. For example, cytochrome c oxidase constitutes approximately 10 of the protein of the mitochondrial inner membrane, and one can purify a membrane fraction of nearly pure cytochrome oxidase by treating beef heart mitochondria with appropriate concentrations of detergents followed by centrifugation to separate detergent solu-bilized components from the membrane residue (30,66,80). This requires 2 to 3 detergent treatments, and in each case, the pellet becomes enriched in cytochrome oxidase while other membrane proteins and excess phospholipid are removed by decanting the supernatants. Depending upon the type and concentration of deter individual detergent molecules the cmc is essentially the concentration of individual molecules in the presence of micelles. Since detergent micelles are too large to pass through the pores of common dialysis tubing, dialysis of detergent solutions proceeds by the movement of individual detergent...

Differential expression of the monocyte innate immunity receptor complex defining monocyte subpopulations

Fig. 2 Flow cytometric differential detergent resistance (FCDR) assay strategy for the detection of DRM association of membrane proteins. A Lipid raft-associated plasma membrane proteins show high resistance to detergent treatment. B Nonraft-associated proteins are rapidly dissolved by even low concentrations of non-ionic detergents in a short time. These processes can be followed by measuring fluorescence means before and after 5 min of detergent treatment by flow cytometry. (Adapted from Gombos et al. 2004 Wolf et al. 2006) Fig. 2 Flow cytometric differential detergent resistance (FCDR) assay strategy for the detection of DRM association of membrane proteins. A Lipid raft-associated plasma membrane proteins show high resistance to detergent treatment. B Nonraft-associated proteins are rapidly dissolved by even low concentrations of non-ionic detergents in a short time. These processes can be followed by measuring fluorescence means before and after 5 min of detergent treatment by...

Xray Crystallography

This section has so far dealt exclusively with the results of X-ray diffraction studies on water-soluble proteins. Although X-ray diffraction studies of membrane proteins have been technically difficult, because these proteins do not readily form three-dimensional crystals, some progress has been made with novel crystallographic approaches using short chain detergents or antibody fragments,50-53 or with highly ordered natural membrane proteins, such as bacteriorhodopsin.24 In the latter case, low-resolution difference analysis of electron density maps with and without the haloalkane anesthetic diiodomethane showed preferential localization in the phos-pholipid-filled center of the naturally occurring protein trimers, suggesting lipid-pro-tein interfacial binding. However, the natural organization of this membrane protein biases the interpretation, since the paucity of phospholipid molecules renders them all essentially interfacial.

Containment

The behavior of the epidermis when distended is also of importance. It is the stratum corneum's role to fend against tearing (2). This tissue is actually stronger per unit mass than the dermal fabric and, as a rule, is sufficiently elastic to adjust to stretching. Its pliability, however, is conditional, and it fissures and cracks if stretched when excessively dry. Arid atmospheres alone can produce this condition (windburn). Detergents and solvents, which extract essential, water-sequestering lipids from the stratum corneum, and diseases such as psoriasis associated with a malformed horny structure render the stratum corneum brittle and prone to Assuring.

Ray Crystallography

A major challenge of X-ray crystallography of trans-membrane proteins is to obtain suitable 3D crystals. Homogeneity and stability at high protein concentrations are important to obtain good results. Different strategies have been used for producing suitable crystals. These strategies include the use of detergents that replace the native membrane lipids and form mixed detergent-membrane protein micelles, crystallization using vapour diffusion, and crystallization using lipid cubic phases and bicelles 29 . The rationale behind the methods using cubic phases 41-43 or bicelles 44,45 is that the solubilized membrane protein is inserted into a native-like environment that is believed to improve the chances of crystallization.

Sample Preparation

Accurate results from amino acid analysis require purified protein and peptide samples. Buffer components (e.g., salts, urea, detergents) can interfere with the amino acid analysis and are removed from the sample before analysis. Methods that utilise post-column derivatisation of the amino acids are generally not affected by buffer components to the extent seen with pre-column derivatisation methods. It is desirable to limit the number of sample manipulations to reduce potential background contamination, to improve analyte recovery, and to reduce labour. Common techniques used to remove buffer components from protein samples include the following methods (1) injecting the protein sample onto a reversed-phase HPLC system, removing the protein with a volatile solvent containing a sufficient organic component, and drying the sample in a vacuum centrifuge (2) dialysis against a volatile buffer or water (3) centrifugal ultrafiltration for buffer replacement with a volatile buffer or water...

B Burst Amplitudes

Limitations The colored product is unstable, and the color-yield depends on protein concentration. Interference has been reported for Lowry assays conducted in the presence of calcium ion-containing salts, carbohydrates, detergents, disulfides (including oxidized DTT), EDTA, glycerol, magnesium ion-containing salts, phenols, potassium ion-containing salts, purines (especially guanine, uric acid, caffeine, and xanthine), as well as Tricine and Tris buffers. Limitations Interference can occur in presence of detergents, membrane-derived lipids, and other surfactants.

Discussion

The importance of GSH for neutralization of toxic products of lipid peroxidation is confirmed by the experiments with SDI, which effectively (by 90.8 ) inhibited increased flux through sorbitol dehydrogenase in the 3-week streptozotocin-diabetic rat model. GSH depletion in 3-week streptozo-tocin-diabetic rats was further exacerbated by the SDI treatment, which is consistent with a further accumulation of HNE over the level in the untreated diabetic group. A more advanced GSH depletion in the SDI-treated diabetic rats compared with the corresponding untreated group is consistent with the findings of Geisen et al. (32) for diabetic lens. These findings implicate sorbitol accumulation-linked osmotic stress in the mechanisms underlying increased vulnerability of diabetic peripheral nerve to oxidative injury and are in accordance with other reports demonstrating the inconsistency between a substantial ( 40 ) depletion of GSH versus a very minor (2) or an absent (12) depletion of GSSG,...

Enhancement

Since the mid 1970's many different structures and classes of lanthanide chelates have been synthesised (Figure 5). For high sensitivity assays the dissociation enhanced system is currently the best approach, however for screening assays where the sensitivity or detection limitation is not an issue the fluorescent chelates can be used to simplify the assay. Some of these chelates can be even used to develop homogeneous and non-separation assays. This assay format is essential for the measurement of a reaction where the components have only a weak binding affinity thus cannot withstand a wash or separation step. Three main assay principles have been developed and applied for homogeneous TRF based assays. The first of these utilizes an energy transfer between a Lanthanide donar (Europium or terbium) chelate and an acceptor molecule (rhodamine, cyaninin dyes). The energy transfer based assay has the advantage that it suits relatively large antigens or ligands. The disadvantage relates to...

A Distribution

In neoxanthin (Block et al., 1983b), but it is not yet known whether carotenoids are present as proteinpigment complexes, as in thylakoids (SiefermannHarms, 1985). When present in protein-pigment complexes, carotenoids are not usually covalently bound to proteins, thus the procedures used to separate envelope membrane proteins involving detergents are not mild enough to preserve the integrity of such complexes if they do occur in envelope membranes (see however, Markwell et al., 1992). Therefore, carotenoids are generally released as 'free' carotenoids during membrane solubilization. This strongly limits our understanding ofthe physiological significance of envelope carotenoids, although a role (as a singlet-oxygen quencher) in preventing (photo)oxidative damage cannot be excluded. The specific distribution of carotenoids among the different protein-pigment complexes in thylakoids has been described extensively by Siefermann-Harms (1985), Cogdell (1988) and Lichtenthaler (1993). The...

Transport Mechanisms

Caco-2 cells can also be used for studies of passive transport mechanisms that are not easily studied in more complex models, such as whole tissue models, or in vivo. For instance, the relative contribution of passive transcellular and paracellular transport 13 , the effect of charge on paracellular transport 37 and the effect of solvent drag 38 . Further, studies of pH-dependent permeability of basic drugs showed that these drugs are transported across the cell monolayers not only in their uncharged but also in their charged form, thus challenging the validity of the pH-partitioning theory for epithelial tissues 39 . Furthermore, Caco-2 cells have been used to investigate the effects of pharmaceutical excipients that may enhance passive drug transport either by enhancing the solubility of the compound e.g. 40-43 or by affecting the epithelial integrity 44-48 . More recently protocols to optimise drug transport have been suggested by inclusion of solubility-enhancing agents...

Plastocyanin

Plant, algal, and cyanobacterial types. Although their sequences differ considerably among these groups, the homologies are high within a group. Well-conserved His42, Cys92, His95, and Met100 (shown by asterisks) are implicated as ligands for coordinating a copper atom. In mechanically broken chloroplast preparations, plastocyanin is usually still bound to thylakoids membranes. High concentrations of salt, sonication, or mild detergents release plastocyanin,

Method 2

This method (commonly referred to as the Lowry assay) is based on the reduction by protein of the phosphomolybdotungstic mixed acid chromogen in the phosphomolybdotungstic reagent, which results in an absorbance maximum at 750 nm. The phosphomolybdotungstic reagent reacts primarily with tyrosine residues in the protein. Colour development reaches a maximum in 20 min to 30 min at room temperature, after which there is a gradual loss of colour. Because the method is sensitive to interfering substances, a procedure for precipitation of the protein from the test sample may be used. Most interfering substances cause a lower colour yield however, some detergents cause a slight increase in colour. A high salt concentration may cause a precipitate to form. Because different protein species may give different colour response intensities, the reference substance and test protein must be the same. Where separation of interfering

Method 3

This method (commonly referred to as the Bradford assay) is based on the absorption shift from 470 nm to 595 nm observed when the acid blue 90 dye binds to protein. The acid blue 90 dye binds most readily to arginine and lysine residues in the protein which can lead to variation in the response of the assay to different proteins. The protein used as reference substance must therefore be the same as the protein to be determined. There are relatively few interfering substances, but it is preferable to avoid detergents and ampholytes in the test sample. Highly alkaline samples may interfere with the acidic reagent.

General Remarks

In contrast to neuronal cells, a number of toxins, which provide interesting tools for the investigation of cell function, do not cross the cell membrane. Controlled cell permeabilization allows the intracellular application of these molecules in a system which retains the exocy-totic response to stimulating or inhibiting agents. This can be achieved by the use either of detergents or of pore-forming toxins (Holz et a ., 1992, Ahnert-Hilger et a ., 1993). The detergent digitonin solubilizes membranes according to their cholesterol content and is therefore in general suitable for the creation of pores, mainly in the plasma membrane. Although this approach has often been used successfully in chromaffin cells (Holz et a ., 1994, Holz et a ., 1992), we did not find it a reliable method for insulin-secreting cells (Wollheim and Ullrich, unpublished). An alternative approach has been described, i.e., intracellular application of toxins by electrophoresis (Boyd et a ., 1995). This method is...

Concluding Remarks

It should, however, be kept in mind that reconstitution itself is quite a striking example of self-organization of a biological structure. A cue as simple as the mixing of 2 different detergents is sufficient to initiate the correct folding of a medium-size membrane protein and the binding of up to 15 or so pigment molecules of several different kinds to their correct binding sites. The effort seems worthwhile to try and understand this self-organization process itself what is the sequence of events between the prereconstitution mixture of components and the fully formed stable complex, what are the structural features involved, and which is the driving force Once we know the answer to these questions, we may understand why Chl -protein complexes appear to reconstitute more easily than other pigment-protein complexes. We may then learn to design biomimetic structures that autonomously form in vitro.

Bio Beads Method

Adsorption of detergents to polystyrene beads (Bio-Beads SM2 Bio-Rad Labora tories) is a simple alternative to conventional dialysis for obtaining 2D crystals of integral membrane proteins (43). Hydrophobic adsorption of detergents onto polystyrene beads can be used for detergent removal independent of the respective CMC and has been used to generate highly ordered 2D crystals of membrane proteins (43). Bio-Beads can be added to very small sample volumes with almost no dilution of the protein or lipids. This property is of great interest for membrane proteins for which purifying large quantities is particularly difficult. However, it should be noted that handling of small quantities of Bio-Beads is difficult, because the beads must not dry in the procedure. According to Rigaud's studies (43), the specific adsorption of lipids is about 100 to 200 times lower than the specific adsorption of detergents (2-4 mg phospholipids g Bio-Beads SM2). Since crystallization reactions are generally...

Oligomerization

Such resistance can often be observed even in SDS when samples are not heated (e.g. alpha-toxin, Vibrio hemolysins, aerolysin). Oligomers formed by the cholesterol-binding toxins dissociate in SDS but persist in non-denaturing detergents (e.g., deoxycholate). E. coli hemolysin displays neither of these two characteristics. Here, data from functional studies appear to indicate formation of small oligomers (Benz et a ., 1989), but stringent proof for their existence has not yet been obtained.

Are Humans at Risk

The general presumption for wildlife is that exposure to environmental oestrogens is via food and water. Whether the same is true for humans is less certain. Food as well as containers for foodstuff and drinks may be important sources of some environmental oestrogens such as phytoestrogens, phthalate esters and bisphenol-A. There may be other more important routes of human exposure to such chemicals from substances such as the many lotions, cosmetics, detergents and shampoos that we now apply to our skins daily. Recent data suggest that that these products are likely to be oestrogenic, as they contain alkyl hydroxybenzoate preservatives. i3 Humans are exposed to a variety of endocrine disrupting chemicals throughout their lifetime establishing the level of exposure accurately presents a difficult task. Furthermore, there is the possibility of additive effects of chemicals in vivo, since this is clearly evident in vitro. i 6 Further information on oestrogenic chemicals and routes of...

Protein Content

A commonly used general protein content method is the Lowry assay. This is based on the biuret reaction of proteins with copper (II) in a basic solution and the Folin-Ciocalteu phosphomolybdic-phosphotungstic acid reduction to heteropolymolybdenum blue by the copper-catalyzed oxidation of the aromatic amino acids tyrosine, tryptophan, and phenylalanine in the protein. The reaction products are blue and are quantitated spectrophotometrically in the visible region between 540 and 560 nm. This reaction is linear at microgram protein levels. The assay, however, is prone to interferences from a number of substances such as alcohols, sugars, and detergents. In some cases, interfering substances or product may be removed prior to analysis, e.g., by precipitation. Also, the preparation of controls containing interfering substances that are in the drug product may correct for their presence. Although bovine serum albumin historically has been used to prepare the standard curve, different...

Protein components

Integral proteins are either deeply embedded in or pass right through the membrane. They can only be displaced from the membrane by disrupting its structure using solvents, disruptive enzymes and detergents. Integral proteins can be roughly divided into two types those where most of the protein is embedded in the bilayer and those where part of the protein is embedded in the lipid layer but the greater part extends into either the extracellular or intracellular fluid or both. The former are often involved in the transport of species across the membrane (see sections 7.3.4 and 7.3.5). The latter usually has ogliosaccharides attached to the section protruding into the extracellular fluid. These oligosaccharides have a variety of functions. For example, the oligosaccharides of the protein glycophorin act as receptors for the influenza virus and also constitute the ABO and MN blood groups.

Shinya Yoshikawa

Cytochrome-c oxidase was first isolated from bovine heart muscle as early as in 1941 by Yakushiji and Okunuki (8). They used a natural detergent, sodium cholate, for solubilizing the enzyme. This enzyme is a multisubunit protein with two extramembrane moieties on both ends of the transmembrane core moiety. The most critical step for isolation of a membrane protein is to extract the protein from the biological membrane by replacing the membrane phospholipid bilayer with detergents. This Crystallization conditions are also greatly influenced by the detergent species used for stabilizing a membrane protein in aqueous solution (10,11). In fact, dodecylmaltoside also stabilizes the absorption spectrum and enzymic activity as effectively as decylmaltoside. However, no crystal has yet been obtained from preparations stabilized with dodecylmaltoside. At present, many kinds of synthetic detergent are commercially available. However, it is desirable to search for the best detergents for...

Prostanoid synthases

As in other P-450 proteins, acidic conditions or prolonged treatment with detergents in the absence of glycerol caused the conversion to 'P-4201, an inactive form which shows the absorbance of the CO complex at 420 nm due to the disrupture of the heme-thiolate bond 20 . Such inactive enzyme preparations are also obtained after immu-noaffinity purification of the enzyme due to the stringent conditions during elution 20,21 . The enzyme bound to the antibody still displayed all the P-450 characteristics.

Buffers and Solvents

The concentrations of detergents, buffers, carrier proteins, reducing agents, and divalent cations can affect the specific signals and apparent potencies of compounds in concentration response curves (Schroter et al., 2000). In general, it is good to stay as close to physiological conditions as possible, The presence of promiscuous inhibitors or aggregating compounds in a chemical library has become a recent area of research. These compounds can cause false positives that can be reduced with certain detergents and can frequently be recognized by steep concentration response curves or enzyme concentration-dependent shifts in the IC50 (Feng and Shoichet, 2006 Shoichet, 2006). The exact mechanism of these false positives is not known but it is possible that these compounds induce the formation of compound-enzyme clusters that reduce enzyme activity. Thus, it is important to have some detergent present in enzyme assays and to re-test hits in orthogonal assays to reduce the possibility of...

Nitrofurantoin

Nitrofurantoin (furadantin, macrobid, others) is a synthetic nitrofuran that is used to prevent and treat urinary tract infections. Bacteria reduce nitrofurantoin to toxic products that apparently mediate cell damage. The antibacterial activity is higher in acidic urine.

Vitellogenesis

The fish liver possesses receptors which specifically respond to estrogen by synthesising the yolk protein vitellogenin. Male, female and juvenile fish all possess such receptors in the liver, but under normal conditions only the female produces sufficient estrogen to induce vitellogenin synthesis. This is usually a seasonal phenomenon and coincides with the need of the developing egg to incorporate yolk protein. Measurement of vitellogenin in plasma therefore provides both a useful indicator of the reproductive status of the female and a method of distinguishing the two sexes. Since vitellogenin is not normally detectable in significant amounts in male, juvenile or sexually regressed female fish, its presence is a clear indication of abnormal stimulation by an environmental estrogen. The extremely high levels of vitellogenin in the plasma of estrogen stimulated fish (up to 10 ), the ease with which it can be measured and the specificity of the response has made this one of the most...

Healthy Chemistry For Optimal Health

Healthy Chemistry For Optimal Health

Thousands Have Used Chemicals To Improve Their Medical Condition. This Book Is one Of The Most Valuable Resources In The World When It Comes To Chemicals. Not All Chemicals Are Harmful For Your Body – Find Out Those That Helps To Maintain Your Health.

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