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Alkaline proteases are used extensively in detergents, the food industry, and leather tanning. Enzymes produced commercially are derived only from microorganisms, and the microorganisms must be able to produce a high enzyme yield from low-cost substrates. The success of alkaline proteases in detergents is depend on whether the enzymes have the following properties 1) a wide pH activity range, 2) stability under high alkaline conditions, 3) high activity and stability in the presence of surfactants, 4) high stability in the presence of builders such as chelating reagents and bleaching agents, 5) high activity over a wide temperature range, 6) long shelf-life, and 7) low production cost. Although many enzymes have been reported, the alkaline proteases described above, however, have several weak points in their enzymatic properties, e.g., they are sensitive in the presence of oxidants and chelating agents. These disadvantages have been overcome by the isolation of new Bacillus strains by...
Isolation of membrane proteins, normally a rather difficult and time-consuming task, has been successfully achieved by the convenient two-phase extraction procedure. Use of nonionic detergents, e.g., Triton X-114, which separate at rather mild temperatures like 20 C into a detergent-rich and detergent-poor phase, provide novel means for obtaining enriched preparations of integral membrane proteins from complex biological systems (20,21). 4. Two-phase partitioning also holds promise for rapid isolation of DNA. By using novel phase systems made of PEG-salt containing chaotropic agents and detergents, it has been shown that nucleic acids partition with high yields to the salt-rich phase, whereas proteins and other cellular material are concentrated in the other phase or precipitates at the interface (22).
In the workplace, all three factors may contribute to dermatitis. For example, a student nurse or trainee hairdresser is exposed to water, detergents, and other factors that will exacerbate any pre-existing eczema. In addition, there may be specific allergies and, as a result of the broken skin, secondary infection can occur making the situation even worse. The following points are helpful in determining the role of occupational causes.
Kobayashi et al. (1995) isolated M-protease that was suitable for use in detergents from alkaliphilic Bacillus sp. KSM-K16. The M-protease was purified to homogeneity from the culture broth by column chromatographies. The N-terminal amino acid sequence was The molecular mass of the protease was 28 kDa, and its isoelectric point was close to pH 10.6. Maximum activity toward casein was observed at 55 C, as shown in Fig. 7.4. The activity was inhibited by phenylmethylsulfonyl fluoride and chymo-statin. The enzyme was very stable in long-term incubation with liquid detergents at 40 C. This protease exhibited good properties as a laundry detergent additive except for oxidant resistance. The gene responsible for the M protein was cloned and sequenced. The accession number is Q99405.
One of the most used kits is the DC protein estimation kits from Bio-Rad. The assay is sensitive and is also reliable in the presence of reducing agents and detergents. The assay is a modified Lowry method. This method is rapid and the reading can be done in 15 min with the color change not varying with time, making the reading stable and reliable. The reaction involves protein with the alkaline copper tartrate and the Folin reagent. The protein residues, especially, tyrosine and tryptophan, and to a lesser extent, cysteine cysteine and histidine, reduces the Folins reagent producing a blue color.
The demand for liquefying a-amylase for use in laundry and automatic dishwashing detergents has been growing for several years. Detergent enzymes must be able to function in washing machines or dishwashers under conditions that are very unfavorable for the stability of the enzyme. The pH is high alkaline in washing conditions. The high temperature (55-60 C) in a dishwasher requires thermostable enzymes. Enzymes are preferentially resistant to various detergent ingredients, such as sufactants, chelators and oxidants. However, most bacterial liquefying a-amylases, such as Bacillus amy loliquefaciens (BAA) and Geobacillus stearothermophilus (BSA), including Bacillus licheniformis (BLA), have optimal pH of between 5 and 7.5. Therefore, they are not practically suitable for use in laundry and dishwashing detergents with high alkalinity.
In designing immunoblotting experiments, it is important to remember that caspases are themselves protease substrates. Zapata et al. (16) have reported that lysis of lymphoid cells in neutral detergents such as Triton X-100 can lead to artefactual caspase activation by granzyme B present in cytotoxic granules. Other serine proteases can also activate certain caspases under cell-free conditions (17). It is, therefore, important that cells be solubilized under conditions that prevent procaspase cleavage and activation after cell lysis. We favour the lysis of cells under strongly denaturing conditions e.g., in 2 sodium dodecyl sulfate (SDS) containing 4 M urea and reducing agent (18) or in 6 M guanidine hydrochloride containing reducing agent (19) . If the latter buffer is used, sample preparation involves more steps (dialysis to remove the positively charged guanidium ions and replace them with dodecyl sulfate as described in ref. 19), but the chances of artefactual proteolysis are...
In screening alkaline cellulases for detergent additives, Ara et al. (1992) and Igarashi et al. (1992) isolated a novel alkaline pullulanase from alkaliphilic Bacillus sp. KSM-1876, which was identified as a relative of Bacillus circulans. The enzyme had an optimum pH for enzyme action of around 10.0-10.5, the highest pH for optimum pullulanase activity. This enzyme is a good candidate for use as detergent additives in dish washers, especially to remove starch from dishes in the presence of detergents. The gene (Accession Number AB049812) was isolated from KSM-1876. However, the plasmid-borne enzyme was not thermostable and not alkaline pullulanase, having an optimum pH of 7.5. The enzyme hydrolyzed pullulan 3.0-fold faster than soluble starch, but hydrolyzed amylose and amylopectin less efficiently. Therefore, the gene cloned was not the major alkaline pullulanase gene, but a minor one.
Very few enzymes are secreted by cells and it is often necessary to disrupt the cells in order to demonstrate enzyme activity. When the total enzyme content of cells is required, it is necessary to disrupt them completely and to remove all particulate material by centrifugation, leaving the dispersed enzyme protein in the supernatant fluid (Figure 8.19). Many enzymes are membrane bound and are only released from fragmented membranes if a suitable detergent is incorporated into the medium. The detergents used include Triton XI00 and the bile salts, e.g. sodium deoxycholate. Enzymes that fail to exhibit maximum activity without the releasing effects of detergents are said to show 'latency'. Many cellular studies, however, require the careful disruption of the cell and the subsequent separation of the various cellular organelles such as mitochondria, nuclei, etc., by a process known as cell fractionation.
The B. burgdorferi outer membrane contains a relatively low density of transmembrane-spanning proteins as determined by freeze-fracture electron microscopy studies (Walker et al., 1991 Radolf et al., 1994 Jones et al., 1995). This may explain why B. burgdorferi is more susceptible to disruption by routine physical manipulations such as centrifugation and resuspension and is more susceptible to detergents compared with Gram-negative bacteria. It is very likely that the unusual surface of this bacterium contributes to the unique properties that B. burgdorferi s.l. possesses, e.g. the ability to survive in both the mammalian host and the tick and to evade the mammalian immune system. Thus, a comprehensive analysis of the membrane architecture and constituents would yield insight into the unique physiological mechanisms by which the bacterium survives in diverse environments and may also elucidate pathogenic mechanisms operative during LB.
Tissue permeabilization is a step taken to increase penetration of immunoreagents, particularly antibodies, into tissue. For antigenic sites embedded within organelles, such as within vesicles, this step appears to be essential. For antigens that are soluble, such as those in the cytosol and for intracellular domains of membranous proteins, including the adrenergic receptors, permeabilization may be kept to a minimum. For EM-ICC, the permeabilization methods involving extraction of lipids from the plasma membrane, such as incubation in nonionic detergents (e.g., Triton X-100), interferes with ultrastructural analysis. Thus, detergent-treatment should be avoided. In cases where tissue penetration is required, methods compatible with EM include the following three
To understand the function of a biological membrane like that of chloroplast thylakoids, it is important to understand the arrangement of its different protein and lipid components. Preparations that have proven to be particularly suited for such studies are those consisting of membrane vesicles that are turned inside-out. Inside-out vesicles from the thylakoid membrane were first obtained from spinach chloroplasts by a combination of mechanical fragmentation and separation by aqueous two-phase partition (1,2). By the same or very similar procedures, inside-out thylakoid vesicles have now also been obtained from other plant sources such as pea (3), barley (4), mangrove (Avicennia marina) (5), lettuce (6), Euglena gracilis (7), cyanobacteria (8,9) and the photosynthetic bacteria Rhodopseudomonas viridis (10). Because the isolation procedure does not involve the use of detergents, the inside-out thyla-koids have a preserved membrane structure and are ideally suited for...
Characterization of serum autoantibodies to the major islet-cell antigens associated with type 1 diabetes has shown that these are of high affinity, but of low capacity, and frequently recognize epitopes that are highly dependent on the native conformation of the molecule. Thus, islet autoantibodies in diabetes tend not to bind antigen on Western blots or fixed tissue sections where antigen conformation is disrupted by ionic detergents or chemical cross-linking 49 . This is in contrast to the situation in stiff-person syndrome, where autoantibodies readily bind GAD on Western blots and epitope characterization has identified major linear epitopes on the molecule. The characteristics of autoantibodies in diabetes place limitations on the type of assays that can be used for autoantibody detection. Procedures where antigen is directly bound to a solid phase, as in a direct ELISA, invariably perform poorly 118 . This is probably the result of conformational changes in the protein on...
The exact processes depend on the nature of the material that is being analyzed. Bone material has the advantage over many other potential samples in that the external surface, which may well contain contaminating DNA, can be removed. After the outer layer has been removed, the bone can be further treated with agents that will destroy DNA on the surface common treatments include washing in strong detergents and sodium hydroxide solutions and treatment with intense ultraviolet (UV) light. When these steps are not possible, then care must be taken to use samples that have a low chance of being contaminated. The extraction method varies depending on the sample but is usually a variant of techniques commonly used when analyzing bone samples. The sample is ground to a fine powder and then dissolved in a 0.5 M EDTA solution the addition of proteinase K aids the process. Nonbone samples are often powered by grinding in the presence of liquid nitrogen and then incubated in solutions...
Requires permeabilization of the cells using non-ionic detergents. Procedures are described here specifically for FACS analysis of BcI-2 but are readily adapted to detection of other Bcl-2 family members by adjustment of the choice and amount of primary antibody employed. The immunodetection of Bcl-2 or other Bcl-2 family proteins can be combined with traditional cell-surface marker analysis, as well as with DNA content analysis or TUNEL assays for detecting cells with fragmented DNA, in two- or three-colour FACS assays.
In 1972, Yoshida and Horikoshi discovered a very- stable alkaline protease in the presence of detergents containing high concentration of perborate in the absence of calcium ion, (Japanese patent JP 740710). Bacillus sp. No. D-6 (FERM No. 1592, later designated Bacillus cohnii D-6, FERM P-1592) produced an alkaline protease, E-l, that was more stable in the presence of detergent additives at 60 C than Bacillus clausii 221 protease. Several properties are presented in Table 7.1. If the enzyme productivity could be increased, the E-l enzyme would be the best enzyme for detergent additives. After our discovery, many researchers tried to industrialize it, but without success. Almost thirty years later, Saeki and our colleagues (2000) dramatically increased the productivity to more than 10 grams liter using gene technology.
Extraction systems based on nonionic surfactants have been described as an alternative to the standard polymer polymer or polymer salt systems. Phase-forming surfactants are, for example, the nonionic polyoxyethylene-type detergents. This kind of aqueous two-phase system (ATPS) is simply induced by a switch in the temperature on the basis of the temperature-dependent reversible hydration dehydration of the polar ethylene oxide headgroups. A single isotropic micellar phase separates into two isotropic phases one of the resulting two aqueous phases, the so-called coazervate phase, is enriched in detergent, whereas the other is depleted (1). The detergent forms micelles in the detergent-depleted phase and is believed to exist in the form of lamellar stacks in the coazervate phase (2). Both phases have a high water content. The temperature at which the phase separation occurs is referred to as the cloud-point. The clouding temperature depends on the structure of the polyoxyethylated...
My second research line was a continuation of the light-scattering work I had started in Boston. Einar Hammarsten had realized the importance of light-scattering for studies of DNA and he helped me to get a grant for a light-scattering photometer. He also arranged a position for me as teaching assistant in medical physical chemistry, which helped our economical situation but also meant more teaching obligations to medical students. With the new light-scattering equipment, I made a more thorough study of the molecular properties of hyaluronans isolated from different tissues. At this time a new technique to isolate and fractionate polyanions had been published by John Scott in Manchester. He used detergents, especially cetylpyridinium chloride (CPC), to precipitate the polymers. Differently charged polymers precipitated at different ionic strengths. The technique suited me well for the isolation of hyaluronan free of sulphated polysaccharides from the tissues. My new results fitted my...
Successful UV-MALDI analysis with solid-state matrices requires an undisturbed crystallization of the sample on the target. Buffers, detergents, and other additives, such as glycerol, interfere with this crystallization even at low concentrations. Therefore, ammonium buffers should replace phosphate and similar buffers in the molecular biological procedures for sample generation. Detergents should be avoided, or non-ionic detergents should be used, if necessary. Proteins, such as polymerases, exo- or endonucleases or restriction enzymes, in the final sample can partially or fully suppress the oligonucleotide signals in the spectrum because of their higher proton affinity. Templates from polymerase chain reaction (PCR) or other enzymatic reactions may also cause problems. All these components need to be removed by precipitation or other suitable methods. Several companies market commercial purification kits. In so-called homogeneous assays (see below) a sample dilution may also...
The solution to this problem takes us back to the great alveolar cells and their surfactant. A surfactant is an agent that disrupts the hydrogen bonds of water and reduces surface tension soaps and detergents are everyday examples. Pulmonary surfactant spreads over the alveolar epithelium and up the alveolar ducts and smallest bronchioles. As these passages contract during expiration, the surfactant molecules are forced closer together as the local concentration of surfactant increases, it exerts a stronger effect. Therefore, as alveoli shrink during expiration, surface tension decreases to nearly zero. Thus, there is little tendency for the alveoli to collapse. The importance of this surfactant is especially apparent when it is lacking. Premature infants often have a deficiency of pulmonary surfactant and experience great difficulty breathing (see chapter 29). The resulting respiratory distress syndrome is often treated by administering artificial surfactant.
In the laboratory these were exciting times, the most exciting I can remember. We learned how to use benign detergents, which, unlike SDS, could solubilize membrane proteins without gross denaturation, in an environment simulating the native state, but an environment where they would also be readily accessible for molecular characterization 42 - an advance in which we were in part anticipated by two likeable young men from Finland, Kai Simons and Ari Helenius 43 .
Patients with mesenteric ischemia often manifest profound metabolic acidosis secondary to sepsis, peripheral hypoperfusion ( lactic acidosis), and toxic products from the bowel itself. This is best managed by aggressive administration of sodium bicarbonate, with frequent monitoring of arterial blood gases. An arterial line facilitates the frequent drawing of arterial blood samples and provides for optimal monitoring of blood pressure. Because of the massive losses
The kidney is opening up and it begins to make more urine, During this period the kidney eliminates water and not toxic products, In other words, the urine increases in quantity but not in quality (Fig. 33). Therefore, the patient is still uremic, but an incneaiie in urine output decreases any fluid overload which, usually, hail complicated the oliguric period in spite of efforts to maintain good fluid balance.
For any radioligand binding experiment, it is important to use fresh incubation solutions prepared in clean plastic or glass bottles. Any contamination with detergents or ions could compromise the experiment. It is also important that the slides are dried quickly and thoroughly after washing to avoid dissociation of the ligand from the binding sites. The binding conditions for the different a2-AR ligands are summarized in Table 1 (see Notes 1-6).
Bcl-2 is notoriously insoluble at neutral pH. However, at pH 3.5, recombinant soluble Bcl-2 protein can be concentrated up to 10-15 mg per ml without detergents. Thus, the procedures described here are for preparation of Bcl-2 in acidic solution. The solubility of Bcl-2 at neutral pH is limited to 50 pig per ml in PBS. High concentrations of glycerol (40 ) afford little benefit at neutral pH for increasing solubility but will slow down protein aggregation. In the presence of high concentrations of non-ionic detergents (4 v v), the concentration of Bcl-2 can reach to 1-2 mg per ml. However, the presence of detergent is not desirable for many experiments.
The condition is prevalent in people in contact with water, soap, detergents and other chemicals (Figure 5.3) it is particularly associated with housework. There is also a high incidence among chefs, bartenders, confectioners and fishmongers. The index and middle finger of the left hand are most often affected, these being the digits most subject to minor trauma such as rubbing during hand-washing
The OR presents a number of environmental hazards to both surgical personnel and patients. Chemical hazards exist from the use of trace anesthetic gases, flammable anesthetic agents, various detergents and antimicrobial solutions, medications, and latex products.18 Other ever-present physical hazards include electrical shock and burns, exposure to radiation from x-ray equipment, and injuries caused by lasers.19 In addition to causing injury directly, the use of lasers can expose OR personnel to papillomavirus in smoke plumes.20 Hazards that are less often considered include noise pollution21 and light hazards from high-intensity illumination.22 The most effective way of minimizing the particular hazards in a given OR is to have an active in-hospital surveillance program run by a multidisciplinary team that includes surgeons.
In 1982, I came to a conclusion that direct identification of the components of paired helical filaments (PHF) was extremely difficult because of their unusual insolubility in various detergents and denaturants (Selkoe et al. 1982b). Thus, I took an indirect immunochemical approach to identification of PHF components. Although we raised excellent polyclonal antibodies to PHF that revealed for the first time the presence of extensive neuropil threads in addition to neurofibrillary tangles (NFT) in the cortex affected by Alzheimer's disease (AD Ihara et al. 1983,1988), we saw no distinct bands. Instead, there was a smear on the blot of AD cortical homogenates (Ihara et al. 1983). I thought that monoclonal antibodies raised against purified PHF might serve to unambiguously identify these components. Thus, I started the PHF monoclonal antibody project as early as 1983, when I returned to University Hospital, as an assistant professor, from Dr. Dennis Selkoe's lab. Every day I was...
The method is suitable for all proteins although the amount of dye bound does vary from one protein to another in a manner that seems to relate to the proportion of basic amino acid residues in the protein. Bovine serum albumin, for instance, gives absorbance values that are 60 greater than the same concentration of egg albumin. Consequently it is important that the standard protein solutions used should be of the same composition as the test protein. The control of pH is important and although the reagent is heavily buffered, any samples that are very alkaline may alter the pH and so affect the result. Some detergents when present show significant interference resulting in increased absorbance values.
Various pollutants and heavy metals have been shown to cause changes in the morphology of the gill in fishes. It is neither within the scope of this paper, nor is it an objective here, to describe in detail the responses of the respiratory and cardiovascular systems to all studied toxicants. However, we describe here some of those effects, emphasizing those responses which are common to a range of different toxicants. Qualitative descriptions of changes in the fish gill in response to toxicant exposure are numerous. In general, the tissue reaction to being exposed to many environmental toxicants resembles an inflammatory response (Fig. 10.10). Fish exposed to heavy metals, detergents, and nitro-phenols show a separation between the epithelial cells and the underlying pillar cell system, which can lead to a collapse of the structural integrity of the secondary lamellae (Skidmore and Tovell, 1972 Fig. 10.10). In response to zinc exposure, for example, Skidmore and Tovell (1972), in...
The nail may be damaged by repeated trauma or by chemical agents such as detergents, alkalis, various solvents, sugar solutions and especially by hot water. The nail plate takes a minimum of 5-6 months to regenerate and therefore it is vulnerable to daily insults. Housework is commonly the cause particularly at risk are the first three fingers of the dominant hand. Anything that slows the rate of nail growth will increase the risk. Cosmetic causes are rare. Some varnishes will damage the superficial layers of the nail. Drying may be enhanced by some nail varnish removers. Soaking fingers in warm soapy solution, for removing the cuticle, is especially dangerous this is common practice among manicurists. It has been shown that climatic and seasonal factors may affect the hydration of the nail plate.
Because the hair shaft contains very low levels of DNA it is prone to contamination but unlike many other types of biological evidence with low levels of DNA it is possible to clean the hair shaft prior to DNA extraction. Several methods have been used to clean hair including washing in mild detergents, water and ethanol and also using a mild lysis step in the same way as is used in the differential extraction of semen 21 .
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