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Nature Of Oxidative Damage

Secondary oxidative damage results from reaction of proteins with products of oxidation of small molecules, including lipids, carbohydrates, and amino acids. The intermediates in this process are reactive carbonyl and dicar-bonyl compounds, such as malondialdehyde, a, -unsaturated and hydroxy-aldehydes, glyoxal, and methylglyoxal (MGO), which react with nucleophilic groups on protein to form lipoxidation (9) and glycoxidation (I0,l l) products (Table 1). Lipoxidation products require oxidation (peroxidation) for their formation from lipids, whereas glycoxidation products are a subclass of advanced glycation end products (AGEs), requiring autoxidation chemistry (oxidation by molecular oxygen) for their formation from reducing sugars or ascorbate. Some AGEs (e.g., pyrraline and imidazolones formed by reaction of 3-deoxy-glucosone 3DG with lysine and arginine residues in protein) do not require oxidation for their formation from reducing sugars. These AGEs are useful indicators of...

Cell Lines and Viruses

OAdV623 formulated in Tris sucrose PEG buffer containing 10 M cationic lipid CS087 (not commercially available) is known as FP253. CS087 is a Tris-conju-gated cationic lipid (trilysine-capryloyl-tris-trilaurate ditrifluoroacetate T-shape empirical formula C66H130N8O10-2C2F3O2 molecular weight 1421.81) (12). Lipid formulation may enhance virus infectivity in cell types that lack the viral receptor (13). FP253 is stored at -80 C in a minimum volume of 200 L in 2-mL borosilicate vials with snap-sealed rubber stoppers.

Homologous Recombination with Circular Ad Vector Genome Plasmids

A kanamycin resistance marker and a conditional suicide gene (the sacB gene of Bacillus subtilis 82 ) is engineered by standard cloning techniques containing the specified modification flanked by appropriate homology sequences to the Ad genome. The colEl-derived shuttle plasmid is transformed into the polA host carrying the incP Ad5 plasmid. Cointegrates are selected by growing the polA host in the presence of both antibiotics. Resolution, leading to loss of the colEl replicon from the recombinant Ad plasmid, of this cointegrate is subsequently selected by growth of the cells in sucrose activating the B. subtilis sacB gene as suicide gene, leading to the concomitant loss of the sacB conditional suicide marker. Consecutive rounds of this two step recombination procedure allow the introduction of multiple independent modifications within the virus genome, with no requirement for an intermediate virus. The potential of this procedure was demonstrated by the recovery of various E1 E30E4...

Cultivation Media of Choice

Stool cultures for Vibrio spp. are plated on the selective medium thiosulfate citrate bile salts sucrose (TCBS) agar. Although some Vibrio spp. grow very poorly on this medium, those that grow well produce either yellow or green colonies, depending on whether they are able to ferment sucrose (and produce yellow colo' nies). Alkaline peptone water (pH 8.4) may be used as an enrichment broth for obtaining growth of vibrios from stool. After inoculation, the broth is incubated for 5 to 8 hours at 35 C and then sub-cultured to TCBS.

Approach to identification

The colonies of these genera resemble those of the family Enterobaderiaceae but notably differ by their positive oxidase test (except V. metschnikovii, which is oxidase-negative). The oxidase test must be performed from 5 sheep blood or another medium without a fermentable sugar (e.g., lactose in MacConkey agar or sucrose in TCBS). The reason for this is that fomentation of a carbohydrate results in acidification of the medium and a false-negative oxidase test may result if the surrounding pH is below 5.1. Likewise, if the violet pigment of a suspected C. violaceum isolate interferes with performance of the oxidase test, the organism should be grown under anaerobic conditions (where it cannot produce pigment) and retested. Fermentation of Sucrose

Preparation of a Microsomal Fraction

Homogenization medium 0.3 M sucrose, 50 mM 3-(-N-morpholino) propanesulfonic acid (MOPS)-KOH, 5 mM Na-EDTA, pH 7.5, 0.2 (w v) casein (enzymatic hydrolysate Sigma Chemical Co., St. Louis, MO, USA C 0626) (see Note 1). 6. Resuspension medium 0.3 M sucrose, 5 mM potassium phosphate, pH 7.8 (see Note 4), 0.1 mM Na-EDTA, 1 mM DTT (see Note 2).

Isolation of Plasma Membranes

Phase mixture 11.70 g of Dextran solution (see item 1), 5.85 g of PEG solution (see item 2), 2.77 g of sucrose (see item 3), 0.675 mL of potassium phosphate, pH 7.8 (see item 4), 0.090 mL of KCl (see item 5), and water to a final weight of 27.00 g in a 50 mL centrifuge tube (see Notes 6 and 7). 8. Bulk-phase system 97.5 g of Dextran solution (see item 1), 48.75 g of PEG solution (see item 2), 30.8 g of sucrose (see item 3), 7.50 mL of potassium phosphate, pH 7.8 (see item 4), 0.75 mL of KCl (see item 5), and water to a final weight of 300 g (see Notes 7 and 8).

Symptoms And Signs

After an incubation period of 2-3 days the illness usually begins with nausea and vomiting, followed by diarrhoea within 24 hours. Vomiting lasts for a short period, and is absent in some cases. In severe cases the stools are frequent, watery, voluminous and foul-smelling. Fever is commonly present and may be high-grade. In children febrile convulsions may occur. Dehydration is most often isotonic, but may be hypotonic, particularly when the patient has been given fluid deficient in electrolytes (e.g. juice). Visible blood in the stools is almost never observed. The white cell count is normal with relative lymphocytosis and transient neutropenia. An important factor in the pathogenesis of a rotavirus infection is lowered disaccharidase function in the gut. Disaccharides (lactose, sucrose) remain unabsorbed in the gut lumen and cause dehydration by osmotic pressure. Many patients with rotavirus gastroenteritis also have symptoms of upper respiratory tract infection, but it is not clear...

Measurement of Apoptosis

Fixation solution 4 paraformaldehyde and 4 sucrose (in PBS, pH 7.4) is prepared immediately before use as described (26). Cells are fixed in fresh 4 paraformaldehyde, 4 sucrose for 20 min and permeabilized with Triton X-100. TUNEL is performed using the in-situ Cell Death Detection Kit I as described by the manufacturer. The percentage of apoptosis is determined by the ratio of the number of DTR-TUNEL-double-positive cells over the total number of DTR-positive cells (4). Here, we briefly describe the TUNEL staining protocol for the in-situ Cell Death Detection Kit I.

S cerevisiae Membrane Vesicles

Two-phase buffer (4 x concentrated) 1.32 M sucrose, 20 mM potassium phosphate buffer, pH 7.8, 4 mM EDTA. 2. Two-phase buffer 0.33 M sucrose, 5 mM potassium phosphate buffer, pH 7.8, 1 mM EDTA. 6. Dilution buffer 0.33 M sucrose, 15 mM MES-Tris, pH 6.5, 1 mM DTT and 1 mM PMSF.

C reinhardtii Membrane Vesicles

Breaking medium 0.25 M sucrose, 50 mM MOPS-KOH, pH 7.8, 3 mM EDTA, 0.5 mM EGTA, 5 mM MgCl2, 1 (w v) BSA, 5 mM mercaptoethanol, and 0.5 mM PMSF. 1. Two-phase buffer (4 x concentrated) 1.0 M sucrose, 20 mM potassium phosphate buffer, pH 7.8. 2. Two-phase buffer 0.25 M sucrose, 5 mM potassium phosphate buffer, pH 7.8. 6. Diluting buffer 0.33 M sucrose, 50 mM MOPS-KOH (pH 7.0), 4 mM MgSO4 and 1 mM PMSF.

Ultrastructural Evaluation Of Apoptosis

Ultrastructural examination of tissues is, in many minds, the gold standard for confirming that a suspected tissue change is indeed apoptosis. With minor changes, the criteria outlined by Kerr et al. (50) still stand as the essential features of apoptosis. While the methodology for standard ultrastructural examination is fairly routine, care should be taken in selecting a fixative (e.g., if one wants to perform immunohistochemistry one must determine if the antigen is glutaraldehyde sensitive), fixative buffer (e.g., dextran instead of sucrose to minimize shrinkage or swelling), method of fixation (immersion vs perfusion), and embedding medium (epoxides for morphology, acrylates for immunohistochemistry).

Hepatitisassociated antigen See Australia antigen

Hepatitis A virus (HAV) Type species of the genus Hepatovirus. Virion diameter 27-29nm, density in CsC1 1.32-1.34 g ml, sedimenting at 160S in sucrose. The genome RNA is positive-sense, 7.48kb in length, with a VPg protein covalently attached at the 5' end, and poly A at the 3' end. There is a single open reading frame encoding a polyprotein of 2235 amino acids. The virus replicates in various primate cell cultures after adaptation, usually without cyto-pathic effects. Causes enterically transmitted 'short incubation' hepatitis (less than 6 weeks) in humans. The virus is present in the feces during the prodromal phase of the disease but usually disappears about the time jaundice appears. Chronic carriers of the virus are not seen and the virus does not cause progressive liver disease. Epidemics of hepatitis A are usually due to water- or food-borne infection. Antibodies may be demonstrated by CFT, immune adherence, hemagglutination and radioimmunoassay. They appear soon after the...

Influence Of Other Chemicals And Physical Environment

No antimicrobial is completely effective against all microorganisms present in a given foodstuff. In theory, one should be able to combine various antimicrobials having different modes of action to compensate for this deficiency. It should then be possible to achieve (1) a broader spectrum of action or (2) an increased antimicrobial action by using this combination (Lueck, 1980). For example, combinations of sorbic acid and benzoic acid inhibit several bacterial strains better than either antimicrobial alone (Rushing and Senn, 1963). Synergistic effects have also been reported using combinations of benzoate and sulfur dioxide, carbon dioxide, sodium chloride, boric acid, or sucrose (Rehm, 1960 Chichester and Tanner, 1972). Additive, synergistic, and antagonistic effects reported in several early studies have been extensively reviewed by Lueck (1980) for benzoic acid as well as for other preservatives. The influence of combinations of chemical antimicrobials on the growth of...

Sweetened Beverage Intake

There has been a clear trend in recent years toward higher per capita consumption of sugar-sweetened beverages, including fruit drinks, carbonated beverages, and sports drinks. Across all age groups, sweetened beverage intake increased by 135 , with a concomitant 38 decrease in milk consumption (28-30). The largest increases in sweetened beverage intake were noted in young adults (ages 19-39) and children (ages 2-18) (27,28). As a result, added sugar now comprises 20 of the total caloric intake in a child's diet (22). Nearly 75 of all children drink at least one 8-oz serving of a sugar-sweetened beverage daily, and the average child consumes 1.4 servings daily. This corresponds to an average annual sugar intake of 14.05 kg yr (22). In addition, one-third of teen males consume more than three soft drinks daily, providing an additional 30 teaspoons of added sugar, equivalent to 120 calories (22,31). Children generally maintain a consistent level of energy intake (36,37) although there...

Rapid Purification of Liver Plasma Membranes

A rat liver is perfused with 0.25 M sucrose in 5 mM Tris-HCl, pH 8.0, to remove as much blood as possible. Squash the liver in a garlic press and transfer maxi 6. Dilute the final bottom phase tenfold with 0.1 M N-acetylglucosamine in 0.25 M sucrose and 5 mM Tris-HCl, pH 8.0, or some other suitable buffer (see Note 7). Mix and pellet membranes by centrifugation at 100,000g for 60 min. Suspend the pellet in a suitable buffer.

Biological Roles for Surface Carbohydrates and Potential Uses

Because polysaccharides represent the predominant structures on the bacterial cell surface, they are important in the interaction between the pathogen, host, and environment. To summarize the reports described in Subheadings 2.-4., C. jejuni carbohydrates are involved in many cellular functions, including assembly of the flagellar filament (73), which we now realize is a type III-like secretory apparatus necessary for the release of invasion colonization proteins (14,15), motility (73), assembly of the TFSS that affects DNA uptake and natural transformation (79), adherence and invasion in vitro (41,54,76), colonization, and disease in vivo (41,64,68,76-78), molecular mimicry of gangliosides (82), autoimmunity leading to GBS (48), maintenance of cell surface charge (41), serum resistance (41,53), antigenic (53,60,83) and phase variation (41,54,57,83,84). Considering the importance of glycoconjugates in C. jejuni biology and based on the hypothesis that glycoconjugates have evolved in...

Starter Culture Propagation A Growth Media

Ingredients commonly used to formulate starter culture media have been described by Whitehead et al. (1993) and are presented in Table 5. Lactose is always used as the major carbohydrate, although low concentrations of maltose, sucrose, or glucose are sometimes added to stimulate growth (Sandine, 1996). Yeast extract is a source of nitrogen as well as a supplier of vitamins, minerals, and other growth stimulants. Casein hydrolysates are added to provide readily available amino acids. Also, addition of whey protein concentrate to whey or UF (ultra filtered) whey permeate broth stimulates growth of lactic acid bacteria (Bury et al., 1998). Heat-stable a-nucleotide, nonprotein nitrogen, or some peptidases could be responsible for this stimulatory effect (Bury et al., 1998). Corn steep liquor, although a good source of vitamins, is not often used, because its supply is limited (Sandine, 1996). Sandine (1996) questioned the need for added antioxi-dants in media formulations, because...

Isolation of structural proteins of the virus

Virus particles also can be separated from cellular components of different density. This is accomplished by generating an equilibrium density gradient of sucrose or other material in an ultracentrifugal field. Virus particles will band or float at a specific location within the gradient corresponding to their equilibrium buoyant density (1.18 g cm3 in the example shown in Fig. 11.1). This position represents a balance of forces on the particle the buoyant force trying to cause the particle to float and the centrifugal force working to cause the particle to sediment lower in the gradient. Fig. 11.1 Equilibrium density gradient centrifugation of virus-infected cell components to isolate virus particles. A preformed sucrose density gradient is layered with a solution of infected cell material and subjected to centrifugation at high g force at 4 C for several days. Virus particles sediment downward until they reach a layer with a density equivalent to their own. At this density, the...

An infant with aplastic anaemia

Booth, unpublished data). A remarkable diversity was observed below the molecular level. PFGE and single-gene RFLP patterns suggested that there were just two major clones, one of which was also isolated from the patient's blood. The minor clone, which was represented by 3 20 faecal isolates, exhibited a completely different PFGE pattern, a unique ompC gene sequence, had acquired sucrose metabolism and a very high MIC for amoxicillin. The pattern of plasmids in this strain was also quite distinct from the major clone, although by transformation experiments it was demonstrated that they also had at least one plasmid in common (J. Park, F. MacKenzie, I. M. Gould, and I. R. Booth, unpublished data). However, the major faecal clone, which was identical to the blood isolate, could be subdivided into two metabolic types with respect to the rate of metabolism of melibiose. In addition, the isolates exhibited distinct MIC values for amoxi-cillin and...

Commercial Starter Culture Preparations

Frozen concentrated cultures contain 1010-1011 cfu g, a sufficient concentration to allow 70 mL to inoculate 1000 L of medium for bulk culture preparation (Sandine, 1996). Preparation of frozen concentrated cultures involves (1) growing cultures under optimal conditions using pH control, (2) harvesting the cells via centrifugation or ultrafiltration, (3) standardizing the cell suspension to a specific activity, (4) adding a cryoprotectant, (5) packaging, and (6) rapid freezing using liquid nitrogen. The pH of the cell concentrate should be 6.6 for lactococci and 5.4-5.8 for lactobacilli (Stadhouders et al., 1971). There are many cryoprotective agents that can be used, including glycerol, monosodium glutamate, sucrose, and lactose (Mayra-Makinen and Bigret, 1993). Rapid freezing can also be accomplished using a dry ice-alcohol mixture (Sandine, 1996). The frozen concentrate should be stored at -196 C (liquid nitrogen) for best retention of activity, although storage at -40 C (dry ice)...

Hypothesis Of Advanced Glycation End Products And Its Receptor

Advanced glycation end products (AGEs) are a heterogeneous group of irreversible adducts resulting from nonenzymatic glycation and oxidation of proteins, lipids, and nucleic acids. Glucose and other reducing sugars react in a nonenzymatic reaction (Maillard reaction) with the N-terminal residues and or e-amino groups of proteins initially forming a Schiff base. Rearrangement of this aldimine leads after a short time to the formation of more stable but still reversible Amadori adducts. The open chain of the resulting ketoamin can react with other amino groups. Oxidation, dehydration, and condensation reactions finally lead to the production of irreversible crosslinks, which are proteinase resistant.

Food Additive Intolerance Reactions

Allergic reactions have been reported to occur to the preservative sulfites (and SO2), sodium benzoate, butylated hydroxyzole (BHA), butylated hydroxytylene (BHT), the sugar substitute aspartame, artificial colors (especially yellow, red, and blue), and the flavor enhancer monosodium glutamate (MSG). The symptoms of principal concern are urticaria and asthma. In most cases, even if it is proven that the food additive is involved in the clinical symptoms, the exact mechanism of the reaction is unknown.

Management options

As long as the above potential problems are considered, anaesthetic management in general is routine. In anaemia, the threshold for transfusion should be lower than normal. New formulations of parenteral iron (iron-sucrose) have been used post-partum to restore haemoglobin concentration more rapidly than oral iron, and are associated with few adverse reactions, unlike iron-dextran and sodium ferric

Signal Transduction via Nuclear Receptors

A second group of coactivators forms chromatin-remodeling complexes that use ATP to make DNA available for transcription. One example of a chromatin-remodeling complex is the switching mating types sucrose nonfermenting (SWI SNF) family of proteins. The actions exerted by this complex are unclear, but they may involve histone sliding or simply a loosening of the association between DNA and histones.

Diet Pharmacological and Behavioral Treatment

The Atkins and Protein Power diets are very high in total and saturated fat compared with current dietary guidelines. Long-term use of these diets for weight maintenance is likely to significantly increase serum cholesterol and risk for coronary heart disease. The Sugar Busters and Zone diets would lower serum cholesterol concentrations and likely reduce risk for coronary heart disease. High-carbohydrate, high-fiber, and low-fat diets would have the greatest effect in decreasing serum cholesterol concentrations and,

Detecting the synthesis of viral genomes

First, many viruses have genomes that can be separated readily from the bulk of cellular DNA using rate zonal centrifugation. For example, the circular genomes of papovaviruses are easily separated from larger cellular material on sucrose density gradients. Detection of RNA virus genomes can be accomplished by virtue of the fact that the viral genome will have a discrete size that is different from any cellular RNA of high abundance, such as ribosomal RNA. Purification of infected cell RNA and size fractionation on a gel or sucrose gradient can then be used to detect the viral genome. If necessary, its identity can be confirmed by specific hybridization.

Characterization of viral mRNA expressed during infection

Fig. 12.8 Separation of HSV DNA from cellular DNA based on differences in base composition. The percentage of G+C residues in a given fragment of dsDNA will determine its buoyant density in CsCl. In the experiment shown, three DNA samples were mixed with a CsCl solution. One sample has a very high G+C content and serves as a density marker. HSV DNA has a lower density, but is significantly higher in G+C content than cellular DNA (approximately 67 versus 48 ). For this reason it has a greater buoyant density in an equilibrium gradient of CsCl. Unlike the equilibrium sucrose gradient shown in Fig. 11.1, CsCl solutions are so dense that the gradient will form under the centrifugal force available in an ultracentrifuge. Therefore, the mixture of DNA and CsCl is made and placed in a centrifuge rotor, and the mixture is allowed to form a density gradient by high-speed centrifugation. Following equilibrium, the various DNA fragments can be isolated by careful dropwise collection of the...

Clinical Box 42 Marijuana Antagonist Blocks Munchies

Recently, researchers have described cannabinoid (CB) receptors, which are the GPCRs that recognize tetrahydrocannabinol found in marijuana. The CB1 receptor is found in areas of the brain that control appetite and addiction to smoking (tobacco), and this receptor may be responsible for the munchies experienced by marijuana smokers. The munchie connection led researchers to develop CBi-receptor antagonists in hopes that they would be useful as aids for weight loss. One antagonist (rimonabant from Sanofi-Aventis) may soon be available as a diet pill and as an antismoking aid.

General Protocol for RAPD Technique to Show Polymorphism

Reagents (all the chemicals, primers and enzymes were obtained from Operon Technology) DNA isolation buffer (Moller et al. 1992), 2 hexadecy-ltrimethyl ammonium bromide (CTAB), NaCl (1.4 M), EDTA (20 mM), Tris HCl (100 mM), chloroform isoamylalcohol (20 1), isopropanol, sodium acetate, ethanol (70 ), Tris EDTA (TE, pH 8.0), Tris-HCl (pH 8.0, 10 mM), EDTA (pH 8.0, 1 mM), DNA amplification mixture for PCR, RNase A, loading buffer, bromophenol blue (0.25 ), sucrose in water (40 , w v store in small aliquots at 4 C), primers (short oligonucleotide), ethidium bromide (caution ethidium bromide is a powerful mutagen wear gloves and masks when handling and weighing). Note all buffers, pipette tips and Eppendorfs should be sterilized at 121 C for 15 min. Sterilize by autoclaving at 15 psi (ca. 103 kPa) for 15 min.

Bacillus sp No RK9 Polygalacturonate Lyase

Kelly and Fogarty (1978) reported that Bacillus sp. No. RK9 isolated from garden soil grew well in alkaline media at pH 9.7 and produced endo-poly-galacturonate lyase. The medium composition suitable for enzyme production was as follows (in grams per liter) sucrose, 10.0 bacteriological peptone, 3.0 yeast extract, 3.0 Na2C03.H20, 10.0 CaCl2-2H20, 3.0 MnS04-4H20, 0.04 MgCl2-6H20, 0.2 K2HP04, 1.0 pH 9.7. Neither pectin nor pectic acid was required for the production of the enzyme, indicating that the enzyme is constitutive. The endo-polvgalacturonate lyase of RK9 was purified 163-fold from culture fluid by precipitation with 50-90 saturated

Membrane Preparation For Receptor Binding Studies

Porcine brains, which were obtained within 30 min after death of the animals from a local slaughterhouse, were used to prepare receptor-rich membranes. The brains were immediately frozen in liquid N2 50 g brain per 200 mL ice-cold buffer (0.32 M sucrose, 10 mM potassium phosphate buffer, pH 7.0, 1 mM EDTA) were homogenized twice for 15 sec in a blender and then for 1 min with an ultraturrax. The homogenate was centrifuged three times for 15 min at 1.400 g and 4 C to separate cellular debris. The supernatant was spun down at 100.000 g for 60 min. The resulting pellet was resuspended in buffer (as above but without sucrose). Aliquots were stored frozen at -80 C. Protein content was determined by the Lowry method, using bovine serum albumin as a standard.32-35

Immunohistochemical Detection of Apoptosis Associated Molecules

Regardless of which apoptosis-associated molecule one wishes to localize, the keys to successful immunohistochemical detection are appropriate tissue fixation, specific antibodies, and sensitive signal amplification. Unfortunately, there is no single fixative or fixation protocol that will work for all antigens and antibodies. If an immunolabeling protocol for the antibody of interest has been published or recommended by the manufacturer, it is usually best to follow the established protocol. When testing new antibodies, we typically begin with paraffin-embedded sections and compare three different tissue fixatives, 4 paraformaldehyde (in 0.01 M phosphate-buffered saline, PBS, pH 7.2), Bouin's solution, and methacarn (6 parts methanol 3 parts chloroform 1 part glacial acetic acid). Paraformaldehyde and Bouin's fixed tissue will also be examined without and with heat-induced epitope retrieval (0.01 M citrate buffer, pH 6.0, for 20 min in a rice steamer or pressure cooker, followed by a...

Nutrient Utilization Genes

That sucrose, fructose, and glucose were the best carbon sources for aflatoxin production. Starch, mannitol, sorbitol, and galactose yielded intermediate amounts of aflatoxin. Davis and Diener25 concluded from their study of A. parasiticus that the majority of carbon compounds that are normally oxidized through both the hexose monophosphate shunt and the glycolytic pathway supported growth and aflatoxin production. Despite the fact that carbon source is one of the most important determinants of aflatoxin biosynthesis, relatively few genes associated with carbon utilization and regulation have been isolated and characterized. Extracellular a-amylase and glucoamylase have been purified from A. flavus,26 but their association with starch degradation and aflatoxin production has not been examined. Yu et al.27 isolated four genes that constitute a gene cluster related to sugar utilization in Aspergillus parasiticus. The nadA, hxtA, glcA, and sugR genes show homology to genes encoding a...

Identification of PrfAControlled Target Genes

Ponents of an ABC transport system predicted to import disaccharides, oligosaccharides, and polyols. The second transcription unit, lmo0278, is monocistronic and codes for the ATPase component of a sugar transporter. It is possible that the products of the two group II transcription units act together to mediate sugar import. lmo0278 has a potential PrfA box located only about four nucleotides upstream of its -10 region. The proximity of these binding sites to the -10 might inhibit transcription by preventing binding of RNAP. In contrast to the situation with lmo0278, lmo0178 lacks an upstream PrfA box near predicted -10 or -35 regions of the promoter, and the mechanism by which PrfA downregulates expression of this gene is unclear. It is also not obvious why it would be advantageous for L. monocytogenes to impair expression of this transport system through PrfA. One possible explanation might be that the group II genes encode a transporter of sugars that are readily available in the...

Inhalation Dosage Form And Delivery System Considerations

Inhalation products, whether intended for intra-pulmonary or systemic delivery, have one fundamental attribute at the basis of dosage form design the aerodynamic particle size distribution. Drugs can be converted to pulmonary formulations by simple size reduction processes such as jet-milling, often followed by admixture with bulking agents such as lactose. More sophisticated approaches involve spray-drying of drug solution or suspension in compositions comprising one or more excipients, such as simple sugars, amino acids, phospholipids, buffering agents and surfactants. While delivering excipients to the lung can require extensive toxicology assessment, there are now a wide range of excipients used in approved inhalation products. The inhalation product manufacturing systems employ technologies and unit operations customized to handling these fine particles, and the pharmaceutical industry is now familiar with a broad range of inhalation product platforms for pulmonary drug delivery.

Issues For The Future

Drying and vitrification have previously been combined for matrix preservation of cardiovascular and skin tissues but not for live cell preservation in tissues or engineered products. However, nature has developed a wide variety of organisms and animals that tolerate dehydration stress by a spectrum of physiological and genetic adaptation mechanisms. Among these adaptive processes, the accumulation of large amounts of disaccharides, especially trehalose and sucrose, are especially noteworthy in almost all anhydrobiotic organisms including plant seeds, bacteria, insects, yeast, brine shrimp, fungi and their spores, cysts of certain crustaceans, and some soil-dwelling ani-mals.216-218 The protective effects of trehalose and sucrose may be classified under two general mechanisms (1) the water replacement hypothesis or stabilization of biological membranes and proteins by direct interaction of sugars with polar residues through hydrogen bonding, and (2) stable glass formation...

Carbohydrate Synthesis 551 C3 plants

Figure 5.18 The Calvin cycle for converting carbon dioxide into carbohydrate. Three molecules of carbon dioxide are fixed to three molecules of ribulose 1,5-bisphosphate (RuBP) in a reaction catalyzed by the enzyme ribulose bisphosphate carboxylase (rubisco). The three 6-carbon intermediates rapidly split into six molecules of 3-phosphoglycerate (PGA) that are reduced to six molecules of glyceraldehyde 3-phosphate (GAP). One GAP is used for synthesis of sucrose or starch. The numbers in the squares indicate the number of molecules of each intermediate. For details, see Sec. 5.5.1 Figure 5.18 The Calvin cycle for converting carbon dioxide into carbohydrate. Three molecules of carbon dioxide are fixed to three molecules of ribulose 1,5-bisphosphate (RuBP) in a reaction catalyzed by the enzyme ribulose bisphosphate carboxylase (rubisco). The three 6-carbon intermediates rapidly split into six molecules of 3-phosphoglycerate (PGA) that are reduced to six molecules of glyceraldehyde...

The Pancreas Butts In

Similarly, the proteases continue the chemical breakdown of proteins into amino acids, which are also absorbed into the bloodstream. And the amy-lases in the small intestine generally finish the chemical breakdown of carbohydrates into simple sugars such as glucose, which are then absorbed. NUTRIENTS Simple sugars + Amino acids + ABSORBED (such as glucose) (from proteins)

Carbohydrates Store Energy Form Cell Walls and Make Rigid Structures

Carbohydrates include sugars, starch, and cellulose. They make up the woody parts of plants and the hard exoskeletons of insects. They are also important components of the cell walls of bacteria and fungi. Simple sugars contain several carbon atoms (glucose has six), hydrogen atoms, and oxygen atoms. These sugars can be joined to form large polymers, such as starch and cellulose. Nucleic acids also contain sugars. For example, each nucleotide (subunit) of DNA and RNA contains a five-carbon sugar (deoxyribose and ribose, respectively). As with other complex polymers, cell walls are built by enzymes that control the process. Some antibiotics, such as penicillin, interfere with the placement of sugars in bacterial cell walls. As a result, bacteria treated with penicillin stop making cell-wall material, leaving holes in the wall. That often causes bacteria to lyse (break apart). Sugars are also important for cellular energy. For example, plants convert the energy of sunlight into chemical...

Development Of Preservation Solutions

The development of preservation solutions for clinical use has resulted from a combination of experiments in tissue biochemistry and experimental transplantation. The work of Keller et al. demonstrated that ion diffusion and edema in renal tissues could be usefully investigated during cold preservation,156 and these techniques were used by many later authors,157 158 resulting in several different preservation solutions being reported over the last 30 years. In particular, prolonged preservation became possible after the development of solutions of intracellular electrolyte composition. These solutions minimized cellular edema and the loss of intracellular potassium. Collins ushered in a new era of simple hypothermic storage by showing that a solution of crystalloid solutes alone, more resembling intracellular than extracellular composition, and quite free of colloid (see Table 9.1), could extend renal preservation reliably for 48 hours and longer.86 Subsequently, solutions of widely...

Studies made on collodion membrane

He also found that dilution of the ether alcohol Collodion with ether gives a thinner and less permeable membrane, whereas thickness and permeability increases with addition of amyl alcohol. Bauer6 found that the addition or 1 ml of glacial acetic acid to 200 ml of diluted collodion solution would reduce average pore size to 200m . The addition of2 ml reduce the pore size to l00 m , and the addition of 3 ml reduces pore size further to 15 m . Field made collodion membranes which held back potassium chloride and sucrose. Kistler9 found that collodion membrane can be made as dense as desired by filtering through them solution of cellulose or of collodion.

Markers of Oxidative Stress

The glycation of proteins by reducing sugars (Maillard reaction) is a key posttranslational event and can result in time-dependent adduct formation that becomes especially susceptible to oxida-tive elaboration. In addition, oxidative stress can result in oxidized sugar derivatives that can subsequently modify proteins through a process known as glycoxidation. Similarly, and of more general importance, oxidative stress results in lipid peroxidation and the production of a range of aldehydes capable of modifying numer We and others (67-69) have determined that AGE modifications are present on both NFT and senile plaques in AD. These studies employed antibodies not only to the specific structures pentosi-dine and pyrraline, but also less defined AGE antibodies raised to carrier proteins treated with reducing sugars for long periods of time. AGE modification is likely an early in-vivo event, since both diffuse senile plaques (67) and paired helical filaments of T (68,69), considered two...

Cryopreservation of Biopsied Embryos

Cryopreservation of biopsied embryos is becoming a necessity with patients who have a greater number of normal embryos than those transferred. Experiments in mice have shown promising results in achieving successful cryopreservation of biopsied embryos (51, 53, 93). Cryopreservation of biopsied human embryos would not be as simple as methods applied for unbiopsied embryos due to the presence of a hole in the zona (which acts as a protective barrier to regulate the inflow of toxic cryoprotectant). To date, few groups have attempted freezing biopsied embryos. One of the first reports by Joris et al. (74) tried freezing biopsied day 3 embryos using a slow freeze thaw procedure with DMSO. Survival in the biopsied group was significantly lower than in intact controls. Nevertheless, in biopsied embryos that did survive the freeze thaw procedure, blasto-cyst formation was seen in a small percentage. Similar results were confirmed by Magli et al. (95). This suggests that although survival...

Extraction and Quantification of Extra Radical Mycelium of AM Fungi in Soils

Of ERM extraction techniques is based on vacuum filtration of a soil suspension through a membrane filter. This membrane filtration technique (MFT) was introduced by Hanssen et al. (1974). The technique was modified recently by several authors and is now widely used for the assessment of ERM lengths of AM fungi (Boddington et al. 1999). Vilarino et al. (1993) developed another extraction technique using a rotating wire frame to retrieve the ERM fragments from an agitated soil suspension. A third set of extraction techniques is based on sucrose flotation and centrifugation (Schubert et al. 1987). All these techniques are suitable for quantification of the lengths of ERM with respect to the spatial distribution of the hyphae in soil (Dodd 1994). Their disadvantage, however, is that they severely disturb the ERM network during sample processing. Thus, the identification of structures such as branched absorbing structures (BAS Bago et al. 1998a, b) or the measurement of morphological...

Processing of the Env Precursor

SU is heavily glycosylated, and the presence of at least some of these sugars is important for virus infectivity. Perhaps the most important function of this heavy glycosylation is to hide the peptides on the surface of Env from neutralizing antibodies that would otherwise have access to the virion surface.

Preservation Of Individual Organs For Clinical Transplantation

UW-derived solutions (HES-free UW) have given successful static storage for 5 days in dogs.198 The gold standard for cadaver kidney preservation thus remains UW. Comparable results are achievable by a variety of other solutions when preservation times do not exceed 24 hours. Thus, for shorter anticipated preservation periods and for living-donor transplantation, UW, sucrose, Collins' solution, hypertonic citrate, and Bretschneider's solution can give equivalent results.172 Perfusion with normal saline would result in transmembrane influx of sodium, loss of potassium, and rapid destruction of cellular integrity. A number of fluids have been designed that retain cellular viability for many hours or days. Cellular edema is reduced by use of impermeant solute such as mannitol, sucrose, or raffinose. Intracellular pH is preserved with buffers such as phosphate, citrate, or lactobionate, and high concentrations of potassium prevent influx of sodium and water. Commonly used solutions are...

StearoylcoA Desaturases

Criture Cursive Flech

Human HEPG2 assay, but lacked oral bioavailability 28 . On the other hand, the quinazolinone CVT-11,563 (8) had excellent bioavailability (F 90 , rat) but only a modest potency for SCD inhibition (IC50 268 nM, rat microsomal assay IC50 68 nM, human HEPG2 cells) 29 . CVT-12,012 (9) is an analog of 7 that incorporates the hydroxyacetyl group of 8. It had good potency for SCD inhibition and good oral bioavailability (IC50 119 nM, human HEPG2 cells, F 78 ). Compound 9 also showed good in vivo efficacy for reduction of the desaturation index and hepatic TGs in high-carbohydrate-fed rats ( 50 reduction, 20 mg kg, po bid, 5 d) 30 . A pyrrolotriazinone analog of 8, CVT-12,805 (10), showed improved potency (SCD IC50 10 nM, rat microsomal assay) and good efficacy for the reduction in desaturation index when dosed orally to high-sucrose-fed mice 31 .

The Properties Of Lipid Rafts

In terms of their role in immune cell signaling, the central feature of lipid rafts is that they provide a mechanism for the lateral segregation of proteins within the plasma membrane (3). This ability to segregate proteins provides a means of concentrating and compartmentalizing certain components of signaling pathway and excluding others. At present, the rules that govern the inclusion and exclusion of proteins from lipid rafts have not been fully delineated, however, analyses of the composition of rafts allows some generalizations to be made. For their characterization, lipid rafts have been separated from the glycerolphospholipid membranes in cells based on their differential solubility in nonionic detergents (3,7). Thus, for example, lipid rafts are isolated from B lymphocytes by sucrose density gradient sedimentation of cell lysates prepared in 1 Triton x 100 at 4 C, conditions under which lipid rafts are insoluble. There are a number of potential pitfalls associated with the...

Inflammation and Immune Response in Human Longevity

Another theory, the network theory,'' suggests that a variety of defense functions or antistress responses, globally acting as antiaging mechanisms, indirectly control the aging process 87 . The stressors include different physical (heat, ultraviolet), chemical (oxygen-free radicals and reducing sugars), and biological (viruses, bacteria) agents. This theory argues that a global reduction in the capability to cope with a variety of stressors and a concomitant progressive increase in the proinflammatory status are major characteristics of the aging process and are provoked by a continuous antigenic load and stress 88 . In fact, assuming that the major characteristic of human immunosenescence is the filling of the immunological space by memory and effector T cell clones as a consequence of the chronic antigenic stress, another major consequence of chronic exposure to antigens is the progressive activation of macrophages and related cells in most organs and tissue 88 . This phenomenon,...

Fungi Are Eukaryotes Having Cell Walls But Not Chloropasts

Yeasts, molds, and mushrooms are fungi. Unlike bacteria, fungal cells store their DNA in true nuclei. Such subcellular structures, called organelles, localize particular cellular functions. For example, mitochondria are power plants that convert chemical energy from sugars into molecules such as ATP. Lysosomes are the cellular equivalent of garbage disposal units. They are filled with enzymes that destroy other macromolecules. Bacterial cells lack such localization of cellular activity.

Disorders of the Kidney

Diabetes and hypertension (high blood pressure) are the two leading causes of kidney disease. In diabetes, blood flow through the kidneys increases, causing the kidneys to enlarge, and the excess sugar in the blood damages the glomeruli (tiny blood vessels that are part of the nephrons). High blood pressure can cause kidney disease by damaging the small blood vessels needed for filtering and reabsorption of fluids. Conversely, hypertension can result from kidney disease if blood flow through the kidneys is obstructed or slowed, resulting in the release of hormones that cause blood pressure to rise. See page 365 for more information about diabetes and page 217 for more information about hypertension.

Laboratory Diagnosis

Urethral specimens should not be collected until 2 hours after the patient has voided. A urogenital swab (or one provided or specified by the manufacturer) is gently inserted into the urethra (females, 1 to 2 cm males, 2 to 4 cm), rotated at least once for 5 seconds and then withdrawn. Again, swabs should be placed into the appropriate transport medium or onto a slide prepared for DFA testing. Because chlamydiae are relatively labile, viability can be maintained by keeping specimens cold and minimizing transport time to the laboratory. For successful culture, specimens should be submitted in a chlamydial transport medium such as 2SP (0.2 M sucrose-phosphate transport medium with antibiotics) a number of commerdal transport media are commercially available. Specimens should be refri

5HT2C Receptor Modulators Progress in Development of New CNS Medicines

The important role of 5-HT in neuropsychiatric maladies has been validated by translation of 5-HT pharmacology to therapeutic benefit in treatment of an array of diseases such as anxiety disorders, depression, schizophrenia, migraine, chemotherapy-induced emesis, and appetite control (9,10). In fact, among the best-selling CNS medicines worldwide are drugs (e.g., selective serotonin reuptake inhibitor (SSRIs), and atypical antipsychotics) that broadly modulate central 5-HT functions (9). tissue lining the cerebral ventricles involved in the production of cerebral spinal fluid (19). 5-HT2c receptor protein and mRNA is also localized to a number of cortical and sub-cortical brain structures including the cortex, hippocampus, nucleus accumbens caudate nucleus, substantia nigra, cerebellum, amygdala, and discrete nuclei of the thalamus and hypothalamus (20-22). This localization reflects the various neural functions with which it is linked, such as cognition, emotional processes,...

Effect of pH on the Dissociation of Benzoic Acid

The effects of pH, NaCl, sucrose, and sorbic and benzoic acids on the growth of 30 strains of food spoilage yeasts have been reported (Praphailong and Fleet, 1997). Zygosaccharomyces bailii and Yarrowia lipolytica were the most resistant to the two preservatives at pH 5.0. Sorbate was more inhibitory than benzoate on the strains that were tested. The combined effects of pH and benzoate on the growth of these strains are presented in Table 2.4. As expected, increasing the pH of the culture medium decreased the effectiveness of this preservative.

Metabolism And Inactivation

In conscious and cooperative patients hypoglycaemia should be treated by the oral administration of a readily absorbable form of carbohydrate, such as sugar lumps or a glucose-based drink. Eating sugar or a sugar-sweetened product will often correct the condition and prevent more serious symptoms. If the reaction becomes more severe, breathing will be shallow and the skin will be pale. In severe hypoglycaemic reactions, intravenous dextrose may be necessary. If hypoglycaemic coma occurs, up to 50 ml of a 50 solution of glucose should be given intravenously and occasionally this may need to be repeated. If after about 1 h, blood glucose concentrations are normal and the patient has failed to regain consciousness, the possibility of cerebral oedema should be considered. In situations where the intravenous administration of glucose is impractical or not feasible, glucagon 0.5 to 1 mg by subcutaneous or intramuscular injection may be given intravenous injection may also be employed. If...


Percent concentrations are easy to prepare, but that unit of measurement is inadequate for many purposes. The physiological effect of a chemical depends on how many molecules of it are present in a given volume, not the weight of the chemical. Five percent glucose, for example, contains almost twice as many glucose molecules as the same volume of 5 sucrose (fig. 2.11a). Each solution contains 50 g of sugar per liter, but glucose has a molecular weight (MW) of 180 and sucrose has a MW of 342. Since each molecule of glucose is lighter, 50 g of glucose contains more molecules than 50 g of sucrose. To produce solutions with a known number of molecules per volume, we must factor in the molecular weight. If we know the MW and weigh out that many grams of the substance, we have a quantity known as its gram molecular weight, or 1 mole. One mole of glucose is 180 g and 1 mole of sucrose is 342 g. Each quantity contains the same number of molecules of the respective sugar a number known as...

Dental Caries

The first step in the origination of caries is the formation of a dental plaque (2). An increase in the amount of plaque is responsible for the ultimate development of gingivitis. A variety of factors interact in the generation of dental plaque and subsequent emergence of caries. These include the presence of a susceptible tooth surface, the proper microflora, and a suitable nutritional substrate for that flora. Several oral acid producing aerobic and anaerobic bacteria, including Streptococcus mutans, Lactobacillus acidophilus, and Actinomyces viscosus, are capable of initiating the carious lesion. However, S. mutans is consistently the only organism recovered from decaying dental fissures and is isolated in greater quantities from carious teeth than in non-carious ones (9). The overwhelming majority of microorganisms isolated from carious dentin are obligate anaerobes (10). The predominant organisms are Propionibac-terium, Eubacteria, Arachnia, Lactobacillus, Bifidobacteria, and...

Human Bodythe Sugars

Individual monosaccharides (such as glucose and fructose) are sweet, simple sugars that are often broken down by the body and used directly for energy. These can be clearly contrasted with the polysaccharides (pahl-e-SACK-uh-rides), which contain ''many'' (poly-) ''sugars'' or smaller saccharides. Within the human body, the main polysaccharide is glycogen. Glycogen is literally a ''producer'' (-gen) of ''sweetness or glucose'' (glyc). Glycogen is stored in large quantities inside our muscle and liver cells. The glycogen molecule consists of a large number of glucose molecules, bonded together in long chains (Figure 4.6). Under conditions of exercise, fasting, or dieting, special enzymes (EN-zighms) - protein ''fermenters or transformers'' - tend to break the stored glycogen back down into individual glucose molecules. These free glucose molecules can then enter cells, and be used to ease any current energy shortage.

Expected Results

Alkaline slant no change in the butt (K NC) glucose, lactose, and sucrose nonutilizer this may also be fermentation only. Add slant add butt (A A) glucose, sucrose, and or lactose fermenter (Figure 13-40, A) Note A black preapitate in the butt indicates production of ferrous sulfide and H2S gas (H2S+) (Figure 13-40, B). Bubbles or cracks in the tube indicate the production of C02 or H2. Drawing a drde around the A for the add butt, that is, A , usually indicates this means the organism ferments glucose and sucrose, glucose and lactose, or glucose, sucrose, and lactose, with the production of gas.

Reverse Genetics

One example of a targeted screen involved testing malvolio (mvl) as a contributing factor affecting foraging behaviour in honey bee workers (Ben-Shahar et al., 2004). In this study, mvl was chosen as a candidate because, as a manganese transporter, it is known to influence responsiveness to sucrose in Drosophila, and variation in sucrose response among individuals is known to influence foraging-related task-specialization in honey bees (see Section 4.3.2). Ben-Shahar and colleagues showed that the levels of mvl mRNA in the brain cells of workers are strongly associated with differences in worker foraging activity pollen foragers tend to have higher levels of mvl transcript than nectar foragers and foragers of either type have higher levels than do non-foraging nurse bees. It appears that some feeding-related genes in Drosophila are also related to feeding in Apis, and in the case of honey bees may be related to age-based task specialization.


Sucrose, dextrose, and fructose are available in both crystalline and syrup forms. Few microorganisms are contained in crystalline sweeteners. Maltodextrins, polydextrose, and corn syrup solids are available in granular form. Some of these materials may carry viable microorganisms, usually yeasts. Bottler's standards for 10-g samples of granulated sugars are less than 200 mesophils, 10 yeasts, and 10 molds (National Soft Drink Association, 1975). In addition to sucrose, dextrose, and fructose, corn sweeteners are available as syrups. Because syrups contain water and provide energy, they may support growth of osmophilic fungi. These microorganisms, usually being highly aerobic, grow on surfaces. They can be killed by exposure to ultraviolet light and their growth can be inhibited by sealing full containers in which they are packed. This is not practicable when the container is a tank into which air must be admitted to displace syrup as it is drawn out during use. For them to flow...

Phase Partition

Phase buffer 50 mM sodium phosphate buffer, pH 7.4 25 mM NaCl 100 mM sucrose. 5. Pure upper and lower phases The pure phases required for the washing steps are obtained as follows. A 250-g two-phase system is prepared in a separatory funnel by mixing 71.3 g of 20 Dextran T500 stock solution, 35.6 g of 40 PEG 3350 stock solution, 50 mL of the phase buffer, and water up to 250 g. The final concentrations of the phase components are 5.7 (w w) dextran, 5.7 (w w) PEG, 5 mM NaCl, 20 mM sucrose, and 10 mM sodium phosphate, pH 7.4.

Other Diseases

Inappropriately regulated MMP expression has been proposed as a mediator of tissue breakdown in the chronic phase of several other disease states. In models of cerebral hemorrhage, increased MMP expression is observed approx 24 h after the initiating injury, and may be responsible for degradation of the blood-brain barrier (118). The MMP inhibitors, Batimastat 3 and BB-1101 4 were shown to reduce the damage to the blood-brain barrier, as monitored by 14C-sucrose uptake (38,118). It has been proposed that such inhibitors may be useful in the treatment of the clinical deterioration that occurs 1-2 d after hemorrhagic brain injury.

Virion Structure

The size of the virions in a given preparation is not highly homogeneous but rather varies over a fairly wide range, suggesting that a discrete, highly ordered structure may not exist. After processing of the Gag precursor during virion maturation, the CA protein collapses to form a more ordered paracrystalline core, and in the case of HIV-1 the conical core is probably a well-de&ned structure (2). But even after maturation the overall diameter of the virion is heterogeneous and suggestive of considerable disorder. The virions exhibit a buoyant density in sucrose in the range of 1.16E1.18 g ml. The sedimentation rate of the particles is typically about 600 S. The virions are sensitive to heat, detergent, and formaldehyde.

Toxin Receptors

The precise nature of the membrane receptors for clostridial glucosylating toxins is not known. In many receptor-binding studies toxin A was used. From these studies and from the proposed properties of CROPs, it was suggested that toxin A binds like a lectin to Gala-1-3Gal 1-4GlcN structures (Krivan et al. 1986 Tucker and Wilkins 1991). A 160-kDa galactose- and N-acetylglucosamine-containing glycoprotein was purified from brush border cells of small intestine of infant hamsters, which was suggested to be a toxin receptor (Rolfe and Song 1993). Furthermore, binding of toxin A is inhibited by lectins specific for Gal and GlcNAc, by immunoglobulin and nonimmunoglobulin components of human milk (Rolfe and Song 1995). It was reported that toxin A binds to the membranous sucrose-isomaltase glycoprotein on rabbit cells (Pothoulakis et al. 1996). This receptor is absent in many toxin A-sensitive cell lines. A human glycosphingolipid was reported to bind to toxin A (Teneberg et al. 1996). It...


Pectin is a major constituent of plant cell walls, and a number of enzymes collectively termed pectinases are the first cell-wall-degrading hydrolases produced by fungal pathogens during the infection process. Isolates of Aspergillus flavus have been found to produce three distinct pectinases designated P1, P3, and P2c,5 as well as the pectin methylesterase.6 P1 and P3 are produced by both low- and high-virulence A. flavus strains isolated from the field, while P2c is produced by only highly virulent field strains.7 The ability of isolates of A. flavus to damage and spread between cotton boll locules was shown to be at least partially related to variations in the production of P2c.8 Strains lacking P2c did not cause as much damage to intercarpillary walls, thus limiting their spread throughout the cotton boll. Interestingly, P2c was the only pectinase produced by A. flavus that was not subject to catabolite repression in culture by simple sugars commonly found in developing cotton...

Pol Gene Expression

In the Golgi the sugar residues are modi&ed by the sequential removal of mannose residues and addition of N-acetyl glucosamine and other sugars to many of the oligosaccharide. O-linked glycosylation and sulfation of Env glycoproteins have also been documented (258,259). The pathway by which Env is transported to the cell surface is not fully understood, but presumably host vesicular transport systems are utilized. There is evidence that clathrin adaptor complexes interact with the cyto-plasmic tail of Env, and direct its movement to the plasma membrane (260). The protein typically becomes a prominent cell surface protein on the infected cell.

In vivo data

In the meantime, other in vitro systems that are not reliant on cerebral cells are being employed to give an early, if approximate, indication of the brain penetrating properties of discovery compounds. MDCK (Madin Darby Canine Kidney) cell monolayers have been proposed as a potential model for the BBB based on their high TEER (Transendothelial Electrical Resistance) value and low permeability to sucrose. However, significant concerns remain over their non-cerebral origin, inter alia, and these are likely to inhibit the use of this cell-line for estimation of BBB permeation 13,19 . Notable among the alternatives is PAMPA-BBB 21 , a modification of the original PAMPA (Parallel Artificial Membrane Permeability Assay) screen developed for predicting intestinal permeability 22 . Other workers have experimented with surface-activity profiling of compounds and shown that the measurements correlate well with passive permeation of the BBB 23 . In summary, the development of a reliable and...

Spore Extraction

AM fungal spores are extracted from soil by wet sieving and decanting, as described by Gerdemann and Nicolson (1963), and by sucrose centrifugation, as described by Smith and Skipper (1979). The soil sample (100 g) is suspended in 1 l water by gentle stirring. A dispersant such as sodium hexametaphosphate can be used with clay soils. Heavier particles are then allowed to settle for a few seconds and the liquid is decanted through a 450 m sieve to remove large particles of organic matter and allow the spores pass through. Next, the suspension is passed through a 100 m sieve and then through a 63 m sieve. The spores and small amount of debris that remain on the 63 m sieve are poured into a centrifuge tube containing water and centrifuged at 1800 rpm. The upper solution is poured off, 40 sucrose is added to the debris at the bottom and the mixture is then centrifuged for 2 min at 1800 rpm. The upper solution is separated for examination under the stereoscopic microscope. The spores which...

And Chromobacterium

This isolate could be either Aeromonas or Vibrio. The string test (see Figure 28-3), growth on TCBS, 0129 disk susceptibility, or salt tolerance can be used to separate these genera. The organism produced yellow colonies on TCBS because it was positive for sucrose. It was accurately identified by a commercial system as Vibrio alginolyticus.

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