Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:APRD00216 (ABC)
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A sensitive chemiluminescence high-performance liquid chromatographic method has been developed for the determination of hyaluronic acid, chondroitin sulphate and dermatan sulphate as their unsaturated disaccharide-dansylhydrazine derivatives involving an effective sample clean-up system. The dansylhydrazones of the unsaturated disaccharides derived from the hyaluronic acid, chondroitin sulphate and dermatan sulphate by chondroitinase ABC and/or chondroitinase ACII, were separated by reversed-phase chromatography using a mixture of 0.1 M sodium acetate buffer (pH 6.0) and 80% acetonitrile on a column (250 mm x 4.0 mm I.D.) packed with amide-80 silica beads (5 microns diameter). For post-column elution in the chemiluminescence system, 1 mM bis[2-(3,6,9-trioxadecanyloxycarbonyl)-4-nitrophenyl]oxalate and 3mM hydrogen peroxide in acetonitrile were used. The detection limit of each glycosaminoglycan was 100 fmol. The method was applicable to the determination of the levels of hyaluronic acid, chondroitin sulphate and dermatan sulphate in rat peritoneal mast cells.
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PMID:Chemiluminescence high-performance liquid chromatography for the determination of hyaluronic acid, chondroitin sulphate and dermatan sulphate. 142 67

Peptides were derived from the large chondroitin sulfate proteoglycan from chick cartilage by clostripain digestion. Using differential chondroitinase ABC and keratanase treatment and direct carbohydrate analysis, three major peptides of 86, 75, and 27 kDa were shown to bear only chondroitin sulfate chains. Another major peptide of 65 kDa was shown to contain both chondroitin sulfate and keratan sulfate chains, allowing it to be separated from the peptides derived from the chondroitin sulfate domain by DEAE-cellulose chromatography. An additional new peptide (100 kDa) containing keratan sulfate chains was found only in clostripain digests of proteoglycan-hyaluronate-link protein aggregates. Unlike any of the other peptides derived from clostripain digestion of proteoglycan monomer or aggregate, this peptide had the properties of a functional hyaluronate binding region. All of these peptides were purified to apparent homogeneity by preparative electroelution from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and deglycosylated with anhydrous hydrogen fluoride. Automated Edman degradation of the two largest chondroitin sulfate peptides revealed that they had unique N termini and several unrecognized residues, which were all subsequently revealed to be modified serine residues following deglycosylation. The keratan sulfate-bearing peptide also had a unique N terminus, which contained a single unrecognized residue, even after HF deglycosylation. Finally, the N terminus of the hyaluronate binding region was blocked. These studies allow estimates of core peptide masses in the absence of carbohydrate as well as provide primary amino acid sequence for O-xylosylated serine residues in the multiply substituted proteoglycans.
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PMID:Chick cartilage chondroitin sulfate proteoglycan core protein. I. Generation and characterization of peptides and specificity for glycosaminoglycan attachment. 236 11

Epimerization of D-glucuronosyl residues to L-iduronosyl ones during biosynthesis of dermatan sulfate involves an abstraction of the C-5 hydrogen of the target sugar residue. After inversion, a hydrogen from the medium is reinserted into the uronosyl residue. In the present study, microsomal enzyme prepared from cultured embryonic skin fibroblasts was incubated with dermatan or chondroitin in the presence of 3H2O of high specific activity. Incubation resulted in incorporation of tritium on C-5 of uronosyl residues of the substrates. The rate of the reaction was highest for dermatan. Incubation of the products with chondroitinase ABC released essentially all the tritium. Dermatan sulfate and chondroitin sulfate were inactive as substrates, which indicates that epimerization takes place before sulfation. Analyses of the product obtained after incubation of chondroitin in 3H2O-containing medium for different incubation times showed that tritium accumulated first in L-iduronosyl residues. Later, tritium was also found in D-glucuronosyl residues. The reverse situation was observed when dermatan was used as substrate. After extended incubation times, the ratio of D-[3H]glucuronic acid to L-[3H]iduronic acid in both dermatan and chondroitin reached a value of 85/15, which may reflect the equilibrium value. Digestion of labeled chondroitin with chondroitinase AC and oxidation of labeled dermatan with periodate showed that after 96 h of incubation with the epimerase and 3H2O, most of the uronic acid residues had been involved in the reaction. Both products were composed of long blocks of D-glucuronic acid-containing disaccharides interrupted by a few L-iduronic acid-containing disaccharides arranged singly of in clusters of two to three. Reincubation of the 3H-labeled products originating from dermatan or chondroitin with the epimerase resulted in release of tritium, which was linear with time and with increasing protein concentration.
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PMID:Biosynthesis of dermatan sulfate. II. Substrate specificity of the C-5 uronosyl epimerase. 670 27

A structure-activity relationship of low molecular weight dermatan sulfate was undertaken to understand better this new non-heparin, glycosaminoglycan-based antithrombotic agent. A dermatan sulfate prepared from bovine intestinal mucosa [average molecular weight (MWavg) 25,000], and currently in clinical trials as an antithrombotic agent, was used in this study. Dermatan sulfate was partially depolymerized using hydrogen peroxide and copper(II) as catalyst to MWavg 5600 to obtain a low molecular weight dermatan sulfate. This low molecular weight dermatan sulfate was then fractionated by gel permeation chromatography to obtain four subfractions having MWavg 7800, 5500, 4200 and 1950. The dermatan sulfate, low molecular weight dermatan sulfate and its subfractions showed substantially different optical rotations. The 1H-NMR spectroscopic analysis of dermatan sulfate samples showed some differences including increased content of GalpNAc4S6S residues and improved resolution in ring resonances for low molecular weight dermatan sulfate fractions, primarily the result of reduced molecular weight and lowered heterogeneity. Saccharide compositional analysis relied on chondroitin ABC lyase treatment followed by capillary electrophoresis. Polyacrylamide gel-based oligosaccharide mapping was also performed by treating dermatan sulfate samples with chondroitin B, AC and ABC lysases. These analyses showed increased amounts of sulfation as the MWavg decreased. In vitro bioassay showed maximum anti-Xa activity in the 4.2 kDa fraction and maximum heparin cofactor II-mediated anti-IIa activity in the 5.5 kDa fraction. The in vivo antithrombotic activity of these fractions was measured using a modified Wessler stasis thrombosis model. The 4.2 kDa fraction showed greater antithrombotic activity than the other low molecular weight dermatan sulfate fractions, dermatan sulfate, and low molecular weight dermatan sulfate. This enhanced activity may result from several structural features of the 4.2 kDa fraction including: a high content of 4,6- and 2,4-disulfated disaccharide sequences; the requirement of specific chain length; a change in the ratio of iduronic to glucuronic acid; and the presence of chondroitin ABC lyase resistant material.
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PMID:Low molecular weight dermatan sulfate as an antithrombotic agent. Structure-activity relationship studies. 816 53

Rambach agar, xylose-lysine-deoxycholate agar (XLD) with different concentrations of Tergitol 4 or 7 ethyl-2 methyl-4 undecanol hydrogen sulphate, sodium salt (XLDT4), Salmonella-Shigella agar (SS) and bismuth sulfite agar according to Wilson-Blair (BS) were evaluated using Salmonella spp. serovars and other bacterial species from the intestinal flora of poultry. Growth of the most common Salmonella serovars isolated from chickens in our country were evaluated using a viable counting technique on the different selective media and these results were compared with those obtained on Columbia base (ABC) agar plus 7% bovine blood (Table 1). Samples from Salmonella experimentally inoculated chickens were also examined. Results showed that Rambach, SS and XLD or XLDT4 were all satisfactory for isolation of Salmonella. Bismuth Sulfite agar was too inhibitory for bacteria important in veterinary practice. The characteristic colonies of Salmonella and other common fecal contaminant bacteria growing on SS, Rambach, XLDT4 and SB are shown in Table 2. Addition of tergitol or novobiocin to XLD agar did not completely inhibit the growth of all Proteus spp. strains examined. None of the Proteus spp. strains able to multiply on SS, XLD or XLDT4 agar grew on the commercial Rambach agar. Several different contaminant bacterial species produced Salmonella-like colonies on Rambach, SS, XLD and XLDT4 agars. Because these contaminant bacterial species are different it is advisable to improve the diagnosis by culturing samples on SS, XLD or XLDT4 agar and also simultaneously on Rambach agar.
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PMID:[Evaluation of selective culture media for the isolation of Salmonella from poultry]. 855 59

High molecular mass-chondroitin sulfate was characterized for M(r), charge density and constituent disaccharides. This glycosaminoglycan was depolymerized by a controlled free-radical process mediated by hydrogen peroxide in the absence or presence of cupric or ferrous ions. Hydrogen peroxide depolymerizes chondroitin sulfate, and the velocity of the reaction increases in the presence of cupric ions and, further, of ferrous ions. Different low molecular mass-chondroitin sulfate fractions were produced and analyzed by high-performance size-exclusion chromatography and polyacrylamide-gel electrophoresis. This last technique strongly supports the hypothesis that the free-radical process proceeds by the destruction of disaccharide units. The treatment of free-radical chondroitin sulfate samples with chondroitinase ABC and testicular hyaluronidase results in a lower capacity of these enzymes to degrade these glycosaminoglycan derivatives with respect to the natural sample. This was confirmed by polyacrylamide-gel electrophoresis and by the time-courses of enzymatic treatment evaluated by spectrophotometric technique (for treatment with chondroitin ABC lyase).
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PMID:Activity of chondroitin ABC lyase and hyaluronidase on free-radical degraded chondroitin sulfate. 859 23

Previous studies of the structure-activity relationships (SAR) for binding of a series of AC-bicyclic cannabinoid structures to the cannabinoid receptors in rat brain (believed to comprise the CB1 subtype) demonstrated the importance of the A-ring aryl C-3 side chain and phenolic hydroxyl substituents, and elucidated the importance of a C-ring hydroxyalkyl substituent [Melvin et al. Mol. Pharmacol. 44, 1008-1015 (1993)]. The present investigation examines the SAR surrounding this region (D-ring) of the molecule that is not present in the structure of delta(9)-THC and other classical cannabinoid compounds. Both rigid fused ring benzo and cyclohexyl derivatives (creating the D-ring) retained binding affinity for the cannabinoid receptor. Extension of ketone or hydroxyl substituents from the C2 position of the D-ring resulted in a 3-fold increase in binding affinity over the unsubstituted structure. However, the fused ring structure is not critical for the interaction with the receptor in as much as opening the ring did not decrease the potency. Extension of the D-ring C-2 alcohol by one carbon in length resulted in a pair of structures, for which the greatest affinity for the CB1 receptor occurred for the hydroxymethyl group in the axial conformation [(+/-)-CP-55,244]. Upon resolution, the latter provided a pair of enantiomers: (-)-CP-55,244 was approximately 3-fold more potent than the racemic mixtures, and (+)-CP-55,244 failed to bind to the CB1 receptor with an IC50 below 1 mM. Opening of the D-ring of these structures resulted in a loss of binding affinity. This study demonstrates that the potency could be optimized in (-)-CP-55,244 for both binding to the CB1 receptor and the biological activity of analgesia. In addition, the rigid positioning of the hydroxypropyl moiety of CP-55,940 enforced by the decalin ring structure of CP-55,244 increased the enantioselectivity by greater than 100-fold. These data define the critical stereochemistry for a region of the nonclassical ACD-tricyclic cannabinoid structure that contributes a potential hydrogen bonding component to the ligand-receptor interaction mechanism. Inasmuch as this region of the molecule is not present on classical ABC-tricyclic cannabinoid compounds, these studies elucidate a unique agonist recognition site on the CB1 receptor.
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PMID:Structure-activity relationships defining the ACD-tricyclic cannabinoids: cannabinoid receptor binding and analgesic activity. 887 58

The Candida albicans CDR1 gene encodes a member of the ABC-type family of multidrug transporters which has been shown to be involved in azole resistance. Using an in-frame gene fusion between the CDR1 open reading frame and the green fluorescent protein allele yEGFP3, an optimized derivative for its use in C. albicans, we show here how the CDR1-yEGFP3 gene expression is induced in response to azoles as well as to other structurally unrelated drugs like cycloheximide. Moderate increases were observed for calcofluor, canavanine, 5'-fluorcytosine, cilofungin and caffeine, while no induction was found for the antifungals benomyl and amphotericin B or hydrogen peroxide at subinhibitory concentrations. The use of confocal microscopy enabled us to localize the Cdr1p fusion protein at the cell periphery, thus suggesting a cytoplasmic membrane localization. These results suggest deregulation of CDR1 gene as a putative mechanism for the generation of azole resistance in this clinically important pathogenic fungus.
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PMID:Induced expression of the Candida albicans multidrug resistance gene CDR1 in response to fluconazole and other antifungals. 960 2

ABC transport systems for import or export of nutrients and other substances across the cell membrane are widely distributed in nature. In most bacterial systems, a periplasmic component is the primary determinant of specificity of the transport complex as a whole. We report here the crystal structure of the periplasmic binding protein for the allose system (ALBP) from Escherichia coli, solved at 1.8 A resolution using the molecular replacement method. As in the other members of the family (especially the ribose binding protein, RBP, with which it shares 35 % sequence homology), this structure consists of two similar domains joined by a three-stranded hinge region. The protein is believed to exist in a dynamic equilibrium of closed and open conformations in solution which is an important part of its function. In the closed ligand-bound form observed here, D-allose is buried at the domain interface. Only the beta-anomer of allopyranose is seen in the crystal structure, although the alpha-anomer can potentially bind with a similar affinity. Details of the ligand-binding cleft reveal the features that determine substrate specificity. Extensive hydrogen bonding as well as hydrophobic interactions are found to be important. Altogether ten residues from both the domains form 14 hydrogen bonds with the sugar. In addition, three aromatic rings, one from each domain with faces parallel to the plane of the sugar ring and a third perpendicular, make up a hydrophobic stacking surface for the ring hydrogen atoms. Our results indicate that the aromatic rings forming the sugar binding cleft can sterically block the binding of any hexose epimer except D-allose, 6-deoxy-allose or 3-deoxy-glucose; the latter two are expected to bind with reduced affinity, due to the loss of some hydrogen bonds. The pyranose form of the pentose, D-ribose, can also fit into the ALBP binding cleft, although with lower binding affinity. Thus, ALBP can function as a low affinity transporter for D-ribose. The significance of these results is discussed in the context of the function of allose and ribose transport systems.
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PMID:Structure of D-allose binding protein from Escherichia coli bound to D-allose at 1.8 A resolution. 1006 13

Bacteria produce and secrete lipases, which can catalyze both the hydrolysis and the synthesis of long-chain acylglycerols. These reactions usually proceed with high regioselectivity and enantioselectivity, and, therefore, lipases have become very important stereoselective biocatalysts used in organic chemistry. High-level production of these biocatalysts requires the understanding of the mechanisms underlying gene expression, folding, and secretion. Transcription of lipase genes may be regulated by quorum sensing and two-component systems; secretion can proceed either via the Sec-dependent general secretory pathway or via ABC transporters. In addition, some lipases need folding catalysts such as the lipase-specific foldases and disulfide-bond-forming proteins to achieve a secretion-competent conformation. Three-dimensional structures of bacterial lipases were solved to understand the catalytic mechanism of lipase reactions. Structural characteristics include an alpha/beta hydrolase fold, a catalytic triad consisting of a nucleophilic serine located in a highly conserved Gly-X-Ser-X-Gly pentapeptide, and an aspartate or glutamate residue that is hydrogen bonded to a histidine. Four substrate binding pockets were identified for triglycerides: an oxyanion hole and three pockets accommodating the fatty acids bound at position sn-1, sn-2, and sn-3. The differences in size and the hydrophilicity/hydrophobicity of these pockets determine the enantiopreference of a lipase. The understanding of structure-function relationships will enable researchers to tailor new lipases for biotechnological applications. At the same time, directed evolution in combination with appropriate screening systems will be used extensively as a novel approach to develop lipases with high stability and enantioselectivity.
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PMID:Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases. 1054 94


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