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41.
Dextranmethylcarboxylate benzylamide sulfate (DMCBSu), a functionalized dextran, exhibits anticoagulant properties. Its synthesis involves three steps: a carboxymethylation with monochloroacetic acid in alkaline water-iso-propanol, a benzylamidification of some of the methylcarboxylate groups with benzylamine in the presence of a water soluble carbodiimide and a partial sulfation of the remaining hydroxyl groups with SO3-pyridine in dimethylformamide. This procedure yields reproducibly DMCBSu with degrees of substitution in methylcarboxylate (MC), benzylamide (B) and sulfate (Su) groups, respectively, up to 1.61, 0.35 and 1.5, each obtained in one step. For a degree of substitution of methylcarboxylate ca. 1, the presence of sulfate groups is absolutely necessary to confer anticoagulant activities to the samples. In addition, the anticoagulant ability is higher for derivatives bearing benzylamide groups. The anticoagulant ability of DMCBSu increases with the degree of sulfation, reaching 20% of heparin activity for a degree of substitution of Su groups about 1.3. 相似文献
42.
Shailubhai K Khai Huynh Q Boddupalli H Yu HH Jacob GS 《Biochemical and biophysical research communications》1999,256(1):170-176
A microsomal galactose-6-O-sulfotransferase (Gal-6-O-Stase) from porcine lymph nodes, able to transfer the sulfate group from adenosine 3'-phosphate 5'-phosphosulphate (PAPS) onto 2'-fucosyllactose (2'-FL) and other sialyl LewisX (sLex)-related sugars, has been purified and characterized. The enzyme was purified to about 35,000-fold by a combination of conventional and affinity chromatographic steps. The purified enzyme preparation exhibited two protein bands at around 80-90 and 170 kDa on 7.5% SDS-PAGE under reducing conditions. Both of these protein bands always comigrated in the gel when peak fractions containing Gal-6-O-Stase activity from the 3',5'-ADP-agarose column were subjected to 6% SDS-PAGE under reducing conditions. These protein bands also showed similar binding patterns to WGA (wheat germ agglutinin), Con A (concanvalin A), and EBA (elderberry agglutinin). Similarly, when the enzyme preparation after the hydroxylapatite step was photolabeled with 8-azido-[32P]-PAPS, both 80-90 and 170 kDa protein bands were labeled in a specific manner. These results suggest a possible association of these two protein bands with the enzyme activity. The carbohydrate substrate specificity of this enzyme suggests that it is well suited to catalyze the sulphonation at the C-6 position of the galactose residues of oligosaccharides that are structurally similar to sLex. Furthermore, a survey of several porcine organs revealed that this enzyme was selectively expressed in lymphoid tissues such as lymph nodes (peripheral and mesenteric) and spleen. These findings suggest that this enzyme may be involved in the assembly of 3'-sialyl-6'-sulfo Lewisx, the major capping group of HEV-ligands for L-selectin. 相似文献
43.
Characterization of Bacillus probiotics available for human use 总被引:4,自引:0,他引:4
Duc le H Hong HA Barbosa TM Henriques AO Cutting SM 《Applied and environmental microbiology》2004,70(4):2161-2171
Bacillus species (Bacillus cereus, Bacillus clausii, Bacillus pumilus) carried in five commercial probiotic products consisting of bacterial spores were characterized for potential attributes (colonization, immunostimulation, and antimicrobial activity) that could account for their claimed probiotic properties. Three B. cereus strains were shown to persist in the mouse gastrointestinal tract for up to 18 days postadministration, demonstrating that these organisms have some ability to colonize. Spores of one B. cereus strain were extremely sensitive to simulated gastric conditions and simulated intestinal fluids. Spores of all strains were immunogenic when they were given orally to mice, but the B. pumilus strain was found to generate particularly high anti-spore immunoglobulin G titers. Spores of B. pumilus and of a laboratory strain of B. subtilis were found to induce the proinflammatory cytokine interleukin-6 in a cultured macrophage cell line, and in vivo, spores of B. pumilus and B. subtilis induced the proinflammatory cytokine tumor necrosis factor alpha and the Th1 cytokine gamma interferon. The B. pumilus strain and one B. cereus strain (B. cereus var. vietnami) were found to produce a bacteriocin-like activity against other Bacillus species. The results that provided evidence of colonization, immunostimulation, and antimicrobial activity support the hypothesis that the organisms have a potential probiotic effect. However, the three B. cereus strains were also found to produce the Hbl and Nhe enterotoxins, which makes them unsafe for human use. 相似文献
44.
Huynh TT Huynh VT Harmon MA Phillips MA 《The Journal of biological chemistry》2003,278(41):39794-39800
The parasitic protozoa Trypanosoma brucei utilizes a novel cofactor (trypanothione, T(SH)2), which is a conjugate of GSH and spermidine, to maintain cellular redox balance. gamma-Glutamylcysteine synthetase (gamma-GCS) catalyzes the first step in the biosynthesis of GSH. To evaluate the importance of thiol metabolism to the parasite, RNAi methods were used to knock down gene expression of gamma-GCS in procyclic T. brucei cells. Induction of gamma-GCS RNAi with tetracycline led to cell death within 4-6 days post-induction. Cell death was preceded by the depletion of the gamma-GCS protein and RNA and by the loss of the cellular pools of GSH and T(SH)2. The addition of GSH (80 microM) to cell cultures rescued the RNAi cell death phenotype and restored the intracellular thiol pools to wild-type levels. Treatment of cells with buthionine sulfoximine (BSO), an enzyme-activated inhibitor of gamma-GCS, also resulted in cell death. However, the toxicity of the inhibitor was not reversed by GSH, suggesting that BSO has more than one cellular target. BSO depletes intracellular thiols to a similar extent as gamma-GCS RNAi; however, addition of GSH did not restore the pools of GSH and T(SH)2. These data suggest that BSO also acts to inhibit the transport of GSH or its peptide metabolites into the cell. The ability of BSO to inhibit both synthesis and transport of GSH likely makes it a more effective cytotoxic agent than an inhibitor with a single mode of action. Finally the potential for the T(SH)2 biosynthetic enzymes to be regulated in response to reduced thiol levels was studied. The expression levels of ornithine decarboxylase and of S-adenosylmethionine decarboxylase, two essential enzymes in spermidine biosynthesis, remained constant in induced gamma-GCS RNAi cell lines. 相似文献
45.
Baldwin J Krebs C Saleh L Stelling M Huynh BH Bollinger JM Riggs-Gelasco P 《Biochemistry》2003,42(45):13269-13279
The diiron(II) cluster in the R2 subunit of Escherichia coli ribonucleotide reductase (RNR) activates oxygen to generate a mu-oxodiiron(III) cluster and the stable tyrosyl radical that is critical for the conversion of ribonucleotides to deoxyribonucleotides. Like those in other diiron carboxylate proteins, such as methane monooxygenase (MMO), the R2 diiron cluster is proposed to activate oxygen by formation of a peroxodiiron(III) intermediate followed by an oxidizing high-valent cluster. Substitution of key active site residues results in perturbations of the normal oxygen activation pathway. Variants in which the active site ligand, aspartate (D) 84, is changed to glutamate (E) are capable of accumulating a mu-peroxodiiron(III) complex in the reaction pathway. Using rapid freeze-quench techniques, this intermediate in a double variant, R2-W48A/D84E, was trapped for characterization by M?ssbauer and X-ray absorption spectroscopy. These samples contained 70% peroxodiiron(III) intermediate and 30% diferrous R2. An Fe-Fe distance of 2.5 A was found to be associated with the peroxo intermediate. As has been proposed for the structures of the higher valent intermediates in both R2 and MMO, carboxylate shifts to a mu-(eta(1),eta(2)) or a mu-1,1 conformation would most likely be required to accommodate the short 2.5 A Fe-Fe distance. In addition, the diferrous form of the enzyme present in the reacted sample has a longer Fe-Fe distance (3.5 A) than does a sample of anaerobically prepared diferrous R2 (3.4 A). Possible explanations for this difference in detected Fe-Fe distance include an O(2)-induced conformational change prior to covalent chemistry or differing O(2) reactivity among multiple diiron(II) forms of the cluster. 相似文献
46.
47.
Huynh QK Kishore N Mathialagan S Donnelly AM Tripp CS 《The Journal of biological chemistry》2002,277(15):12550-12558
Nuclear factor-kappaB activation depends on phosphorylation and degradation of its inhibitor protein, IkappaB. The phosphorylation of IkappaBalpha on Ser(32) and Ser(36) is initiated by an IkappaB kinase (IKK) complex that includes a catalytic heterodimer composed of IkappaB kinase 1 (IKK-1) and IkappaB kinase 2 (IKK-2) as well as a regulatory adaptor subunit, NF-kappaB essential modulator. Recently, two related IkappaB kinases, TBK-1 and IKK-i, have been described. TBK-1 and IKK-i show sequence and structural homology to IKK-1 and IKK-2. TBK-1 and IKK-i phosphorylate Ser(36) of IkappaBalpha. We describe the kinetic mechanisms in terms of substrate and product inhibition of the recombinant human (rh) proteins, rhTBK-1, rhIKK-I, and rhIKK-1/rhIKK-2 heterodimers. The results indicate that although each of these enzymes exhibits a random sequential kinetic mechanism, the effect of the binding of one substrate on the affinity of the other substrate is significantly different. ATP has no effect on the binding of an IkappaBalpha peptide for the rhIKK-1/rhIKK-2 heterodimer (alpha = 0.99), whereas the binding of ATP decreased the affinity of the IkappaBalpha peptide for both rhTBK-1 (alpha = 10.16) and rhIKK-i (alpha = 62.28). Furthermore, the dissociation constants of ATP for rhTBK-1 and rhIKK-i are between the expected values for kinases, whereas the dissociation constants of the IkappaBalpha peptide for each IKK isoforms is unique with rhTBK-1 being the highest (K(IkappaBalpha) = 69.87 microm), followed by rhIKK-i (K(IkappaBalpha) = 5.47 microm) and rhIKK-1/rhIKK-2 heterodimers (K(IkappaBalpha) = 0.12 microm). Thus this family of IkappaB kinases has very unique kinetic properties. 相似文献
48.
Hsu S Borke JL Lewis JB Singh B Aiken AC Huynh CT Schuster GS Caughman GB Dickinson DP Smith AK Osaki T Wang XF 《Cell proliferation》2002,35(3):183-192
A human oral tumour progression model was established that consists of normal epithelial cells and three cell lines representing stages from dysplastic to metastatic cells. To investigate the impact of exogenous transforming growth factor-beta 1 on this model system, we analysed the responsiveness of those cells to transforming growth factor-beta 1 and explored the potential mechanism underlying the transforming growth factor-beta 1 activity. We found that the growth of all cell types, regardless of their stage of tumour progression, is inhibited by transforming growth factor-beta 1, although to different degrees. Transforming growth factor-beta 1 induced the expression of cyclin-dependent kinase inhibitors p15(INK4B), p21WAF1/(CIP1) and p27(KIP1). In contrast, transforming growth factor-beta 1 was found to stimulate the invasive potential of one cell type that represents the most advanced stage of tumour phenotype, suggesting that the impact of transforming growth factor-beta 1 on functional features of tumour cells other than cellular proliferation may play a significant role in the process of oral tumour progression. 相似文献
49.
50.
Neelaredoxin, an iron-binding protein from the syphilis spirochete, Treponema pallidum, is a superoxide reductase 总被引:4,自引:0,他引:4