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1

Modulation of Alternaria infectoria Cell Wall Chitin and Glucan Synthesis by Cell Wall Synthase Inhibitors  

PubMed Central

The present work reports the effects of caspofungin, a ?-1,3-glucan synthase inhibitor, and nikkomycin Z, an inhibitor of chitin synthases, on two strains of Alternaria infectoria, a melanized fungus involved in opportunistic human infections and respiratory allergies. One of the strains tested, IMF006, bore phenotypic traits that conferred advantages in resisting antifungal treatment. First, the resting cell wall chitin content was higher and in response to caspofungin, the chitin level remained constant. In the other strain, IMF001, the chitin content increased upon caspofungin treatment to values similar to basal IMF006 levels. Moreover, upon caspofungin treatment, the FKS1 gene was upregulated in IMF006 and downregulated in IMF001. In addition, the resting ?-glucan content was also different in both strains, with higher levels in IMF001 than in IMF006. However, this did not provide any advantage with respect to echinocandin resistance. We identified eight different chitin synthase genes and studied relative gene expression when the fungus was exposed to the antifungals under study. In both strains, exposure to caspofungin and nikkomycin Z led to modulation of the expression of class V and VII chitin synthase genes, suggesting its importance in the robustness of A. infectoria. The pattern of A. infectoria phagocytosis and activation of murine macrophages by spores was not affected by caspofungin. Monotherapy with nikkomycin Z and caspofungin provided only fungistatic inhibition, while a combination of both led to fungal cell lysis, revealing a strong synergistic action between the chitin synthase inhibitor and the ?-glucan synthase inhibitor against this fungus. PMID:24614372

Fernandes, Chantal; Anjos, Jorge; Walker, Louise A.; Silva, Branca M. A.; Cortes, Luisa; Mota, Marta; Munro, Carol A.; Gow, Neil A. R.

2014-01-01

2

Chitin synthase 2 inhibitory activity of O-methyl pisiferic acid and 8,20-dihydroxy-9(11),13-abietadien-12-one, isolated from Chamaecyparis pisifera.  

PubMed

In the course of search for potent chitin synthase inhibitors from plant extracts, the chitin synthase 2 inhibitors, O-methyl pisiferic acid and 8,20-dihydroxy-9(11),13-abietadien-12-one which have diterpene skeleton, were isolated from the leaves of Chamaecyparis pisifera. These compounds inhibited chitin synthase 2 of Saccharomyces cerevisiae with the IC50 values of 5.8 and 226.4 microM, respectively. Especially, O-methyl pisiferic acid showed 15.3-fold stronger inhibitory activity than polyoxin D (IC50=88.6 microM), a well-known chitin synthase inhibitor. These compounds exhibited weaker inhibitory activities against chitin synthase 1 than chitin synthase 2, whereas it showed no inhibitory activity for chitin synthase 3. The compound exhibited mixed competitive inhibition with respect to UDP-N-acetyl-D-glucosamine as substrate (Ki=5 microM). These results indicated that O-methyl pisiferic acid is a specific inhibitor of chitin synthase 2. The compound also inhibited chitin synthase 1 of Candida albicans, which represents analogues to chitin synthase 2 of S. cerevisiae, with an IC50 of 75.6 microM, which represents 1.8-fold weaker activity than that of polyoxin D. Although O-methyl pisiferic acid has been reported for antibacterial and insecticidal activities, the present study is the first report on its inhibitory activity against chitin synthase 2. PMID:18379078

Kang, Tae Hoon; Hwang, Eui Il; Yun, Bong Sik; Shin, Chul Soo; Kim, Sung Uk

2008-04-01

3

Inhibitory activity for chitin synthase II from Saccharomyces cerevisiae by tannins and related compounds.  

PubMed

In the course of search for potent inhibitors of chitin synthase II from natural resources, seven tannins and related compounds were isolated from the aerial part of Euphorbia pekinensis and identified as gallic acid (1), methyl gallate (2), 3-O-galloyl-(-)-shikimic acid (3), corilagin (4), geraniin (5), quercetin-3-O-(2"-O-galloyl)-beta-D-glucoside (6), and kaempferol-3-O-(2"-O-galloyl)-beta-D-glucoside (7). These and nine related compounds, (-)-quinic acid (8), (-)-shikimic acid (9), ellagic acid (10), kaempferol (11), quercetin (12), quercitrin (13), rutin (14), quercetin-3-O-(2"-O-galloyl)-beta-D-rutinoside (15) and 1,3,4,6-tetra-O-galloyl-beta-D-glucose (16), were evaluated for the inhibitory activity against chitin synthase II and III. They inhibited chitin synthase II with IC(50) values of 18-206 microM, except for two organic acids, (-)-quinic acid (8) and (-)-shikimic acid (9). Among them, 3-O-galloyl-(-)-shikimic acid (3) was the most potent inhibitor against chitin synthase II of Saccharomyces cerevisiae with an IC(50) value of 18 microM. The inhibition appears to be selective for chitin synthase II, as they did not appreciably inhibit chitin synthase III. PMID:11509967

Hwang, E I; Ahn, B T; Lee, H B; Kim, Y K; Lee, K S; Bok, S H; Kim, Y T; Kim, S U

2001-08-01

4

Potent inhibition of chitin synthase by an azasugar--investigation of synergistic effect with UDP.  

PubMed

We identified 6-deoxy-homoDMDP as a potent inhibitor of chitin synthase (Ki = 38 microM), displaying an uncompetitive inhibition pattern. Dual inhibition was also performed with the enzymatic reaction product uridine 5'-diphosphate in order to evaluate the concurrent effect of both inhibitors. An interaction coefficient alpha of 0.9 was found, revealing synergistic inhibition. PMID:15968816

Djebaili, Mounira; Behr, Jean-Bernard

2005-04-01

5

Chitin synthase A: a novel epidermal development regulation gene in the larvae of Bombyx mori.  

PubMed

Chitin synthase is the key regulatory enzyme for chitin synthesis and excretion in insects, as well as a specific target of insecticides. The chitin synthase A gene (BmChsA) cloned from Bombyx mori, the model species of lepidopteran, is an epidermis-specific expressed gene during the molting stage. Knockdown BmChsA gene in 3rd instar larvae increased the number of non-molting and abnormal molting larvae. Exposure to nikkomycin Z, a chitin synthase inhibitor downregulated the expression of BmChsA and decreased the amount of epidermis chitin during the molting process. The thickness of the new epidermis and its dense structure varied greatly. The exogenous hormones significantly upregulated the expression of BmChsA with low levels of endogenous MH and high levels of endogenous JH immediately after molting. With low levels of endogenous hormones during the mulberry intake process, BmChsA was rarely upregulated by exogenous hormones. With high levels of endogenous MH and low levels of endogenous JH during the molting stage, we did not detect the upregulation of BmChsA by exogenous hormones. The expression of BmChsA was regulated by endocrine hormones, which directly affected the chitin synthesis-dependent epidermal regeneration and molting process. PMID:24577751

Zhuo, Weiwei; Fang, Yan; Kong, Lingfei; Li, Xi; Sima, Yanghu; Xu, Shiqing

2014-07-01

6

Chitin synthase 2 is essential for septum formation and cell division in Saccharomyces cerevisiae.  

PubMed Central

Previous work led to the puzzling conclusion that chitin synthase 1, the major chitin synthase activity in Saccharomyces cerevisiae, is not required for synthesis of the chitinous primary septum. The mechanism of in vivo synthesis of chitin has now been clarified by cloning the structural gene for the newly found chitin synthase 2, a relatively minor activity in yeast. Disruption of the chitin synthase 2 gene results in the loss of well-defined septa and in growth arrest, establishing that the gene product is essential for both septum formation and cell division. Images PMID:2968606

Silverman, S J; Sburlati, A; Slater, M L; Cabib, E

1988-01-01

7

Chitin synthase 2 is essential for septum formation and cell division in Saccharomyces cerevisiae.  

PubMed

Previous work led to the puzzling conclusion that chitin synthase 1, the major chitin synthase activity in Saccharomyces cerevisiae, is not required for synthesis of the chitinous primary septum. The mechanism of in vivo synthesis of chitin has now been clarified by cloning the structural gene for the newly found chitin synthase 2, a relatively minor activity in yeast. Disruption of the chitin synthase 2 gene results in the loss of well-defined septa and in growth arrest, establishing that the gene product is essential for both septum formation and cell division. PMID:2968606

Silverman, S J; Sburlati, A; Slater, M L; Cabib, E

1988-07-01

8

Functional differentiation of chitin synthases in Yarrowia lipolytica.  

PubMed

In this study, we identified seven chitin synthase-encoding genes in the genome of the dimorphic yeast Yarrowia lipolytica. Three encoded chitin synthases with myosin motor-like domains at their N-termini, and we designated these CSM1 to CSM3, whereas four were identified as CHS1 to CHS4. To investigate the functions of these seven genes, we constructed and characterized their deletion mutants. The chs2? mutant formed chained cells in which daughter cells were connected with mother cells and had abnormally thick septa at the bud neck. The chs4? mutant showed remarkably reduced chitin content in its cell wall. The chs2?, csm1?, and csm2? mutants were found to be highly sensitive to chitin binding dyes, calcofluor white (CFW) and Congo red, whereas the chs4? mutant was resistant to CFW. These results suggest that Chs2 and Chs4 play major roles in septum formation and cell wall chitin synthesis respectively, whereas Csm1 and Csm2 are involved in the maintenance of cell wall architecture and/or cell wall integrity. The populations of filamentous cells, a type of cell population that are defined by the lengths of the cellular long and short axes, decreased in the chs3? mutant, suggesting that Chs3 is involved in cellular morphogenesis. PMID:23748777

Sheng, Wei; Yamashita, Shuichi; Ohta, Akinori; Horiuchi, Hiroyuki

2013-01-01

9

2-Acylamido Analogues of N-Acetylglucosamine Prime Formation of Chitin Oligosaccharides by Yeast Chitin Synthase 2*  

PubMed Central

Chitin, a homopolymer of ?1,4-linked N-acetylglucosamine (GlcNAc) residues, is a key component of the cell walls of fungi and the exoskeletons of arthropods. Chitin synthases transfer GlcNAc from UDP-GlcNAc to preexisting chitin chains in reactions that are typically stimulated by free GlcNAc. The effect of GlcNAc was probed by using a yeast strain expressing a single chitin synthase, Chs2, by examining formation of chitin oligosaccharides (COs) and insoluble chitin, and by replacing GlcNAc with 2-acylamido analogues of GlcNAc. Synthesis of COs was strongly dependent on inclusion of GlcNAc in chitin synthase incubations, and N,N?-diacetylchitobiose (GlcNAc2) was the major reaction product. Formation of both COs and insoluble chitin was also stimulated by GlcNAc2 and by N-propanoyl-, N-butanoyl-, and N-glycolylglucosamine. MALDI analyses of the COs made in the presence of 2-acylamido analogues of GlcNAc showed they that contained a single GlcNAc analogue and one or more additional GlcNAc residues. These results indicate that Chs2 can use certain 2-acylamido analogues of GlcNAc, and likely free GlcNAc and GlcNAc2 as well, as GlcNAc acceptors in a UDP-GlcNAc-dependent glycosyltransfer reaction. Further, formation of modified disaccharides indicates that CSs can transfer single GlcNAc residues. PMID:24619411

Gyore, Jacob; Parameswar, Archana R.; Hebbard, Carleigh F. F.; Oh, Younghoon; Bi, Erfei; Demchenko, Alexei V.; Price, Neil P.; Orlean, Peter

2014-01-01

10

2-Acylamido analogues of N-acetylglucosamine prime formation of chitin oligosaccharides by yeast chitin synthase 2.  

PubMed

Chitin, a homopolymer of ?1,4-linked N-acetylglucosamine (GlcNAc) residues, is a key component of the cell walls of fungi and the exoskeletons of arthropods. Chitin synthases transfer GlcNAc from UDP-GlcNAc to preexisting chitin chains in reactions that are typically stimulated by free GlcNAc. The effect of GlcNAc was probed by using a yeast strain expressing a single chitin synthase, Chs2, by examining formation of chitin oligosaccharides (COs) and insoluble chitin, and by replacing GlcNAc with 2-acylamido analogues of GlcNAc. Synthesis of COs was strongly dependent on inclusion of GlcNAc in chitin synthase incubations, and N,N'-diacetylchitobiose (GlcNAc2) was the major reaction product. Formation of both COs and insoluble chitin was also stimulated by GlcNAc2 and by N-propanoyl-, N-butanoyl-, and N-glycolylglucosamine. MALDI analyses of the COs made in the presence of 2-acylamido analogues of GlcNAc showed they that contained a single GlcNAc analogue and one or more additional GlcNAc residues. These results indicate that Chs2 can use certain 2-acylamido analogues of GlcNAc, and likely free GlcNAc and GlcNAc2 as well, as GlcNAc acceptors in a UDP-GlcNAc-dependent glycosyltransfer reaction. Further, formation of modified disaccharides indicates that CSs can transfer single GlcNAc residues. PMID:24619411

Gyore, Jacob; Parameswar, Archana R; Hebbard, Carleigh F F; Oh, Younghoon; Bi, Erfei; Demchenko, Alexei V; Price, Neil P; Orlean, Peter

2014-05-01

11

Chitin synthase in the filarial parasite, Brugia malayi.  

PubMed

Fragments of putative chitin synthase (chs) genes from two filarial species (Brugia malayi and Dirofilaria immitis) were amplified by PCR using degenerate primers. The full genomic and cDNA sequences were obtained for the B. malayi chs gene (Bm-chs-1); the predicted amino acid sequence is highly similar, over a large region, to two CHS sequences of the nematode Caenorhabditis elegans and also to two insect CHS sequences. Bm-chs-1 is abundantly transcribed in B. malayi adult females, independent of their fertilization status, but is also expressed in males and microfilariae. Oocytes and early embryos contain large amounts of Bm-chs-1 transcript by in situ hybridization, but later stage embryos within the maternal uterus show little or no Bm-chs-1 transcript. No specific hybridization could be demonstrated in maternal somatic tissues. Polyclonal antibodies were raised against a peptide expressed from a recombinant cDNA fragment of Bm-chs-1; immunostaining detected CHS protein in oocytes and early to midstage embryos. These studies characterize a gene that is likely to be essential to oogenesis and embryonic development in a parasitic nematode. Because chitin synthesis and eggshell formation begin after fertilization, the presence of CHS protein in early oocytes suggests that the enzyme must be activated as a result of fertilization. These studies also demonstrate that chitin synthesis may not be restricted to eggshell formation in nematodes, as the Bm-chs-1 gene is transcribed in life cycle stages other than adult females. PMID:11163442

Harris, M T; Lai, K; Arnold, K; Martinez, H F; Specht, C A; Fuhrman, J A

2000-12-01

12

Early divergence, broad distribution, and high diversity of animal chitin synthases.  

PubMed

Even though chitin is one of the most abundant biopolymers in nature, current knowledge on chitin formation is largely based only on data from fungi and insects. This study reveals unanticipated broad taxonomic distribution and extensive diversification of chitin synthases (CSs) in Metazoa, shedding new light on the relevance of chitin in animals and suggesting unforeseen complexity of chitin synthesis in many groups. We uncovered robust orthologs to insect type CSs in several representatives of deuterostomes, which generally are not thought to possess chitin. This suggests a broader distribution and function of chitin in this branch of the animal kingdom. We characterize a new CS type present not only in basal metazoans such as sponges and cnidarians but also in several bilaterian representatives. The most extensive diversification of CSs took place during emergence of lophotrochozoans, the third large group of protostomes next to arthropods and nematodes, resulting in coexistence of up to ten CS paralogs in molluscs. Independent fusion to different kinds of myosin motor domains in fungi and lophotrochozoans points toward high relevance of CS interaction with the cytoskeleton for fine-tuned chitin secretion. Given the fundamental role that chitin plays in the morphology of many animals, the here presented CS diversification reveals many evolutionary complexities. Our findings strongly suggest a very broad and multifarious occurrence of chitin and question an ancestral role as cuticular component. The molecular mechanisms underlying regulation of animal chitin synthesis are most likely far more complex and diverse than existing data from insects suggest. PMID:24443419

Zakrzewski, Anne-C; Weigert, Anne; Helm, Conrad; Adamski, Marcin; Adamska, Maja; Bleidorn, Christoph; Raible, Florian; Hausen, Harald

2014-01-01

13

Inhibitors of the fungal cell wall. Synthesis of 4-aryl-4-N-arylamine-1-butenes and related compounds with inhibitory activities on beta(1-3) glucan and chitin synthases.  

PubMed

As part of our project devoted to the search for antifungal agents, which act via a selective mode of action, we synthesized a series of new 4-aryl- or 4-alkyl-N-arylamine-1-butenes and transformed some of them into 2-substituted 4-methyl-tetrahydroquinolines and quinolines by using a novel three-step synthesis. Results obtained in agar dilution assays have shown that 4-aryl homoallylamines not possessing halogen in their structures, tetrahydroquinolines and quinolines, display a range of antifungal properties in particular against Epidermophyton floccosum and Microsporum canis. Regarding the mode of action, all active compounds showed in vitro inhibitory activities against beta(1-3) glucan-synthase and mainly against chitin-synthase. These enzymes catalyze the synthesis of beta(1-3) glucan and chitin, respectively, major polymers of the fungal cell wall. Since fungal but not mammalian cells are encased in a cell wall, its inhibition may represent a useful mode of action for these antifungal compounds. PMID:10819157

Urbina, J M; Cortés, J C; Palma, A; López, S N; Zacchino, S A; Enriz, R D; Ribas, J C; Kouznetzov, V V

2000-04-01

14

Population bulk segregant mapping uncovers resistance mutations and the mode of action of a chitin synthesis inhibitor in arthropods  

PubMed Central

Because of its importance to the arthropod exoskeleton, chitin biogenesis is an attractive target for pest control. This point is demonstrated by the economically important benzoylurea compounds that are in wide use as highly specific agents to control insect populations. Nevertheless, the target sites of compounds that inhibit chitin biogenesis have remained elusive, likely preventing the full exploitation of the underlying mode of action in pest management. Here, we show that the acaricide etoxazole inhibits chitin biogenesis in Tetranychus urticae (the two-spotted spider mite), an economically important pest. We then developed a population-level bulk segregant mapping method, based on high-throughput genome sequencing, to identify a locus for monogenic, recessive resistance to etoxazole in a field-collected population. As supported by additional genetic studies, including sequencing across multiple resistant strains and genetic complementation tests, we associated a nonsynonymous mutation in the major T. urticae chitin synthase (CHS1) with resistance. The change is in a C-terminal transmembrane domain of CHS1 in a highly conserved region that may serve a noncatalytic but essential function. Our finding of a target-site resistance mutation in CHS1 shows that at least one highly specific chitin biosynthesis inhibitor acts directly to inhibit chitin synthase. Our work also raises the possibility that other chitin biogenesis inhibitors, such as the benzoylurea compounds, may also act by inhibition of chitin synthases. More generally, our genetic mapping approach should be powerful for high-resolution mapping of simple traits (resistance or otherwise) in arthropods. PMID:22393009

Van Leeuwen, Thomas; Demaeght, Peter; Osborne, Edward J.; Dermauw, Wannes; Gohlke, Simon; Nauen, Ralf; Grbic, Miodrag; Tirry, Luc; Merzendorfer, Hans; Clark, Richard M.

2012-01-01

15

Chitin Synthases with a Myosin Motor-Like Domain Control the Resistance of Aspergillus fumigatus to Echinocandins  

PubMed Central

Aspergillus fumigatus has two chitin synthases (CSMA and CSMB) with a myosin motor-like domain (MMD) arranged in a head-to-head configuration. To understand the function of these chitin synthases, single and double csm mutant strains were constructed and analyzed. Although there was a slight reduction in mycelial growth of the mutants, the total chitin synthase activity and the cell wall chitin content were similar in the mycelium of all of the mutants and the parental strain. In the conidia, chitin content in the ?csmA strain cell wall was less than half the amount found in the parental strain. In contrast, the ?csmB mutant strain and, unexpectedly, the ?csmA/?csmB mutant strain did not show any modification of chitin content in their conidial cell walls. In contrast to the hydrophobic conidia of the parental strain, conidia of all of the csm mutants were hydrophilic due to the presence of an amorphous material covering the hydrophobic surface-rodlet layer. The deletion of CSM genes also resulted in an increased susceptibility of resting and germinating conidia to echinocandins. These results show that the deletion of the CSMA and CSMB genes induced a significant disorganization of the cell wall structure, even though they contribute only weakly to the overall cell wall chitin synthesis. PMID:22964252

Jimenez-Ortigosa, Cristina; Aimanianda, Vishukumar; Muszkieta, Laetitia; Mouyna, Isabelle; Alsteens, David; Pire, Stephane; Beau, Remi; Krappmann, Sven; Beauvais, Anne; Dufrene, Yves F.

2012-01-01

16

An epidermis-specific chitin synthase CDNA in Choristoneura fumiferana: cloning, characterization, developmental and hormonal-regulated expression.  

PubMed

Chitin synthase catalyzes chitin synthesis in the exoskeleton, tracheal system and gut during insect development. A chitin synthase 1 (CfCHS1) cDNA was identified and cloned from the spruce budworm, Choristoneura fumiferana. The CfCHS1 cDNA is 5,300 bp in length and codes a 1,564-amino acid protein with a molecular mass of 178 kDa. The deduced protein contains 16 transmembrane helixes in its domains A and C. The single copy CfCHS1 gene expressed during each of the larval molts from the 3rd to the 6th instar. The gene expressed highly and periodically in the epidermis during each of molts, whereas no transcripts were detected in the midgut and fat body. 20-hydroxyecdysone and the ecdysone agonist RH5992 suppressed CfCHS1 expression, whereas the juvenile hormone analog methoprene induced CfCHS1 expression. These results implicate that CfCHS1 is involved in the chitin synthase and new chitin formation during molting in the insect. PMID:21181720

Ampasala, Dinakar R; Zheng, Sichun; Zhang, Dayu; Ladd, Tim; Doucet, Daniel; Krell, Peter J; Retnakaran, Arthur; Feng, Qili

2011-02-01

17

Physiological and Morphological Aspects of Aedes aegypti Developing Larvae: Effects of the Chitin Synthesis Inhibitor Novaluron  

PubMed Central

Population control of the dengue vector mosquito, Aedes aegypti, is difficult due to many reasons, one being the development of resistance to neurotoxic insecticides employed. The biosynthesis of chitin, a major constituent of insect cuticle, is a novel target for population control. Novaluron is a benzoylphenylurea (BPU) that acts as a chitin synthesis inhibitor, already used against mosquitoes. However, information regarding BPU effects on immature mosquito stages and physiological parameters related with mosquito larval development are scarce. A set of physiological parameters were recorded in control developing larvae and novaluron was administered continuously to Ae. aegypti larvae, since early third instar. Larval instar period duration was recorded from third instar until pupation. Chitin content was measured during third and fourth instars. Fourth instars were processed histochemically at the mesothorax region, stained with hematoxylin and eosin (HE) for assessment of internal tissues, and labeled with WGA-FITC to reveal chitinized structures. In control larvae: i) there is a chitin content increase during both third and fourth instars where late third instars contain more chitin than early fourth instars; ii) thoracic organs and a continuous cuticle, closely associated with the underlying epidermis were observed; iii) chitin was continuously present throughout integument cuticle. Novaluron treatment inhibited adult emergence, induced immature mortality, altered adult sex ratio and caused delay in larval development. Moreover, novaluron: i) significantly affected chitin content during larval development; ii) induced a discontinuous and altered cuticle in some regions while epidermis was often thinner or missing; iii) rendered chitin cuticle presence discontinuous and less evident. In both control and novaluron larvae, chitin was present in the peritrophic matrix. This study showed quantitatively and qualitatively evidences of novaluron effects on Ae. aegypti larval development. To our knowledge, this is the first report describing histological alterations produced by a BPU in immature vector mosquitoes. PMID:22291942

Farnesi, Luana C.; Brito, Jose M.; Linss, Jutta G.; Pelajo-Machado, Marcelo; Valle, Denise; Rezende, Gustavo L.

2012-01-01

18

Botrytis cinerea chitin synthase BcChsVI is required for normal growth and pathogenicity.  

PubMed

Fungal chitin synthase of classes V and VI (or VII), which contain an additional N-terminal myosin motor domain, have been shown to play important roles in pathogenesis. To study the function of BcChsVI in Botrytis cinerea, BcChs6 gene was disrupted through Agrobacterium tumefaciens-mediated transformation. The Bcchs6 disruption mutant exhibited a 45.5 % increasing in its chitin content when compared with wild strain. The qRT-PCR analysis revealed that in Bcchs6 mutant the expression of BcChs6 was significantly decreased, while the expression of BcChs2 and BcChs3a was increased when compared with wild type. It is probable that the disruption of this gene provoked a compensatory mechanism regulating the cellular response to cell wall damage. Interestingly, the radial growth of Bcchs6 mutant was drastically reduced when 50 % solute was removed from the regular PDA medium, and they were more sensitive to Calcofluor white and other cell wall disturbing chemicals. Pathogenicity assays on tomato leaves indicated that they were significantly reduced in their ability to cause disease. Our results demonstrated that BcChs6 is necessary for proper hyphal growth and pathogenicity of B. cinerea on tomato leaves. PMID:23722656

Cui, Zhifeng; Wang, Yanhua; Lei, Na; Wang, Kun; Zhu, Tingheng

2013-08-01

19

Cloning and sequence analysis of chitin synthase gene fragments of Demodex mites.  

PubMed

To our knowledge, few reports on Demodex studied at the molecular level are available at present. In this study our group, for the first time, cloned, sequenced and analyzed the chitin synthase (CHS) gene fragments of Demodex folliculorum, Demodex brevis, and Demodex canis (three isolates from each species) from Xi'an China, by designing specific primers based on the only partial sequence of the CHS gene of D. canis from Japan, retrieved from GenBank. Results show that amplification was successful only in three D. canis isolates and one D. brevis isolate out of the nine Demodex isolates. The obtained fragments were sequenced to be 339 bp for D. canis and 338 bp for D. brevis. The CHS gene sequence similarities between the three Xi'an D. canis isolates and one Japanese D. canis isolate ranged from 99.7% to 100.0%, and those between four D. canis isolates and one D. brevis isolate were 99.1%-99.4%. Phylogenetic trees based on maximum parsimony (MP) and maximum likelihood (ML) methods shared the same clusters, according with the traditional classification. Two open reading frames (ORFs) were identified in each CHS gene sequenced, and their corresponding amino acid sequences were located at the catalytic domain. The relatively conserved sequences could be deduced to be a CHS class A gene, which is associated with chitin synthesis in the integument of Demodex mites. PMID:23024043

Zhao, Ya-e; Wang, Zheng-hang; Xu, Yang; Xu, Ji-ru; Liu, Wen-yan; Wei, Meng; Wang, Chu-ying

2012-10-01

20

Cloning and sequence analysis of chitin synthase gene fragments of Demodex mites*  

PubMed Central

To our knowledge, few reports on Demodex studied at the molecular level are available at present. In this study our group, for the first time, cloned, sequenced and analyzed the chitin synthase (CHS) gene fragments of Demodex folliculorum, Demodex brevis, and Demodex canis (three isolates from each species) from Xi’an China, by designing specific primers based on the only partial sequence of the CHS gene of D. canis from Japan, retrieved from GenBank. Results show that amplification was successful only in three D. canis isolates and one D. brevis isolate out of the nine Demodex isolates. The obtained fragments were sequenced to be 339 bp for D. canis and 338 bp for D. brevis. The CHS gene sequence similarities between the three Xi’an D. canis isolates and one Japanese D. canis isolate ranged from 99.7% to 100.0%, and those between four D. canis isolates and one D. brevis isolate were 99.1%–99.4%. Phylogenetic trees based on maximum parsimony (MP) and maximum likelihood (ML) methods shared the same clusters, according with the traditional classification. Two open reading frames (ORFs) were identified in each CHS gene sequenced, and their corresponding amino acid sequences were located at the catalytic domain. The relatively conserved sequences could be deduced to be a CHS class A gene, which is associated with chitin synthesis in the integument of Demodex mites. PMID:23024043

Zhao, Ya-e; Wang, Zheng-hang; Xu, Yang; Xu, Ji-ru; Liu, Wen-yan; Wei, Meng; Wang, Chu-ying

2012-01-01

21

Chitin synthases are required for survival, fecundity and egg hatch in the red flour beetle, Tribolium castaneum.  

PubMed

The synthesis of chitin, the beta-1,4-linked polymer of N-acetylglucosamine, is catalyzed by chitin synthase (CHS). Chitin is essential for the structural integrity of the exoskeletal cuticle and midgut peritrophic membrane (PM) of insects. To study the functions of the two chitin synthase genes, TcCHS-A and TcCHS-B, during embryonic and adult development in the red flour beetle, Tribolium castaneum, RNA interference (RNAi) experiments were carried out. When dsRNA for TcCHS-A was injected into male or female pharate adults, all insects died 5-7 d after the adult molt, and the females failed to oviposit prior to death. When dsTcCHS-A was injected into young adults 1-2 d post-eclosion, a similar lethal phenotype was obtained after 5 d and no oviposition occurred. When dsTcCHS-A injections were delayed until after adult maturation (7-10 d post-eclosion), the treated females did oviposit and the resulting embryos appeared to develop normally. However, the chitin content of the eggs was dramatically reduced, the embryos became twisted and enlarged, and the eggs did not hatch. Adults treated with dsRNA for TcCHS-B exhibited little or no chitin in their PM and died about 2 wk after injection. None of the TcCHS-B-treated females oviposited, which was probably a secondary effect caused by starvation. These results extend our previous findings that CHS genes are required for all types of molt. The present study also demonstrates that these genes have additional roles in embryonic and adult development. PMID:18718535

Arakane, Yasuyuki; Specht, Charles A; Kramer, Karl J; Muthukrishnan, Subbaratnam; Beeman, Richard W

2008-10-01

22

THE EFFECT OF AN INSECT CHITIN SYNTHESIS INHIBITOR ON HONEY BEES  

E-print Network

THE EFFECT OF AN INSECT CHITIN SYNTHESIS INHIBITOR ON HONEY BEES E.W. HERBERT, Jr. R.J. ARGAUER * H. SHIMANUKI * U.S. Department of Agriculture, Agricultural Research Service """ Bioenvironmental Bee-flying colonies of honey bees. Brood rearing was temporarily terminated for a period of 2-3 weeks depending

Paris-Sud XI, Université de

23

WdCHS3, a Gene That Encodes a Class III Chitin Synthase in Wangiella (Exophiala) dermatitidis, Is Expressed Differentially under Stress Conditions  

PubMed Central

Class III chitin synthases are important for hyphal growth in some filamentous fungi but are not found in yeasts. Using a specific PCR product that encodes a portion of the class III chitin synthase of W. dermatitidis as a probe, we isolated the chitin synthase gene, WdCHS3, from this polymorphic melanized pathogen of humans. Northern blotting showed that WdCHS3 was highly expressed under stress conditions, such as the shift of cells to temperatures commensurate with infection, or to conditions that induce cellular morphogenesis in this fungus. Analysis of the 5? upstream sequence of WdCHS3 provided evidence for a negative regulatory element at between ?780 and ?1600 bp. Western blotting indicated that the production of the WdChs3p was temperature dependent and temporally regulated. Disruption of WdCHS3 in a wild-type strain and in two temperature-sensitive morphological mutants resulted in significantly reduced chitin synthase activities but did not obviously affect their morphologies, growth rates, chitin contents, or virulence. This paradox suggested that the contributions of the high levels of WdCHS3 gene expression and WdChs3p production in strains subjected to stress reside in unknown or unexamined parts of the life cycle of this ecologically poorly known member of the Fungi Imperfecti. Nonetheless, this report presents the first evidence that transcription of a chitin synthase gene is regulated by a negative regulatory element in its 5? upstream sequence. PMID:10648509

Wang, Zheng; Szaniszlo, Paul J.

2000-01-01

24

Oligomerization of the chitin synthase Chs3 is monitored at the Golgi and affects its endocytic recycling.  

PubMed

Chs3, the catalytic subunit of chitin synthase III in Saccharomyces cerevisiae, is a complex polytopic membrane protein whose plasma membrane expression is tightly controlled: export from the ER requires interaction with Chs7; exit from the Golgi is dependent on the exomer complex, and precise bud neck localization relies on endocytosis. Moreover, Chs3 is efficiently recycled from endosomes to the TGN in an AP-1-dependent manner. Here we show that the export of Chs3 requires the cargo receptor Erv14, in a step that is independent of Chs7. Chs3 oligomerized in the ER through its N-terminal cytosolic region. However, the truncated (?126)Chs3 was still exported by Erv14, but was sent back from the Golgi to the ER in a COPI- and Rer1-dependent manner. A subset of the oligomerization-deficient Chs3 proteins evaded Golgi quality control and reached the plasma membrane, where they were enzymatically active but poorly endocytosed. This resulted in high CSIII levels, but calcofluor white resistance, explained by the reduced intercalation of calcofluor white between nascent chitin fibres. Our data show that the oligomerization of Chs3 through its N-terminus is essential for proper protein trafficking and chitin synthesis and is therefore monitored intracellularly. PMID:23926947

Sacristan, Carlos; Manzano-Lopez, Javier; Reyes, Abigail; Spang, Anne; Muñiz, Manuel; Roncero, Cesar

2013-10-01

25

Molecular Cloning, Characterization and mRNA Expression of a Chitin Synthase 2 Gene from the Oriental Fruit Fly, Bactrocera dorsalis (Diptera: Tephritidae)  

PubMed Central

Chitin synthase (CHS), a potential target for eco-friendly insecticides, plays an essential role in chitin formation in insects. In this study, a full-length cDNA encoding chitin synthase 2 (BdCHS2) was cloned and characterized in the oriental fruit fly, Bactrocera dorsalis. The BdCHS2 cDNA had 4417 nucleotides, containing an open reading frame of 4122 nucleotides, which encoded 1373 amino acid residues with a predicted molecular weight of 158.5 kDa. Phylogenetic analysis with other insect CHSs suggested that BdCHS2 belongs to insect CHS2. The BdCHS2 transcript was predominately found in midgut but was detected at low levels in fat body, Malpighian tubules, integument, and trachea. Moreover, BdCHS2 was expressed in all developmental stages, and highly expressed in the feeding stages. There was a positive relationship between BdCHS2 expression and total chitin content during development. Furthermore, both the gene expression and chitin content in midgut decreased when the insect was fed for 24 h, then starved for 24 h, while they increased dramatically and rapidly under the condition of starvation for 24 h then feeding for 24 h. These results suggest that BdCHS2 may play an important role in regulating chitin content of the midgut, and subsequently affect the growth and development of B. dorsalis. PMID:23965972

Chen, Li; Yang, Wen-Jia; Cong, Lin; Xu, Kang-Kang; Wang, Jin-Jun

2013-01-01

26

Undecaprenyl diphosphate synthase inhibitors: antibacterial drug leads.  

PubMed

There is a significant need for new antibiotics due to the rise in drug resistance. Drugs such as methicillin and vancomycin target bacterial cell wall biosynthesis, but methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) have now arisen and are of major concern. Inhibitors acting on new targets in cell wall biosynthesis are thus of particular interest since they might also restore sensitivity to existing drugs, and the cis-prenyl transferase undecaprenyl diphosphate synthase (UPPS), essential for lipid I, lipid II, and thus, peptidoglycan biosynthesis, is one such target. We used 12 UPPS crystal structures to validate virtual screening models and then assayed 100 virtual hits (from 450,000 compounds) against UPPS from S. aureus and Escherichia coli. The most promising inhibitors (IC50 ?2 ?M, Ki ?300 nM) had activity against MRSA, Listeria monocytogenes, Bacillus anthracis, and a vancomycin-resistant Enterococcus sp. with MIC or IC50 values in the 0.25-4 ?g/mL range. Moreover, one compound (1), a rhodanine with close structural similarity to the commercial diabetes drug epalrestat, exhibited good activity as well as a fractional inhibitory concentration index (FICI) of 0.1 with methicillin against the community-acquired MRSA USA300 strain, indicating strong synergism. PMID:24827744

Sinko, William; Wang, Yang; Zhu, Wei; Zhang, Yonghui; Feixas, Ferran; Cox, Courtney L; Mitchell, Douglas A; Oldfield, Eric; McCammon, J Andrew

2014-07-10

27

Large-Scale Phylogenetic Classification of Fungal Chitin Synthases and Identification of a Putative Cell-Wall Metabolism Gene Cluster in Aspergillus Genomes  

PubMed Central

The cell wall is a protective and versatile structure distributed in all fungi. The component responsible for its rigidity is chitin, a product of chitin synthase (Chsp) enzymes. There are seven classes of chitin synthase genes (CHS) and the amount and type encoded in fungal genomes varies considerably from one species to another. Previous Chsp sequence analyses focused on their study as individual units, regardless of genomic context. The identification of blocks of conserved genes between genomes can provide important clues about the interactions and localization of chitin synthases. On the present study, we carried out an in silico search of all putative Chsp encoded in 54 full fungal genomes, encompassing 21 orders from five phyla. Phylogenetic studies of these Chsp were able to confidently classify 347 out of the 369 Chsp identified (94%). Patterns in the distribution of Chsp related to taxonomy were identified, the most prominent being related to the type of fungal growth. More importantly, a synteny analysis for genomic blocks centered on class IV Chsp (the most abundant and widely distributed Chsp class) identified a putative cell wall metabolism gene cluster in members of the genus Aspergillus, the first such association reported for any fungal genome. PMID:25148134

Pacheco-Arjona, Jose Ramon; Ramirez-Prado, Jorge Humberto

2014-01-01

28

Laboratory evaluation of five chitin synthesis inhibitors against the colorado potato beetle, Leptinotarsa decemlineata.  

PubMed

Results of laboratory experiments are reported that tested the effects of five chitin synthesis inhibitors, diflubenzuron, cyromazine, lufenuron, hexaflumuron and triflumuron. on second instars of the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Crysomelidae), originally collected from potato fields of Bostanabaad, a town 66 km southeast of Tabriz, Iran. In bioassays, the larvae were fed potato leaves dipped in aqueous solutions containing chitin synthesis inhibitors. The mortalities and abnormalities of the treated larvae were recorded 72 hours after treatments. LC(50) values were 58.6, 69.6, 27.3, 0.79 and 81.4 mg ai/ L for diflubenzuron, cyromazine, lufenuron, hexaflumuron and triflumuron, respectively. Compared with phosalone, which is one of the common insecticides used for controlling this pest in Iran, lufenuron and hexaflumuron seem to be much more potent, and if they perform equally well in the field, they would be suitable candidates to be considered as reduced risk insecticides in management programs for L. decemlineata due to much wider margin of safety for mammals and considerably fewer undesirable environmental side effects. PMID:20345285

Karimzadeh, R; Hejazi, M J; Rahimzadeh Khoei, F; Moghaddam, M

2007-01-01

29

Laboratory Evaluation of Five Chitin Synthesis Inhibitors Against the Colorado Potato Beetle, Leptinotarsa decemlineata  

PubMed Central

Results of laboratory experiments are reported that tested the effects of five chitin synthesis inhibitors, diflubenzuron, cyromazine, lufenuron, hexaflumuron and triflumuron. on second instars of the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Crysomelidae), originally collected from potato fields of Bostanabaad, a town 66 km southeast of Tabriz, Iran. In bioassays, the larvae were fed potato leaves dipped in aqueous solutions containing chitin synthesis inhibitors. The mortalities and abnormalities of the treated larvae were recorded 72 hours after treatments. LC50 values were 58.6, 69.6, 27.3, 0.79 and 81.4 mg ai/ L for diflubenzuron, cyromazine, lufenuron, hexaflumuron and triflumuron, respectively. Compared with phosalone, which is one of the common insecticides used for controlling this pest in Iran, lufenuron and hexaflumuron seem to be much more potent, and if they perform equally well in the field, they would be suitable candidates to be considered as reduced risk insecticides in management programs for L. decemlineata due to much wider margin of safety for mammals and considerably fewer undesirable environmental side effects. PMID:20345285

Karimzadeh, R.; Hejazi, M. J.; Rahimzadeh Khoei, F.; Moghaddam, M.

2007-01-01

30

La chitine synthase de Neocallimastix frontalis, un marqueur enzymatique de la biomasse fongique  

E-print Network

est donc possible de calculer la quantité de mycélium contenu dans une culture. Ainsi, une expérience dans une culture de champignon du rumen avec une marge d'erreur inférieure à 10 %. De plus ce dosage ne prend en compte que le mycélium vivant alors que le dosage de chitine (Akin, 1987) peu

Paris-Sud XI, Université de

31

Farnesyl diphosphate synthase inhibitors from in silico screening.  

PubMed

The relaxed complex scheme is an in silico drug screening method that accounts for receptor flexibility using molecular dynamics simulations. Here, we used this approach combined with similarity searches and experimental inhibition assays to identify several low micromolar, non-bisphosphonate inhibitors, bisamidines, of farnesyl diphosphate synthase (FPPS), an enzyme targeted by some anticancer and antimicrobial agents and for the treatment of bone resorption diseases. This novel class of farnesyl diphosphate synthase inhibitors have more drug-like properties than existing bisphosphonate inhibitors, making them interesting pharmaceutical leads. PMID:23421555

Lindert, Steffen; Zhu, Wei; Liu, Yi-Liang; Pang, Ran; Oldfield, Eric; McCammon, J Andrew

2013-06-01

32

Farnesyl Diphosphate Synthase Inhibitors from In Silico Screening  

PubMed Central

The relaxed complex scheme is an in silico drug screening method that accounts for receptor flexibility using molecular dynamics simulations. Here, we used this approach combined with similarity searches and experimental inhibition assays to identify several low micromolar, non-bisphosphonate inhibitors, bisamidines, of farnesyl diphosphate synthase (FPPS), an enzyme targeted by some anticancer and antimicrobial agents and for the treatment of bone resorption diseases. This novel class of farnesyl diphosphate synthase inhibitors have more drug-like properties than existing bisphosphonate inhibitors, making them interesting pharmaceutical leads. PMID:23421555

Lindert, Steffen; Zhu, Wei; Liu, Yi-Liang; Pang, Ran; Oldfield, Eric; McCammon, J Andrew

2013-01-01

33

Laboratory Evaluation of Flurox, a Chitin Synthesis Inhibitor, on the Termite, Microcerotermes diversus  

PubMed Central

Microcerotermes diversus (Silvestri) (Isoptera: Termitidae) is the most economically destructive termite in structures in southwest Iran. One sustainable control strategy that usually helps to reduce subterranean termite damage in buildings, is the use of insect growth regualtors in a suitable bait matrix that are safe to the user and the environment. In the laboratory assays described here, the delayed toxicity of Flurox, a chitin synthesis inhibitor, to M. diversus was evaluated under force-feeding and choice trials. Flurox induced worker and nymph mortality and incomplete ecdysis in nymphs of M. diversus under no-choice and two-choice feeding tests. These adverse effects may cause disruption of the caste balance in M. diversus, leading to the collapse of the colony. These assays determined concentrations of Flurox that can be used in bait formulations. PMID:20569123

Habibpour, Behzad

2010-01-01

34

Physiological, biochemical, and microscopic analyses of weakened walls of conidia in Colletotrichum graminicola with a disrupted class V chitin synthase gene, chsA  

Microsoft Academic Search

Colletotrichum graminicola (teleomorph, Glomerella graminicola) mutant T30 has a disruption in chsA, which is a class V or a class VI chitin synthase (class VI if recognized). The mutant conidia were significantly easier to fracture with glass beads than conidia of the wild type and two T30-derived strains that were complemented with chsA. Similarly, after extraction in alkali, the walls

A. Amnuaykanjanasin; L. Epstein; J. M. Labavitch

2003-01-01

35

Morphological changes of the filamentous fungus Mucor mucedo and inhibition of chitin synthase activity induced by anethole.  

PubMed

trans-Anethole (anethole), a major component of anise oil, has a broad antimicrobial spectrum with antimicrobial activity relatively weaker than those of well-known antibiotics, and significantly enhances the antifungal activity of polygodial and dodecanol against the baker's yeast Saccharomyces cerevisiae and human pathogenic yeast Candida albicans. However, the antifungal mechanism of anethole is unresolved. Anethole demonstrated antifungal activity against the filamentous fungus, Mucor mucedo IFO 7684, accompanied by hyphal morphological changes such as swollen hyphae at the tips. Its minimum growth inhibitory concentration was 0.625?mM. A hyperosmotic condition (1.2?M sorbitol) restricted the induction of morphological changes, while hypoosmotic treatment (distilled water) induced bursting of hyphal tips and leakage of cytoplasmic constituents. Furthermore, anethole dose-dependently inhibited chitin synthase (CHS) activity in permeabilized hyphae in an uncompetitive manner. These results suggest that the morphological changes of M. mucedo could be explained by the fragility of cell walls caused by CHS inhibition. PMID:21721062

Yutani, Masahiro; Hashimoto, Yukie; Ogita, Akira; Kubo, Isao; Tanaka, Toshio; Fujita, Ken-ichi

2011-11-01

36

Prostaglandin H synthase-2 inhibitors interfere with prostaglandin H synthase-1 inhibition by nonsteroidal anti-inflammatory drugs  

Microsoft Academic Search

Ram seminal vesicle microsomes, a rich source of prostaglandin H synthase-1, were incubated with 100 nM of the prostaglandin H synthase-2 inhibitors N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide (NS-398) and 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonyl) thiophene (DuP-697) prior to exposure to the prostaglandin H synthase inhibitors aspirin, indomethacin, ibuprofen or naproxen. Activity of the enzyme was measured by following the conversion of arachidonic acid to prostaglandin E2 and

Moti Rosenstock; Abraham Danon; Mazal Rubin; Gilad Rimon

2001-01-01

37

Evaluation of Two Formulated Chitin Synthesis Inhibitors, Hexaflumuron and Lufenuron Against the Raisin Moth, Ephestia figulilella  

PubMed Central

The raisin moth, Ephestia figulilella Gregson (Lepidoptera: Pyralidae), has a nearly cosmopolitan distribution, and causes severe quantitative and qualitative losses throughout the world. The larvae attack various drying and dried fruits, fallen figs, and damaged or moldy clusters of grapes on vines. Control of this pest in storage depends mostly on synthetic pesticides with several adverse side effects. To mitigate the adverse effects of these pesticides, investigations have focused on the development of compounds with more selectivity, and short residual life. In this research, insecticidal effects of two chitin synthesis inhibitors, hexaflumuron and lufenuron, were investigated against E. figulilella. Graded concentrations of each pesticide were prepared with distilled water. One-day-old fifth instar were sprayed by Potter's precision spray tower. Application of hexaflumuron and lufenuron on last instar larvae of E. figulilella caused not only mortality in larval stage, but also caused defects in pupal and adult stages. Larval mortality increased as concentration increased. The longevity of the fifth instars in both hexaflumuron and lufenuron treatments, in comparison with the controls, increased by more than 12 days. The longevity of adults decreased by about 10 days. Probit analysis data revealed that the sensitivity of the test insect to hexaflumuron (EC50 = 95.38 ppm) was greater than lufenuron (EC50= 379.21 ppm). PMID:23425138

Khajepour, Simin; Izadi, Hamzeh; Asari, Mohammad Javad

2012-01-01

38

Inhibitors of glycogen synthase 3 kinase  

DOEpatents

Compounds of formula 1: ##STR1## wherein R.sub.1 is alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl, substituted with 0-3 substituents selected from lower alkyl, halo, hydroxy, lower alkoxy, amino, lower alkyl-amino, and nitro; R.sub.2 is hydroxy, amino, or lower alkoxy; R.sub.3 is H, lower alkyl, lower acyl, lower alkoxy-acyl, or amnino-acyl; R.sub.4 is H or lower alkyl; and pharmaceutically acceptable salts and esters thereof; are effective inhibitors of GSK3.

Schultz, Peter (Oakland, CA); Ring, David B. (Palo Alto, CA); Harrison, Stephen D. (Berkeley, CA); Bray, Andrew M. (Victoria, AU)

2000-01-01

39

The myosin motor domain-containing chitin synthase PdChsVII is required for development, cell wall integrity and virulence in the citrus postharvest pathogen Penicillium digitatum.  

PubMed

Chitin is an essential component of the fungal cell wall and a potential target in the development of new antifungal compounds, due to its presence in fungi and not in plants or vertebrates. Chitin synthase genes (chs) constitute a complex family in filamentous fungi and are involved in fungal development, morphogenesis, pathogenesis and virulence. In this study, additional chs genes in the citrus postharvest pathogen Penicillium digitatum have been identified. Comparative analyses included each PdChs in each one of the classes I to VII previously established, and support the grouping of these into three divisions. Disruption of the gene coding PdChsVII, which contains a short version of a myosin motor domain, has been achieved by using Agrobacterium tumefaciens-mediated transformation and revealed its role in the life cycle of the fungus. Disruption strains were viable but showed reduced growth and conidia production. Moreover, Pdchs mutants developed morphological defects as balloon-like enlarged cells and increased chitin content, indicative of an altered cell wall structure. Gene disruption also increased susceptibility to antifungal compounds such as calcofluor white (CFW), sodium dodecyl sulfate (SDS), hydroxide peroxide (H2O2) and commercial fungicides, but significantly no change was observed in the sensitivity to antifungal peptides. The PdchsVII mutants were able to infect citrus fruit and produced tissue maceration, although had reduced virulence and most importantly were greatly impaired in the production of visible mycelium and conidia on the fruit. PMID:24727399

Gandía, Mónica; Harries, Eleonora; Marcos, Jose F

2014-06-01

40

Antifolate inhibitors of thymidylate synthase as anticancer drugs.  

PubMed

Inhibitors of thymidylate synthase (TS) play an essential role in the pharmacological management of several tumors. Two antifolates, Raltitrexed and Pemetrexed, are licensed anticancer drugs, with Pemetrexed, unlike Raltitrexed, undergoing further intense clinical development. Other antifolate TS inhibitors, recently/currently tested in clinical studies, that show encouraging anticancer activities are Plevitrexed, GW7904L and Nolatrexed. A new prospect among antifolates, demonstrating a very desirable pattern of pharmacological properties, is BGC 945 that showed promising antitumor activities and has been nominated for clinical development. In this paper, apart from reviewing their biochemical and pharmacological properties, up-to-date characteristics of clinical development of all the mentioned agents are presented. In addition, trends and perspectives for developing improved antifolate inhibitors of TS and future drugs are discussed. Drug resistance is the main barrier to more effective treatment of cancers with antifolates; therefore, mechanisms of antifolate resistance and currently applied approaches to overcome it are also pointed out in the review. PMID:20854257

Jarmu?a, A

2010-11-01

41

Aldosterone synthase inhibitors in hypertension: current status and future possibilities.  

PubMed

The renin-angiotensin aldosterone system is a critical mechanism for controlling blood pressure, and exerts most of its physiological effects through the action of angiotensin II. In addition to increasing blood pressure by increasing vascular resistance, angiotensin II also stimulates aldosterone secretion from the adrenal gland. Aldosterone acts to cause an increase in sodium and water reabsorption, thus elevating blood pressure. Although treatment with angiotensin converting enzyme inhibitors initially lowers circulating aldosterone, with chronic treatment aldosterone levels increase back to baseline, a phenomenon termed aldosterone escape; aldosterone blockade may therefore give added value in the treatment of hypertension. The first mineralocorticoid receptor antagonist developed was spironolactone, but its use has been severely hampered by adverse (notably oestrogenic) effects. The more recently developed mineralocorticoid receptor antagonist eplerenone exhibits a better adverse effect profile, although it is not devoid of effects similar to spironolactone. In addition, aldosterone activates non-genomic receptors that are not inhibited by either eplerenone or spironolactone. It is believed that deleterious organ remodelling is mediated by aldosterone via such non-genomic pathways. A new class of drugs, the aldosterone synthase inhibitors, is currently under development. These may offer a novel therapeutic approach for both lowering blood pressure and preventing the non-genomic effects of aldosterone. Here, we will review the cardiovascular effects of aldosterone and review the drugs available that target this hormone, with a particular focus on the aldosterone synthase inhibitors. PMID:24570839

Hargovan, Milan; Ferro, Albert

2014-01-01

42

Structural Studies of Pterin-Based Inhibitors of Dihydropteroate Synthase  

PubMed Central

Dihydropteroate synthase (DHPS) is a key enzyme in bacterial folate synthesis and the target of the sulfonamide class of antibacterials. Resistance and toxicities associated with sulfonamides have led to a decrease in their clinical use. Compounds that bind to the pterin binding site of DHPS, as opposed to the p-amino benzoic acid (pABA) binding site targeted by the sulfonamide agents, are anticipated to bypass sulfonamide resistance. To identify such inhibitors and map the pterin binding pocket, we have performed virtual screening, synthetic, and structural studies using Bacillus anthracis DHPS. Several compounds with inhibitory activity have been identified, and crystal structures have been determined that show how the compounds engage the pterin site. The structural studies identify the key binding elements and have been used to generate a structure-activity based pharmacophore map that will facilitate the development of the next generation of DHPS inhibitors which specifically target the pterin site. PMID:19899766

Hevener, Kirk E.; Yun, Mi-Kyung; Qi, Jianjun; Kerr, Iain D.; Babaoglu, Kerim; Hurdle, Julian G.; Balakrishna, Kanya; White, Stephen W.; Lee, Richard E.

2009-01-01

43

Suppression of arthritis by an inhibitor of nitric oxide synthase  

PubMed Central

Nitric oxide (NO), a toxic radical gas produced during the metabolism of L-arginine by NO synthase (NOS), has been implicated as a mediator of immune and inflammatory responses. A single injection of streptococcal cell wall fragments (SCW) induces the accumulation of inflammatory cells within the synovial tissue and a cell-mediated immune response that leads destructive lesions. We show here that NO production is elevated in the inflamed joints of SCW-treated rats. Administration of NG-monomethyl-L-arginine, an inhibitor of NOS, profoundly reduced the synovial inflammation and tissue damage as measured by an articular index and reflected in the histopathology. These studies implicate the NO pathway in the pathogenesis of an inflammatory arthritis and demonstrate the ability of a NOS inhibitor to modulate the disease. PMID:7688035

1993-01-01

44

Glycogen synthase kinase-3 inhibitors: Rescuers of cognitive impairments.  

PubMed

Impairment of cognitive processes is a devastating outcome of many diseases, injuries, and drugs affecting the central nervous system (CNS). Most often, very little can be done by available therapeutic interventions to improve cognitive functions. Here we review evidence that inhibition of glycogen synthase kinase-3 (GSK3) ameliorates cognitive deficits in a wide variety of animal models of CNS diseases, including Alzheimer's disease, Fragile X syndrome, Down syndrome, Parkinson's disease, spinocerebellar ataxia type 1, traumatic brain injury, and others. GSK3 inhibitors also improve cognition following impairments caused by therapeutic interventions, such as cranial irradiation for brain tumors. These findings demonstrate that GSK3 inhibitors are able to ameliorate cognitive impairments caused by a diverse array of diseases, injury, and treatments. The improvements in impaired cognition instilled by administration of GSK3 inhibitors appear to involve a variety of different mechanisms, such as supporting long-term potentiation and diminishing long-term depression, promotion of neurogenesis, reduction of inflammation, and increasing a number of neuroprotective mechanisms. The potential for GSK3 inhibitors to repair cognitive deficits associated with many conditions warrants further investigation of their potential for therapeutic interventions, particularly considering the current dearth of treatments available to reduce loss of cognitive functions. PMID:23916593

King, Margaret K; Pardo, Marta; Cheng, Yuyan; Downey, Kimberlee; Jope, Richard S; Beurel, Eléonore

2014-01-01

45

Molecular docking studies to map the binding site of squalene synthase inhibitors on dehydrosqualene synthase of Staphylococcus aureus.  

PubMed

Dehydrosqualene synthase of Staphylococcus aureus is involved in the synthesis of golden carotenoid pigment staphyloxanthin. This pigment of S. aureus provides the antioxidant property to this bacterium to survive inside the host cell. Dehydrosqualene synthase (CrtM) is having structural similarity with the human squalene synthase enzyme which is involved in the cholesterol synthesis pathway in humans (Liu et al., 2008). Cholesterol lowering drugs were found to have inhibitory effect on dehydrosqualene synthase enzyme of S. aureus. The present study attempts to focus on squalene synthase inhibitors, lapaquistat acetate and squalestatins reported as cholesterol lowering agents in vitro and in vivo but not studied in context to dehydrosqualene synthase of S. aureus. Mode of binding of lapaquistat acetate and squalestatin analogs on dehydrosqualene synthase (CrtM) enzyme of S. aureus was identified by performing docking analysis with Scigress Explorer Ultra 7.7 docking software. Based on the molecular docking analysis, it was found that the His18, Arg45, Asp48, Asp52, Tyr129, Gln165, Asn168 and Asp172 residues interacted with comparatively high frequency with the inhibitors studied. Comparative docking study with Discovery studio 2.0 also confirmed the involvement of these residues of dehydrosqualene synthase enzyme with the inhibitors studied. This further confirms the importance of these residues in the enzyme function. In silico ADMET analysis was done to predict the ADMET properties of the standard drugs and test compounds. This might provide insights to develop new drugs to target the virulence factor, dehydrosqualene synthase of S. aureus. PMID:20645653

Kahlon, Amandeep Kaur; Roy, Sudeep; Sharma, Ashok

2010-10-01

46

Synthesis of Novel ?-Lactone Inhibitors of Fatty Acid Synthase  

PubMed Central

Fatty acid synthase (FAS) is necessary for growth and survival of tumor cells and is a promising drug target for oncology. Here, we report on the syntheses and activity of novel inhibitors of the thioesterase domain of FAS. Using the structure of orlistat as a starting point, which contains a ?-lactone as the central pharmacophore, 28 novel congeners were synthesized and examined. Structural features such as the length of the ?- and ?-alkyl chains, their chemical composition, and amino ester substitutions were altered and the resulting compounds explored for inhibitory activity toward the thioesterase domain of FAS. Nineteen congeners show improved potency for FAS in biochemical assays relative to orlistat. Three of that subset, including the natural product valilactone, also display an increased potency in inducing tumor cell death and improved solubility compared to orlistat. These findings support the idea that an orlistat congener can be optimized for use in a preclinical drug design and for clinical drug development. PMID:18710210

Richardson, Robyn D.; Ma, Gil; Oyola, Yatsandra; Zancanella, Manuel; Knowles, Lynn M.; Cieplak, Piotr; Romo, Daniel; Smith, Jeffrey W.

2011-01-01

47

Isolation and structural determination of squalene synthase inhibitor from Prunus mume fruit.  

PubMed

Squalene synthase plays an important role in the cholesterol biosynthetic pathway. Inhibiting this enzyme in hypercholesterolemia can lower not only plasma cholesterol but also plasma triglyceride levels. A squalene synthase inhibitor was screened from Prunus mume fruit, and then purified via sequential processes of ethanol extraction, HP-20 column chromatography, ethyl acetate extraction, silica gel column chromatography, and crystallization. The squalene synthase inhibitor was identified as chlorogenic acid with a molecular mass of 354 Da and a molecular formula of C16H18O9 based on UV spectrophotometry, 1H and 13C NMRs, and mass spectrometry. Chlorogenic acid inhibited the squalene synthase of pig liver with an IC50 level of 100 nM. Since chlorogenic acid was an effective inhibitor against the squalene synthase of an animal source, it may be a potential therapeutic agent for hypercholesterolemia. PMID:18167444

Choi, Sung-Won; Hur, Nam-Yoon; Ahn, Soon-Cheol; Kim, Dong-Seob; Lee, Jae-Kwon; Kim, Dae-Ok; Park, Seung-Kook; Kim, Byung-Yong; Baik, Moo-Yeol

2007-12-01

48

Short Communication The nitric oxide synthase inhibitor l-NAME suppresses androgen-induced  

E-print Network

Short Communication The nitric oxide synthase inhibitor l-NAME suppresses androgen-induced male anesthesia and each implanted with a Silastic tube (Helix Medical, 10 mm long, internal diameter 1.47 mm

Crews, David

49

Design of more potent squalene synthase inhibitors with multiple activities.  

PubMed

With the increasing realization that modulating a multiplicity of targets can be an asset in the treatment of multifactorial disorders, we hereby report the synthesis and evaluation of the first compounds in which antioxidant, anti-inflammatory as well as squalene synthase (SQS) inhibitory activities are combined by design, in a series of simple molecules, extending their potential range of activities against the multifactorial disease of atherosclerosis. The activity of the initially synthesized antihyperlipidemic morpholine derivatives (1-6), in which we combined several pharmacophore moieties, was evaluated in vitro (antioxidant, inhibition of SQS and lipoxygenase) and in vivo (anti-dyslipidemic and anti-inflammatory effect). We further compared the in vitro SQS inhibitory action of these derivatives with theoretically derived molecular interactions by performing an in silico docking study using the X-ray crystal structure of human SQS. Based on low energy preferred binding modes, we designed potentially more potent SQS ligands. We proceeded with synthesizing and evaluating these new structures (7-12) in vitro and in vivo, to show that the new derivatives were significantly more active than formerly developed congeners, both as SQS inhibitors (20-70-fold increase in activity) and antioxidants (4-30-fold increase in activity). A significant correlation between experimental activity [Log(1/IC(50))] and the corresponding binding free energy (?G(b)) of the docked compounds was shown. These results, taken together, show a promising alternative and novel approach for the design and development of multifunctional antiatherosclerosis agents. PMID:20888243

Kourounakis, Angeliki P; Matralis, Alexios N; Nikitakis, Anastasios

2010-11-01

50

Behavioral and histological changes in the Formosan subterranean termite (Isoptera: Rhinotermitidae) induced by the chitin synthesis inhibitor noviflumuron.  

PubMed

This study describes the behavioral and histological changes of the molting process in Coptotermes formosanus Shiraki caused by the chitin synthesis inhibitor noviflumuron. Termites exposed to noviflumuron initiated ecdysis as untreated individuals did; however, peristalsis contractions were weak and the expansion of the dorsal breach of the exoskeleton did not occur. Treated termites could not complete their molting process and died after the initiation of the ecdysis. Histological observations showed that the process of voiding the gut protozoa during premolting was not affected by the noviflumuron treatment. However, the formation of the new cuticle was disrupted resulting in the loss of integrity of the cuticle. The alteration of the cuticle was visible in the gizzard (foregut), the thoracic pleurons, and most of the exoskeleton. Muscles were partially able to reattach to the incompletely formed new cuticle, and muscle contractions resulted in tearing off the cuticle. Because the integrity of the newly formed cuticle was compromised by the noviflumuron treatment, we concluded that termites' death was caused primarily by the loss of hemolymph as a result of the damage done by the muscle contractions on the exoskeleton during the peristalsis. As the physiological homeostasis was disrupted, termites were too weak to shed their old cuticle, ultimately resulting in termite dying during the molting process. PMID:24772556

Xing, Lin; Chouvenc, Thomas; Su, Nan-Yao

2014-04-01

51

The efficacy of a chitin synthesis inhibitor against field populations of organophosphate-resistant Aedes aegypti in Brazil.  

PubMed

The mosquito Aedes aegypti is the main focus of dengue control campaigns. Because of widespread resistance against conventional chemical insecticides, chitin synthesis inhibitors (CSIs) are considered control alternatives. We evaluated the resistance status of four Brazilian Ae. aegypti populations to both the organophosphate temephos and the pyrethroid deltamethrin, which are used in Brazil to control larvae and adults, respectively. All vector populations exhibited high levels of temephos resistance and varying rates of alterations in their susceptibility to pyrethroids. The effect of the CSI novaluron on these populations was also investigated. Novaluron was effective against all populations under laboratory conditions. Field-simulated assays with partial water replacement were conducted to evaluate novaluron persistence. Bioassays were continued until an adult emergence inhibition of at least 70% was attained. We found a residual effect of eight weeks under indoor conditions and novaluron persisted for five-six weeks in assays conducted in an external area. Our data show that novaluron is effective against the Ae. aegypti populations tested, regardless of their resistance to conventional chemical insecticides. PMID:22510835

Fontoura, Nathalia Giglio; Bellinato, Diogo Fernandes; Valle, Denise; Lima, José Bento Pereira

2012-05-01

52

SHORT REPORT Open Access Saccharomyces cerevisiae chitin biosynthesis  

E-print Network

SHORT REPORT Open Access Saccharomyces cerevisiae chitin biosynthesis activation by N added to a chitin synthase assay performed on a Saccharomyces cerevisiae membrane fraction. Findings: N]. In Saccharomyces cerevisiae, the organism in which chitin biosynthesis has been most studied, three differentially

Paris-Sud XI, Université de

53

Chloropropionyl-CoA: a mechanism-based inhibitor of HMG-CoA synthase and fatty acid synthase  

SciTech Connect

Recent work on the mechanisms of inactivation of HMG-CoA synthase and fatty acid synthase by chloropropionyl-CoA (Cl-prop-CoA) suggests that this analog is a mechanism-based (suicide) inhibitor; the acyl group is enzymatically converted to an acrylyl derivative prior to alkylation of the target proteins. When Cl-(/sup 3/H)prop-CoA is incubated with the target enzymes, /sup 3/H/sub 2/O is produced concomitantly with enzyme inactivation; this suggests that deprotonation and chloride elimination to form an acrylyl moiety occurs. Difficulty in cleanly synthesizing acrylyl-CoA complicates direct demonstration of the intermediacy of this species. However, synthesis of a functionally equivalent reactive substrate analog, S-acrylyl-N-acetylcysteamine has been accomplished. This analog irreversibly inhibits both HMG-CoA synthase and fatty acid synthase in a site directed fashion. Concentrations required for effective inhibition (K/sub i/ values of 1.9 mM and 3.6 mM, respectively) are much higher than observed with Cl-prop-CoA. Maximal rates of inactivation (as vertical bar ..-->.. infinity) are comparable to those measured with Cl-prop-CoA, indicating that an acrylyl derivative is kinetically competent to function as an intermediate, as required if Cl-prop-CoA is a mechanism-based inhibitor. S-acrylyl-N-acetylcysteamine also inactivates HMG-CoA lyase. In this case, kinetic studies indicate that a bimolecular process is involved (k/sub 2/ = 86.7M/sup -1/min/sup -1/ at 30/sup 0/, pH 7.0).

Miziorko, H.M.; Ahmad, F.; Behnke, C.E.

1986-05-01

54

MULTI-ANALYTE CHEMISTRY METHODS FOR PESTICIDES WHICH ARE ACETOLACTATE SYNTHASE (ALS) INHIBITORS IN SOIL  

EPA Science Inventory

A joint EPA/state/industry working group has developed several multi-analyte methods to analyze soils for low ppb (parts per billion) levels of herbicides (such as sulfonylureas, imidazolinones, and sulfonamides) that are acetolactate synthase (ALS) inhibitors and may cause phyto...

55

Genetic rearrangements on the Chlorovirus genome that switch between hyaluronan synthesis and chitin synthesis  

Microsoft Academic Search

Chlorella viruses or chloroviruses form polysaccharide fibers on the cell wall of host Chlorella cells after infection. Such polysaccharides are either hyaluronan synthesized by virus-encoded hyaluronan synthase (HAS) or chitin synthesized by viral chitin synthase (CHS). Some chloroviruses synthesize both hyaluronan (HA) and chitin simultaneously. To understand the relationship between “HA-synthesizing” and “chitin-synthesizing” viruses, we characterized the CVK2 genomic regions,

Ali Mohammed Mohammed Ali; Takeru Kawasaki; Takashi Yamada

2005-01-01

56

Identification, mRNA Expression, and Functional Analysis of Chitin Synthase 1 Gene and Its Two Alternative Splicing Variants in Oriental Fruit Fly, Bactrocera dorsalis  

PubMed Central

Two alternative splicing variants of chitin synthase 1 gene (BdCHS1) were cloned and characterized from the oriental fruit fly, Bactrocera dorsalis (Hendel). The cDNA of both variants (BdCHS1a and BdCHS1b) consisted of 5,552 nucleotides (nt), with an open reading frame (ORF) of 4,776 nt, encoding a protein of 1,592 amino acid residues, plus 685- and 88-nt of 5?- and 3?-noncoding regions, respectively. The alternative splicing site was located between positions 3,784-3,960 and formed a pair of mutually exclusive exons (a/b) that were same in size (177 nt), but showed only 65% identity at the nucleotide level. During B. dorsalis growth and development, BdCHS1 and BdCHS1a were both mainly expressed during the larval-pupal and pupal-adult transitions, while BdCHS1b was mainly expressed during pupal-adult metamorphosis and in the middle of the pupal stage. BdCHS1a was predominately expressed in the integument whereas BdCHS1b was mainly expressed in the trachea. The 20-hydroxyecdysone (20E) induced the expression of BdCHS1 and its variants. Injection of dsRNA of BdCHS1, BdCHS1a, and BdCHS1b into third-instar larvae significantly reduced the expression levels of the corresponding variants, generated phenotypic defects, and killed most of the treated larvae. Furthermore, silencing of BdCHS1 and BdCHS1a had a similar result in that the larva was trapped in old cuticle and died without tanning completely, while silencing of BdCHS1b has no effect on insect morphology. These results demonstrated that BdCHS1 plays an important role in the larval-pupal transition and the expression of BdCHS1 in B. dorsalis is regulated by 20E. PMID:23569438

Yang, Wen-Jia; Xu, Kang-Kang; Cong, Lin; Wang, Jin-Jun

2013-01-01

57

Small-molecule inhibitor binding to an N-acyl-homoserine lactone synthase.  

PubMed

Quorum sensing (QS) controls certain behaviors of bacteria in response to population density. In gram-negative bacteria, QS is often mediated by N-acyl-L-homoserine lactones (acyl-HSLs). Because QS influences the virulence of many pathogenic bacteria, synthetic inhibitors of acyl-HSL synthases might be useful therapeutically for controlling pathogens. However, rational design of a potent QS antagonist has been thwarted by the lack of information concerning the binding interactions between acyl-HSL synthases and their ligands. In the gram-negative bacterium Burkholderia glumae, QS controls virulence, motility, and protein secretion and is mediated by the binding of N-octanoyl-L-HSL (C8-HSL) to its cognate receptor, TofR. C8-HSL is synthesized by the acyl-HSL synthase TofI. In this study, we characterized two previously unknown QS inhibitors identified in a focused library of acyl-HSL analogs. Our functional and X-ray crystal structure analyses show that the first inhibitor, J8-C8, binds to TofI, occupying the binding site for the acyl chain of the TofI cognate substrate, acylated acyl-carrier protein. Moreover, the reaction byproduct, 5'-methylthioadenosine, independently binds to the binding site for a second substrate, S-adenosyl-L-methionine. Closer inspection of the mode of J8-C8 binding to TofI provides a likely molecular basis for the various substrate specificities of acyl-HSL synthases. The second inhibitor, E9C-3oxoC6, competitively inhibits C8-HSL binding to TofR. Our analysis of the binding of an inhibitor and a reaction byproduct to an acyl-HSL synthase may facilitate the design of a new class of QS-inhibiting therapeutic agents. PMID:21730159

Chung, Jiwoung; Goo, Eunhye; Yu, Sangheon; Choi, Okhee; Lee, Jeehyun; Kim, Jinwoo; Kim, Hongsup; Igarashi, Jun; Suga, Hiroaki; Moon, Jae Sun; Hwang, Ingyu; Rhee, Sangkee

2011-07-19

58

Synthesis of Bi-substrate State Mimics of Dihydropteroate Synthase as Potential Inhibitors and Molecular Probes  

PubMed Central

The increasing emergence of resistant bacteria drives us to design and develop new antimicrobial agents. Pursuant to that goal, a new targeting approach of the dihydropteroate synthase enzyme, which serves as the site of action for the sulfonamide class of antimicrobial agents, is being explored. Using structural information, a new class of transition state mimics has been designed and synthesized that have the capacity to bind to the pterin, phosphate and para-amino binding sites. The design, synthesis and evaluation of these compounds as inhibitors of Bacillus anthracis dihydropteroate synthase is described herein. Outcomes from this work have identified the first trivalent inhibitors of dihydropteroate synthase whose activity displayed slow binding inhibition. The most active compounds in this series contained an oxidized pterin ring. The binding of these inhibitors was modeled into the dihydropteroate synthase active site and demonstrated a good correlation with the observed bioassay data, as well as provided important insight for the future design of higher affinity transition state mimics. PMID:21216602

Qi, Jianjun; Virga, Kristopher G.; Das, Sourav; Zhao, Ying; Yun, Mi-Kyung; White, Stephen W.; Lee, Richard E.

2010-01-01

59

Serotonergic mediation of the antidepressant-like effects of nitric oxide synthase inhibitors.  

PubMed

Recent studies indicate that nitric oxide (NO) synthase inhibitors have antidepressant-like potential in various animal models. In the present study the behavioural activity of the NO synthase inhibitors, N(G)-nitro-L-arginine (L-NA) and 7-nitroindazole (7-NI), were assessed in a modified rat forced swimming test (FST). Both L-NA and 7-NI, dose dependently reduced immobility and increased swimming behaviour in the rat FST. This behavioural profile parallels the one previously shown with selective serotonin re-uptake inhibitors and serotonergic agonists. Thus, we examined the role of serotonin mediating the behavioural effects of L-NA and 7-NI in the rat FST. Depletion of endogenous serotonin using para-chlorophenylalanine (pCPA; 3 x 150 mg/kg, i.p.) completely blocked L-NA (20 mg/kg, i.p.) and 7-NI (20 mg/kg, i.p.)-induced reductions in immobility and increases in swimming behaviour during the FST. In conclusion these observations suggest that NO synthase inhibitors elicit their antidepressant-like activity in the modified swimming test through a serotonin dependent mechanism. PMID:12668047

Harkin, A; Connor, T J; Walsh, M; St John, N; Kelly, J P

2003-04-01

60

Intramolecular Hydrogen Bonding: A Potential Strategy for More Bioavailable Inhibitors of Neuronal Nitric Oxide Synthase  

PubMed Central

Selective neuronal nitric oxide synthase (nNOS) inhibitors have therapeutic applications in the treatment of numerous neurodegenerative diseases. Here we report the synthesis and evaluation of a series of inhibitors designed to have increased cell membrane permeability via intramolecular hydrogen bonding. Their potencies were examined in both purified enzyme and cell-based assays; a comparison of these results demonstrates that two of the new inhibitors display significantly increased membrane permeability over previous analogs. NMR spectroscopy provides evidence of intramolecular hydrogen bonding under physiological conditions in two of the inhibitors. Crystal structures of the inhibitors in the nNOS active site confirm the predicted non-intramolecular hydrogen bonded binding mode. Intramolecular hydrogen bonding may be an effective approach for increasing cell membrane permeability without affecting target protein binding. PMID:22370337

Labby, Kristin Jansen; Xue, Fengtian; Kraus, James M.; Ji, Haitao; Mataka, Jan; Li, Huiying; Martasek, Pavel; Roman, Linda J.; Poulos, Thomas L.; Silverman, Richard B.

2012-01-01

61

Cyclopropyl- and Methyl-Containing Inhibitors of Neuronal Nitric Oxide Synthase  

PubMed Central

Inhibitors of neuronal nitric oxide synthase have been proposed as therapeutics for the treatment of different types of neurological disorders. On the basis of a cis-3,4-pyrrolidine scaffold, a series of trans-cyclopropyl- and methyl-containing nNOS inhibitors have been synthesized. The insertion of a rigid electron-withdrawing cyclopropyl ring decreases the basicity of the adjacent amino group, which resulted in decreased inhibitory activity of these inhibitors compared to the parent compound. Nonetheless, three of them exhibited double-digit nanomolar inhibition with high nNOS selectivity on the basis of in vitro enzyme assays. Crystal structures of nNOS and eNOS with these inhibitors bound provide a basis for detailed structure-activity relationship (SAR) studies. The conclusions from these studies will be used as a guide in the future development of selective NOS inhibitors. PMID:23352768

Li, Huiying; Xue, Fengtian; Kraus, James M.; Ji, Haitao; Labby, Kristin Jansen; Mataka, Jan; Delker, Silvia L.; Martasek, Pavel; Roman, Linda J.; Poulos, Thomas L.; Silverman, Richard B.

2013-01-01

62

Squalene synthase inhibitors: An update on the search for new antihyperlipidemic and antiatherosclerotic agents.  

PubMed

Atherosclerosis and related heart disease is strongly associated with elevated blood levels of total (and LDL) cholesterol. Due to the widespread incidence as well as severity of this pathological condition, major efforts have been made for the discovery and development of hypocholesteroleamic agents. In the past few decades, HMG-CoA reductase inhibitors (statins) are being extensively used as lipid lowering drugs. These agents act predominantly by inhibiting the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) that is the rate limiting step of cholesterol biosynthesis. Both the success as well as drawbacks of HMGRIs, have led to the investigation and design of inhibitors of other (downstream) enzymes involved in the multistep cholesterol biosynthetic pathway. One such class of agents consists of the squalene sythase inhibitors which act at the first and solely committed step towards the biosynthesis of the cholesterol nucleus. This target is considered not to interfere with the biosynthesis of other biologically important molecules and thus a better side-effect profile is expected for these inhibitors. Several classes of squalene synthase inhibitors (SQSIs), such as substrate or transition-state analogues, zaragozic acids or 2,8- dioxabicyclo[3.2.1]octane derivatives, dicarboxylic acid and quinuclidine derivatives, 4,1-benzoxazepine as well as substituted morpholine derivatives, have been studied as potent inhibitors of squalene synthase. So far only one benzoxazepine derivative (TAK-475) has been evaluated in advanced clinical trials. In this article we review the up to date research and literature on the therapeutic potential of this relatively new class of compounds, the drug discovery efforts towards the development of active squalene synthase inhibitors, their activity profile and effectiveness, as well as their structure-activity relationships. PMID:21864285

Kourounakis, A P; Katselou, M G; Matralis, A N; Ladopoulou, E M; Bavavea, E

2011-01-01

63

Antimicrobial natural products as ?-ketoacyl-acyl carrier protein synthase III inhibitors  

Microsoft Academic Search

?-Ketoacyl-acyl carrier protein synthase III (KAS III) initiates bacterial fatty acid biosynthesis, making it one of the most promising condensing enzymes. In a previous study, we developed three pharmacophore maps from receptor-oriented pharmacophore-based in silico screening and proposed a potent antimicrobial inhibitor for KAS III. Using these pharmacophore maps, we examined a natural product database and chose 4 natural compounds

Jee-Young Lee; Ki-Woong Jeong; Soyoung Shin; Ju-Un Lee; Yangmee Kim

2009-01-01

64

Upregulation of Endothelial Nitric Oxide Synthase by HMG CoA Reductase Inhibitors  

Microsoft Academic Search

Background—Oxidized low-density lipoprotein (ox-LDL) causes endothelial dysfunction in part by decreasing the availability of endothelial nitric oxide (NO). Although HMG CoA reductase inhibitors restore endothelial function by reducing serum cholesterol levels, it is not known whether they can also directly upregulate endothelial NO synthase (ecNOS) activity. Methods and Results—Human saphenous vein endothelial cells were treated with ox-LDL (50 mg\\/mL thiobarbituric

Ulrich Laufs; Vito La Fata; Jorge Plutzky; James K. Liao

65

Possible mechanisms of action of nitric oxide synthase inhibitors in chronic tension-type headache  

Microsoft Academic Search

Summary It has been demonstrated recently that nitric oxide synthase (NOS) inhibition has an analgesic effect in patients with chronic tension-type headache. The aim of the present study was to investigate the influence of the NOS inhibitor, L-NG methyl arginine hydrochloride (L-NMMA), on two of the most prominent features of chronic tension-type headache, i.e. increased muscle hardness and increased myofascial

M. Ashina; L. Bendtsen; R. Jensen; L. H. Lassen; F. Sakai; J. Olesen

1999-01-01

66

Visualizing inducible nitric-oxide synthase in living cells with a heme-binding fluorescent inhibitor  

Microsoft Academic Search

The study of nitric-oxide synthase (NOS) physiology is constrained by the lack of suitable probes to detect NOS in living cells or animals. Here, we characterized a fluorescent inducible NOS (iNOS) inhibitor called PIF (pyrimidine imidazole FITC) and examined its utility for microscopic imaging of iNOS in living cells. PIF binding to iNOS displayed high affinity, isoform selectivity, and heme

Koustubh Panda; Mamta Chawla-Sarkar; Cecile Santos; Thomas Koeck; Serpil C. Erzurum; John F. Parkinson; Dennis J. Stuehr

2005-01-01

67

Syntheses and herbicidal activity of new triazolopyrimidine-2-sulfonamides as acetohydroxyacid synthase inhibitor  

Microsoft Academic Search

The triazolopyrimidine-2-sulfonanilide, discovered from preparing bioisosteres of the sulfonylurea herbicides, is an important class of acetohydroxyacid synthase (AHAS, EC 4.1.3.18) inhibitors. At least over ten triazolopyrimidine sulfonanilides have been commercialized as herbicides for the control of broadleaf weeds and grass with cereal crop selectivity. Herein, a series of triazolopyrimidine-2-sulfonanilides were designed and synthesized with the aim of discovery of new

Chao-Nan Chen; Qiong Chen; Yu-Chao Liu; Xiao-Lei Zhu; Cong-Wei Niu; Zhen Xi; Guang-Fu Yang

2010-01-01

68

Biological activity of a novel rationally designed lipophilic thymidylate synthase inhibitor  

Microsoft Academic Search

AG-331 {N 6[4-( N-morpholinosulfonyl)benzyl]- N 6-methyl-2,6-diamino-benz[ cd]indole glucuronate} is a novel lipophilic thymidylate synthase (TS) inhibitor. The properties of this compound were investigated in H35 rat hepatoma cells and in three variant cell lines resistant to antifolates by differing mechanisms. There was no evidence for any intracellular effect of AG-331 on dihydrofolate reductase (DHFR); however, the low degree of cross-resistance

Brigid M. O’Connor; Stephanie Webber; Robert C. Jackson; John Galivan; Myung S. Rhee

1994-01-01

69

Zaragozic acids: a family of fungal metabolites that are picomolar competitive inhibitors of squalene synthase.  

PubMed Central

Three closely related fungal metabolites, zaragozic acids A, B, and C, that are potent inhibitors of squalene synthase have been isolated and characterized. Zaragozic acids A, B, and C were produced from an unidentified sterile fungal culture, Sporormiella intermedia, and Leptodontium elatius, respectively. The structures of the zaragozic acids and their trimethyl esters were determined by a combination of physical and chemical techniques. The zaragozic acids are characterized by a novel 2,8-dioxobicyclo[3.2.1]octane-4,6,7- trihydroxyl-3,4,5-tricarboxylic acid core and differ from each other in the structures of the 6-acyl and 1-alkyl side chains. They were found to be potent competitive inhibitors of rat liver squalene synthase with apparent Ki values of 78 pM, 29 pM, and 45 pM, respectively. They inhibited cholesterol synthesis in Hep G2 cells, and zaragozic acid A was an inhibitor of acute hepatic cholesterol synthesis in the mouse (50% inhibitory dose of 200 micrograms/kg of body weight). Inhibition of squalene synthase in cells and in vivo was accompanied by an accumulation of label from [3H]mevalonate into farnesyl diphosphate, farnesol, and organic acids. These data indicate that the zaragozic acids are a previously unreported class of therapeutic agents with potential for the treatment of hypercholesterolemia. PMID:8419946

Bergstrom, J D; Kurtz, M M; Rew, D J; Amend, A M; Karkas, J D; Bostedor, R G; Bansal, V S; Dufresne, C; VanMiddlesworth, F L; Hensens, O D

1993-01-01

70

Chitin research revisited.  

PubMed

Two centuries after the discovery of chitin, it is widely accepted that this biopolymer is an important biomaterial in many aspects. Numerous studies on chitin have focused on its biomedical applications. In this review, various aspects of chitin research including sources, structure, biosynthesis, chitinolytic enzyme, chitin binding protein, genetic engineering approach to produce chitin, chitin and evolution, and a wide range of applications in bio- and nanotechnology will be dealt with. PMID:20714419

Khoushab, Feisal; Yamabhai, Montarop

2010-01-01

71

Chitin Research Revisited  

PubMed Central

Two centuries after the discovery of chitin, it is widely accepted that this biopolymer is an important biomaterial in many aspects. Numerous studies on chitin have focused on its biomedical applications. In this review, various aspects of chitin research including sources, structure, biosynthesis, chitinolytic enzyme, chitin binding protein, genetic engineering approach to produce chitin, chitin and evolution, and a wide range of applications in bio- and nanotechnology will be dealt with. PMID:20714419

Khoushab, Feisal; Yamabhai, Montarop

2010-01-01

72

Nanocrystalline chitin thin films.  

PubMed

Elucidating the interactions between crystalline chitin and various biomacromolecules is of fundamental importance for designing and fabricating chitin-based biomaterials. This work highlights a simple method to prepare ultrathin films of chitin nanocrystals (chitin NC) by spincoating chitin NCs from a colloidal suspension onto a gold surface modified by an amine-terminated self-assembled monolayer. Atomic force microscopy confirmed that chitin NC films are reasonably smooth and homogeneous, and quartz crystal microbalance with dissipation monitoring (QCM-D) solvent exchange experiments demonstrated that chitin NC films have twice as much water as amorphous regenerated chitin (RChitin) films of similar thickness. QCM-D data also showed that chitinase-catalyzed hydrolysis of chitin NC films was much slower than that of RChitin films. Chitinase not only degraded, but also caused the swelling of the chitin nanocrystals. BSA adsorption studies demonstrated that chitin NC films have high protein loading capacity, and thus show potential applications for enzyme immobilization. PMID:24507267

Wang, Chao; Esker, Alan R

2014-02-15

73

Effect of a selective thromboxane synthase inhibitor on arterial graft patency and platelet deposition in dogs  

SciTech Connect

This study examined the effect of selective thromboxane synthase inhibition and nonselective cyclooxygenase inhibition on vascular graft patency and indium 111-labeled platelet deposition in 35 mongrel dogs undergoing carotid artery replacement with 4 mm X 4 cm polytetrafluoroethylene (PTFE) (one side) and Dacron (opposite side) end-to-end grafts. Aspirin-dipyridamole therapy improved one-week graft patency, from 46% in untreated dogs to 93% in treated dogs. Thromboxane synthase inhibition (U-63557A) improved graft patency in these dogs to 81%. Both drug treatments reduced platelet deposition on Dacron and PTFE grafts by 48% to 68% compared with control dogs. Dacron grafts accumulated significantly more platelets than PTFE grafts but had comparable patency rates. Low-dose aspirin therapy had no significant effect on either graft patency or platelet deposition. All treatment groups showed a 60% to 76% reduction in serum thromboxane B2, but only thromboxane synthase inhibitor treatment increased plasma 6-keto-prostaglandin F1 alpha by 100%. Selective thromboxane synthase inhibition improved small-caliber prosthetic graft patency to the same extent as did conventional cyclooxygenase inhibition in this preliminary study.

McDaniel, M.D.; Huntsman, W.T.; Miett, T.O.; Cronenwett, J.L.

1987-08-01

74

Location of Inhibitor Binding Sites in the Human Inducible Prostaglandin E Synthase, MPGES1  

PubMed Central

The inducible microsomal prostaglandin E2 synthase 1 (MPGES1) is an integral membrane protein co-expressed with and functionally coupled to cyclooxygenase 2 (COX-2) generating the pro-inflammatory molecule PGE2. The development of effective inhibitors of MPGES1 holds promise as a highly selective route to control inflammation. In this paper we describe the use of backbone amide H/D exchange mass spectrometry to map the binding sites of different types of inhibitors of MPGES1. The results reveal the locations of specific inhibitor binding sites which include the GSH binding site and a hydrophobic cleft in the protein thought to accommodate the prostaglandin H2 substrate. In the absence of three-dimensional crystal structures of the enzyme-bound inhibitors, the results provide clear physical evidence that three pharmacologically active inhibitors bind in a hydrophobic cleft composed of sections of trans-membrane helices Ia, IIb, IIIb and IVb at the interface of subunits in the trimer. In principle, the H/D exchange behavior of the protein can be used as a preliminary guide for optimization of inhibitor efficacy. Finally, a comparison of the structures and H/D exchange behavior of MPGES1 and the related enzyme MGST1 in the presence of glutathione and the inhibitor glutathione sulfonate confirm the unusual observation that two proteins from the same superfamily harbor GSH binding sites in different locations. PMID:21805999

Prage, Edward B.; Pawelzik, Sven-Christian; Busenlehner, Laura S.; Kim, Kwangho; Morgenstern, Ralf; Jakobsson, Per-Johan; Armstrong, Richard N.

2011-01-01

75

ATP Synthase and the Actions of Inhibitors Utilized To Study Its Roles in Human Health, Disease, and Other Scientific Areas  

PubMed Central

Summary: ATP synthase, a double-motor enzyme, plays various roles in the cell, participating not only in ATP synthesis but in ATP hydrolysis-dependent processes and in the regulation of a proton gradient across some membrane-dependent systems. Recent studies of ATP synthase as a potential molecular target for the treatment of some human diseases have displayed promising results, and this enzyme is now emerging as an attractive molecular target for the development of new therapies for a variety of diseases. Significantly, ATP synthase, because of its complex structure, is inhibited by a number of different inhibitors and provides diverse possibilities in the development of new ATP synthase-directed agents. In this review, we classify over 250 natural and synthetic inhibitors of ATP synthase reported to date and present their inhibitory sites and their known or proposed modes of action. The rich source of ATP synthase inhibitors and their known or purported sites of action presented in this review should provide valuable insights into their applications as potential scaffolds for new therapeutics for human and animal diseases as well as for the discovery of new pesticides and herbicides to help protect the world's food supply. Finally, as ATP synthase is now known to consist of two unique nanomotors involved in making ATP from ADP and Pi, the information provided in this review may greatly assist those investigators entering the emerging field of nanotechnology. PMID:19052322

Hong, Sangjin; Pedersen, Peter L.

2008-01-01

76

Hepatic 5-aminolevulinic acid synthase mRNA stability is modulated by inhibitors of heme biosynthesis and by metalloporphyrins.  

PubMed

Hepatic 5-aminolevulinic acid synthase, the first and normally rate-controlling enzyme of heme biosynthesis, is regulated by heme. One of the known mechanisms whereby increased cellular heme regulates 5-aminolevulinic acid synthase is by decreasing the stability of its mRNA. In primary cultures of chick embryo liver cells, we tested whether a decrease in cellular heme might increase 5-aminolevulinic acid synthase mRNA stability and whether heme or other metalloporphyrins could reverse this stabilization. We found that: (a) The stability of 5-aminolevulinic acid synthase mRNA was markedly increased by inhibitors of heme biosynthesis, namely, 4,6-dioxoheptanoic acid or deferoxamine; (b) This increased stability of 5-aminolevulinic acid synthase mRNA was reversed by the addition of heme (10 microM) or by the combination of zinc mesoporphyrin (50 nM), an inhibitor of heme oxygenase, and heme (200 nM); (c) Repression of 5-aminolevulinic acid synthase mRNA levels by zinc mesoporphyrin (10 microM) was due to inhibition of heme oxygenase, rather than a direct, heme-like, effect of zinc mesoporphyrin on 5-aminolevulinic acid synthase mRNA; (d) Among the several non-heme metalloporphyrins tested, only zinc mesoporphyrin and chromium mesoporphyrin significantly decreased 5-aminolevulinic acid synthase mRNA without increasing heme oxygenase mRNA. PMID:8797843

Cable, E E; Gildemeister, O S; Pepe, J A; Donohue, S E; Lambrecht, R W; Bonkovsky, H L

1996-08-15

77

Substituted pyrrolo[2,3-d]pyrimidines as Cryptosporidium hominis thymidylate synthase inhibitors.  

PubMed

Cryptosporidiosis, a gastrointestinal disease caused by a protozoan Cryptosporidium hominis is often fatal in immunocompromised individuals. There is little clinical data to show that the existing treatment by nitazoxanide and paromomycin is effective in immunocompromised individuals. Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer and malaria. A novel series of classical antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines have been evaluated as Cryptosporidium hominis thymidylate synthase (ChTS) inhibitors. Crystal structure in complex with the most potent compound, a 2'-chlorophenyl with a sulfur bridge with a Ki of 8.83±0.67 nM is discussed in terms of several Van der Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate. Of these interactions, two interactions with the non-conserved residues (A287 and S290) offer an opportunity to develop ChTS specific inhibitors. Compound 6 serves as a lead compound for analog design and its crystal structure provides clues for the design of ChTS specific inhibitors. PMID:23927969

Kumar, Vidya P; Frey, Kathleen M; Wang, Yiqiang; Jain, Hitesh K; Gangjee, Aleem; Anderson, Karen S

2013-10-01

78

Identification and development of biphenyl substituted iminosugars as improved dual glucosylceramide synthase/neutral glucosylceramidase inhibitors.  

PubMed

This work details the evaluation of a number of N-alkylated deoxynojirimycin derivatives on their merits as dual glucosylceramide synthase/neutral glucosylceramidase inhibitors. Building on our previous work, we synthesized a series of d-gluco and l-ido-configured iminosugars N-modified with a variety of hydrophobic functional groups. We found that iminosugars featuring N-pentyloxymethylaryl substituents are considerably more potent inhibitors of glucosylceramide synthase than their aliphatic counterparts. In a next optimization round, we explored a series of biphenyl-substituted iminosugars of both configurations (d-gluco and l-ido) with the aim to introduce structural features known to confer metabolic stability to drug-like molecules. From these series, two sets of molecules emerge as lead series for further profiling. Biphenyl-substituted l-ido-configured deoxynojirimycin derivatives are selective for glucosylceramidase and the nonlysosomal glucosylceramidase, and we consider these as leads for the treatment of neuropathological lysosomal storage disorders. Their d-gluco-counterparts are also potent inhibitors of intestinal glycosidases, and because of this characteristic, we regard these as the prime candidates for type 2 diabetes therapeutics. PMID:25250725

Ghisaidoobe, Amar T; van den Berg, Richard J B H N; Butt, Saleem S; Strijland, Anneke; Donker-Koopman, Wilma E; Scheij, Saskia; van den Nieuwendijk, Adrianus M C H; Koomen, Gerrit-Jan; van Loevezijn, Arnold; Leemhuis, Mark; Wennekes, Tom; van der Stelt, Mario; van der Marel, Gijsbert A; van Boeckel, Constant A A; Aerts, Johannes M F G; Overkleeft, Herman S

2014-11-13

79

[NO-synthase inhibitor prevents flight-induced activation of agressiveness and courtship in male].  

PubMed

Previous experience of flying enhances the aggressiveness (Hofmann and Stevenson, 2000, Nature, 403: 613) and accelerates the courtship behaviour (Dyakonova and Krushinski, 2003, DAN, 390: 709-712) of crickets Gryllus bimacultus. We present evidence that these effects may be mediated by activation of nitric oxide synthesis. The effects of flying on fighting and courtship were largely abolished in crickets who received haemocoel injections of a nonspecific NO-synthase inhibitor LNNA. Unlike this, LNNA exerted no significant effects on aggressive and courtship behaviour of nonflown males. PMID:16119442

D'iakonova, V E; Krushinski?, A L

2005-06-01

80

Biological activity of a novel rationally designed lipophilic thymidylate synthase inhibitor  

Microsoft Academic Search

AG-331 {N6[4-(N-morpholinosulfonyl)benzyl]-N6-methyl-2,6-diamino-benz[cd]indole glucuronate} is a novel lipophilic thymidylate synthase (TS) inhibitor. The properties of this compound were investigated in H35 rat hepatoma cells and in three variant cell lines resistant to antifolates by differing mechanisms. There was no evidence for any intracellular effect of AG-331 on dihydrofolate reductase (DHFR); however, the low degree of cross-resistance found for the H35FF line,

Brigid M. O'Connor; Stephanie Webber; Robert C. Jackson; John Galivanl; Myung S. Rhee

1994-01-01

81

Potent, Highly Selective, and Orally Bioavailable Gem-Difluorinated Monocationic Inhibitors of Neuronal Nitric Oxide Synthase  

PubMed Central

In our efforts to discover neuronal isoform selective nitric oxide synthase (NOS) inhibitors we have developed a series of compounds containing a pyrrolidine ring with two stereogenic centers. The enantiomerically pure compounds, (S,S) vs. (R,R), exhibited two different binding orientations, with (R,R) inhibitors showing much better potency and selectivity. To improve the bioavailability of these inhibitors we have introduced a CF2 moiety geminal to an amino group in the long tail of one of these inhibitors, which reduced its basicity, resulting in compounds with monocationic character under physiological pH conditions. Biological evaluations have led to a nNOS inhibitor with a Ki of 36 nM and high selectivity for nNOS over eNOS (3800-fold) and iNOS (1400-fold). MM-PBSA calculations indicated that the low pKa NH is, at least, partially protonated when bound to the active site. A comparison of rat oral bioavailability of the difluorinated compound to the parent molecule shows 22% for the difluorinated compound versus essentially no oral bioavailability for the parent compound. This indicates that the goal of this research to make compounds with only one protonated nitrogen atom at physiological pH to allow for membrane permeability, but which can become protonated when bound to NOS, has been accomplished. PMID:20843082

Xue, Fengtian; Li, Huiying; Delker, Silvia L.; Fang, Jianguo; Martasek, Pavel; Roman, Linda J.; Poulos, Thomas L.; Silverman, Richard B.

2010-01-01

82

DXP synthase-catalyzed C-N bond formation: Nitroso substrate specificity studies guide selective inhibitor design  

PubMed Central

1-Deoxy-d-xylulose 5-phosphate (DXP) synthase catalyzes the first step in the non-mammalian isoprenoid biosynthetic pathway to form DXP from pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) in a thiamin diphosphate-dependent manner. Its unique structure and mechanism distinguish DXP synthase from its homologs, suggesting it should be pursued as an anti-infective drug target. However, few reports describe development of selective inhibitors of this enzyme. Here, we reveal a function of DXP synthase that catalyzes C-N bond formation and exploit aromatic nitroso substrates as active site probes. Substrate specificity studies reveal high affinity of DXP synthase for aromatic nitroso substrates compared to the related ThDP-dependent enzyme Pyruvate Dehydrogenase (PDH). Results from inhibition and mutagenesis studies indicate nitroso substrates bind to E. coli DXP synthase in a manner distinct from d-GAP. Our results suggest that incorporation of aryl acceptor substrate mimics into unnatural bisubstrate analogs will impart selectivity to DXP synthase inhibitors. As proof of concept, we show selective inhibition of DXP synthase by benzylacetylphosphonate (BnAP). PMID:23824585

Morris, Francine; Vierling, Ryan; Boucher, Lauren; Bosch, Jurgen; Meyers, Caren L. Freel

2013-01-01

83

Structure-guided Design of Selective Inhibitors of Neuronal Nitric Oxide Synthase  

PubMed Central

Nitric oxide synthases (NOSs) comprise three closely related isoforms that catalyze the oxidation of l-arginine to l-citrulline and the important second messenger nitric oxide (NO). Pharmacological selective inhibition of neuronal NOS (nNOS) has the potential to be therapeutically beneficial in various neurodegenerative diseases. Here we present a structure-guided, selective nNOS inhibitor design based on the crystal structure of lead compound 1 in nNOS. The best inhibitor, 7, exhibited low nanomolar inhibitory potency and good isoform selectivities (nNOS over eNOS and iNOS are 472-fold and 239-fold, respectively). Consistent with the good selectivity, 7 binds to nNOS and eNOS with different binding modes. The distinctly different binding modes of 7, driven by the critical residue Asp597 in nNOS, offers compelling insight to explain its isozyme selectivity, which should guide future drug design programs. PMID:23451760

Huang, He; Li, Huiying; Martasek, Pavel; Roman, Linda J.; Poulos, Thomas L.; Silverman, Richard B.

2013-01-01

84

A high-throughput screen for quorum-sensing inhibitors that target acyl-homoserine lactone synthases.  

PubMed

Many Proteobacteria use N-acyl-homoserine lactone (acyl-HSL) quorum sensing to control specific genes. Acyl-HSL synthesis requires unique enzymes that use S-adenosyl methionine as an acyl acceptor and amino acid donor. We developed and executed an enzyme-coupled high-throughput cell-free screen to discover acyl-HSL synthase inhibitors. The three strongest inhibitors were equally active against two different acyl-HSL synthases: Burkholderia mallei BmaI1 and Yersinia pestis YspI. Two of these inhibitors showed activity in whole cells. The most potent compound behaves as a noncompetitive inhibitor with a Ki of 0.7 µM and showed activity in a cell-based assay. Quorum-sensing signal synthesis inhibitors will be useful in attempts to understand acyl-HSL synthase catalysis and as a tool in studies of quorum-sensing control of gene expression. Because acyl-HSL quorum-sensing controls virulence of some bacterial pathogens, anti-quorum-sensing chemicals have been sought as potential therapeutic agents. Our screen and identification of acyl-HSL synthase inhibitors serve as a basis for efforts to target quorum-sensing signal synthesis as an antivirulence approach. PMID:23924613

Christensen, Quin H; Grove, Tyler L; Booker, Squire J; Greenberg, E Peter

2013-08-20

85

[Hematopoietic prostaglandin D synthase inhibitors for the treatment of duchenne muscular dystrophy].  

PubMed

Duchenne muscular dystrophy (DMD) is a severe X-linked muscle disease, characterized by progressive skeletal muscle atrophy and weakness. DMD is caused by mutations in the dystrophin gene, which encodes for the cytoskeletal protein dystrophin. DMD is one of the most common types of muscular dystrophies, affecting approximately 1 in 3,500 boys. There is no complete cure for this disease. Clinical trials for gene transfer therapy as a treatment for DMD have been performed but mainly in animal models. Hematopoietic prostaglandin (PG) D synthase (H-PGDS) was found to be induced in grouped necrotic muscle fibers of DMD patients and animal models, mdx mice, and DMD dogs. We found an orally active H-PGDS inhibitor (HQL-79) and determined the 3D structure of the inhibitor-human H-PGDS complex by X-ray crystallography. Oral administration of HQL-79 markedly suppressed prostaglandin D2 (PGD2) production, reduced necrotic muscle volume, and improved muscle strength in mdx dystrophic mice. Based on the high-resolution 3D structures of the inhibitor-H-PGDS complex, we designed alternative H-PGDS inhibitors, which were 100- to 3000-times more potent than HQL-79, as assessed by in vitro and in vivo analyses. We used these novel inhibitors for the treatment of DMD dogs and confirmed that oral administration of these inhibitors prevented skeletal muscle atrophy and weakness by decreasing PGD2 production. These results indicate that PGD2, synthesized by H-PGDS, is involved in the expansion of muscle necrosis in DMD. Thus, inhibition of H-PGDS by using inhibitors is a novel therapy for DMD. PMID:22068479

Kamauchi, Shinya; Urade, Yoshihiro

2011-11-01

86

Furegrelate, a thromboxane synthase inhibitor, blunts the development of pulmonary arterial hypertension in neonatal piglets  

PubMed Central

The development of pulmonary arterial hypertension (PAH) in pediatric patients has been linked to the production of the arachidonic acid metabolite, thromboxane A2 (TxA2). The present study evaluated the therapeutic effect of furegrelate sodium, a thromboxane synthase inhibitor, on the development of PAH in a neonatal piglet model. Three-day-old piglets were exposed to 21 days of normoxia (N; 21% FIO2) or chronic hypoxia (CH; 10% FIO2). A third group of piglets received the oral TxA2 synthase inhibitor, furegrelate (3 mg/kg, 2 or 3 times daily) at the induction of CH. In vivo hemodynamics confirmed a 2.55-fold increase of the pulmonary vascular resistance index (PVRI) in CH piglets (104±7 WU) compared to N piglets (40±2 WU). The CH piglets treated twice daily with furegrelate failed to show improved PVRI, but furegrelate three times daily lowered the elevated PVRI in CH piglets by 34% to 69±5 WU and ameliorated the development of right ventricular hypertrophy. Microfocal X-ray computed tomography (CT) scanning was used to estimate the diameter-independent distensibility term, ? (% change in diameter per Torr). Pulmonary arterial distensibility in isolated lungs of CH piglets (?=1.0±0.1% per Torr) was lower than that of N piglets (?=1.5±0.1% per Torr) indicative of vascular remodeling. Arterial distensibility was partially restored in furegrelate-treated CH piglets (? =1.2±0.1% per Torr) and microscopic evidence showing muscularization of small pulmonary arteries also was less prominent in these animals. Finally, isolated lungs of furegrelate-treated piglets showed lower basal and vasodilator-induced transpulmonary pressures compared to CH animals. These findings suggest that pharmacological inhibition of TxA2 synthase activity by furegrelate blunts the development of hypoxia-induced PAH in an established neonatal piglet model primarily by preserving the structural integrity of the pulmonary vasculature. PMID:22837860

Hirenallur-S., Dinesh K.; Detweiler, Neil D.; Haworth, Steven T.; Leming, Jeaninne T.; Gordon, John B.; Rusch, Nancy J.

2012-01-01

87

Inhibitors of the inducible microsomal prostaglandin E2 synthase (mPGES-1) derived from MK-886.  

PubMed

A series of potent and selective inhibitors of the inducible microsomal PGE2 synthase (mPGES-1) has been developed based on the indole FLAP inhibitor MK-886. Compounds 23 and 30 inhibit mPGES-1 with potencies in the low nanomolar range and with selectivities of at least 100-fold compared to their inhibition of mPGES-2, thromboxane synthase and binding affinity to FLAP. They also block the production of PGE2 in cell based assays but with a decreased potency and more limited selectivity compared to the enzyme assays. PMID:15953724

Riendeau, Denis; Aspiotis, Renee; Ethier, Diane; Gareau, Yves; Grimm, Erich L; Guay, Jocelyne; Guiral, Sébastien; Juteau, Hélène; Mancini, Joseph A; Méthot, Nathalie; Rubin, Joel; Friesen, Richard W

2005-07-15

88

Novel prostaglandin D synthase inhibitors generated by fragment-based drug design.  

PubMed

We describe the discovery of novel inhibitors of prostaglandin D2 synthase (PGDS) through fragment-based lead generation and structure-based drug design. A library of 2500 low-molecular-weight compounds was screened using 2D nuclear magnetic resonance (NMR), leading to the identification of 24 primary hits. Structure determination of protein-ligand complexes with the hits enabled a hit optimization process, whereby we harvested increasingly more potent inhibitors out of our corporate compound collection. Two iterative cycles were carried out, comprising NMR screening, molecular modeling, X-ray crystallography, and in vitro biochemical testing. Six novel high-resolution PGDS complex structures were determined, and 300 hit analogues were tested. This rational drug design procedure culminated in the discovery of 24 compounds with an IC 50 below 1 microM in the in vitro assay. The best inhibitor (IC 50 = 21 nM) is one of the most potent inhibitors of PGDS to date. As such, it may enable new functional in vivo studies of PGDS and the prostaglandin metabolism pathway. PMID:18341273

Hohwy, Morten; Spadola, Loredana; Lundquist, Britta; Hawtin, Paul; Dahmén, Jan; Groth-Clausen, Ib; Nilsson, Ewa; Persdotter, Sofia; von Wachenfeldt, Karin; Folmer, Rutger H A; Edman, Karl

2008-04-10

89

Conformationally-Restricted Dipeptide Amides as Potent and Selective Neuronal Nitric Oxide Synthase Inhibitors  

PubMed Central

Four new conformationally-restricted analogues of the potent and selective neuronal nitric oxide synthase inhibitor, L-nitroargininyl-L-2,4-diaminobutyramide (1), have been synthesized. N?-Methyl and N?-benzyl derivatives (3 and 4, respectively) of 4N-(L-ArgNO2)-trans-4-amino-L-prolineamide (2) are also selective inhibitors, but the potency and selectivity of 3 are weak. Analogue 4 has only one-third the potency and one-half to one-third the selectivity of 2 against iNOS and eNOS, respectively. 3-N-(L-ArgNO2)-trans-3-amino-L-prolineamide (6) is as potent an inhibitor of nNOS as is 2; selectivity for nNOS over iNOS is half of that for 2 but the selectivity for nNOS over eNOS is almost double that for 2. The corresponding cis-isomer (5) is a weak inhibitor of nNOS. These results are supported by computer modeling. PMID:17034131

Ji, Haitao; Gomez-Vidal, Jose A.; Martasek, Pavel; Roman, Linda J.; Silverman, Richard B.

2008-01-01

90

Stroke Protection by 3-hydroxy-3-methylglutaryl (HMG)CoA Reductase Inhibitors Mediated by Endothelial Nitric Oxide Synthase  

Microsoft Academic Search

The treatment of ischemic strokes is limited to prophylactic agents that block the coagulation cascade. Here, we show that cholesterol-lowering agents, 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, protect against cerebral injury by a previously unidentified mechanism involving the selective up-regulation of endothelial NO synthase (eNOS). Prophylactic treatment with HMG-CoA reductase inhibitors augments cerebral blood flow, reduces cerebral infarct size, and improves neurological

Matthias Endres; Ulrich Laufs; Zhihong Huang; Tadashi Nakamura; Paul Huang; Michael A. Moskowitz; James K. Liao

1998-01-01

91

Visualizing inducible nitric-oxide synthase in living cells with a heme-binding fluorescent inhibitor.  

PubMed

The study of nitric-oxide synthase (NOS) physiology is constrained by the lack of suitable probes to detect NOS in living cells or animals. Here, we characterized a fluorescent inducible NOS (iNOS) inhibitor called PIF (pyrimidine imidazole FITC) and examined its utility for microscopic imaging of iNOS in living cells. PIF binding to iNOS displayed high affinity, isoform selectivity, and heme specificity, and was essentially irreversible. PIF was used to successfully image iNOS expressed in RAW264.7 cells, HEK293T cells, human A549 epithelial cells, and freshly obtained human lung epithelium. PIF was used to estimate a half-life for iNOS of 1.8 h in HEK293T cells. Our work reveals that fluorescent probes like PIF will be valuable for studying iNOS cell biology and in understanding the pathophysiology of diseases that involve dysfunctional iNOS expression. PMID:16006534

Panda, Koustubh; Chawla-Sarkar, Mamta; Santos, Cecile; Koeck, Thomas; Erzurum, Serpil C; Parkinson, John F; Stuehr, Dennis J

2005-07-19

92

ELIGLUSTAT TARTRATE: Glucosylceramide Synthase Inhibitor Treatment of Type 1 Gaucher Disease.  

PubMed

Eliglustat tartrate (Genz-112638) is a novel, orally administered agent currently in development for the treatment of lysosomal storage disorders, including type 1 Gaucher disease and Fabry disease. This glucosylceramide analogue acts as an inhibitor of glucosylceramide synthase, a Golgi complex enzyme that catalyzes the formation of glucosylceramide from ceramide and UDP-glucose and is the first step in the formation of glucocerebroside-based glycosphingolipids. Pre-clinical pharmacological studies demonstrate that the agent has a high therapeutic index, excellent oral bioavailability and limited toxicity. Phase I studies in healthy volunteers revealed limited toxicity with an excellent pharmacodynamic response, as measured by decreased plasma glucosylceramide concentrations. Phase II studies in patients with type 1 Gaucher disease have demonstrated promising clinical responses, as measured by decreases in spleen size, improvement in hemoglobin concentrations and increased platelet counts. Two randomized phase III trials testing the efficacy and safety of eliglustat tartrate are currently in progress. PMID:22563139

Shayman, J A

2010-08-01

93

Discovery of Highly Potent and Selective Inhibitors of Neuronal Nitric Oxide Synthase by Fragment Hopping  

PubMed Central

Selective inhibition of neuronal nitric oxide synthase (nNOS) has been shown to prevent brain injury and is important for the treatment of various neurodegenerative disorders. This study shows that not only greater inhibitory potency and isozyme selectivity, but more drug-like properties can be achieved by fragment hopping. Based on the structure of lead molecule 6, fragment hopping effectively extracted the minimal pharmacophoric elements in the active site of nNOS for ligand hydrophobic and steric interactions and generated appropriate lipophilic fragments for lead optimization. More potent and selective inhibitors with better drug-like properties were obtained within the design of 20 derivatives (compounds 7-26). Our structure-based inhibitor design for nNOS and SAR analysis reveal the robustness and efficiency of fragment hopping in lead discovery and structural optimization, which implicates a broad application of this approach to many other therapeutic targets for which known drug-like small-molecule modulators are still limited. PMID:19125620

Ji, Haitao; Li, Huiying; Martasek, Pavel; Roman, Linda J.; Poulos, Thomas L.; Silverman, Richard B.

2009-01-01

94

2-alkylaminoethyl-1,1-bisphosphonic acids are potent inhibitors of the enzymatic activity of Trypanosoma cruzi squalene synthase.  

PubMed

As part of our efforts aimed at searching for new antiparasitic agents, the effect of representative 2-alkylaminoethyl-1,1-bisphosphonic acids on Trypanosoma cruzi squalene synthase (TcSQS) was investigated. These compounds had proven to be potent inhibitors of T. cruzi. This cellular activity had been associated with an inhibition of the enzymatic activity of T. cruzi farnesyl diphosphate synthase. 2-Alkylaminoethyl-1,1-bisphosphonic acids appear to have a dual action, since they also inhibit TcSQS at the nanomolar range. PMID:22585217

Rodrígues-Poveda, Carlos A; González-Pacanowska, Dolores; Szajnman, Sergio H; Rodríguez, Juan B

2012-08-01

95

In vivo study of radioprotective effect of NO-synthase inhibitors and acetyl-L-carnitine.  

PubMed

This study investigated the protective effect of two nitric oxide synthase inhibitors N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 mg/kg i.p.) and aminoguanidine (AG, 400 mg/kg i.p.), and an antioxidant acetyl-L-carnitine (ALC, 250 mg/kg i.p., once daily for five days) against radiation-induced damage in Wistar rats. Blood samples were collected 6 h after whole-body irradiation with 8 Gy. Plasma concentrations of nitrite+nitrate (NO(x)) and malondialdehyde (MDA) were measured by high-performance liquid chromatography. A single injection of L-NAME one hour before exposure effectively prevented the radiation-induced elevation of plasma NO(x) and it reduced 2.6-fold the risk for death during the subsequent 30-day period. Pretreatment with ALC prevented the radiation-induced increase in plasma MDA and it had similar effect on mortality as L-NAME did. Presumably due to its short half-life, the partially iNOS-selective inhibitor and antioxidant AG given in a single dose before exposure did not attenuate MDA and NO(x) and it failed to significantly improve the 30-day survival. In conclusion, pretreatment with both the nonspecific NOS inhibitor L-NAME and the antioxidant ALC markedly reduce mortality to radiation sickness in rats. The radioprotective effect may be directly related to effective attenuation of the radiation-induced elevation of NO production by L-NAME and of oxidative stress by ALC. PMID:23869893

Babicová, A; Havlínová, Z; Hroch, M; Rezá?ová, M; Pejchal, J; Vávrová, J; Chládek, J

2013-12-20

96

Selectivity of commonly used pharmacological inhibitors for cystathionine ? synthase (CBS) and cystathionine ? lyase (CSE)  

PubMed Central

Background and Purpose Hydrogen sulfide (H2S) is a signalling molecule that belongs to the gasotransmitter family. Two major sources for endogenous enzymatic production of H2S are cystathionine ? synthase (CBS) and cystathionine ? lyase (CSE). In the present study, we examined the selectivity of commonly used pharmacological inhibitors of H2S biosynthesis towards CSE and CBS. Experimental Approach To address this question, human CSE or CBS enzymes were expressed and purified from Escherichia coli as fusion proteins with GSH-S-transferase. After purification, the activity of the recombinant enzymes was tested using the methylene blue method. Key Results ?-cyanoalanine (BCA) was more potent in inhibiting CSE than propargylglycine (PAG) (IC50 14 ± 0.2 ?M vs. 40 ± 8 ?M respectively). Similar to PAG, L-aminoethoxyvinylglycine (AVG) only inhibited CSE, but did so at much lower concentrations. On the other hand, aminooxyacetic acid (AOAA), a frequently used CBS inhibitor, was more potent in inhibiting CSE compared with BCA and PAG (IC50 1.1 ± 0.1 ?M); the IC50 for AOAA for inhibiting CBS was 8.5 ± 0.7 ?M. In line with our biochemical observations, relaxation to L-cysteine was blocked by AOAA in aortic rings that lacked CBS expression. Trifluoroalanine and hydroxylamine, two compounds that have also been used to block H2S biosynthesis, blocked the activity of CBS and CSE. Trifluoroalanine had a fourfold lower IC50 for CBS versus CSE, while hydroxylamine was 60-fold more selective against CSE. Conclusions and Implications In conclusion, although PAG, AVG and BCA exhibit selectivity in inhibiting CSE versus CBS, no selective pharmacological CBS inhibitor is currently available. PMID:23488457

Asimakopoulou, Antonia; Panopoulos, Panagiotis; Chasapis, Christos T; Coletta, Ciro; Zhou, Zongmin; Cirino, Giuseppe; Giannis, Athanassios; Szabo, Csaba; Spyroulias, Georgios A; Papapetropoulos, Andreas

2013-01-01

97

Syntheses and herbicidal activity of new triazolopyrimidine-2-sulfonamides as acetohydroxyacid synthase inhibitor.  

PubMed

The triazolopyrimidine-2-sulfonanilide, discovered from preparing bioisosteres of the sulfonylurea herbicides, is an important class of acetohydroxyacid synthase (AHAS, EC 4.1.3.18) inhibitors. At least over ten triazolopyrimidine sulfonanilides have been commercialized as herbicides for the control of broadleaf weeds and grass with cereal crop selectivity. Herein, a series of triazolopyrimidine-2-sulfonanilides were designed and synthesized with the aim of discovery of new herbicides with higher activity. The assay results of the inhibition activity of the synthesized compounds against Arabidopsis thatiana AHAS indicated that some compounds showed a little higher activity against flumetsulam (FS), the first commercial triazolopyrimidine-2-sulfonanilide-type herbicide. The ki values of two promising compounds 3d and 8h are respectively, 1.61 and 1.29 microM, while that of FS is 1.85 microM. Computational simulation results indicated the ester group of compound 3d formed hydrogen bonds with the surrounding residues Arg'198 and Ser653, which accounts for its 11.5-folds higher AHAS inhibition activity than Y6610. Further green house assay showed that compound 3d has comparable herbicidal activity as FS. Even at the concentration of 37.5g.ai/ha, 3d showed excellent herbicidal activity against Galium aparine, Cerastium arvense, Chenopodium album, Amaranthus retroflexus, and Rmumex acetasa, moderate herbicidal activity against Polygonum humifusum, Cyperus iria, and Eclipta prostrate. The combination of in vitro and in vivo assay indicated that 3d could be regarded as a new potential acetohydroxyacid synthase-inhibiting herbicide candidate for further study. PMID:20598554

Chen, Chao-Nan; Chen, Qiong; Liu, Yu-Chao; Zhu, Xiao-Lei; Niu, Cong-Wei; Xi, Zhen; Yang, Guang-Fu

2010-07-15

98

Non-antibiotic quorum sensing inhibitors acting against N-acyl homoserine lactone synthase as druggable target.  

PubMed

N-acylhomoserine lactone (AHL)-based quorum sensing (QS) is important for the regulation of proteobacterial virulence determinants. Thus, the inhibition of AHL synthases offers non-antibiotics-based therapeutic potentials against QS-mediated bacterial infections. In this work, functional AHL synthases of Pseudomonas aeruginosa LasI and RhlI were heterologously expressed in an AHL-negative Escherichia coli followed by assessments on their AHLs production using AHL biosensors and high resolution liquid chromatography-mass spectrometry (LCMS). These AHL-producing E. coli served as tools for screening AHL synthase inhibitors. Based on a campaign of screening synthetic molecules and natural products using our approach, three strongest inhibitors namely are salicylic acid, tannic acid and trans-cinnamaldehyde have been identified. LCMS analysis further confirmed tannic acid and trans-cinnemaldehyde efficiently inhibited AHL production by RhlI. We further demonstrated the application of trans-cinnemaldehyde inhibiting Rhl QS system regulated pyocyanin production in P. aeruginosa up to 42.06%. Molecular docking analysis suggested that trans-cinnemaldehyde binds to the LasI and EsaI with known structures mainly interacting with their substrate binding sites. Our data suggested a new class of QS-inhibiting agents from natural products targeting AHL synthase and provided a potential approach for facilitating the discovery of anti-QS signal synthesis as basis of novel anti-infective approach. PMID:25430794

Chang, Chien-Yi; Krishnan, Thiba; Wang, Hao; Chen, Ye; Yin, Wai-Fong; Chong, Yee-Meng; Tan, Li Ying; Chong, Teik Min; Chan, Kok-Gan

2014-01-01

99

Identification of a Glycogen Synthase Kinase-3[beta] Inhibitor that Attenuates Hyperactivity in CLOCK Mutant Mice  

SciTech Connect

Bipolar disorder is characterized by a cycle of mania and depression, which affects approximately 5 million people in the United States. Current treatment regimes include the so-called 'mood-stabilizing drugs', such as lithium and valproate that are relatively dated drugs with various known side effects. Glycogen synthase kinase-3{beta} (GSK-3{beta}) plays a central role in regulating circadian rhythms, and lithium is known to be a direct inhibitor of GSK-3{beta}. We designed a series of second generation benzofuran-3-yl-(indol-3-yl)maleimides containing a piperidine ring that possess IC{sub 50} values in the range of 4 to 680 nM against human GSK-3{beta}. One of these compounds exhibits reasonable kinase selectivity and promising preliminary absorption, distribution, metabolism, and excretion (ADME) data. The administration of this compound at doses of 10 to 25 mg kg{sup -1} resulted in the attenuation of hyperactivity in amphetamine/chlordiazepoxide-induced manic-like mice together with enhancement of prepulse inhibition, similar to the effects found for valproate (400 mg kg{sup -1}) and the antipsychotic haloperidol (1 mg kg{sup -1}). We also tested this compound in mice carrying a mutation in the central transcriptional activator of molecular rhythms, the CLOCK gene, and found that the same compound attenuates locomotor hyperactivity in response to novelty. This study further demonstrates the use of inhibitors of GSK-3{beta} in the treatment of manic episodes of bipolar/mood disorders, thus further validating GSK-3{beta} as a relevant therapeutic target in the identification of new therapies for bipolar patients.

Kozikowski, Alan P.; Gunosewoyo, Hendra; Guo, Songpo; Gaisina, Irina N.; Walter, Richard L.; Ketcherside, Ariel; McClung, Colleen A.; Mesecar, Andrew D.; Caldarone, Barbara (Psychogenics); (Purdue); (UIC); (UTSMC)

2012-05-02

100

Quinoline antifolate thymidylate synthase inhibitors: variation of the C2- and C4-substituents.  

PubMed

Modifications to the bicyclic ring system of the potent thymidylate synthase (TS) inhibitor N-[4-[N-[(2-amino-3,4-dihydro-4-oxo-6- quinazolinyl)methyl]-N-prop-2-ynylamino]benzoyl]-L-glutamic acid (1, CB3717) have led to the synthesis of a series of quinoline antifolates bearing a variety of substituents at the C2 and C4 positions. In general the synthetic route involved the coupling of the appropriate diethyl N-[4-(prop-2-ynylamino)benzoyl]-L-glutamate with a disubstituted 6-(bromomethyl)quinoline followed by deprotection using mild alkali. The compounds were tested as inhibitors of partially purified L1210 TS. As a measure of cytotoxicity, the compounds were tested for their inhibition of the growth of L1210 cells in culture. Good enzyme inhibition and cytotoxicity were found for compounds containing chloro, amino, or methyl substituents at the C2 position with chloro or bromo substituents at C4. The effect on enzyme inhibition of varying the N10 substituent of 2h was similar to that observed in the quinazolinone-containing antifolates, indicating that the quinoline compounds may be interacting with the enzyme in a similar way to the quinazolinones. Also, the introduction of a 2'-fluoro substituent into the benzoyl ring of several of the quinoline antifolates led to an increase in both TS inhibition and the inhibition of L1210 cell growth. These data demonstrate that the N3-H of the pyrimidine ring of the quinazolinone antifolates is not required for binding to TS if appropriate substituents are placed at the C2 and C4 positions of the bicyclic ring system. PMID:1495009

Warner, P; Barker, A J; Jackman, A L; Burrows, K D; Roberts, N; Bishop, J A; O'Connor, B M; Hughes, L R

1992-07-24

101

Biological activity of a novel rationally designed lipophilic thymidylate synthase inhibitor.  

PubMed

AG-331 (N6[4-(N-morpholinosulfonyl)benzyl]-N6-methyl-2,6-diamino- benz[cd]indole glucuronate) is a novel lipophilic thymidylate synthase (TS) inhibitor. The properties of this compound were investigated in H35 rat hepatoma cells and in three variant cell lines resistant to antifolates by differing mechanisms. There was no evidence for any intracellular effect of AG-331 on dihydrofolate reductase (DHFR); however, the low degree of cross-resistance found for the H35FF line, which has elevated TS levels, suggested that TS may not be the sole locus of action of AG-331 in hepatoma cells. TS-directed effects of AG-331 were suggested by the pattern of its inhibition of deoxyuridine incorporation into DNA and the lesser effects of purine incorporation. In addition, H35 cells treated with 10 microM AG-331 were shown to accumulate in the S phase of the cell cycle, and this effect could be reversed by coadministration of thymidine. However, when treatments were conducted at a 5-fold higher concentration of AG-331, no S-phase block was apparent, suggesting the loss of a TS-directed effect at high inhibitor concentrations. Thymidine and folinic acid also failed to protect cells against AG-331 cytotoxicity, suggesting an alternate mode of action. Similar results were also obtained in protection experiments with a human hepatoma cell line, HEPG2, although previous results obtained in colon- and breast-cancer cell lines have suggested TS specific effects for AG-331. The possibility that biotransformation of AG-331 to other toxic species may occur in liver-derived cell lines has yet to be investigated. PMID:8004755

O'Connor, B M; Webber, S; Jackson, R C; Galivan, J; Rhee, M S

1994-01-01

102

Effects of an NO synthase inhibitor on aggressive and sexual behavior in male crickets.  

PubMed

The mechanisms of the effects of some types of behavior on others have received little study. The present investigation addresses the phenomenon present in male crickets of the species Gryllus bimaculatus, consisting of the powerful activation by transient flight (3 min) of aggression to another male and of the female courtship program. We found that flight did not evoke these behavioral changes in males injected with the NO synthase inhibitor LNNA. The intensity and duration of fights with another male, the frequency of ritual singing by the victor, and the intensity with which the victor pursued the vanquished only increased significantly after flight in control male crickets injected with Ringer's solution, but not in experimental crickets. Similarly, flown males injected with LNNA were no different from unflown males in terms of the intensity of female courtship (the latent period and relative duration of courtship singing); in controls, the latent period was significantly shorter and the duration of singing was significantly greater in flown crickets. LNNA had no effect on aggressive or sexual behavior in unflown males. These results demonstrate that flight may increase NO synthesis, making a significant contribution to the formation of the flight-evoked behavioral state. PMID:16645775

D'yakonova, V E; Krushinskii, A L

2006-06-01

103

Property-based design of a glucosylceramide synthase inhibitor that reduces glucosylceramide in the brain.  

PubMed

Synthesis inhibition is the basis for the treatment of type 1 Gaucher disease by the glucosylceramide synthase (GCS) inhibitor eliglustat tartrate. However, the extended use of eliglustat and related compounds for the treatment of glycosphingolipid storage diseases with CNS manifestations is limited by the lack of brain penetration of this drug. Property modeling around the D-threo-1-phenyl-2-decanoylamino-3-morpholino-propanol (PDMP) pharmacophore was employed in a search for compounds of comparable activity against the GCS but lacking P-glycoprotein (MDR1) recognition. Modifications of the carboxamide N-acyl group were made to lower total polar surface area and rotatable bond number. Compounds were screened for inhibition of GCS in crude enzyme and whole cell assays and for MDR1 substrate recognition. One analog, 2-(2,3-dihydro-1H-inden-2-yl)-N-((1R,2R)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-hydroxy-3-(pyrrolidin-1-yl)propan-2-yl)acetamide (CCG-203586), was identified that inhibited GCS at low nanomolar concentrations with little to no apparent recognition by MDR1. Intraperitoneal administration of this compound to mice for 3 days resulted in a significant dose dependent decrease in brain glucosylceramide content, an effect not seen in mice dosed in parallel with eliglustat tartrate. PMID:22058426

Larsen, Scott D; Wilson, Michael W; Abe, Akira; Shu, Liming; George, Christopher H; Kirchhoff, Paul; Showalter, H D Hollis; Xiang, Jianming; Keep, Richard F; Shayman, James A

2012-02-01

104

Structural studies provide clues for analog design of specific inhibitors of Cryptosporidium hominis thymidylate synthase-dihydrofolate reductase.  

PubMed

Cryptosporidium is the causative agent of a gastrointestinal disease, cryptosporidiosis, which is often fatal in immunocompromised individuals and children. Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer, bacterial infections, and malaria. Cryptosporidium hominis has a bifunctional thymidylate synthase and dihydrofolate reductase enzyme, compared to separate enzymes in the host. We evaluated lead compound 1 from a novel series of antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines as an inhibitor of Cryptosporidium hominis thymidylate synthase with selectivity over the human enzyme. Complementing the enzyme inhibition compound 1 also has anti-cryptosporidial activity in cell culture. A crystal structure with compound 1 bound to the TS active site is discussed in terms of several van der Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate (TS), cofactor NADPH and inhibitor methotrexate (DHFR). Another crystal structure in complex with compound 1 bound in both the TS and DHFR active sites is also reported here. The crystal structures provide clues for analog design and for the design of ChTS-DHFR specific inhibitors. PMID:25127103

Kumar, Vidya P; Cisneros, Jose A; Frey, Kathleen M; Castellanos-Gonzalez, Alejandro; Wang, Yiqiang; Gangjee, Aleem; White, A Clinton; Jorgensen, William L; Anderson, Karen S

2014-09-01

105

Indirubin core structure of glycogen synthase kinase-3 inhibitors as novel chemotype for intervention with 5-lipoxygenase.  

PubMed

The enzymes 5-lipoxygenase (5-LO) and glycogen synthase kinase (GSK)-3 represent promising drug targets in inflammation. We made use of the bisindole core of indirubin, present in GSK-3 inhibitors, to innovatively target 5-LO at the ATP-binding site for the design of dual 5-LO/GSK-3 inhibitors. Evaluation of substituted indirubin derivatives led to the identification of (3Z)-6-bromo-3-[(3E)-3-hydroxyiminoindolin-2-ylidene]indolin-2-one (15) as a potent, direct, and reversible 5-LO inhibitor (IC50 = 1.5 ?M), with comparable cellular effectiveness on 5-LO and GSK-3. Together, we present indirubins as novel chemotypes for the development of 5-LO inhibitors, the interference with the ATP-binding site as a novel strategy for 5-LO targeting, and dual 5-LO/GSK-3 inhibition as an unconventional and promising concept for anti-inflammatory intervention. PMID:24697244

Pergola, Carlo; Gaboriaud-Kolar, Nicolas; Jestädt, Nadine; König, Stefanie; Kritsanida, Marina; Schaible, Anja M; Li, Haokun; Garscha, Ulrike; Weinigel, Christina; Barz, Dagmar; Albring, Kai F; Huber, Otmar; Skaltsounis, Alexios L; Werz, Oliver

2014-05-01

106

Discovery and development of the covalent hydrates of trifluoromethylated pyrazoles as riboflavin synthase inhibitors with antibiotic activity against Mycobacterium tuberculosis.  

PubMed

A high-throughput screening (HTS) hit compound displayed moderate inhibition of Mycobacterium tuberculosis and Escherichia coli riboflavin synthases. The structure of the hit compound provided by the commercial vendor was reassigned as [3-(4-chlorophenyl)-5-hydroxy-5-(trifluoromethyl)-4,5-dihydro-1H-pyrazol-1-yl](o-tolyl)methanone (18). The hit compound had a k(is) of 8.7 microM vs. M. tuberculosis riboflavin synthase and moderate antibiotic activity against both M. tuberculosis replicating phenotype and nonreplicating persistent phenotype. Molecular modeling studies suggest that two inhibitor molecules bind in the active site of the enzyme, and that the binding is stabilized by stacking between the benzene rings of two adjacent ligands. The most potent antibiotic in the series proved to be [5-(4-chlorophenyl)-5-hydroxy-3-(trifluoromethyl)-4,5-dihydro-1H-pyrazol-1-yl](m-tolyl)methanone (16), which displayed a minimum inhibitory concentration (MIC) of 36.6 microM vs. M. tuberculosis replicating phenotype and 48.9 microM vs. M. tuberculosis nonreplicating phenotype. The HTS hit compound and its analogues provide the first examples of riboflavin synthase inhibitors with antibiotic activity. PMID:19545132

Zhao, Yujie; Bacher, Adelbert; Illarionov, Boris; Fischer, Markus; Georg, Gunda; Ye, Qi-Zhuang; Fanwick, Phillip E; Franzblau, Scott G; Wan, Baojie; Cushman, Mark

2009-08-01

107

First report on chitinous holdfast in sponges (Porifera)  

PubMed Central

A holdfast is a root- or basal plate-like structure of principal importance that anchors aquatic sessile organisms, including sponges, to hard substrates. There is to date little information about the nature and origin of sponges’ holdfasts in both marine and freshwater environments. This work, to our knowledge, demonstrates for the first time that chitin is an important structural component within holdfasts of the endemic freshwater demosponge Lubomirskia baicalensis. Using a variety of techniques (near-edge X-ray absorption fine structure, Raman, electrospray ionization mas spectrometry, Morgan–Elson assay and Calcofluor White staining), we show that chitin from the sponge holdfast is much closer to ?-chitin than to ?-chitin. Most of the three-dimensional fibrous skeleton of this sponge consists of spicule-containing proteinaceous spongin. Intriguingly, the chitinous holdfast is not spongin-based, and is ontogenetically the oldest part of the sponge body. Sequencing revealed the presence of four previously undescribed genes encoding chitin synthases in the L. baicalensis sponge. This discovery of chitin within freshwater sponge holdfasts highlights the novel and specific functions of this biopolymer within these ancient sessile invertebrates. PMID:23677340

Ehrlich, Hermann; Kaluzhnaya, Oksana V.; Tsurkan, Mikhail V.; Ereskovsky, Alexander; Tabachnick, Konstantin R.; Ilan, Micha; Stelling, Allison; Galli, Roberta; Petrova, Olga V.; Nekipelov, Serguei V.; Sivkov, Victor N.; Vyalikh, Denis; Born, Rene; Behm, Thomas; Ehrlich, Andre; Chernogor, Lubov I.; Belikov, Sergei; Janussen, Dorte; Bazhenov, Vasilii V.; Worheide, Gert

2013-01-01

108

First report on chitinous holdfast in sponges (Porifera).  

PubMed

A holdfast is a root- or basal plate-like structure of principal importance that anchors aquatic sessile organisms, including sponges, to hard substrates. There is to date little information about the nature and origin of sponges' holdfasts in both marine and freshwater environments. This work, to our knowledge, demonstrates for the first time that chitin is an important structural component within holdfasts of the endemic freshwater demosponge Lubomirskia baicalensis. Using a variety of techniques (near-edge X-ray absorption fine structure, Raman, electrospray ionization mas spectrometry, Morgan-Elson assay and Calcofluor White staining), we show that chitin from the sponge holdfast is much closer to ?-chitin than to ?-chitin. Most of the three-dimensional fibrous skeleton of this sponge consists of spicule-containing proteinaceous spongin. Intriguingly, the chitinous holdfast is not spongin-based, and is ontogenetically the oldest part of the sponge body. Sequencing revealed the presence of four previously undescribed genes encoding chitin synthases in the L. baicalensis sponge. This discovery of chitin within freshwater sponge holdfasts highlights the novel and specific functions of this biopolymer within these ancient sessile invertebrates. PMID:23677340

Ehrlich, Hermann; Kaluzhnaya, Oksana V; Tsurkan, Mikhail V; Ereskovsky, Alexander; Tabachnick, Konstantin R; Ilan, Micha; Stelling, Allison; Galli, Roberta; Petrova, Olga V; Nekipelov, Serguei V; Sivkov, Victor N; Vyalikh, Denis; Born, René; Behm, Thomas; Ehrlich, Andre; Chernogor, Lubov I; Belikov, Sergei; Janussen, Dorte; Bazhenov, Vasilii V; Wörheide, Gert

2013-07-01

109

X-ray Structures and Absolute Configurations of the Antibiotics Oligomycins A, B and C: Inhibitors of ATP Synthase  

Microsoft Academic Search

\\u000a Abstract  Detailed X-ray structures are presented of the three chemical variants of the antibiotic Oligomycin (Oligomycins A, B and\\u000a C), all inhibitors of the enzyme ATP synthase, which has itself been the subject of intensive studies in recent years. All\\u000a three oligomycins crystallized in space group P212121 with Z = 4 molecules per unit cell. Oligomycin A crystallized as the methanol solvate C45H72O11 · CH3OH

Rex A. Palmer; Brian S. Potter

2008-01-01

110

The occurrence of chitin in the hemocytes of invertebrates  

PubMed Central

The light-organ symbiosis of Euprymna scolopes, the Hawaiian bobtail squid, is a useful model for the study of animal–microbe interactions. Recent analyses have demonstrated that chitin breakdown products play a role in communication between E. scolopes and its bacterial symbiont Vibrio fischeri. In this study, we sought to determine the source of chitin in the symbiotic organ. We used a commercially available chitin-binding protein (CBP) conjugated to fluorescein to label the polymeric chitin in host tissues. Confocal microscopy revealed that the only cells in contact with the symbionts that labeled with the probe were the macrophage-like hemocytes, which traffic into the light-organ crypts where the bacteria reside. Labeling of extracted hemocytes by CBP was markedly decreased following treatment with purified chitinase, providing further evidence that the labeled molecule is polymeric chitin. Further, CBP-positive areas co-localized with both a halide peroxidase antibody and Lysotracker, a lysosomal marker, suggesting that the chitin-like biomolecule occurs in the lysosome or acidic vacuoles. Reverse transcriptase polymerase chain reaction (PCR) of hemocytes revealed mRNA coding for a chitin synthase, suggesting that the hemocytes synthesize chitin de novo. Finally, upon surveying blood cells from other invertebrate species, we observed CBP-positive regions in all granular blood cells examined, suggesting that this feature is a shared character among the invertebrates; the vertebrate blood cells that we sampled did not label with CBP. Although the function of the chitin-like material remains undetermined, its presence and subcellular location in invertebrate hemocytes suggests a conserved role for this polysaccharide in the immune system of diverse animals. PMID:21723107

Heath-Heckman, Elizabeth A.C.; McFall-Ngai, Margaret J.

2011-01-01

111

Sulfa and trimethoprim-like drugs - antimetabolites acting as carbonic anhydrase, dihydropteroate synthase and dihydrofolate reductase inhibitors.  

PubMed

Recent advances in microbial genomics, synthetic organic chemistry and X-ray crystallography provided opportunities to identify novel antibacterial targets for the development of new classes of antibiotics and to design more potent antimicrobial compounds derived from existing antibiotics in clinical use for decades. The antimetabolites, sulfa drugs and trimethoprim (TMP)-like agents, are inhibitors of three families of enzymes. One family belongs to the carbonic anhydrases, which catalyze a simple but physiologically relevant reaction in all life kingdoms, carbon dioxide hydration to bicarbonate and protons. The other two enzyme families are involved in the synthesis of tetrahydrofolate (THF), i.e. dihydropteroate synthase (DHPS) and dihydrofolate reductase. The antibacterial agents belonging to the THF and DHPS inhibitors were developed decades ago and present significant bacterial resistance problems. However, the molecular mechanisms of drug resistance both to sulfa drugs and TMP-like inhibitors were understood in detail only recently, when several X-ray crystal structures of such enzymes in complex with their inhibitors were reported. Here, we revue the state of the art in the field of antibacterials based on inhibitors of these three enzyme families. PMID:23627736

Capasso, Clemente; Supuran, Claudiu T

2014-06-01

112

Discovery and in Vivo Evaluation of Potent Dual CYP11B2 (Aldosterone Synthase) and CYP11B1 Inhibitors.  

PubMed

Aldosterone is a key signaling component of the renin-angiotensin-aldosterone system and as such has been shown to contribute to cardiovascular pathology such as hypertension and heart failure. Aldosterone synthase (CYP11B2) is responsible for the final three steps of aldosterone synthesis and thus is a viable therapeutic target. A series of imidazole derived inhibitors, including clinical candidate 7n, have been identified through design and structure-activity relationship studies both in vitro and in vivo. Compound 7n was also found to be a potent inhibitor of 11?-hydroxylase (CYP11B1), which is responsible for cortisol production. Inhibition of CYP11B1 is being evaluated in the clinic for potential treatment of hypercortisol diseases such as Cushing's syndrome. PMID:24900631

Meredith, Erik L; Ksander, Gary; Monovich, Lauren G; Papillon, Julien P N; Liu, Qian; Miranda, Karl; Morris, Patrick; Rao, Chang; Burgis, Robin; Capparelli, Michael; Hu, Qi-Ying; Singh, Alok; Rigel, Dean F; Jeng, Arco Y; Beil, Michael; Fu, Fumin; Hu, Chii-Whei; LaSala, Daniel

2013-12-12

113

Structure-based design, synthesis, and biological evaluation of lipophilic-tailed monocationic inhibitors of neuronal nitric oxide synthase  

PubMed Central

Selective inhibitors of neuronal nitric oxide synthase (nNOS) have the potential to develop into new neurodegenerative therapeutics. Recently, we described the discovery of novel nNOS inhibitors (1a and 1b) based on a cis-pyrrolidine pharmacophore. These compounds and related ones were found to have poor blood-brain barrier permeability, presumably because of the basic nitrogens in the molecule. Here, a series of monocationic compounds was designed on the basis of docking experiments using the crystal structures of 1a,b bound to nNOS. These compounds were synthesized and evaluated for their ability to inhibit neuronal nitric oxide synthase. Despite the excellent overlap of these compounds with 1a,b bound to nNOS, they exhibited low potency. This is because they bound in the nNOS active site in the normal orientation rather than the expected flipped orientation used in the computer modeling. The biphenyl or phenoxyphenyl tail is disordered and does not form good protein-ligand interactions. These studies demonstrate the importance of the size and rigidity of the side chain tail and the second basic amino group for nNOS binding efficiency and the importance of the hydrophobic tail for conformational orientation in the active site of nNOS. PMID:20673724

Xue, Fengtian; Huang, Jinwen; Ji, Haitao; Fang, Jianguo; Li, Huiying; Martasek, Pavel; Roman, Linda J.; Poulos, Thomas L.; Silverman, Richard B.

2010-01-01

114

L-NAME, a nitric oxide synthase inhibitor, as a potential countermeasure to post-suspension hypotension in rats  

NASA Technical Reports Server (NTRS)

A large number of astronauts returning from spaceflight experience orthostatic hypotension. This hypotension may be due to overproduction of vasodilatory mediators, such as nitric oxide (NO) and prostaglandins. To evaluate the role of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) as a countermeasure against the post-suspension reduction in mean arterial pressure (MAP), we assessed the cardiovascular responses and vascular reactivity to 7-day 30 degrees tail-suspension and a subsequent 6 hr post-suspension period in conscious rats. After a pre-suspension reading, direct MAP and heart rate (HR) were measured daily and every 2 hrs post-suspension. The NO synthase inhibitor L-NAME (20 mg/kg, i.v.), or saline, were administered after the 7th day reading prior to release from suspension and at 2 and 4 hrs post-suspension. At 6 hrs post-suspension, vascular reactivity was assessed. While MAP did not change during the suspension period, it was reduced post-suspension. Heart rate was not significantly altered. L-NAME administration reversed the post-suspension reduction in MAP. In addition, the baroreflex sensitivity for heart rate was modified by L-NAME. Thus, the post-suspension reduction in MAP may be due to overproduction of NO and altered baroreflex activity.

Bayorh, M. A.; Socci, R. R.; Watts, S.; Wang, M.; Eatman, D.; Emmett, N.; Thierry-Palmer, M.

2001-01-01

115

Protective effects of a squalene synthase inhibitor, lapaquistat acetate (TAK-475), on statin-induced myotoxicity in guinea pigs  

SciTech Connect

High-dose statin treatment has been recommended as a primary strategy for aggressive reduction of LDL cholesterol levels and protection against coronary artery disease. The effectiveness of high-dose statins may be limited by their potential for myotoxic side effects. There is currently little known about the molecular mechanisms of statin-induced myotoxicity. Previously we showed that T-91485, an active metabolite of the squalene synthase inhibitor lapaquistat acetate (lapaquistat: a previous name is TAK-475), attenuated statin-induced cytotoxicity in human skeletal muscle cells [Nishimoto, T., Tozawa, R., Amano, Y., Wada, T., Imura, Y., Sugiyama, Y., 2003a. Comparing myotoxic effects of squalene synthase inhibitor, T-91485, and 3-hydroxy-3-methylglutaryl coenzyme A. Biochem. Pharmacol. 66, 2133-2139]. In the current study, we investigated the effects of lapaquistat administration on statin-induced myotoxicity in vivo. Guinea pigs were treated with either high-dose cerivastatin (1 mg/kg) or cerivastatin together with lapaquistat (30 mg/kg) for 14 days. Treatment with cerivastatin alone decreased plasma cholesterol levels by 45% and increased creatine kinase (CK) levels by more than 10-fold (a marker of myotoxicity). The plasma CK levels positively correlated with the severity of skeletal muscle lesions as assessed by histopathology. Co-administration of lapaquistat almost completely prevented the cerivastatin-induced myotoxicity. Administration of mevalonolactone (100 mg/kg b.i.d.) prevented the cerivastatin-induced myotoxicity, confirming that this effect is directly related to HMG-CoA reductase inhibition. These results strongly suggest that cerivastatin-induced myotoxicity is due to depletion of mevalonate derived isoprenoids. In addition, squalene synthase inhibition could potentially be used clinically to prevent statin-induced myopathy.

Nishimoto, Tomoyuki; Ishikawa, Eiichiro [Pharmacology Research Laboratories I, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka 532-8686 (Japan); Anayama, Hisashi; Hamajyo, Hitomi; Nagai, Hirofumi [Development Research Center, Takeda Pharmaceutical Company Limited, 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka 532-8686 (Japan); Hirakata, Masao [Pharmacology Research Laboratories I, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka 532-8686 (Japan); Tozawa, Ryuichi [Pharmacology Research Laboratories I, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka 532-8686 (Japan)], E-mail: Ryuichi_Tozawa@takeda.co.jp

2007-08-15

116

NOpiates: Novel Dual Action Neuronal Nitric Oxide Synthase Inhibitors with ?-Opioid Agonist Activity  

PubMed Central

A novel series of benzimidazole designed multiple ligands (DMLs) with activity at the neuronal nitric oxide synthase (nNOS) enzyme and the ?-opioid receptor was developed. Targeting of the structurally dissimilar heme-containing enzyme and the ?-opioid GPCR was predicated on the modulatory role of nitric oxide on ?-opioid receptor function. Structure–activity relationship studies yielded lead compound 24 with excellent nNOS inhibitory activity (IC50 = 0.44 ?M), selectivity over both endothelial nitric oxide synthase (10-fold) and inducible nitric oxide synthase (125-fold), and potent ?-opioid binding affinity, Ki = 5.4 nM. The functional activity as measured in the cyclic adenosine monosphospate secondary messenger assay resulted in full agonist activity (EC50 = 0.34 ?M). This work represents a novel approach in the development of new analgesics for the treatment of pain. PMID:24900459

2012-01-01

117

Arsenic toxicity induced endothelial dysfunction and dementia: pharmacological interdiction by histone deacetylase and inducible nitric oxide synthase inhibitors.  

PubMed

Arsenic toxicity has been reported to damage all the major organs including the brain and vasculature. Dementia including Alzheimer's disease (AD) and vascular dementia (VaD) are posing greater risk to the world population as it is now increasing at a faster rate. We have investigated the role of sodium butyrate, a selective histone deacetylase (HDAC) inhibitor and aminoguanidine, a selective inducible nitric oxide synthase (iNOS) inhibitor in pharmacological interdiction of arsenic toxicity induced vascular endothelial dysfunction and dementia in rats. Arsenic toxicity was done by administering arsenic drinking water to rats. Morris water-maze (MWM) test was used for assessment of learning and memory. Endothelial function was assessed using student physiograph. Oxidative stress (aortic superoxide anion, serum and brain thiobarbituric acid reactive species, brain glutathione) and nitric oxide levels (serum nitrite/nitrate) were also measured. Arsenic treated rats have shown impairment of endothelial function, learning and memory, reduction in serum nitrite/nitrate & brain GSH levels along with increase in serum & brain TBARS. Sodium butyrate as well as aminoguanidine significantly convalesce arsenic induced impairment of learning, memory, endothelial function, and alterations in various biochemical parameters. It may be concluded that arsenic induces endothelial dysfunction and dementia, whereas, sodium butyrate, a HDAC inhibitor as well as aminoguanidine, a selective iNOS inhibitor may be considered as potential agents for the management of arsenic induced endothelial dysfunction and dementia. PMID:23921152

Sharma, Bhupesh; Sharma, P M

2013-11-15

118

The mobility of a conserved tyrosine residue controls isoform-dependent enzyme-inhibitor interactions in nitric oxide synthases.  

PubMed

Many pyrrolidine-based inhibitors highly selective for neuronal nitric oxide synthase (nNOS) over endothelial NOS (eNOS) exhibit dramatically different binding modes. In some cases, the inhibitor binds in a 180° flipped orientation in nNOS relative to eNOS. From the several crystal structures we have determined, we know that isoform selectivity correlates with the rotamer position of a conserved tyrosine residue that H-bonds with a heme propionate. In nNOS, this Tyr more readily adopts the out-rotamer conformation, while in eNOS, the Tyr tends to remain fixed in the original in-rotamer conformation. In the out-rotamer conformation, inhibitors are able to form better H-bonds with the protein and heme, thus increasing inhibitor potency. A segment of polypeptide that runs along the surface near the conserved Tyr has long been thought to be the reason for the difference in Tyr mobility. Although this segment is usually disordered in both eNOS and nNOS, sequence comparisons and modeling from a few structures show that this segment is structured quite differently in eNOS and nNOS. In this study, we have probed the importance of this surface segment near the Tyr by making a few mutants in the region followed by crystal structure determinations. In addition, because the segment near the conserved Tyr is highly ordered in iNOS, we also determined the structure of an iNOS-inhibitor complex. This new structure provides further insight into the critical role that mobility plays in isoform selectivity. PMID:25089924

Li, Huiying; Jamal, Joumana; Delker, Silvia; Plaza, Carla; Ji, Haitao; Jing, Qing; Huang, He; Kang, Soosung; Silverman, Richard B; Poulos, Thomas L

2014-08-19

119

The crystal structure of spermidine synthase with a multisubstrate adduct inhibitor  

Microsoft Academic Search

Polyamines are essential in all branches of life. Spermidine synthase (putrescine aminopropyltransferase, PAPT) catalyzes the biosynthesis of spermidine, a ubiquitous polyamine. The crystal structure of the PAPT from Thermotoga maritima (TmPAPT) has been solved to 1.5 Angstroms resolution in the presence and absence of AdoDATO (S-adenosyl-1,8-diamino-3-thiooctane), a compound containing both substrate and product moieties. This, the first structure of an

Sergey Korolev; Yoshihiko Ikeguchi; Tatiana Skarina; Steven Beasley; Cheryl Arrowsmith; Alexei Savchenko; Aled Edwards; Andrzej Joachimiak; Anthony E. Pegg

2001-01-01

120

Determination of chitin content in fungal cell wall: an alternative flow cytometric method.  

PubMed

The conventional methods used to evaluate chitin content in fungi, such as biochemical assessment of glucosamine release after acid hydrolysis or epifluorescence microscopy, are low throughput, laborious, time-consuming, and cannot evaluate a large number of cells. We developed a flow cytometric assay, efficient, and fast, based on Calcofluor White staining to measure chitin content in yeast cells. A staining index was defined, its value was directly related to chitin amount and taking into consideration the different levels of autofluorecence. Twenty-two Candida spp. and four Cryptococcus neoformans clinical isolates with distinct susceptibility profiles to caspofungin were evaluated. Candida albicans clinical isolate SC5314, and isogenic strains with deletions in chitin synthase 3 (chs3?/chs3?) and genes encoding predicted GlycosylPhosphatidylInositol (GPI)-anchored proteins (pga31?/? and pga62?/?), were used as controls. As expected, the wild-type strain displayed a significant higher chitin content (P < 0.001) than chs3?/chs3? and pga31?/? especially in the presence of caspofungin. Ca. parapsilosis, Ca. tropicalis, and Ca. albicans showed higher cell wall chitin content. Although no relationship between chitin content and antifungal drug susceptibility phenotype was found, an association was established between the paradoxical growth effect in the presence of high caspofungin concentrations and the chitin content. This novel flow cytometry protocol revealed to be a simple and reliable assay to estimate cell wall chitin content of fungi. PMID:23359335

Costa-de-Oliveira, Sofia; Silva, Ana P; Miranda, Isabel M; Salvador, Alexandre; Azevedo, Maria M; Munro, Carol A; Rodrigues, Acácio G; Pina-Vaz, Cidália

2013-03-01

121

Iminosugar-Based Inhibitors of Glucosylceramide Synthase Increase Brain Glycosphingolipids and Survival in a Mouse Model of Sandhoff Disease  

PubMed Central

The neuropathic glycosphingolipidoses are a subgroup of lysosomal storage disorders for which there are no effective therapies. A potential approach is substrate reduction therapy using inhibitors of glucosylceramide synthase (GCS) to decrease the synthesis of glucosylceramide and related glycosphingolipids that accumulate in the lysosomes. Genz-529468, a blood-brain barrier-permeant iminosugar-based GCS inhibitor, was used to evaluate this concept in a mouse model of Sandhoff disease, which accumulates the glycosphingolipid GM2 in the visceral organs and CNS. As expected, oral administration of the drug inhibited hepatic GM2 accumulation. Paradoxically, in the brain, treatment resulted in a slight increase in GM2 levels and a 20-fold increase in glucosylceramide levels. The increase in brain glucosylceramide levels might be due to concurrent inhibition of the non-lysosomal glucosylceramidase, Gba2. Similar results were observed with NB-DNJ, another iminosugar-based GCS inhibitor. Despite these unanticipated increases in glycosphingolipids in the CNS, treatment nevertheless delayed the loss of motor function and coordination and extended the lifespan of the Sandhoff mice. These results suggest that the CNS benefits observed in the Sandhoff mice might not necessarily be due to substrate reduction therapy but rather to off-target effects. PMID:21738789

Ashe, Karen M.; Bangari, Dinesh; Li, Lingyun; Cabrera-Salazar, Mario A.; Bercury, Scott D.; Nietupski, Jennifer B.; Cooper, Christopher G. F.; Aerts, Johannes M. F. G.; Lee, Edward R.; Copeland, Diane P.; Cheng, Seng H.; Scheule, Ronald K.; Marshall, John

2011-01-01

122

Antiinflammatory Effects of Mercaptoethylguanidine, a Combined Inhibitor of Nitric Oxide Synthase and Peroxynitrite Scavenger, in Carrageenan-induced Models of Inflammation  

Microsoft Academic Search

In vitro studies have demonstrated that mercaptoethylguanidine (MEG), a selective inhibitor of the inducible NO synthase (iNOS), is also effective as a scavenger of peroxynitrite (a potent cytotoxic oxidant produced by the reaction of NO and superoxide). In the present study, we evaluated the antiinflammatory potential of MEG treatment in two models of acute inflammation (carrageenan-induced paw edema and pleurisy),

Salvatore Cuzzocrea; Basilia Zingarelli; Paul Hake; Andrew L Salzman; Csaba Szabo

1998-01-01

123

Effect of Potential Amine Prodrugs of Selective Neuronal Nitric Oxide Synthase Inhibitors on Blood-Brain Barrier Penetration  

PubMed Central

Several prodrug approaches were taken to mask amino groups in two potent and selective neuronal nitric oxide synthase (nNOS) inhibitors containing either a primary or secondary amino group to lower the charge and improve blood-brain barrier (BBB) penetration. The primary amine was masked as an azide and the secondary amine as an amide or carbamate. The azide was not reduced to the amine under a variety of in vitro and ex vivo conditions. Despite the decrease in charge of the amino group as an amide and as carbamates, BBB penetration did not increase. It appears that the use of azides as prodrugs for primary amines or amides and carbamates as prodrugs for secondary amines are not universally effective approaches for CNS applications. PMID:19796958

Silverman, Richard B.; Lawton, Graham R; Ranaivo, Hantamalala Ralay; Seo, Jiwon; Watterson, D. Martin

2009-01-01

124

Inhibitors of the Salicylate Synthase (MbtI) from Mycobacterium tuberculosis Discovered by High-Throughput Screening  

PubMed Central

A simple steady-state kinetic high-throughput assay was developed for the salicylate synthase MbtI from Mycobacterium tuberculosis, which catalyzes the first committed step of mycobactin biosynthesis. The mycobactins are small-molecule iron chelators produced by M. tuberculosis, and their biosynthesis has been identified as a promising target for the development of new antitubercular agents. The assay was miniaturized to a 384-well plate format and high-throughput screening was performed at the National Screening Laboratory for the Regional Centers of Excellence in Biodefense and Emerging Infectious Diseases (NSRB). Three classes of compounds were identified comprising the benzisothiazolones (class I), diarylsulfones (class II), and benzimidazole-2-thiones (class III). Each of these compound series was further pursued to investigate their biochemical mechanism and structure–activity relationships. Benzimidazole-2-thione 4 emerged as the most promising inhibitor owing to its potent reversible inhibition. PMID:21053346

Vasan, Mahalakshmi; Neres, Joao; Williams, Jessica; Wilson, Daniel J.; Teitelbaum, Aaron M.; Remmel, Rory P.; Aldrich, Courtney C.

2010-01-01

125

Chitin nanowhisker aerogels.  

PubMed

Chitin nanowhiskers are structured into mesoporous aerogels by using the same benign process used previously in our group to make cellulose nanowhisker aerogels. The nanowhiskers are sonicated in water to form a hydrogel before solvent-exchange with ethanol and drying under supercritical CO2 (scCO2 ). Aerogels are prepared with various densities and porosities, relating directly to the initial chitin nanowhisker content. scCO2 drying enables the mesoporous network structure to be retained as well as allowing the gel to retain its initial dimensions. The chitin aerogels have low densities (0.043-0.113?g?cm(-3) ), high porosities (up to 97?%), surface areas of up to 261?m(2) ?g(-1) , and mechanical properties at the high end of other reported values (modulus between 7 and 9.3?MPa). The aerogels were further characterized by using X-ray diffraction, BET analysis, electron microscopy, FTIR, and thermogravimetric analysis. Characterization showed that the rod-like crystalline nature of the nanowhiskers was retained during the aerogel production process, making the aerogel truly an assembled structure of chitin nanocrystals. These aerogels also showed the lowest reported shrinkage during drying to date, with an average shrinkage of only 4?%. PMID:23335426

Heath, Lindy; Zhu, Lifan; Thielemans, Wim

2013-03-01

126

Genetic rearrangements on the Chlorovirus genome that switch between hyaluronan synthesis and chitin synthesis.  

PubMed

Chlorella viruses or chloroviruses form polysaccharide fibers on the cell wall of host Chlorella cells after infection. Such polysaccharides are either hyaluronan synthesized by virus-encoded hyaluronan synthase (HAS) or chitin synthesized by viral chitin synthase (CHS). Some chloroviruses synthesize both hyaluronan (HA) and chitin simultaneously. To understand the relationship between "HA-synthesizing" and "chitin-synthesizing" viruses, we characterized the CVK2 genomic regions, one flanking chs and the other corresponding to PBCV-1 has and found that on CVK2 DNA, a single ORF (PBCV-1 A330R) was replaced with a 5 kbp region containing chs, ugdh2 (the second gene for UDP-glucose dehydrogenase) and two other ORFs, and that has was replaced with another chs gene. In some chloroviruses, ugdh was lost. These results suggest that chlorovirus types changed from "has viruses" to "chs viruses" or from "chs viruses" to "has viruses" by exchanging the genes. PMID:16112160

Mohammed Ali, Ali Mohammed; Kawasaki, Takeru; Yamada, Takashi

2005-11-10

127

The Design and Synthesis of Potent and Selective Inhibitors of Trypanosoma brucei Glycogen Synthase Kinase 3 for the Treatment of Human African Trypanosomiasis  

PubMed Central

Glycogen synthase kinase 3 (GSK3) is a genetically validated drug target for human African trypanosomiasis (HAT), also called African sleeping sickness. We report the synthesis and biological evaluation of aminopyrazole derivatives as Trypanosoma brucei GSK3 short inhibitors. Low nanomolar inhibitors, which had high selectivity over the off-target human CDK2 and good selectivity over human GSK3? enzyme, have been prepared. These potent kinase inhibitors demonstrated low micromolar levels of inhibition of the Trypanosoma brucei brucei parasite grown in culture. PMID:25198388

2014-01-01

128

Peripheral enzymatic deacetylation of chitin and reprecipitated chitin particles.  

PubMed

The enzymatic deacetylation of various chitin preparations was investigated using the fungal chitin deacetylase (CDA) isolated from Rhizopus oryzae growth medium. Specific extracellular enzyme activity after solid state fermentation was 10 times higher than that after submerged fermentation. Natural crystalline chitin is a very poor substrate for the enzyme, but showed a five-time better deacetylation after dissolution and reprecipitation. Chitin particles, enzymatically deacetylated for only 1% exhibited a strongly increased binding capacity towards ovalbumin, while maintaining the rigidity and insolubility of chitin in a moderate acidic environment. Because of the unique combination of properties, these CDA treated chitin materials were named "chit-in-osan". Chitinosan was shown to be an attractive matrix for column chromatography because no hydrogel formation was observed, that impaired the flow of eluent. Under the same conditions, partially deacetylated chitosan swelled and blocked the flow in the column. PMID:15919204

Aye, Kyaw Nyein; Karuppuswamy, Renuka; Ahamed, Tangir; Stevens, Willem F

2006-03-01

129

Identification and first insights into the structure and biosynthesis of chitin from the freshwater sponge Spongilla lacustris.  

PubMed

This work demonstrates that chitin is an important structural component within the skeletal fibers of the freshwater sponge Spongilla lacustris. Using a variety of analytical techniques ((13)C solid state NMR, FT-IR, Raman, NEXAFS, ESI-MS, Morgan-Elson assay and Calcofluor White Staining); we show that this sponge chitin is much closer to ?-chitin, known to be present in other animals, than to ?-chitin. Genetic analysis confirmed the presence of chitin synthases, which are described for the first time in a sponge. The presence of chitin in both marine (demosponges and hexactinellids) and freshwater sponges indicates that this important structural biopolymer was already present in their common ancestor. PMID:23831449

Ehrlich, Hermann; Kaluzhnaya, Oksana V; Brunner, Eike; Tsurkan, Mikhail V; Ereskovsky, Alexander; Ilan, Micha; Tabachnick, Konstantin R; Bazhenov, Vasilii V; Paasch, Silvia; Kammer, Martin; Born, René; Stelling, Allison; Galli, Roberta; Belikov, Sergei; Petrova, Olga V; Sivkov, Victor V; Vyalikh, Denis; Hunoldt, Sebastian; Wörheide, Gert

2013-09-01

130

Chitin synthesis in chlorovirus CVK2-infected chlorella cells.  

PubMed

Hyaluronan synthesis in chlorovirus PBCV-1-infected Chlorella cells was previously reported (DeAngelis et al., 1997). In contrast, we report here on the detection, characterization, and expression of a gene for chitin synthase (chs) encoded by chlorovirus CVK2 isolated in Kyoto, Japan. The CVK2 chs gene encoding an open reading frame of 516 aa was expressed as early as 10 min postinfection (p.i.), peaked at 20-40 min p.i., and disappeared at 120-180 min p.i. The chitin polysaccharide began to accumulate as chitinase-sensitive, hair-like fibers on the outside of the virus-infected Chlorella cell wall by 30 min p.i. All chloroviruses without the gene for hyaluronan synthase (has) alternatively contained the chs gene, suggesting the importance of polysaccharide production in the course of virus infection. A few chloroviruses possessed both the chs and has genes and produced chitin and hyaluronan simultaneously. Polysaccharide accumulation on the algal surface may protect virus-infected algae from uptake by other organisms, such as protozoa. Since CVK2 was reported to encode two chitinases and one chitosanase, CVK2 is a very peculiar virus that encodes enzymes required for both the synthesis and the degradation of chitin materials. PMID:12429521

Kawasaki, Takeru; Tanaka, Masahiro; Fujie, Makoto; Usami, Shoji; Sakai, Kazuo; Yamada, Takashi

2002-10-10

131

Pharmacological characterization of KLYP961, a dual inhibitor of inducible and neuronal nitric-oxide synthases.  

PubMed

Nitric oxide (NO) derived from neuronal nitric-oxide synthase (nNOS) and inducible nitric-oxide synthase (iNOS) plays a key role in various pain and inflammatory states. KLYP961 (4-((2-cyclobutyl-1H-imidazo[4,5-b]pyrazin-1-yl)methyl)-7,8-difluoroquinolin-2(1H)-one) inhibits the dimerization, and hence the enzymatic activity of human, primate, and murine iNOS and nNOS (IC(50) values 50-400 nM), with marked selectivity against endothelial nitric-oxide synthase (IC(50) >15,000 nM). It has ideal drug like-properties, including excellent rodent and primate pharmacokinetics coupled with a minimal off-target activity profile. In mice, KLYP961 attenuated endotoxin-evoked increases in plasma nitrates, a surrogate marker of iNOS activity in vivo, in a sustained manner (ED(50) 1 mg/kg p.o.). KLYP961 attenuated pain behaviors in a mouse formalin model (ED(50) 13 mg/kg p.o.), cold allodynia in the chronic constriction injury model (ED(50) 25 mg/kg p.o.), or tactile allodynia in the spinal nerve ligation model (ED(50) 30 mg/kg p.o.) with similar efficacy, but superior potency relative to gabapentin, pregabalin, or duloxetine. Unlike morphine, the antiallodynic activity of KLYP961 did not diminish upon repeated dosing. KLYP961 also attenuated carrageenin-induced edema and inflammatory hyperalgesia and writhing response elicited by phenylbenzoquinone with efficacy and potency similar to those of celecoxib. In contrast to gabapentin, KLYP961 did not impair motor coordination at doses as high as 1000 mg/kg p.o. KLYP961 also attenuated capsaicin-induced thermal allodynia in rhesus primates in a dose-related manner with a minimal effective dose (? 10 mg/kg p.o.) and a greater potency than gabapentin. In summary, KLYP961 represents an ideal tool with which to probe the physiological role of NO derived from iNOS and nNOS in human pain and inflammatory states. PMID:21036913

Symons, Kent T; Nguyen, Phan M; Massari, Mark E; Anzola, John V; Staszewski, Lena M; Wang, Li; Yazdani, Nahid; Dorow, Steven; Muhammad, Jerry; Sablad, Marciano; Rozenkrants, Natasha; Bonefous, Celine; Payne, Joseph E; Rix, Peter J; Shiau, Andrew K; Noble, Stewart A; Smith, Nicholas D; Hassig, Christian A; Zhang, Yan; Rao, Tadimeti S

2011-02-01

132

Developing dual and specific inhibitors of dimethylarginine dimethylaminohydrolase-1 and nitric oxide synthase: Toward a targeted polypharmacology to control nitric oxide†  

PubMed Central

Molecules that block nitric oxide's (NO) biosynthesis are of significant interest. For example, nitric oxide synthase (NOS) inhibitors have been suggested as anti-tumor therapeutics, as have inhibitors of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that catabolizes endogenous NOS inhibitors. Dual-targeted inhibitors hold promise as more effective reagents to block NO biosynthesis than single-targeted compounds. In this study, a small set of known NOS inhibitors are surveyed as inhibitors of recombinant human DDAH-1. From these, an alkylamidine scaffold is selected for homologation. Stepwise lengthening of one substituent converts an NOS-selective inhibitor into a dual-targeted NOS/DDAH-1 inhibitor and then into a DDAH-1 selective inhibitor, as seen in the inhibition constants of N5-(1-iminoethyl)-, N5-(1-iminopropyl)-, N5-(1-iminopentyl)- and N5-(1-iminohexyl)-l-ornithine for neuronal NOS (1.7, 3, 20, >1,900 ?M, respectively) and DDAH-1 (990, 52, 7.5, 110 ?M, respectively). A 1.9Å X-ray crystal structure of the N5-(1-iminopropyl)-l-ornithine : DDAH-1 complex indicates covalent bond formation between the inhibitor's amidino carbon and the active-site Cys274, and solution studies show reversible competitive inhibition, consistent with a reversible covalent mode of DDAH inhibition by alkylamidine inhibitors. These represent a versatile scaffold for the development of a targeted polypharmacological approach to control NO biosynthesis. PMID:19663506

Wang, Yun; Monzingo, Arthur F.; Hu, Shougang; Schaller, Tera H.; Robertus, Jon D.; Fast, Walter

2009-01-01

133

CT2108A and B: New fatty acid synthase inhibitors as antifungal agents.  

PubMed

A systematic screen for new natural products that displayed antifungal activity by inhibition of fungal fatty acid synthase (FAS) led to the discovery of two new fungal metabolites, designated CT2108A (1) and CT2108B (2). The metabolites were produced by Penicillium solitum (Westling) strain CT2108 and were classified as azaphilones. The structures of these new metabolites were determined using a variety of 1D and 2D NMR experiments, including COSY, HMQC, and HMBC. The chemical conversion of CT2108A to CT2108B was effected using WCl(6). The related metabolite, patulodin (3), was also isolated from the fermentation culture of this P. solitum isolate. Both new compounds inhibited fungal FAS, and neither was found to significantly inhibit human FAS activity. PMID:12932120

Laakso, Jodi A; Raulli, Robert; McElhaney-Feser, Gail E; Actor, Paul; Underiner, Ted L; Hotovec, Brian J; Mocek, Ursula; Cihlar, Ronald L; Broedel, Sheldon E

2003-08-01

134

Glycogen Synthase Kinase 3 Inhibitors in the Next Horizon for Alzheimer's Disease Treatment  

PubMed Central

Glycogen synthase kinase 3 (GSK-3), a proline/serine protein kinase ubiquitously expressed and involved in many cellular signaling pathways, plays a key role in the pathogenesis of Alzheimer's disease (AD) being probably the link between ?-amyloid and tau pathology. A great effort has recently been done in the discovery and development of different new molecules, of synthetic and natural origin, able to inhibit this enzyme, and several kinetics mechanisms of binding have been described. The small molecule called tideglusib belonging to the thiadiazolidindione family is currently on phase IIb clinical trials for AD. The potential risks and benefits of this new kind of disease modifying drugs for the future therapy of AD are discussed in this paper. PMID:21760986

Martinez, Ana; Gil, Carmen; Perez, Daniel I.

2011-01-01

135

DOI: 10.1002/cmdc.201100589 Dual Dehydrosqualene/Squalene Synthase Inhibitors: Leads for Innate  

E-print Network

-targeting ap- proach to antibacterial therapy. Here, we report the discovery of such lead compounds. First, we inhibitors such as compound 1 are poor NET inducers (Table 1). Likewise, the use of statins to block pigment School of Pharmacy & Pharma- ceutical Sciences, University of California, San Diego 9500 Gilman Drive, La

Nizet, Victor

136

Structureactivity relationship of the 7-hydroxy benzimidazole analogs as glycogen synthase kinase 3b inhibitor  

E-print Network

b inhibitors have been proposed with a wide range of potencies at the discovery stage. In addition. Ro), ygsuh@snu.ac.kr (Y.-G. Suh). Staurosporine Indirubin AR-AO014418 Tideglusib (NP-12) O N N S O H

Suh, Young-Ger

137

Chitin Synthesis in Chlorovirus CVK2Infected Chlorella Cells  

Microsoft Academic Search

Hyaluronan synthesis in chlorovirus PBCV-1-infected Chlorella cells was previously reported (DeAngelis et al., 1997). In contrast, we report here on the detection, characterization, and expression of a gene for chitin synthase (chs) encoded by chlorovirus CVK2 isolated in Kyoto, Japan. The CVK2 chs gene encoding an open reading frame of 516 aa was expressed as early as 10 min postinfection

Takeru Kawasaki; Masahiro Tanaka; Makoto Fujie; Shoji Usami; Kazuo Sakai; Takashi Yamada

2002-01-01

138

The fatty acid synthase inhibitor triclosan: repurposing an anti-microbial agent for targeting prostate cancer.  

PubMed

Inhibition of FASN has emerged as a promising therapeutic target in cancer, and numerous inhibitors have been investigated. However, severe pharmacological limitations have challenged their clinical testing. The synthetic FASN inhibitor triclosan, which was initially developed as a topical antibacterial agent, is merely affected by these pharmacological limitations. Yet, little is known about its mechanism in inhibiting the growth of cancer cells. Here we compared the cellular and molecular effects of triclosan in a panel of eight malignant and non-malignant prostate cell lines to the well-known FASN inhibitors C75 and orlistat, which target different partial catalytic activities of FASN. Triclosan displayed a superior cytotoxic profile with a several-fold lower IC50 than C75 or orlistat. Structure-function analysis revealed that alcohol functionality of the parent phenol is critical for inhibitory action. Rescue experiments confirmed that end product starvation was a major cause of cytotoxicity. Importantly, triclosan, C75 and orlistat induced distinct changes to morphology, cell cycle, lipid content and the expression of key enzymes of lipid metabolism, demonstrating that inhibition of different partial catalytic activities of FASN activates different metabolic pathways. These finding combined with its well-documented pharmacological safety profile make triclosan a promising drug candidate for the treatment of prostate cancer. PMID:25313139

Sadowski, Martin C; Pouwer, Rebecca H; Gunter, Jennifer H; Lubik, Amy A; Quinn, Ronald J; Nelson, Colleen C

2014-10-15

139

The crystal structure of spermidine synthase with a multisubstrate adduct inhibitor.  

SciTech Connect

Polyamines are essential in all branches of life. Spermidine synthase (putrescine aminopropyltransferase, PAPT) catalyzes the biosynthesis of spermidine, a ubiquitous polyamine. The crystal structure of the PAPT from Thermotoga maritima (TmPAPT) has been solved to 1.5 Angstroms resolution in the presence and absence of AdoDATO (S-adenosyl-1,8-diamino-3-thiooctane), a compound containing both substrate and product moieties. This, the first structure of an aminopropyltransferase, reveals deep cavities for binding substrate and cofactor, and a loop that envelops the active site. The AdoDATO binding site is lined with residues conserved in PAPT enzymes from bacteria to humans, suggesting a universal catalytic mechanism. Other conserved residues act sterically to provide a structural basis for polyamine specificity. The enzyme is tetrameric; each monomer consists of a C-terminal domain with a Rossmann-like fold and an N-terminal {beta}-stranded domain. The tetramer is assembled using a novel barrel-type oligomerization motif.

Korolev, S.; Ikeguchi, Y.; Skarina, T.; Beasley, S.; Arrowsmith, C.; Edwards, A.; Joachimiak, A.; Pegg, A. E.; Savchenko, A.; Pennsylvania State Univ. Coll. of Medicine; Milton S. Hershey Medical Center; Banting and Best Department of Medical Research; Univ. of Health Network

2002-01-01

140

Fatty acid synthase inhibitors from plants: isolation, structure elucidation, and SAR studies.  

PubMed

Fatty acid synthase (FAS) has been identified as a potential antifungal target. FAS prepared from Saccharomyces cerevisiae was employed for bioactivity-guided fractionation of Chlorophora tinctoria,Paspalum conjugatum, Symphonia globulifera, Buchenavia parviflora, and Miconia pilgeriana. Thirteen compounds (1-13), including three new natural products (1, 4, 12), were isolated and their structures identified by spectroscopic interpretation. They represented five chemotypes, namely, isoflavones, flavones, biflavonoids, hydrolyzable tannin-related derivatives, and triterpenoids. 3'-Formylgenistein (1) and ellagic acid 4-O-alpha-l-rhamnopyranoside (9) were the most potent compounds against FAS, with IC(50) values of 2.3 and 7.5 microg/mL, respectively. Furthermore, 43 (14-56) analogues of the five chemotypes from our natural product repository and commercial sources were tested for their FAS inhibitory activity. Structure-activity relationships for some chemotypes were investigated. All these compounds were further evaluated for antifungal activity against Candida albicans and Cryptococcus neoformans. Although there were several antifungal compounds in the set, correlation between the FAS inhibitory activity and antifungal activity could not be defined. PMID:12502337

Li, Xing-Cong; Joshi, Alpana S; ElSohly, Hala N; Khan, Shabana I; Jacob, Melissa R; Zhang, Zhizheng; Khan, Ikhlas A; Ferreira, Daneel; Walker, Larry A; Broedel, Sheldon E; Raulli, Robert E; Cihlar, Ronald L

2002-12-01

141

Allosteric Inhibitors at the Heterodimer Interface of Imidazole Glycerol Phosphate Synthase  

NASA Astrophysics Data System (ADS)

Imidazole glycerol phosphate synthase (IGPS) from Thermotoga maritima is a heterodimeric enzyme composed of the HisH and HisF proteins. It is attractive as a pathological target since it is absent in mammals but found in plant and opportunistic human pathogens. IGPS was experimentally determined to be a V-type allosteric enzyme that is involved in an essential biosynthetic pathway of microorganisms. The enzyme catalyzes the hydrolysis of glutamine to form NH3 in the HisH protein, followed by cyclization of NH3 with N'-[(5'-phosphoribulosyl)imino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) in the HisF subunit, forming imidazole glycerol phosphate (IGP) and 5-aminoimidazole-4-carboxamide ribotide (AICAR) that enter the histidine and purine biosynthetic pathways. Allosteric motions induced upon the binding of the effector PRFAR to HisF propagate through the non-covalent HisH/HisF interface and synchronize catalytic activity at the two distant active sites. However, the nature of the allosteric pathway and the feasibility of manipulating signal transduction by using allosteric drug-like molecules remain to be established. Molecular docking studies of commercial drugs at the HisH/HisF interface were used to identify stable candidates with a potential allosteric effect on the reaction mechanism. Molecular dynamic simulations and calculations of NMR chemical shifts were combined to elucidate the allosteric pathway of IGPS.

Snoeberger, Ning-Shiuan Nicole

142

Bcl2L13 is a ceramide synthase inhibitor in glioblastoma  

PubMed Central

Therapy resistance is a major limitation to the successful treatment of cancer. Here, we identify Bcl2-like 13 (Bcl2L13), an atypical member of the Bcl-2 family, as a therapy susceptibility gene with elevated expression in solid and blood cancers, including glioblastoma (GBM). We demonstrate that mitochondria-associated Bcl2L13 inhibits apoptosis induced by a wide spectrum of chemo- and targeted therapies upstream of Bcl2-associated X protein activation and mitochondrial outer membrane permeabilization in vitro and promotes GBM tumor growth in vivo. Mechanistically, Bcl2L13 binds to proapoptotic ceramide synthases 2 (CerS2) and 6 (CerS6) via a unique C-terminal 250-aa sequence located between its Bcl-2 homology and membrane anchor domains and blocks homo- and heteromeric CerS2/6 complex formation and activity. Correspondingly, CerS2/6 activity and Bcl2L13 abundance are inversely correlated in GBM tumors. Thus, our genetic and functional studies identify Bcl2L13 as a regulator of therapy susceptibility and point to the Bcl2L13–CerS axis as a promising target to enhance responses of therapy-refractory cancers toward conventional and targeted regimens currently in clinical use. PMID:24706805

Jensen, Samuel A.; Calvert, Andrea E.; Volpert, Giora; Kouri, Fotini M.; Hurley, Lisa A.; Luciano, Janina P.; Wu, Yongfei; Chalastanis, Alexandra; Futerman, Anthony H.; Stegh, Alexander H.

2014-01-01

143

Bcl2L13 is a ceramide synthase inhibitor in glioblastoma.  

PubMed

Therapy resistance is a major limitation to the successful treatment of cancer. Here, we identify Bcl2-like 13 (Bcl2L13), an atypical member of the Bcl-2 family, as a therapy susceptibility gene with elevated expression in solid and blood cancers, including glioblastoma (GBM). We demonstrate that mitochondria-associated Bcl2L13 inhibits apoptosis induced by a wide spectrum of chemo- and targeted therapies upstream of Bcl2-associated X protein activation and mitochondrial outer membrane permeabilization in vitro and promotes GBM tumor growth in vivo. Mechanistically, Bcl2L13 binds to proapoptotic ceramide synthases 2 (CerS2) and 6 (CerS6) via a unique C-terminal 250-aa sequence located between its Bcl-2 homology and membrane anchor domains and blocks homo- and heteromeric CerS2/6 complex formation and activity. Correspondingly, CerS2/6 activity and Bcl2L13 abundance are inversely correlated in GBM tumors. Thus, our genetic and functional studies identify Bcl2L13 as a regulator of therapy susceptibility and point to the Bcl2L13-CerS axis as a promising target to enhance responses of therapy-refractory cancers toward conventional and targeted regimens currently in clinical use. PMID:24706805

Jensen, Samuel A; Calvert, Andrea E; Volpert, Giora; Kouri, Fotini M; Hurley, Lisa A; Luciano, Janina P; Wu, Yongfei; Chalastanis, Alexandra; Futerman, Anthony H; Stegh, Alexander H

2014-04-15

144

The chsD and chsE genes of Aspergillus nidulans and their roles in chitin synthesis.  

PubMed

Two chitin synthase genes, chsD and chsE, were identified from the filamentous ascomycete Aspergillus nidulans. In a region that is conserved among chitin synthases, the deduced amino acid sequences of chsD and chsE have greater sequence identity to the polypeptides encoded by the Saccharomyces cerevisiae CHS3 gene (also named CSD2, CAL1, DIT101, and KTI1) and the Candida albicans CHSE gene than to other chitin synthases. chsE is more closely related to the CHS3 genes, and this group constitutes the class IV chitin synthases. chsD differs sufficiently from the other classes of fungal chitin synthase genes to constitute a new class, class V. Each of the wild-type A. nidulans genes was replaced by a copy that had a substantial fraction of its coding region replaced by the A. nidulans argB gene. Hyphae from both chsD and chsE disruptants contain about 60-70% of the chitin content of wild-type hyphae. The morphology and development of chsE disruptants are indistinguishable from those of wild type. Nearly all of the conidia of chsD disruption strains swell excessively and lyse when germinated in low osmotic strength medium. Conidia that do not lyse produce hyphae that initially have normal morphology but subsequently lyse at subapical locations and show ballooned walls along their length. The lysis of germinating conidia and hyphae of chsD disruptants is prevented by the presence of osmotic stabilizers in the medium. Conidiophore vesicles from chsD disruption strains frequently swell excessively and lyse, resulting in colonies that show reduced conidiation. These properties indicate that chitin synthesized by the chsD-encoded isozyme contributes to the rigidity of the walls of germinating conidia, of the subapical region of hyphae, and of conidiophore vesicles, but is not necessary for normal morphology of these cells. The phenotypes of chsD and chsE disruptants indicate that the chitin synthesized by each isozyme serves a distinct function. The propensity of a chsD disruptant for osmotically induced lysis was compared to that of strains carrying two other mutations (tsE6 and orlA::trpC) which also result in reduced chitin content vegetative cell lysis. The concentration of osmotic stabilizer necessary to remedy the lysis of strains carrying the three mutations is inversely related to the chitin content of each strain. This finding directly demonstrates the importance of chitin to the integrity of the cell wall and indicates that agents that inhibit the chsD-encoded chitin synthase could be useful anti-Aspergillus drugs. PMID:8810520

Specht, C A; Liu, Y; Robbins, P W; Bulawa, C E; Iartchouk, N; Winter, K R; Riggle, P J; Rhodes, J C; Dodge, C L; Culp, D W; Borgia, P T

1996-06-01

145

Glucocorticoid-induced insulin resistance in skeletal muscles: defects in insulin signalling and the effects of a selective glycogen synthase kinase-3 inhibitor  

Microsoft Academic Search

Aims\\/hypothesis  Treatment with glucocorticoids, especially at high doses, induces insulin resistance. The aims of the present study were to identify the potential defects in insulin signalling that contribute to dexamethasone-induced insulin resistance in skeletal muscles, and to investigate whether the glycogen synthase-3 (GSK-3) inhibitor CHIR-637 could restore insulin-stimulated glucose metabolism.Materials and methods  Skeletal muscles were made insulin-resistant by treating male Wistar rats

J. Ruzzin; A. S. Wagman; J. Jensen

2005-01-01

146

Cerebroprotective Effect of the Nitric Oxide Synthase Inhibitors, 1-(2-Trifluoromethylphenyl) Imidazole and 7Nitro Indazole, After Transient Focal Cerebral Ischemia in the Rat  

Microsoft Academic Search

The novel neuronal nitric oxide synthase inhibitors, 1-(2-trifluoromethylphenyl)imidazole (TRIM) and 7-nitro indazole (7-NI), were used to investigate the role of nitric oxide in a model of transient focal cerebral ischemia in vivo. In halothane-anesthetized rats, the middle cerebral artery (MCA) was occluded for 2 hours using an intravascular thread and then reperfused for 22 hours before histologic evaluation. TRIM(10, 20,

Katherine J. Escott; John S. Beech; Kristin K. Haga; Steve C. R. Williams; Brian S. Meldrum; Philip M. W. Bath; Philip MW Bath

1998-01-01

147

Effects of sigma receptor agonists on the impairment of spontaneous alternation behavior and decrease of cyclic GMP level induced by nitric oxide synthase inhibitors in mice  

Microsoft Academic Search

In this study, we investigated the involvement of the interaction between sigma receptors and the nitric oxide\\/cyclic GMP pathway in short term memory in mice, assessed through spontaneous alternation behavior in a Y-maze. NG-Nitro-L-arginine methyl ester and 7-nitro indazole, both nitric oxide synthase inhibitors, impaired the spontaneous alternation behavior. These impairments were attenuated by (+) SKF 10,047 and (+) pentazocine,

Takayoshi Mamiya; Yukihiro Noda; Akihiro Noda; Masayuki Hiramatsu; Katsuhiro Karasawa; Tsutomu Kameyama; Shoei Furukawa; Kiyofumi Yamada; Toshitaka Nabeshima

2000-01-01

148

N?-Nitro-L-Arginine, a Nitric Oxide Synthase Inhibitor, Antagonizes Quinolinic Acid-Induced Neurotoxicity and Oxidative Stress in Rat Striatal Slices  

Microsoft Academic Search

Nitric oxide (NO) is a potential contributor to neurotoxicity following overactivation of N-methyl-D-aspartate (NMDA) receptors. In this work we investigated the effect of N?-nitro-L-arginine (L-NARG 25, 50, or 100 ?M), a selective inhibitor of nitric oxide synthase (NOS) -the synthetic enzyme of NO- on quinolinic acid (QUIN 100 ?M)-induced neurotoxicity (measured as lactate dehydrogenase (LDH) leakage) in rat striatal slices.

Daniel Santamaría; Velia Espinoza-González; Camilo Ríos; Abel Santamaría

1999-01-01

149

The effect of the nitric oxide synthase inhibitor, l?NMMA, on sodium metabisulphite?induced bronchoconstriction and refractoriness in asthma  

Microsoft Academic Search

The effect of the nitric oxide synthase inhibitor, l-NMMA, on sodium metabisulphite- induced bronchoconstriction and refractoriness in asthma. A.M. Hamad, A.Wisniewski, S.P. Range, T. Small, F. Holland, A.J. Knox. #ERS Journals Ltd 1999. ABSTRACT: Refractoriness to indirect bronchoconstrictor stimuli, is a feature of asthma but the mechanism is poorly understood. This study tested the hypothesis that endogenous nitric oxide (NO)

A. m. Hamad; A. Wisniewski; S. p. Range; T. Small; F. Holland; A. j. Knox

1999-01-01

150

Identification of Inhibitors against Mycobacterium tuberculosis Thiamin Phosphate Synthase, an Important Target for the Development of Anti-TB Drugs  

PubMed Central

Tuberculosis (TB) continues to pose a serious challenge to human health afflicting a large number of people throughout the world. In spite of the availability of drugs for the treatment of TB, the non-compliance to 6–9 months long chemotherapeutic regimens often results in the emergence of multidrug resistant strains of Mycobacterium tuberculosis adding to the precariousness of the situation. This has necessitated the development of more effective drugs. Thiamin biosynthesis, an important metabolic pathway of M.tuberculosis, is shown to be essential for the intracellular growth of this pathogen and hence, it is believed that inhibition of this pathway would severely affect the growth of M.tuberculosis. In this study, a comparative homology model of M.tuberculosis thiamin phosphate synthase (MtTPS) was generated and employed for virtual screening of NCI diversity set II to select potential inhibitors. The best 39 compounds based on the docking results were evaluated for their potential to inhibit the MtTPS activity. Seven compounds inhibited MtTPS activity with IC50 values ranging from 20 – 100 µg/ml and two of these exhibited weak inhibition of M.tuberculosis growth with MIC99 values being 125 µg/ml and 162.5 µg/ml while one compound was identified as a very potent inhibitor of M.tuberculosis growth with an MIC99 value of 6 µg/ml. This study establishes MtTPS as a novel drug target against M.tuberculosis leading to the identification of new lead molecules for the development of antitubercular drugs. Further optimization of these lead compounds could result in more potent therapeutic molecules against Tuberculosis. PMID:21818324

Khare, Garima; Kar, Ritika; Tyagi, Anil K.

2011-01-01

151

Role of Tyr348 in Tyr385 Radical Dynamics and Cyclooxygenase Inhibitor Interactions in Prostaglandin H Synthase-2†  

PubMed Central

Both prostaglandin H synthase (PGHS) isoforms utilize a radical at Tyr385 to abstract a hydrogen atom from arachidonic acid, initializing prostaglandin synthesis. A Tyr348–Tyr385 hydrogen bond appears to be conserved in both isoforms; this hydrogen bonding has the potential to modulate the positioning and reactivity of the Tyr385 side chain. The EPR signal from the Tyr385 radical undergoes a time-dependent transition from a wide doublet to a wide singlet species in both isoforms. In PGHS-2, this transition results from radical migration from Tyr385 to Tyr504. Localization of the radical to Tyr385 in the recombinant human PGHS-2 Y504F mutant was exploited in examining the effects of blocking Tyr385 hydrogen bonding by introduction of a further Y348F mutation. Cyclooxygenase and peroxidase activities were found to be maintained in the Y348F/Y504F mutant, but the Tyr385 radical was formed more slowly and had greater rotational freedom, as evidenced by observation of a transition from an initial wide doublet species to a narrow singlet species, a transition not seen in the parent Y504F mutant. The effect of disrupting Tyr385 hydrogen bonding on the cyclooxygenase active site structure was probed by examination of cyclooxygenase inhibitor kinetics. Aspirin treatment eliminated all oxygenase activity in the Y348F/Y504F double mutant, with no indication of the lipoxygenase activity observed in aspirin-treated wild-type PGHS-2. Introduction of the Y348F mutation also strengthened the time-dependent inhibitory action of nimesulide. These results suggest that removal of Tyr348–Tyr385 hydrogen bonding in PGHS-2 allows greater conformational flexibility in the cyclooxygenase active site, resulting in altered interactions with inhibitors and altered Tyr385 radical behavior. PMID:16401081

Rogge, Corina E.; Ho, Bryant; Liu, Wen; Kulmacz, Richard J.; Tsai, Ah-Lim

2010-01-01

152

Nitroarginine, an inhibitor of nitric oxide synthase, prevents changes in superoxide radical and antioxidant enzymes induced by ammonia intoxication.  

PubMed

Injection of large doses of ammonium salts leads to the rapid death of animals. However, the molecular mechanisms involved in ammonia toxicity remain to be clarified. We reported that injecting ammonium acetate (7 mmol/kg) to rats increases the production of superoxide and reduces the activities of some antioxidant enzymes in rat liver and brain. We proposed that these effects induced by ammonia intoxication would be mediated by formation of nitric oxide. To test this possibility we tested whether injection of nitroarginine, an inhibitor of nitric oxide synthase, prevents the effects of ammonia intoxication on antioxidant enzymes and superoxide formation. Following injection of ammonia, glutathione peroxidase, superoxide dismutase and catalase activities were decreased in liver by 42%, 54% and 44%, respectively. In brain these activities were reduced by 35%, 46% and 65%, respectively. Glutathione reductase remained unchanged. Superoxide production in submitochondrial particles from liver and brain was increased by more than 100% in both tissues. Both reduction of activity of antioxidant enzymes and increased superoxide radical production were prevented by previous injection of 45 mg/kg of nitroarginine, indicating that ammonia induces increased formation of nitric oxide, which in turn reduces the activity of antioxidant enzymes, leading to increased formation of superoxide. PMID:9570638

Kosenko, E; Kaminsky, Y; Lopata, O; Muravyov, N; Kaminsky, A; Hermenegildo, C; Felipo, V

1998-03-01

153

Phytotoxicity of Acetohydroxyacid Synthase Inhibitors Is Not Due to Accumulation of 2-Ketobutyrate and/or 2-Aminobutyrate.  

PubMed Central

Acetohydroxyacid synthase (AHAS) is the site of action of herbicides of different chemical classes, such as imidazolinones, sulfonylureas, and triazolopyrimidines. Inhibition of AHAS causes the accumulation of 2-ketobutyrate (2-KB) and 2-aminobutyrate (2-AB) (the transamination product of 2-KB), and it has been proposed that the phytotoxicity of these inhibitors is due to this accumulation. Experiments were done to determine the relationship between accumulation of 2-KB and 2-AB and the phytotoxicity of imazaquin to maize (Zea mays). Imazaquin concentrations that inhibit growth of maize plants also cause the accumulation of 2-KB and 2-AB in the shoots. Supplementation of imazaquin-treated plants with isoleucine reduced the pools of 2-KB and 2-AB in the plant but did not protect plants from the growth inhibitory effects of imazaquin. Conversely, feeding 2-AB to maize plants increased 2-KB and 2-AB pools to much higher levels than those observed in imazaquin-treated plants, yet such high pools of 2-KB and 2-AB in the plant had no significant effect on growth. These results conclusively demonstrate that growth inhibition following imazaquin treatment is not due to accumulation of 2-KB and/or 2-AB in plants. Changes in the amino acid profiles after treatment with imazaquin suggest that starvation for the branched-chain amino acids may be the primary cause of growth retardation of maize. PMID:12232015

Shaner, D. L.; Singh, B. K.

1993-01-01

154

Phytotoxicity of Acetohydroxyacid Synthase Inhibitors Is Not Due to Accumulation of 2-Ketobutyrate and/or 2-Aminobutyrate.  

PubMed

Acetohydroxyacid synthase (AHAS) is the site of action of herbicides of different chemical classes, such as imidazolinones, sulfonylureas, and triazolopyrimidines. Inhibition of AHAS causes the accumulation of 2-ketobutyrate (2-KB) and 2-aminobutyrate (2-AB) (the transamination product of 2-KB), and it has been proposed that the phytotoxicity of these inhibitors is due to this accumulation. Experiments were done to determine the relationship between accumulation of 2-KB and 2-AB and the phytotoxicity of imazaquin to maize (Zea mays). Imazaquin concentrations that inhibit growth of maize plants also cause the accumulation of 2-KB and 2-AB in the shoots. Supplementation of imazaquin-treated plants with isoleucine reduced the pools of 2-KB and 2-AB in the plant but did not protect plants from the growth inhibitory effects of imazaquin. Conversely, feeding 2-AB to maize plants increased 2-KB and 2-AB pools to much higher levels than those observed in imazaquin-treated plants, yet such high pools of 2-KB and 2-AB in the plant had no significant effect on growth. These results conclusively demonstrate that growth inhibition following imazaquin treatment is not due to accumulation of 2-KB and/or 2-AB in plants. Changes in the amino acid profiles after treatment with imazaquin suggest that starvation for the branched-chain amino acids may be the primary cause of growth retardation of maize. PMID:12232015

Shaner, D. L.; Singh, B. K.

1993-12-01

155

Effect of nitric oxide synthase inhibitor L-NAME on fear extinction in rats: a task-dependent effect.  

PubMed

There is increasing evidence that nitric oxide may be involved in learning and memory. However, there remain comparatively few studies that have explored the relationship between nitric oxide signaling and fear extinction, an inhibitory learning model. In the present study, we tested the effects of nitric oxide synthase inhibitor l-NAME on three tone fear extinction tasks in rats. In task 1, rats received fear conditioning, extinction training and extinction test in the same context (AAA design). In task 2, rats received fear conditioning in context A, extinction training in context B and extinction test in context A (ABA design). In task 3, rats received fear conditioning in context A, extinction training and extinction test in context B (ABB design). l-NAME (10, 20 and 40 mg/kg) was injected intraperitoneally 30 min prior to extinction training in each task. Percent of time spent freezing was used to measure conditioned fear response. We found that l-NAME administrations had no effect on freezing in task 1 and 2 but produced a dose-dependent increase in task 3. Further results indicated that the increased freezing in task 3 was not attributed to state-dependency effects or nonspecific changes of locomotor activity that followed l-NAME injection. These results showed that l-NAME produced a task-dependent impairment of fear extinction, and implied that nitric oxide signaling was involved in memory process of certain extinction tasks. PMID:24792396

Luo, Huaiqing; Han, Li; Tian, Shaowen

2014-06-20

156

Influence of the acetolactate synthase inhibitor metsulfuron-methyl on the operation, regulation and organisation of photosynthesis in Solanum nigrum.  

PubMed

The influence of the acetolactate synthase inhibitor metsulfuron-methyl on the operation of the photosynthetic apparatus was examined on 4-weeks-old climate chamber-grown Solanum nigrum plant. To have an indication on the relative performance of the photosynthetic apparatus of ALS-treated plants, the level of carbon dioxide (CO(2)) fixation, the relative quantum efficiency of photosystem I (Phi(PSI)) or photosystem II (Phi(PSII)) electron transport and leaf chlorophyll content were assessed for both control and treated plants at 2, 4 and 7 days after application of the herbicide. Results indicated a progressive inhibition of the level of CO(2) fixation, the relative quantum efficiency of photosystem I (Phi(PSI)) and II (Phi(PSII)) electron transport and the leaf chlorophyll content already 2 days after application of the herbicide. The linear relationship between the photosystem I and II was unaltered by herbicidal treatment and was sustained under conditions where large changes in pigment composition of the leaves occurred. It appears that the stress-induced loss of leaf chlorophyll is not a catastrophic process but rather is the consequence of a well-organised breakdown of components. Under photorespiratory and non-photorespiratory conditions, the relationship between the index of electron transport flow through photosystem I and II and the rate of CO(2) fixation is altered so that electron transport becomes less efficient at driving CO(2) fixation. PMID:16691366

Riethmuller-Haage, Ingrid; Bastiaans, Lammert; Harbinson, Jeremy; Kempenaar, Corné; Kropff, Martin J

2006-06-01

157

Hyaluronan Synthase 2 (HAS2) Promotes Breast Cancer Cell Invasion by Suppression of Tissue Metalloproteinase Inhibitor 1 (TIMP-1)*  

PubMed Central

Invasion and metastasis are the primary causes of breast cancer mortality, and increased knowledge about the molecular mechanisms involved in these processes is highly desirable. High levels of hyaluronan in breast tumors have been correlated with poor patient survival. The involvement of hyaluronan in the early invasive phase of a clone of breast cancer cell line MDA-MB-231 that forms bone metastases was studied using an in vivo-like basement membrane model. The metastatic to bone tumor cells exhibited a 7-fold higher hyaluronan-synthesizing capacity compared with MDA-MB-231 cells predominately due to an increased expression of hyaluronan synthase 2 (HAS2). We found that knockdown of HAS2 completely suppressed the invasive capability of these cells by the induction of tissue metalloproteinase inhibitor 1 (TIMP-1) and dephosphorylation of focal adhesion kinase. HAS2 knockdown-mediated inhibition of basement membrane remodeling was rescued by HAS2 overexpression, transfection with TIMP-1 siRNA, or addition of TIMP-1-blocking antibodies. Moreover, knockdown of HAS2 suppressed the EGF-mediated induction of the focal adhesion kinase/PI3K/Akt signaling pathway. Thus, this study provides new insights into a possible mechanism whereby HAS2 enhances breast cancer invasion. PMID:22016393

Bernert, Berit; Porsch, Helena; Heldin, Paraskevi

2011-01-01

158

Property-based design of a glucosylceramide synthase inhibitor that reduces glucosylceramide in the brain[S  

PubMed Central

Synthesis inhibition is the basis for the treatment of type 1 Gaucher disease by the glucosylceramide synthase (GCS) inhibitor eliglustat tartrate. However, the extended use of eliglustat and related compounds for the treatment of glycosphingolipid storage diseases with CNS manifestations is limited by the lack of brain penetration of this drug. Property modeling around the D-threo-1-phenyl-2-decanoylamino-3-morpholino-propanol (PDMP) pharmacophore was employed in a search for compounds of comparable activity against the GCS but lacking P-glycoprotein (MDR1) recognition. Modifications of the carboxamide N-acyl group were made to lower total polar surface area and rotatable bond number. Compounds were screened for inhibition of GCS in crude enzyme and whole cell assays and for MDR1 substrate recognition. One analog, 2-(2,3-dihydro-1H-inden-2-yl)-N-((1R,2R)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-hydroxy-3-(pyrrolidin-1-yl)propan-2-yl)acetamide (CCG-203586), was identified that inhibited GCS at low nanomolar concentrations with little to no apparent recognition by MDR1. Intraperitoneal administration of this compound to mice for 3 days resulted in a significant dose dependent decrease in brain glucosylceramide content, an effect not seen in mice dosed in parallel with eliglustat tartrate. PMID:22058426

Larsen, Scott D.; Wilson, Michael W.; Abe, Akira; Shu, Liming; George, Christopher H.; Kirchhoff, Paul; Showalter, H. D. Hollis; Xiang, Jianming; Keep, Richard F.; Shayman, James A.

2012-01-01

159

Eliglustat tartrate, an orally active glucocerebroside synthase inhibitor for the potential treatment of Gaucher disease and other lysosomal storage diseases.  

PubMed

Eliglustat tartrate (Genz-112638), currently under development by Genzyme Corp, is a glucocerebroside (glucosylceramide) synthase inhibitor for the treatment of Gaucher disease and other lysosomal storage disorders. Gaucher disease is an inherited defect of lysosomal functions caused by mutations in the GBA1 gene leading to accumulation of glucocerebroside, primarily in macrophages. Gaucher disease is characterized by visceromegaly and skeletal complications, including osteoporosis and painful episodes of osteonecrosis. In vitro studies demonstrated that, following exposure to eliglustat tartrate, the abundance of GM1 and GM3 gangliosides in cultured human erythroleukemia cells and murine melanoma cells was decreased. In vivo, eliglustat tartrate administered to Asp409Val/null mice lowered the concentrations of glucocerebroside in the liver, lung and spleen and reduced the number of Gaucher cells in the liver. In a phase Ib clinical trial in healthy volunteers, plasma glucocerebroside concentrations were decreased after dosing with eliglustat tartrate, and in phase II clinical trials in patients with type 1 (non-neuronopathic) Gaucher disease, spleen and liver volumes were diminished. Patients also demonstrated improved bone mineral density, correction of abnormal bone marrow signal with MRI and normalization of glucocerebroside and ganglioside GM3 levels. Eliglustat tartrate is orally active and, with potent effects on the primary identified molecular target for type 1 Gaucher disease and other glycosphingolipidoses, appears likely to fulfill high expectations for clinical efficacy. PMID:20872320

Cox, Timothy M

2010-10-01

160

Discovery of a Novel Class of Orally Active Antifungal ?-1,3-d-Glucan Synthase Inhibitors?  

PubMed Central

The echinocandins are a class of semisynthetic natural products that target ?-1,3-glucan synthase (GS). Their proven clinical efficacy combined with minimal safety issues has made the echinocandins an important asset in the management of fungal infection in a variety of patient populations. However, the echinocandins are delivered only parenterally. A screen for antifungal bioactivities combined with mechanism-of-action studies identified a class of piperazinyl-pyridazinones that target GS. The compounds exhibited in vitro activity comparable, and in some cases superior, to that of the echinocandins. The compounds inhibit GS in vitro, and there was a strong correlation between enzyme inhibition and in vitro antifungal activity. In addition, like the echinocandins, the compounds caused a leakage of cytoplasmic contents from yeast and produced a morphological response in molds characteristic of GS inhibitors. Spontaneous mutants of Saccharomyces cerevisiae with reduced susceptibility to the piperazinyl-pyridazinones had substitutions in FKS1. The sites of these substitutions were distinct from those conferring resistance to echinocandins; likewise, echinocandin-resistant isolates remained susceptible to the test compounds. Finally, we present efficacy and pharmacokinetic data on an example of the piperazinyl-pyridazinone compounds that demonstrated efficacy in a murine model of Candida glabrata infection. PMID:21844320

Walker, Scott S.; Xu, Yiming; Triantafyllou, Ilias; Waldman, Michelle F.; Mendrick, Cara; Brown, Nathaniel; Mann, Paul; Chau, Andrew; Patel, Reena; Bauman, Nicholas; Norris, Christine; Antonacci, Barry; Gurnani, Maya; Cacciapuoti, Anthony; McNicholas, Paul M.; Wainhaus, Samuel; Herr, R. Jason; Kuang, Rongze; Aslanian, Robert G.; Ting, Pauline C.; Black, Todd A.

2011-01-01

161

Design and syntheses of novel phthalazin-1(2H)-one derivatives as acetohydroxyacid synthase inhibitors.  

PubMed

A series of 2-substituted-8-(4,6-dimethoxypyrimidin-2-yloxy)-4-methylphthalazin-1-one derivatives, 7a-7w, were designed via an ortho-substituent cyclization strategy to discover a new herbicidal lead structure. These compounds were synthesized by a seven-step route using 3-hydroxy-acetophenone as a starting material. Determination of the Ki values against wild-type A. thaliana acetohydroxyacid synthase (AHAS) (EC 4.1.3.18) indicated that some of the compounds displayed good enzyme inhibition activity comparable to that of KIH-6127. The further preliminary bioassay data on weeds showed that the synthesized compounds exhibited typical injury symptoms of AHAS-inhibiting herbicides, and some of them showed broad-spectrum and high herbicidal activities in postemergence treatments against Echinochloa crusgalli, Digitaria sanguinalis, Setaria viridis, Brassica juncea, Amaranthus retroflexus, and Chenopodium album at an application rate of 150 g ai/ha. To our knowledge, this is the first report of methylphthalazin-1-one derivatives as AHAS inhibitors. PMID:17117801

Li, Yuan-Xiang; Luo, Yan-Ping; Xi, Zhen; Niu, Congwei; He, Yan-Zhen; Yang, Guang-Fu

2006-11-29

162

Chitin production by arthropods in the hydrosphere  

Microsoft Academic Search

Chitin is widely distributed in nature and its annual production is thought to be huge. However, the chitin production has been rarely estimated in aquatic ecosystems, despite the growing economic interest in this polymer. Arthropods are one of the main chitin producers in the hydrosphere and a correct evaluation of the chitin production by these organisms in the different marine

Henry-Michel Cauchie

2002-01-01

163

Synthesis of potent inhibitors of ?-ketoacyl-acyl carrier protein synthase III as potential antimicrobial agents.  

PubMed

Mycobacterium tuberculosis FabH, an essential enzyme in the mycolic acid biosynthetic pathway, is an attractive target for novel anti-tubercolosis agents. Structure-based design and synthesis of 1-(4-carboxybutyl)-4-(4-(substituted benzyloxy)phenyl)-1H-pyrrole-2-carboxylic acid derivatives 7a-h, a subset of eight potential FabH inhibitors, is described in this paper. The Vilsmeier-Haack reaction was employed as a key step. The structures of all the newly synthesized compounds were identified by IR, ¹H-NMR, ¹³C-NMR, ESI-MS and HRMS. The alamarBlue™ microassay was employed to evaluate the compounds 7a-h against Mycobacterium tuberculosis H??Rv. The results demonstrate that the compound 7d possesses good in vitro antimycobacterial activity against Mycobacterium tuberculosis H??Rv (Minimum Inhibitory Concentration value [MIC], 12.5 µg/mL).These compounds may prove useful in the discovery and development of new anti-tuberculosis drugs. PMID:22534662

Liu, Yan; Zhong, Wu; Li, Rui-Juan; Li, Song

2012-01-01

164

Amino-acid substitutions at the domain interface affect substrate and allosteric inhibitor binding in ?-isopropylmalate synthase from Mycobacterium tuberculosis.  

PubMed

?-Isopropylmalate synthase (?-IPMS) is a multi-domain protein catalysing the condensation of ?-ketoisovalerate (?-KIV) and acetyl coenzyme A (AcCoA) to form ?-isopropylmalate. This reaction is the first committed step in the leucine biosynthetic pathway in bacteria and plants, and ?-IPMS is allosterically regulated by this amino acid. Existing crystal structures of ?-IPMS from Mycobacterium tuberculosis (MtuIPMS) indicate that this enzyme has a strikingly different domain arrangement in each monomer of the homodimeric protein. This asymmetry results in two distinct interfaces between the N-terminal catalytic domains and the C-terminal regulatory domains in the dimer. In this study, residues Arg97 and Asp444 across one of these unequal domain interfaces were substituted to evaluate the importance of protein asymmetry and salt bridge formation between this pair of residues. Analysis of solution-phase structures of wild-type and variant MtuIPMS indicates that substitutions of these residues have little effect on overall protein conformation, a result also observed for addition of the feedback inhibitor leucine to the wild-type enzyme. All variants had increased catalytic efficiency relative to wild-type MtuIPMS, and those with an Asp444 substitution displayed increased affinity for the substrate AcCoA. All variants also showed reduced sensitivity to leucine and altered biphasic reaction kinetics when compared with those of the wild-type enzyme. It is proposed that substituting residues at the asymmetric domain interface increases flexibility in the protein, particularly affecting the AcCoA binding site and the response to leucine, without penalty on catalysis. PMID:23500460

Huisman, Frances H A; Squire, Christopher J; Parker, Emily J

2013-04-01

165

Intranasal delivery of insulin and a nitric oxide synthase inhibitor in an experimental model of amyotrophic lateral sclerosis.  

PubMed

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disorder in which motor neurons may be targeted by oxidative and nitrergic stress without sufficient compensation by intrinsic support mechanisms. In this work, we addressed two key tenets of this hypothesis for the pathogenesis of ALS. Using superoxide dismutase (SOD) 1G93A mice, we studied the impact of reduction of nitrergic stress within the CNS with the use of a broad spectrum nitric oxide synthase (NOS) inhibitor, NG-nitro-l-arginine methyl ester. A separate cohort of SOD1G93A mice received direct insulin neurotrophic support, ligating receptors expressed upon motor neurons, to attempt protection against neuronal and functional motor dropout. For direct access, we used a novel form of intranasal delivery that provides peak concentration levels in the CNS within 1 h of delivery without systemic side effects at doses which previously rescued retrograde loss of motor axons after axotomy. To identify even minor impacts of these interventions on the outcome, we utilized an intensive program of serial behavioral and electrophysiological testing weekly, combined with endpoint quantitative morphometry and molecular analysis. This intensive evaluation enhanced our knowledge of the time course in SOD1G93A mice and impact of the SOD1G93A mutation upon motor neurons and their function. Neither intervention had even minimal impact upon slowing progression of disease in SOD1G93A mice. Our data argue against significant roles for nitrergic stress in promoting motor neuron loss and the importance of alternative neurotrophic support mechanisms that might support motor neurons and prevent disease progression in SOD1G93A mice. PMID:18951954

Martinez, J A; Francis, G J; Liu, W Q; Pradzinsky, N; Fine, J; Wilson, M; Hanson, L R; Frey, W H; Zochodne, D; Gordon, T; Toth, C

2008-12-10

166

MICROBIOLOGY: Chitin, Cholera, and Competence  

NSDL National Science Digital Library

Access to the article is free, however registration and sign-in are required. Vibrio cholerae, a human pathogen, inhabits aquatic environments and is often associated with chitin-containing organisms. In their Perspective, Bartlett and Azam discuss the findings of Meibom et al. in the same issue of chitin-mediated natural DNA transformation in V. cholerae. This finding opens a new window for understanding conditions that influence the evolution of this bacterium in aquatic habitats.

Douglas H. Bartlett (cripps Institution of Oceanography, University of California;Marine Biology Research Division); Farooq Azam (cripps Institution of Oceanography, University of California;Marine Biology Research Division)

2005-12-16

167

Systemic Delivery of a Glucosylceramide Synthase Inhibitor Reduces CNS Substrates and Increases Lifespan in a Mouse Model of Type 2 Gaucher Disease  

PubMed Central

Neuropathic Gaucher disease (nGD), also known as type 2 or type 3 Gaucher disease, is caused by a deficiency of the enzyme glucocerebrosidase (GC). This deficiency impairs the degradation of glucosylceramide (GluCer) and glucosylsphingosine (GluSph), leading to their accumulation in the brains of patients and mouse models of the disease. These accumulated substrates have been thought to cause the severe neuropathology and early death observed in patients with nGD and mouse models. Substrate accumulation is evident at birth in both nGD mouse models and humans affected with the most severe type of the disease. Current treatment of non-nGD relies on the intravenous delivery of recombinant human glucocerebrosidase to replace the missing enzyme or the administration of glucosylceramide synthase inhibitors to attenuate GluCer production. However, the currently approved drugs that use these mechanisms do not cross the blood brain barrier, and thus are not expected to provide a benefit for the neurological complications in nGD patients. Here we report the successful reduction of substrate accumulation and CNS pathology together with a significant increase in lifespan after systemic administration of a novel glucosylceramide synthase inhibitor to a mouse model of nGD. To our knowledge this is the first compound shown to cross the blood brain barrier and reduce substrates in this animal model while significantly enhancing its lifespan. These results reinforce the concept that systemically administered glucosylceramide synthase inhibitors could hold enhanced therapeutic promise for patients afflicted with neuropathic lysosomal storage diseases. PMID:22912851

Cabrera-Salazar, Mario A.; DeRiso, Matthew; Bercury, Scott D.; Li, Lingyun; Lydon, John T.; Weber, William; Pande, Nilesh; Cromwell, Mandy A.; Copeland, Diane; Leonard, John; Cheng, Seng H.; Scheule, Ronald K.

2012-01-01

168

Anti-malarial drug targets: screening for inhibitors of 2C-methyl-D-erythritol 4-phosphate synthase (IspC protein) in Mediterranean plants.  

PubMed

The recently discovered non-mevalonate pathway of isoprenoid biosynthesis serves as the unique source of terpenoids in numerous pathogenic eubacteria and in apicoplast-type protozoa, most notably Plasmodium, but is absent in mammalian cells. It is therefore an attractive target for anti-infective chemotherapy. The first committed step of the non-mevalonate pathway is catalyzed by 2C-methyl-D-erythritol 4-phosphate synthase (IspC). Using photometric and NMR spectroscopic assays, we screened extracts of Mediterranean plants for inhibitors of the enzyme. Strongest inhibitory activity was found in leaf extracts of Cercis siliquastrum. PMID:17293098

Kaiser, J; Yassin, M; Prakash, S; Safi, N; Agami, M; Lauw, S; Ostrozhenkova, E; Bacher, A; Rohdich, F; Eisenreich, W; Safi, J; Golan-Goldhirsh, A

2007-04-01

169

Effect of diabetic duration on serum concentrations of endogenous inhibitor of nitric oxide synthase in patients and rats with diabetes.  

PubMed

This study was designed to investigate the effect of diabetic duration on serum concentrations of endogenous inhibitor of nitric oxide synthase N(G), N(G)-asymmetric dimethylarginine (ADMA) in patients and rats with diabetes, and to determine whether elevated endogenous ADMA is implicated in endothelial dysfunction or macroangiopathy in diabetes. Experimental diabetic model was induced by a single intraperitoneal injection of streptozotocin to male Sprague-Dawley rats and fed for 2-, 4- and 8-week, respectively. Type 2 diabetic patients with different diabetic duration were recruited from Xiangya Hospital. Plasma glucose and serum ADMA levels were measured in both patients and rats. Moreover, endothelium-dependent relaxation of thoracic aortas and some parameters of metabolic control were examined in rats. Serum ADMA concentrations were significantly elevated in type 2 diabetic patients compared with healthy subjects (3.44 +/- 0.40 vs 1.08 +/- 0.14 micromol/L, n = 50 in diabetic patients and n = 40 in healthy subjects, P < 0.01). The serum levels of ADMA in patients with macroangiopathy were higher than the patients without macroangiopathy (P < 0.01). But no difference was observed in serum ADMA concentrations between groups of patients with different diabetic duration. Similarly, serum levels of ADMA in diabetic rats were also significantly elevated at 2-week duration compared with duration-matched control (3.71 +/- 0.20 vs 1.04 +/- 0.23 micromol/L, n = 5 approximately 6, P < 0.01). This elevation of ADMA was retained to 4- and 8-week (3.54 +/- 0.76 vs 0.95 +/- 0.06 micromol/L for 4-week, 3.21 +/- 0.50 vs 1.03 +/- 0. 09 micromol/L for 8-week, n = 5 approximately 6, all P < 0.01) and remained unchanged among three diabetic groups. The elevation of ADMA was accompanied by impairment of endothelium-dependent relaxation and poor metabolic control in diabetic rat. These results first reveal that the extent of elevation in serum ADMA in both rats and patients with diabetes is not proportion with the length of their diabetic duration but rather with the metabolic control of this disease. Elevated endogenous ADMA may be implicated in diabetes-induced endothelial dysfunction and macroangiopathy. This study is helpful to prevention and treatment of diabetic-induced endothelial dysfunction or macroangiopathy. PMID:15862600

Xiong, Yan; Lei, Minxiang; Fu, Sihai; Fu, Yunfeng

2005-05-27

170

Development of a high-throughput assay for aldosterone synthase inhibitors using high-performance liquid chromatography-tandem mass spectrometry.  

PubMed

Aldosterone plays a key role in the pathogenesis of hypertension, congestive heart failure, and chronic kidney disease. Aldosterone biosynthesis involves three membrane-bound enzymes: aldosterone synthase, adrenodoxin, and adrenodoxin reductase. Here, we report the development of a mass spectrometry-based high-throughput whole cell-based assay for aldosterone synthesis. A human adrenal carcinoma cell line (H295R) overexpressing human aldosterone synthase cDNA was established. The production of aldosterone in these cells was initiated with the addition of 11-deoxycorticosterone, the immediate substrate of aldosterone synthase. An automatic liquid handler was used to gently distribute cells uniformly to well plates. The adaption of a second automated liquid handling system to extract aldosterone from the cell culture medium into organic solvent enabled the development of 96- and 384-well plate formats for this cellular assay. A high-performance liquid chromatography-tandem mass spectrometry method was established for the detection of aldosterone. Production of aldosterone was linear with time and saturable with increasing substrate concentration. The assay was highly reproducible with an overall average Z' value=0.49. This high-throughput assay would enable high-throughput screening for inhibitors of aldosterone biosynthesis. PMID:24959941

Yurek, David; Yu, Lan; Schrementi, James; Bell, Michael G; McGee, James; Kowala, Mark; Kuo, Ming-shang; Wang, Jian

2014-10-01

171

Different nitric oxide synthase inhibitors cause rapid and differential alterations in the ligand-binding capacity of transmitter receptors in the rat cerebral cortex.  

PubMed

Inhibitors of nitric oxide (NO) synthesis reduce postlesional neuronal death during reperfusion injury by reducing the NO-mediated increase in excitatory neurotransmitter-release. The protective effects of various NO-synthase (NOS) inhibitors differ due to their isoform selectivity. The effects of NO-mediated excessive neurotransmitter supply are transmitted via specific neurotransmitter receptors expressed by the target cells. We report changes in the ligand-binding of different excitatory and inhibitory neurotransmitter-receptors studied by in vitro receptor autoradiography after in vivo-application of NOS-inhibitors. Since the constitutively expressed neuronal NOS-I is area-specifically distributed within the rat cortex, numerous cortical areas were studied in non-lesioned rats, in order to analyze the area-specific effects of NOS-inhibitors. The results showed that the NOS-I-specific inhibitor 7-nitroindazole increased binding of 3H-muscimol, 3H-pirenzepine and 3H-kainate, whereas the less isoform-specific, general NOS-inhibitor L-nitroarginine increased binding of 3H-muscimol and 3H-AMPA in most cortical areas, leaving 3H-kainate binding almost unchanged. The water soluble L-nitroarginine-methylester caused similar effects to those of L-nitroarginine which changed over a period of chronic treatment. The inhibitory GABAA-receptors were increased after NOS-inhibition in most cortical areas, whereas binding of 3H-Oxotremorine-M (acetylcholine receptors), 3H-MK-801 (NMDA-receptors) and 3H-AMPA (AMPA receptors) was affected differently among the cortical areas. Strongest alterations of ligand-binding capacity after administration of NOS-inhibitors were seen in cortical areas known to contain the highest packing densities of NOS-I-positive interneurons such as the piriform and entorhinal cortices, indicating that, in normal animals, neurotransmission and probably cognitive information processing would be affected by the pharmacological modulation of nitric oxide production. PMID:10427371

Bidmon, H J; Wu, J; Palomero-Gallagher, N; Oermann, E; Mayer, B; Schleicher, A; Zilles, K

1999-07-01

172

Synthesis of isoprenoid bisphosphonate ethers through C-P bond formations: Potential inhibitors of geranylgeranyl diphosphate synthase  

PubMed Central

Summary A set of bisphosphonate ethers has been prepared through sequential phosphonylation and alkylation of monophosphonate ethers. After formation of the corresponding phosphonic acid salts, these compounds were tested for their ability to inhibit the enzyme geranylgeranyl diphosphate synthase (GGDPS). Five of the new compounds show IC50 values of less than 1 ?M against GGDPS with little to no activity against the related enzyme farnesyl diphosphate synthase (FDPS). The most active compound displayed an IC50 value of 82 nM when assayed with GGDPS, and no activity against FDPS even at a 10 ?M concentration. PMID:25161722

Zhou, Xiang; Reilly, Jacqueline E; Loerch, Kathleen A; Hohl, Raymond J

2014-01-01

173

Effects of estrous synchronization on response to nitric oxide donors, nitric oxide synthase inhibitors, and endothelin-1 in vitro.  

PubMed

Two experiments were conducted to determine the effects of nitric oxide (NO) donors, endothelin-(ET-1), and NO synthase (NOS) inhibitors on bovine luteal function in vitro. In experiment 1, estrus in Brahman cows was synchronized with Synchro-Mate-B (SMB) and day-13-14 corpora luteal slices were weighed, diced and incubated in vitro. Treatments (100 ng/ml) were: vehicle, N[see symbol in text]-nitro-L-arginine-L-methyl ester (L-NAME), N(G)-monomethyl-L-arginine acetate (L-NMMA), diethylenetriamine (DETA), DETA-NONOate, sodium nitroprusside (SNP), or ET-1. In experiment 2, estrus was synchronized with Lutalyse, a Controlled Intravaginal Progesterone Releasing Device (CIDR), or cows were not synchronized. Corpora lutea were collected, weighed, and luteal slices were weighed, diced and incubated in vitro with treatments. Treatments (100ng/ml) were: vehicle, L- NAME, L-NMMA, DETA, DETA-NONOate, sodium nitroprusside, S-nitroso-N-acetylpenicillamine (SNAP) or endothelin-1. Tissues were incubated in M- 199 for 1 h without treatments and for 4 and 8 h in both experiments with treatments in both experiments. Media were analyzed for progesterone, prostaglandins E2 and F2alpha (PGE2, PGF2alpha) by radioimmunoassay (RIA). Hormone data in experiments 1 and 2 were analyzed by 2 x 7 and 3 x 2 x 8 factorial design for analysis of variance (ANOVA), respectively. Luteal weights in experiment 2 were analyzed by a one-way ANOVA. Concentrations of progesterone in media were similar (P > or = 0.05) among treatments within experiments. Concentrations of PGE2 in media in experiment 1 were undetectable in 90 and 57% of the samples at 4 and 8 h, respectively. PGF2alpha increased (P < or = 0.05) with time, but did not differ (P > or = 0.05) among treatments. Secretion of PGF2alpha was not affected by treatments (P > or = 0.05). In experiment 2, luteal weights of the induced estrous cycle were decreased (P < or = 0.05) by Lutalyse. Concentrations of PGE2 and PGF2alpha increased (P < or = 0.05) with time in control of all three synchronization regimens. DETA-NONOate, SNAP, sodium nitroprusside (NO donors) and ET-1 increased (P < or = 0.05) PGE2 except in the CIDR synchronized group (P > or = 0.05). No treatment increased (P > or = 0.05) PGF2alpha in any synchronization regimen. It is concluded that either SMB containing norgestomet or a CIDR containing progesterone alters luteal secretion of PGE2, Lutalyse lowers luteal weights in the induced estrous cycle, and NO or ET-1 given alone are not luteolytic agents. It is suggested that NO and ET-1 could have indirect antiluteolytic/luteotropic effects via increasing PGE2 secretion by luteal tissue rather than being luteolytic. PMID:15560115

Weems, Y S; Randel, R D; Tatman, S; Lewis, A W; Neuendorff, D A; Weems, C W

2004-10-01

174

Different strains of rats show different sensitivity to block of long-term potentiation by nitric oxide synthase inhibitors  

Microsoft Academic Search

Nitric oxide is presumed to play important roles in the induction of synaptic plasticity and learning. Previous publications, however, reported contradictory results. Block of nitric oxide synthase (NOS) has been shown to impair the induction of long-term potentiation of synaptic transmission in some studies. Other studies observed a partial block of long-term potentiation depending on experimental conditions, while yet other

Christian Hölscher

2002-01-01

175

Inhibitors of dihydropteroate synthase: substituent effects in the side-chain aromatic ring of 6-[[3-(aryloxy)propyl]amino]-5-nitrosoisocytosines and synthesis and inhibitory potency of bridged 5-nitrosoisocytosine-p-aminobenzoic acid analogues.  

PubMed

We previously reported that 6-(methylamino)-5-nitrosoisocytosine (5) is a potent inhibitor (I50 = 1.6 microM) of Escherichia coli dihydropteroate synthase. It was noted that 6-amino substituents larger than methyl were detrimental to binding, although the adverse steric effect could be overcome by a positive ancillary binding contribution of a phenyl ring attached at the terminus of certain 6-alkylamino substituents. We selected the 6-[[3-(aryloxy)propyl]amino]-5-nitrosoisocytosine structure as a parent system and explored the effects of aromatic substituents on synthase inhibition. The nature of the aryl substitution influences binding, as shown by a 30-fold range of inhibitory potencies observed for the 15 aryl analogues (I50 values = 0.6-18 microM), although there is no apparent correlation between synthase inhibition and the electronic or hydrophobic characteristics of the aryl substituents. To explore the possibility that the aryl ring of these inhibitors might interact with the synthase binding site for the substrate p-aminobenzoic acid (PABA), three compounds were synthesized in which a PABA analogue is bridged to the nitrosoisocytosine moiety by linkage to an amino group at C-6 of the isocytosine. The bridged analogues significantly inhibited the synthase (I50 values = 2.5-8.9 microM) but were of unexceptional potency compared with other members of the (aryloxy)propyl series. Structure-activity considerations and inhibition kinetics did not support the PABA binding site as the synthase region that interacts with the aryl ring of these inhibitors. Despite the potent synthase inhibition exhibited by many of the nitrosoisocytosines studied, none of the 18 new analogues showed significant antibacterial activity. PMID:3486292

Lever, O W; Bell, L N; Hyman, C; McGuire, H M; Ferone, R

1986-05-01

176

Ceramide synthase inhibitor fumonisin B1 inhibits apoptotic cell death in SCC17B human head and neck squamous carcinoma cells after Pc4 photosensitization.  

PubMed

The sphingolipid ceramide modulates stress-induced cell death and apoptosis. We have shown that ceramide generated via de novo sphingolipid biosynthesis is required to initiate apoptosis after photodynamic therapy (PDT). The objective of this study was to define the role of ceramide synthase (CERS) in PDT-induced cell death and apoptosis using fumonisin B1 (FB), a CERS inhibitor. We used the silicon phthalocyanine Pc4 for PDT, and SCC17B cells, as a clinically-relevant model of human head and neck squamous carcinoma. zVAD-fmk, a pan-caspase inhibitor, as well as FB, protected cells from death after PDT. In contrast, ABT199, an inhibitor of the anti-apoptotic protein Bcl2, enhanced cell killing after PDT. PDT-induced accumulation of ceramide in the endoplasmic reticulum and mitochondria was inhibited by FB. PDT-induced Bax translocation to the mitochondria and cytochrome c release were also inhibited by FB. These novel data suggest that PDT-induced cell death via apoptosis is CERS/ceramide-dependent. PMID:25266739

Boppana, Nithin B; Kodiha, Mohamed; Stochaj, Ursula; Lin, Ho-Sheng; Haimovitz-Friedman, Adriana; Bielawska, Alicja; Bielawski, Jacek; Divine, George W; Boyd, John A; Korbelik, Mladen; Separovic, Duska

2014-10-15

177

[Expression of acetohydroxyacid synthase isozyme genes ilvBN, ilvGM, ilvIH and their resistance to AHAS-inhibitor herbicides].  

PubMed

Acetohydroxyacid synthase (AHAS) catalyses the first reaction in the pathway for synthesis of the branched-chain amino acids. AHAS is the target for sulfonylurea, imidazolinone and other AHAS-inhibitor herbicides. Herbicides-resistant AHAS genes have potential application in plant transgenetic engineering and development of new generation herbicide. The AHAS isozyme genes ilvBN, ilvGM and ilvIH were cloned from metsulfuron-methyl resistant strain Klebsiella sp. HR11 and metsulfuron-methyl sensitive strain Klebsiella pneumoniae MGH 78578. Homologous sequences comparison indicated that the differences in AHAS isozyme genes at amino acid levels between strain HR11 and strain MGH 78578 were mainly on the large subunits of ilvBN and ilvGM. The three AHAS isozyme genes from HR11 and MGH 78578 were ligated into the expression vector pET29a(+) and expressed in Escherichia coli BL21, respectively. The results of enzyme inhibition assay showed that only ilvBN and ilvGM from strain HR11 showed strong resistance to AHAS-inhibitor herbicides, while ilvIH from strain HR11 and ilvBN, ilvGM and ilvIH from strain MGH78578 were sensitive to AHAS-inhibitor herbicides. PMID:19835141

Shen, Jingjing; Li, Yongfeng; Huang, Xing; Yu, Xinyan; He, Jian; Li, Shunpeng

2009-07-01

178

Glycogen synthase kinase 3 inhibitors induce the canonical WNT/?-catenin pathway to suppress growth and self-renewal in embryonal rhabdomyosarcoma  

PubMed Central

Embryonal rhabdomyosarcoma (ERMS) is a common pediatric malignancy of muscle, with relapse being the major clinical challenge. Self-renewing tumor-propagating cells (TPCs) drive cancer relapse and are confined to a molecularly definable subset of ERMS cells. To identify drugs that suppress ERMS self-renewal and induce differentiation of TPCs, a large-scale chemical screen was completed. Glycogen synthase kinase 3 (GSK3) inhibitors were identified as potent suppressors of ERMS growth through inhibiting proliferation and inducing terminal differentiation of TPCs into myosin-expressing cells. In support of GSK3 inhibitors functioning through activation of the canonical WNT/?-catenin pathway, recombinant WNT3A and stabilized ?-catenin also enhanced terminal differentiation of human ERMS cells. Treatment of ERMS-bearing zebrafish with GSK3 inhibitors activated the WNT/?-catenin pathway, resulting in suppressed ERMS growth, depleted TPCs, and diminished self-renewal capacity in vivo. Activation of the canonical WNT/?-catenin pathway also significantly reduced self-renewal of human ERMS, indicating a conserved function for this pathway in modulating ERMS self-renewal. In total, we have identified an unconventional tumor suppressive role for the canonical WNT/?-catenin pathway in regulating self-renewal of ERMS and revealed therapeutic strategies to target differentiation of TPCs in ERMS. PMID:24706870

Chen, Eleanor Y.; DeRan, Michael T.; Ignatius, Myron S.; Grandinetti, Kathryn Brooke; Clagg, Ryan; McCarthy, Karin M.; Lobbardi, Riadh M.; Brockmann, Jillian; Keller, Charles; Wu, Xu; Langenau, David M.

2014-01-01

179

Molecular Dynamic Simulation and Inhibitor Prediction of Cysteine Synthase Structured Model as a Potential Drug Target for Trichomoniasis  

PubMed Central

In our presented research, we made an attempt to predict the 3D model for cysteine synthase (A2GMG5_TRIVA) using homology-modeling approaches. To investigate deeper into the predicted structure, we further performed a molecular dynamics simulation for 10?ns and calculated several supporting analysis for structural properties such as RMSF, radius of gyration, and the total energy calculation to support the predicted structured model of cysteine synthase. The present findings led us to conclude that the proposed model is stereochemically stable. The overall PROCHECK G factor for the homology-modeled structure was ?0.04. On the basis of the virtual screening for cysteine synthase against the NCI subset II molecule, we present the molecule 1-N, 4-N-bis [3-(1H-benzimidazol-2-yl) phenyl] benzene-1,4-dicarboxamide (ZINC01690699) having the minimum energy score (?13.0?Kcal/Mol) and a log?P value of 6 as a potential inhibitory molecule used to inhibit the growth of T. vaginalis infection. PMID:24073401

Singh, Satendra; Singh, Atul Kumar; Gautam, Budhayash

2013-01-01

180

Structure of N-acetyl-L-glutamate synthase/kinase from Maricaulis maris with the allosteric inhibitor L-arginine bound.  

PubMed

Maricaulis maris N-acetylglutamate synthase/kinase (mmNAGS/K) catalyzes the first two steps in L-arginine biosynthesis and has a high degree of sequence and structural homology to human N-acetylglutamate synthase, a regulator of the urea cycle. The synthase activity of both mmNAGS/K and human NAGS are regulated by L-arginine, although L-arginine is an allosteric inhibitor of mmNAGS/K, but an activator of human NAGS. To investigate the mechanism of allosteric inhibition of mmNAGS/K by L-arginine, we have determined the structure of the mmNAGS/K complexed with L-arginine at 2.8 Å resolution. In contrast to the structure of mmNAGS/K in the absence of L-arginine where there are conformational differences between the four subunits in the asymmetric unit, all four subunits in the L-arginine liganded structure have very similar conformations. In this conformation, the AcCoA binding site in the N-acetyltransferase (NAT) domain is blocked by a loop from the amino acid kinase (AAK) domain, as a result of a domain rotation that occurs when L-arginine binds. This structural change provides an explanation for the allosteric inhibition of mmNAGS/K and related enzymes by L-arginine. The allosterically regulated mechanism for mmNAGS/K differs significantly from that for Neisseria gonorrhoeae NAGS (ngNAGS). To define the active site, several residues near the putative active site were mutated and their activities determined. These experiments identify roles for Lys356, Arg386, Asn391 and Tyr397 in the catalytic mechanism. PMID:23850694

Zhao, Gengxiang; Haskins, Nantaporn; Jin, Zhongmin; M Allewell, Norma; Tuchman, Mendel; Shi, Dashuang

2013-08-01

181

Structure of N-acetyl-L-glutamate synthase/kinase from Maricaulis maris with the allosteric inhibitor L-arginine bound  

PubMed Central

Maricaulis maris N-acetylglutamate synthase/kinase (mmNAGS/K) catalyzes the first two steps in L-arginine biosynthesis and has a high degree of sequence and structural homology to human N-acetylglutamate synthase, a regulator of the urea cycle. The synthase activity of both mmNAGS/K and human NAGS are regulated by L-arginine, although L-arginine is an allosteric inhibitor of mmNAGS/K, but an activator of human NAGS. To investigate the mechanism of allosteric inhibition of mmNAGS/K by L-arginine, we have determined the structure of the mmNAGS/K complexed with L-arginine at 2.8 Å resolution. In contrast to the structure of mmNAGS/K in the absence of L-arginine where there are conformational differences between the four subunits in the asymmetric unit, all four subunits in the L-arginine liganded structure have very similar conformations. In this conformation, the AcCoA binding site in the N-acetyltransferase (NAT) domain is blocked by a loop from the amino acid kinase (AAK) domain, as a result of a domain rotation that occurs when L-arginine binds. This structural change provides an explanation for the allosteric inhibition of mmNAGS/K and related enzymes by L-arginine. The allosterically regulated mechanism for mmNAGS/K differs significantly from that for Neisseria gonorrhoeae NAGS (ngNAGS). To define the active site, several residues near the putative active site were mutated and their activities determined. These experiments identify roles for Lys356, Arg386, Asn391 and Tyr397 in the catalytic mechanism. PMID:23850694

Zhao, Gengxiang; Haskins, Nantaporn; Jin, Zhongmin; Allewell, Norma M.; Tuchman, Mendel; Shi, Dashuang

2013-01-01

182

Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells  

SciTech Connect

Highlights: •EV-077 reduced TNF-? induced inflammation in endothelial cells. •The thromboxane mimetic U69915 enhanced vascular smooth muscle cell proliferation. •EV-077 inhibited smooth muscle cell proliferation. -- Abstract: The prothrombotic mediator thromboxane A{sub 2} is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNF? incubation, whereas concentrations of 6-keto PGF1? in supernatants of endothelial cells incubated with TNF? were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNF?-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy.

Petri, Marcelo H. [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden)] [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden); Tellier, Céline; Michiels, Carine [NARILIS, URBC, University of Namur, Namur (Belgium)] [NARILIS, URBC, University of Namur, Namur (Belgium); Ellertsen, Ingvill [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden)] [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden); Dogné, Jean-Michel [Department of Pharmacy, Namur Thrombosis and Hemostasis Center, University of Namur, Namur (Belgium)] [Department of Pharmacy, Namur Thrombosis and Hemostasis Center, University of Namur, Namur (Belgium); Bäck, Magnus, E-mail: Magnus.Back@ki.se [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden)] [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden)

2013-11-15

183

Inhibition of prostaglandin D2 clearance in rat hepatocytes by the thromboxane receptor antagonists daltroban and ifetroban and the thromboxane synthase inhibitor furegrelate.  

PubMed

Prostanoids, i.e. prostaglandins and thromboxane, regulate liver-specific functions both in homeostasis and during defense reactions. For example, prostanoids are released from Kupffer cells, the resident liver macrophages, in response to the inflammatory mediator anaphylatoxin C5a, and mediate an enhanced glucose output from hepatocytes as energy supply. In perfused rat livers, the thromboxane receptor antagonist daltroban enhanced C5a-induced prostanoid overflow and reduced glucose output. It was the aim of this study to elucidate whether daltroban interfered with prostanoid release from Kupffer cells or prostanoid clearance by hepatocytes, and/or whether it directly influenced prostanoid-dependent glucose metabolism in these cells. In perfused rat livers, daltroban enhanced prostaglandin (PG)D(2) overflow not only after infusion of C5a (15-fold), but also after PGD(2) (10-fold). Neither daltroban nor another receptor antagonist, ifetroban, or the thromboxane synthase inhibitor furegrelate enhanced prostanoid release from Kupffer cells. In contrast, all inhibitors reduced clearance, i.e. uptake and degradation, of PGD(2) by hepatocytes: within 5 min uptake of 1 nmol/L PGD(2) was reduced from 43+/-5 fmol (controls) to 22+/-6 fmol (daltroban), 24+/-6 fmol (ifetroban) and 21+/-6 fmol (furegrelate). PGD(2) in the medium was reduced to 39+/-7% in the controls, but remained at 93+/-9%, 93+/-11% and 60+/-3% in the presence of the inhibitors. PGD(2)-dependent glucose output in the perfused liver or activation of glycogen phosphorylase in isolated hepatocytes remained unaffected by daltroban. These data clearly demonstrate that the thromboxane-inhibitors reduced PGD(2) clearance by hepatocytes, presumably by inhibition of prostanoid transport into the cells. In contrast, they did not interfere with PGD(2)-dependent glucose metabolism, suggesting an independent mechanism for the inhibition of glucose output from the liver. PMID:12906929

Pestel, Sabine; Nath, Annegret; Jungermann, Kurt; Schieferdecker, Henrike L

2003-08-15

184

Utilisation of chitinous materials in pigment adsorption.  

PubMed

The effect of adding the cells of four lactobacilli to a squid pen powder (SPP)-containing medium on prodigiosin (PG) production by Serratia marcescens TKU011 is examined. The best increase in PG productivity was shown by strain TKU012. Among the samples of strain TKU012 and the chitinous materials of cicada casting powder (CCP), shrimp shell powder (SSP), squid pen powder (SPP), ?-chitin, and ?-chitin, TKU012 cells displayed the best adsorption rate (84%) for PG, followed by CCP, SSP, SPP, ?-chitin, and ?-chitin. As for the water-soluble food colourants, Allura Red AC (R40) and Tartrazne (Y4), SPP and SSP had better adsorptive powers than pure chitin preparations, strain TKU012, and CCP. Treatment with organic solvents, hot alkali, or proteases (papain, bromelain) diminished the adsorption rates of the biosorbents. PMID:22953835

Wang, San-Lang; Chen, Yan-Cheng; Yen, Yue-Horng; Liang, Tzu-Wen

2012-12-01

185

Chitin, Chitinase Responses, and Invasive Fungal Infections  

PubMed Central

The human immune system is capable of recognizing and degrading chitin, an important cell wall component of pathogenic fungi. In the context of host-immune responses to fungal infections, herein we review the particular contributions and interplay of fungus and chitin recognition, and chitin-degrading enzymes, known as chitinases. The mechanisms of host chitinase responses may have implications for diagnostic assays as well as novel therapeutic approaches for patients that are at risk of contracting fatal fungal infections. PMID:22187561

Vega, Karina; Kalkum, Markus

2012-01-01

186

Comparative modeling and virtual screening for the identification of novel inhibitors for myo-inositol-1-phosphate synthase.  

PubMed

Myo-inositol-1-phosphate (MIP) synthase is a key enzyme in the myo-inositol biosynthesis pathway. Disruption of the inositol signaling pathway is associated with bipolar disorders. Previous work suggested that MIP synthase could be an attractive target for the development of anti-bipolar drugs. Inhibition of this enzyme could possibly help in reducing the risk of a disease in patients. With this objective, three dimensional structure of the protein was modeled followed by the active site prediction. For the first time, computational studies were carried out to obtain structural insights into the interactive behavior of this enzyme with ligands. Virtual screening was carried out using FILTER, ROCS and EON modules of the OpenEye scientific software. Natural products from the ZINC database were used for the screening process. Resulting compounds were docked into active site of the target protein using FRED (Fast Rigid Exhaustive Docking) and GOLD (Genetic Optimization for Ligand Docking) docking programs. The analysis indicated extensive hydrogen bonding network and hydrophobic interactions which play a significant role in ligand binding. Four compounds are shortlisted and their binding assay analysis is underway. PMID:24752405

Azam, Syed Sikander; Sarfaraz, Sara; Abro, Asma

2014-08-01

187

Chitin Degradation Proteins Produced by the Marine Bacterium Vibrio harveyi Growing on Different Forms of Chitin  

PubMed Central

Relatively little is known about the number, diversity, and function of chitinases produced by bacteria, even though chitin is one of the most abundant polymers in nature. Because of the importance of chitin, especially in marine environments, we examined chitin-degrading proteins in the marine bacterium Vibrio harveyi. This bacterium had a higher growth rate and more chitinase activity when grown on (beta)-chitin (isolated from squid pen) than on (alpha)-chitin (isolated from snow crab), probably because of the more open structure of (beta)-chitin. When exposed to different types of chitin, V. harveyi excreted several chitin-degrading proteins into the culture media. Some chitinases were present with all of the tested chitins, while others were unique to a particular chitin. We cloned and identified six separate chitinase genes from V. harveyi. These chitinases appear to be unique based on DNA restriction patterns, immunological data, and enzyme activity. This marine bacterium and probably others appear to synthesize separate chitinases for efficient utilization of different forms of chitin and chitin by-products. PMID:16535505

Svitil, A. L.; Chadhain, S.; Moore, J. A.; Kirchman, D. L.

1997-01-01

188

Structure-Based Design of Novel Pyrimido[4,5-c]pyridazine Derivatives as Dihydropteroate Synthase Inhibitors with Increased Affinity  

SciTech Connect

Dihydropteroate synthase (DHPS) is the validated drug target for sulfonamide antimicrobial therapy. However, due to widespread drug resistance and poor tolerance, the use of sulfonamide antibiotics is now limited. The pterin binding pocket in DHPS is highly conserved and is distinct from the sulfonamide binding site. It therefore represents an attractive alternative target for the design of novel antibacterial agents. We previously carried out the structural characterization of a known pyridazine inhibitor in the Bacillus anthracis DHPS pterin site and identified a number of unfavorable interactions that appear to compromise binding. With this structural information, a series of 4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]pyridazines were designed to improve binding affinity. Most importantly, the N-methyl ring substitution was removed to improve binding within the pterin pocket, and the length of the side chain carboxylic acid was optimized to fully engage the pyrophosphate binding site. These inhibitors were synthesized and evaluated by an enzyme activity assay, X-ray crystallography, isothermal calorimetry, and surface plasmon resonance to obtain a comprehensive understanding of the binding interactions from structural, kinetic, and thermodynamic perspectives. This study clearly demonstrates that compounds lacking the N-methyl substitution exhibit increased inhibition of DHPS, but the beneficial effects of optimizing the side chain length are less apparent.

Zhao, Ying; Hammoudeh, Dalia; Yun, Mi-Kyung; Qi, Jianjun; White, Stephen W.; Lee, Richard E. (Tennessee-HSC); (SJCH)

2012-05-29

189

A leukotriene C4 synthase inhibitor with the backbone of 5-(5-methylene-4-oxo-4,5-dihydrothiazol-2-ylamino) isophthalic acid  

PubMed Central

The cysteinyl leukotrienes (cys-LTs), leukotriene C4 (LTC4) and its metabolites, LTD4 and LTE4, are proinflammatory lipid mediators in asthma and other inflammatory diseases. They are generated through the 5-lipoxygenase/LTC4 synthase (LTC4S) pathway and act via at least two distinct G protein-coupled receptors. The inhibition of human LTC4S will make a simple way to treat the cys-LT relevant inflammatory diseases. Here, we show that compounds having 5-(5-methylene-4-oxo-4,5-dihydrothiazol-2-ylamino) isophthalic acid moiety suppress LTC4 synthesis, glutathione conjugation to the precursor LTA4, in both an enzyme assay and a whole-cell assay. Hierarchical in silico screenings of 6 million compounds provided 300,000 dataset for docking, and after energy minimization based on the crystal structure of LTC4S, 111 compounds were selected as candidates for a competitive inhibitor to glutathione. One of those compounds showed significant inhibitory activity, and subsequently, its derivative 5-((Z)-5-((E)-2-methyl-3-phenylallylidene)-4-oxo-4,5-dihydrothiazol-2-ylamino) isophthalic acid (compound 1) was found to be the most potent inhibitor. The enzyme assay showed the IC50 was 1.9 µM and the corresponding 95% confidence interval was from 1.7 to 2.2 µM. The whole-cell assay showed that compound 1 was cell permeable and inhibited LTC4 synthesis in a concentration dependent manner. PMID:23378248

Ago, Hideo; Okimoto, Noriaki; Kanaoka, Yoshihide; Morimoto, Gentaro; Ukita, Yoko; Saino, Hiromichi; Miyano, Masashi

2013-01-01

190

Potent Dual Thymidylate Synthase and Dihydrofolate Reductase Inhibitors: Classical and Nonclassical 2-Amino-4-oxo-5-arylthio-substituted-6-methylthieno[2,3-d]pyrimidine Antifolates  

PubMed Central

N-{4-[(2-Amino-6-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-5-yl)sulfanyl]benzoyl}-L-glutamic acid (4) and nine nonclassical analogues 5–13 were synthesized as potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors. The key intermediate in the synthesis was 2-amino-6-methylthieno[2,3-d]pyrimidin-4(3H)-one (16), which was converted to the 5-bromo-substituted compound 17 followed by an Ullmann reaction to afford 5–13. The classical analogue 4 was synthesized by coupling the benzoic acid derivative 19 with diethyl l-glutamate and saponification. Compound 4 is the most potent dual inhibitor of human TS (IC50 = 40 nM) and human DHFR (IC50 = 20 nM) known to date. The nonclassical analogues 5–13 were moderately potent against human TS with IC50 values ranging from 0.11 to 4.6 µM. The 4-nitrophenyl analogue 7 was the most potent compound in the nonclassical series, demonstrating potent dual inhibitory activities against human TS and DHFR. This study indicated that the 5-substituted 2-amino-4-oxo-6-methylthieno[2,3-d]pyrimidine scaffold is highly conducive to dual human TS-DHFR inhibitory activity. PMID:18800768

Gangjee, Aleem; Qiu, Yibin; Li, Wei; Kisliuk, Roy L.

2013-01-01

191

The effects of NMDA receptor antagonists and nitric oxide synthase inhibitors on opioid tolerance and withdrawal. Medication development issues for opiate addiction.  

PubMed

This article is an exploration of the National Institute on Drug Abuse (NIDA) Technical Review on the role of glutamatergic systems in the development of opiate addiction. The effects of "glutamate antagonist" medications on opioid tolerance and withdrawal are examined. In rodents, mu opioid tolerance can be inhibited by noncompetitive N-methyl D-aspartate (NMDA) receptor antagonists [MK801, dextromethorphan (DM), ketamine, phencyclidine (PCP)], competitive NMDA receptor antagonists (LY274614, NPC17742, LY235959), partial glycine agonists (ACPC), glycine antagonists (ACEA-1328), and nitric oxide synthase (NOS) inhibitors [L-NNA, L-NMMA, methylene blue (MB)]. Similarly, some of the symptoms of opioid withdrawal observed in opioid-dependent rodents also can be inhibited by noncompetitive NMDA receptor antagonists (MK801, DM, ketamine), competitive NMDA receptor antagonists (LY274614), glycine antagonists (felbamate), and NOS inhibitors (L-NNA, L-NMMA, L-NAME, L-NIO, 7-NI, MB). There are some serious toxicological effects associated with the administration of some of the noncompetitive NMDA receptor antagonists in rodent but not in squirrel monkey brain, and some medications induce PCP-like behavioral effects. The medications with the most immediate clinical appeal are those that could be coadministered with methadone to decrease mu opioid tolerance and dependence; they include DM, MB, 7-NI, ACPC, and ACEA-1328. PMID:8747752

Herman, B H; Vocci, F; Bridge, P

1995-12-01

192

Characterization of acetohydroxyacid synthase from Mycobacterium tuberculosis and the identification of its new inhibitor from the screening of a chemical library.  

PubMed

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate- (ThDP-) and FAD-dependent enzyme that catalyzes the first common step in the biosynthetic pathway of the branched-amino acids such as leucine, isoleucine, and valine. The genes of AHAS from Mycobacterium tuberculosis were cloned, and overexpressed in E. coli and purified to homogeneity. The purified AHAS from M. tuberculosis is effectively inhibited by pyrazosulfuron ethyl (PSE), an inhibitor of plant AHAS enzyme, with the IC(50) (inhibitory concentration 50%) of 0.87 microM. The kinetic parameters of M. tuberculosis AHAS were determined, and an enzyme activity assay system using 96-well microplate was designed. After screening of a chemical library composed of 5600 compounds using the assay system, a new class of AHAS inhibitor was identified with the IC(50) in the range of 1.8-2.6 microM. One of the identified compounds (KHG20612) further showed growth inhibition activity against various strains of M. tuberculosis. The correlation of the inhibitory activity of the identified compound against AHAS to the cell growth inhibition activity suggested that AHAS might be served as a target protein for the development of novel anti-tuberculosis therapeutics. PMID:16111681

Choi, Kyoung-Jae; Yu, Yeon Gyu; Hahn, Hoh Gyu; Choi, Jung-Do; Yoon, Moon-Young

2005-08-29

193

Aminothiazole-featured pirinixic acid derivatives as dual 5-lipoxygenase and microsomal prostaglandin E2 synthase-1 inhibitors with improved potency and efficiency in vivo.  

PubMed

Dual inhibition of microsomal prostaglandin E2 synthase-1 (mPGES-1) and 5-lipoxygenase (5-LO) is currently pursued as potential pharmacological strategy for treatment of inflammation and cancer. Here we present a series of 26 novel 2-aminothiazole-featured pirinixic acid derivatives as dual 5-LO/mPGES-1 inhibitors with improved potency (exemplified by compound 16 (2-[(4-chloro-6-{[4-(naphthalen-2-yl)-1,3-thiazol-2-yl]amino}pyrimidin-2-yl)sulfanyl]octanoic acid) with IC50 = 0.3 and 0.4 ?M, respectively) and bioactivity in vivo. Computational analysis presumes binding sites of 16 at the tip of the 5-LO catalytic domain and within a subpocket of the mPGES-1 active site. Compound 16 (10 ?M) hardly suppressed cyclooxygenase (COX)-1/2 activities, failed to inhibit 12/15-LOs, and is devoid of radical scavenger properties. Finally, compound 16 reduced vascular permeability and inflammatory cell infiltration in a zymosan-induced mouse peritonitis model accompanied by impaired levels of cysteinyl-leukotrienes and prostaglandin E2. Together, 2-aminothiazole-featured pirinixic acids represent potent dual 5-LO/mPGES-1 inhibitors with an attractive pharmacological profile as anti-inflammatory drugs. PMID:24171493

Hanke, Thomas; Dehm, Friederike; Liening, Stefanie; Popella, Sven-Desiderius; Maczewsky, Jonas; Pillong, Max; Kunze, Jens; Weinigel, Christina; Barz, Dagmar; Kaiser, Astrid; Wurglics, Mario; Lämmerhofer, Michael; Schneider, Gisbert; Sautebin, Lidia; Schubert-Zsilavecz, Manfred; Werz, Oliver

2013-11-27

194

Activation of the Wnt pathway through use of AR79, a glycogen synthase kinase 3? inhibitor, promotes prostate cancer growth in soft tissue and bone  

PubMed Central

Due to its bone anabolic activity, methods to increase Wnt activity, such as inhibitors of dickkopf-1 and sclerostin, are being clinically explored. Glycogen synthase kinase (GSK3?) inhibits Wnt signaling through inducing ?-catenin degradation. Therefore, AR79, an inhibitor of GSK3?, is being evaluated as a bone anabolic agent. However, Wnt activation has potential to promote tumor growth. The goal of this study was to determine if AR79 impacted progression of prostate cancer (PCa). PCa tumors were established in subcutaneous and bone sites of mice followed by AR79 administration. Tumor growth, ?-catenin activation, proliferation (Ki67 expression) and apoptosis (caspase 3 activity) were measured. Additionally, PCa and osteoblast cell lines were treated with AR79 and ?-catenin status, proliferation (with ?-catenin knocked down in some cases) and proportion of the ALDH+CD133+ stem-like cells was determined. AR79 promoted PCa growth, decreased phospho-?-catenin expression and increased total and nuclear ?-catenin expression in tumors and increased tumor-induced bone remodeling. Additionally, it decreased caspase 3 and increased Ki67 expression. In addition, AR79 increased bone formation in normal mouse tibiae. AR79 inhibited ?-catenin phosphorylation, increased nuclear ?-catenin accumulation in PCa and osteoblast cell lines and increased proliferation of PCa cells in vitro through ?-catenin. Furthermore, AR79 increased the ALDH+CD133+ cancer stem cell-like proportion of the PCa cell lines. We conclude that AR79, while being bone anabolic, promotes PCa cell growth through Wnt pathway activation. PMID:24088787

Jiang, Yuan; Dai, Jinlu; Zhang, Honglai; Sottnik, Joe L.; Keller, Jill M.; Escott, Katherine J.; Sanganee, Hitesh J.; Yao, Zhi; McCauley, Laurie K.; Keller, Evan T.

2013-01-01

195

Role of nitric oxide in sleep regulation: effects of L-NAME, an inhibitor of nitric oxide synthase, on sleep in rats.  

PubMed

The effect of N(G)-nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of enzyme nitric oxide synthase (NOS), on spontaneous sleep during the light period, was studied in adult rats implanted for chronic sleep recordings. L-NAME was injected by subcutaneous (s.c.) or intracerebroventricular (i.c.v.) routes or was infused directly into the dorsal raphe nuclei (DRN). Subcutaneous (1.25-5.0 mg/kg) or i.c.v. (0.25-1.0 mg) administration of L-NAME increased waking (W) and reduced slow wave sleep (SWS) and rapid-eye-movement sleep (REMS) during the first 3 h of recording. On the other hand, direct application of L-NAME into the DRN (50.0-150.0 microg) induced an increment of W and a reduction of SWS without suppressing REMS. Values of W and SWS were significantly different compared with those of controls during the 6-h recording period. The effects of L-NAME observed after s.c. or i.c.v. administration confirm previous studies in rabbits and rats, in which the NOS inhibitor reduced sleep and increased W in a dose-dependent manner. It is possible that REMS suppression after L-NAME could be related to a reduction of acetylcholine release in areas critical for REMS promotion. A decrease in gamma-aminobutyric acid (GABA) release after nitric oxide synthesis inhibition could play a role in the reduction of SWS. PMID:10212067

Monti, J M; Hantos, H; Ponzoni, A; Monti, D; Banchero, P

1999-04-01

196

Lenticular mitoprotection. Part A: Monitoring mitochondrial depolarization with JC-1 and artifactual fluorescence by the glycogen synthase kinase-3? inhibitor, SB216763  

PubMed Central

Purpose Dissipation of the electrochemical gradient across the inner mitochondrial membrane results in mitochondrial membrane permeability transition (mMPT), a potential early marker for the onset of apoptosis. In this study, we demonstrate a role for glycogen synthase kinase-3? (GSK-3?) in regulating mMPT. Using direct inhibition of GSK-3? with the GSK-3? inhibitor SB216763, mitochondria may be prevented from depolarizing (hereafter referred to as mitoprotection). Cells treated with SB216763 showed an artifact of fluorescence similar to the green emission spectrum of the JC-1 dye. We demonstrate the novel use of spectral deconvolution to negate the interfering contributing fluorescence by SB216763, thus allowing an unfettered analysis of the JC-1 dye to determine the mitochondrial membrane potential. Methods Secondary cultures of virally transfected human lens epithelial cells (HLE-B3) were exposed to acute hypoxic conditions (approximately 1% O2) followed by exposure to atmospheric oxygen (approximately 21% O2). The fluorescent dye JC-1 was used to monitor the extent of mitochondrial depolarization upon exposure of inhibitor treatment relative to the control cells (mock inhibition) in atmospheric oxygen. Annexin V-fluorescein isothiocyanate/propidium iodide staining was implemented to determine cell viability. Results Treatment of HLE-B3 cells with SB216763 (12 µM), when challenged by oxidative stress, suppressed mitochondrial depolarization relative to control cells as demonstrated with JC-1 fluorescent dye analysis. Neither the control nor the SB216763-treated HLE-B3 cells tested positive with annexin V-fluorescein isothiocyanate/propidium iodide staining under the conditions of the experiment. Conclusions Inhibition of GSK-3? activity by SB216763 blocked mMPT relative to the slow but consistent depolarization observed with the control cells. We conclude that inhibition of GSK-3? activity by the GSK-3? inhibitor SB216763 provides positive protection against mitochondrial depolarization. PMID:23825920

Brooks, Morgan M.; Neelam, Sudha; Fudala, Rafal; Gryczynski, Ignacy

2013-01-01

197

Iminosugar-based inhibitors of glucosylceramide synthase prolong survival but paradoxically increase brain glucosylceramide levels in Niemann-Pick C mice.  

PubMed

Niemann Pick type C (NPC) disease is a progressive neurodegenerative disease caused by mutations in NPC1 or NPC2, the gene products of which are involved in cholesterol transport in late endosomes. NPC is characterized by an accumulation of cholesterol, sphingomyelin and glycosphingolipids in the visceral organs, primarily the liver and spleen. In the brain, there is a redistribution of unesterified cholesterol and a concomitant accumulation of glycosphingolipids. It has been suggested that reducing the aberrant lysosomal storage of glycosphingolipids in the brain by a substrate reduction therapy (SRT) approach may prove beneficial. Inhibiting glucosylceramide synthase (GCS) using the iminosugar-based inhibitor miglustat (NB-DNJ) has been reported to increase the survival of NPC mice. Here, we tested the effects of Genz-529468, a more potent iminosugar-based inhibitor of GCS, in the NPC mouse. Oral administration of Genz-529468 or NB-DNJ to NPC mice improved their motor function, reduced CNS inflammation, and increased their longevity. However, Genz-529468 offered a wider therapeutic window and better therapeutic index than NB-DNJ. Analysis of the glycolipids in the CNS of the iminosugar-treated NPC mouse revealed that the glucosylceramide (GL1) but not the ganglioside levels were highly elevated. This increase in GL1 was likely caused by the off-target inhibition of the murine non-lysosomal glucosylceramidase, Gba2. Hence, the basis for the observed effects of these inhibitors in NPC mice might be related to their inhibition of Gba2 or another unintended target rather than a result of substrate reduction. PMID:22366055

Nietupski, Jennifer B; Pacheco, Joshua J; Chuang, Wei-Lien; Maratea, Kimberly; Li, Lingyun; Foley, Joseph; Ashe, Karen M; Cooper, Christopher G F; Aerts, Johannes M F G; Copeland, Diane P; Scheule, Ronald K; Cheng, Seng H; Marshall, John

2012-04-01

198

Structure determination of glycogen synthase kinase-3 from Leishmania major and comparative inhibitor structure?activity relationships with Trypanosoma brucei GSK-3  

SciTech Connect

Glycogen synthase kinase-3 (GSK-3) is a drug target under intense investigation in pharmaceutical companies and constitutes an attractive piggyback target for eukaryotic pathogens. Two different GSKs are found in trypanosomatids, one about 150 residues shorter than the other. GSK-3 short (GeneDB: Tb927.10.13780) has previously been validated genetically as a drug target in Trypanosoma brucei by RNAi induced growth retardation; and chemically by correlation between enzyme and in vitro growth inhibition. Here, we report investigation of the equivalent GSK-3 short enzymes of L. major (LmjF18.0270) and L. infantum (LinJ18{_}V3.0270, identical in amino acid sequences to LdonGSK-3 short) and a crystal structure of LmajGSK-3 short at 2 {angstrom} resolution. The inhibitor structure-activity relationships (SARs) of L. major and L. infantum are virtually identical, suggesting that inhibitors could be useful for both cutaneous and visceral leishmaniasis. Leishmania spp. GSK-3 short has different inhibitor SARs than TbruGSK-3 short, which can be explained mostly by two variant residues in the ATP-binding pocket. Indeed, mutating these residues in the ATP-binding site of LmajGSK-3 short to the TbruGSK-3 short equivalents results in a mutant LmajGSK-3 short enzyme with SAR more similar to that of TbruGSK-3 short. The differences between human GSK-3{beta} (HsGSK-3{beta}) and LmajGSK-3 short SAR suggest that compounds which selectively inhibit LmajGSK-3 short may be found.

Ojo, Kayode K.; Arakaki, Tracy L.; Napuli, Alberto J.; Inampudi, Krishna K.; Keyloun, Katelyn R.; Zhang, Li; Hol, Wim G.J.; Verlind, Christophe L.M.J.; Merritt, Ethan A.; Van Voorhis, Wesley C. (UWASH)

2012-04-24

199

Structure Determination of Glycogen Synthase Kinase-3 from Leishmania major and Comparative Inhibitor Structure-Activity Relationships with Trypanosoma brucei GSK-3  

PubMed Central

Glycogen synthase kinase-3 (GSK-3) is a drug target under intense investigation in pharmaceutical companies and constitutes an attractive piggyback target for eukaryotic pathogens. Two different GSKs are found in trypanosomatids, one about 150 residues shorter than the other. GSK-3 short (GeneDB: Tb927.10.13780) has previously been validated genetically as a drug target in Trypanosoma brucei by RNAi induced growth retardation; and chemically by correlation between enzyme and in vitro growth inhibition. Here, we report investigation of the equivalent GSK-3 short enzymes of L. major (LmjF18.0270) and L. infantum (LinJ18_V3.0270, identical in amino acid sequences to LdonGSK-3 short) and a crystal structure of LmajGSK-3 short at 2 Å resolution. The inhibitor structure-activity relationships (SARs) of L. major and L. infantum are virtually identical, suggesting that inhibitors could be useful for both cutaneous and visceral leishmaniasis. Leishmania spp. GSK-3 short has different inhibitor SARs than TbruGSK-3 short, which can be explained mostly by two variant residues in the ATP-binding pocket. Indeed, mutating these residues in the ATP-binding site of LmajGSK-3 short to the TbruGSK-3 short equivalents results in a mutant LmajGSK-3 short enzyme with SAR more similar to that of TbruGSK-3 short. The differences between human GSK-3? (HsGSK-3?) and LmajGSK-3 short SAR suggest that compounds which selectively inhibit LmajGSK-3 short may be found. PMID:21195115

Ojo, Kayode K.; Arakaki, Tracy L.; Napuli, Alberto J.; Inampudi, Krishna K.; Keyloun, Katelyn R.; Zhang, Li; Hol, Wim G.J.; Verlinde, Christophe L.M.J.; Merritt, Ethan A.; Van Voorhis, Wesley C.

2011-01-01

200

Synthesis and structure-activity relationship of 4-quinolone-3-carboxylic acid based inhibitors of glycogen synthase kinase-3?.  

PubMed

The synthesis, GSK-3? inhibitory activity, and anti-microbial activity of bicyclic and tricyclic derivatives of the 5,7-diamino-6-fluoro-4-quinolone-3-carboxylic acid scaffold were studied. Kinase selectivity profiling indicated that members of this class were potent and highly selective GSK-3 inhibitors. PMID:21873061

Cociorva, Oana M; Li, Bei; Nomanbhoy, Tyzoon; Li, Qiang; Nakamura, Ayako; Nakamura, Kai; Nomura, Masahiro; Okada, Kyoko; Seto, Shigeki; Yumoto, Kazuhiro; Liyanage, Marek; Zhang, Melissa C; Aban, Arwin; Leen, Brandon; Szardenings, Anna Katrin; Rosenblum, Jonathan S; Kozarich, John W; Kohno, Yasushi; Shreder, Kevin R

2011-10-01

201

Chitin-natural clay nanotubes hybrid hydrogel.  

PubMed

Novel hybrid hydrogel was synthesized from chitin NaOH/urea aqueous solution in presence of halloysite nanotubes (HNTs) via crosslinking with epichlorohydrin. Fourier transform infrared (FT-IR) spectra and atomic force microscopy (AFM) results confirmed the interfacial interactions in the chitin-HNTs hybrid hydrogel. The compressive strength and shear modulus of chitin hydrogel were significantly increased by HNTs as shown in the static compressive experiment and rheology measurement. The hybrid hydrogels showed highly porous microstructures by scanning electron microscopy (SEM). The swelling ratio of chitin hydrogel decreased because of the addition of HNTs. The malachite green's absorption experiment result showed that the hybrid hydrogel exhibited much higher absorption rate than the pure chitin hydrogel. The prepared hybrid hydrogel had potential applications in waste treatment and biomedical areas. PMID:23535366

Liu, Mingxian; Zhang, Yun; Li, Jingjing; Zhou, Changren

2013-07-01

202

Plasma concentration of asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, is elevated in hyperthyroid patients.  

PubMed

Cardiovascular manifestations are frequent findings in patients with thyroid hormone disorders. Nitric oxide (NO) plays a key role in vascular, endothelial-mediated relaxation. NO is synthesized from L-arginine by NO synthase, an enzyme inhibited by endogenous compounds, mainly asymmetric dimethylarginine [asymmetric N(G),N(G)-dimethyl-L-arginine (ADMA)]. The aim of our work was to investigate whether plasma L-arginine and dimethylarginine concentrations and NO production are altered in hypo- and hyperthyroid patients, compared with control subjects. L-arginine, ADMA and symmetric dimethylarginine were analyzed by HPLC. NO was measured as plasma nitrite plus nitrate (NO(x)) concentration by a colorimetric method based on Griess reagent. L-arginine, ADMA, and symmetric dimethylarginine plasma levels in the hypothyroid group were similar to those of the control group; whereas in hyperthyroidism, these values were significantly increased. However, the L-arginine/ADMA ratio was decreased in hyperthyroid patients, resulting in diminished NO(x) production. When all subjects were analyzed together, free T(4) levels were directly correlated with ADMA and inversely correlated with NO(x). PMID:12466365

Hermenegildo, Carlos; Medina, Pascual; Peiró, Marta; Segarra, Gloria; Vila, José M; Ortega, Joaquín; Lluch, Salvador

2002-12-01

203

Melanin Externalization in Candida albicans Depends on Cell Wall Chitin Structures?  

PubMed Central

The fungal pathogen Candida albicans produces dark-pigmented melanin after 3 to 4 days of incubation in medium containing l-3,4-dihydroxyphenylalanine (l-DOPA) as a substrate. Expression profiling of C. albicans revealed very few genes significantly up- or downregulated by growth in l-DOPA. We were unable to determine a possible role for melanin in the virulence of C. albicans. However, we showed that melanin was externalized from the fungal cells in the form of electron-dense melanosomes that were free or often loosely bound to the cell wall exterior. Melanin production was boosted by the addition of N-acetylglucosamine to the medium, indicating a possible association between melanin production and chitin synthesis. Melanin externalization was blocked in a mutant specifically disrupted in the chitin synthase-encoding gene CHS2. Melanosomes remained within the outermost cell wall layers in chs3? and chs2? chs3? mutants but were fully externalized in chs8? and chs2? chs8? mutants. All the CHS mutants synthesized dark pigment at equivalent rates from mixed membrane fractions in vitro, suggesting it was the form of chitin structure produced by the enzymes, not the enzymes themselves, that was involved in the melanin externalization process. Mutants with single and double disruptions of the chitinase genes CHT2 and CHT3 and the chitin pathway regulator ECM33 also showed impaired melanin externalization. We hypothesize that the chitin product of Chs3 forms a scaffold essential for normal externalization of melanosomes, while the Chs8 chitin product, probably produced in cell walls in greater quantity in the absence of CHS2, impedes externalization. PMID:20543065

Walker, Claire A.; Gomez, Beatriz L.; Mora-Montes, Hector M.; Mackenzie, Kevin S.; Munro, Carol A.; Brown, Alistair J. P.; Gow, Neil A. R.; Kibbler, Christopher C.; Odds, Frank C.

2010-01-01

204

Quinolone derivatives containing strained spirocycle as orally active glycogen synthase kinase 3? (GSK-3?) inhibitors for type 2 diabetics.  

PubMed

The design, synthesis, and evaluation of 6-6-7 tricyclic quinolones containing the strained spirocycle moiety aiming at the GSK-3? inhibitor were described. Among the synthesized compounds, 44, having a cyclobutane ring on a spirocycle, showed excellent GSK-3? inhibitory activity in both cell-free and cell-based assays (IC(50) = 36nM, EC(50) = 3.2?M, respectively). Additionally, 44 decreased the plasma glucose concentration dose-dependently after an oral glucose tolerance test in mice. PMID:22261023

Seto, Shigeki; Yumoto, Kazuhiko; Okada, Kyoko; Asahina, Yoshikazu; Iwane, Aya; Iwago, Maki; Terasawa, Reiko; Shreder, Kevin R; Murakami, Koji; Kohno, Yasushi

2012-02-01

205

Reference Genes to Study Herbicide Stress Response in Lolium sp.: Up-Regulation of P450 Genes in Plants Resistant to Acetolactate-Synthase Inhibitors  

PubMed Central

Variation in the expression of numerous genes is at the basis of plant response to environmental stresses. Non-target-site-based resistance to herbicides (NTSR), the major threat to grass weed chemical control, is governed by a subset of the genes involved in herbicide stress response. Quantitative PCR assays allowing reliable comparison of gene expression are thus key to identify genes governing NTSR. This work aimed at identifying a set of reference genes with a stable expression to be used as an internal standard for the normalisation of quantitative PCR data in studies investigating NTSR to herbicides inhibiting acetolactate synthase (ALS) in the major grass weed Lolium sp. Gene expression stability was assessed in plants resistant or sensitive to two ALS inhibitors, subjected or not to herbicide stress. Using three complementary approaches implemented in the programs BestKeeper, NormFinder and geNorm, cap-binding protein, glyceraldehyde-3-phosphate-dehydrogenase and ubiquitin were identified as the most suitable reference genes. This reference gene set can probably be used to study herbicide response in other weed species. It was used to compare the expression of the genes encoding two herbicide target enzymes (ALS and acetyl-coenzyme A carboxylase) and five cytochromes P450 (CYP) with potential herbicide-degrading activity between plants resistant or sensitive to ALS inhibitors. Overall, herbicide application enhanced CYP gene expression. Constitutive up-regulation of all CYP genes observed in resistant plants compared to sensitive plants suggested enhanced secondary metabolism in the resistant plants. Comprehensive transcriptome studies associated to gene expression analyses using the reference gene set validated here are required to unravel NTSR genetic determinants. PMID:23696834

Duhoux, Arnaud; Delye, Christophe

2013-01-01

206

Elevated levels of the serum endogenous inhibitor of nitric oxide synthase and metabolic control in rats with streptozotocin-induced diabetes.  

PubMed

This study was designed to determine the relationship between elevated levels of the endogenous inhibitor of nitric oxide synthase, asymmetric dimethylarginine (ADMA), and metabolic control in rats with streptozotocin-induced diabetes. Serum levels of ADMA were measured by high-performance liquid chromatography at 8 weeks after diabetes was induced. Endothelium-dependent relaxation to acetylcholine was tested in aortic rings from nondiabetic age-matched control, untreated diabetic, and insulin-treated diabetic rats to evaluate endothelial function. Serum concentrations of glucose, glycosylated serum protein, and malondialdehyde were examined to estimate metabolic control. Serum levels of ADMA increased dramatically in untreated diabetic rats compared with control rats. This elevation in ADMA levels was accompanied by impairment of the endothelium-dependent relaxation response to acetylcholine in aortic rings. Long-term insulin treatment not only prevented the elevation of serum ADMA levels, but also improved the impairment of endothelium-dependent relaxation in diabetic rats. Serum levels of glucose, glycosylated serum protein, and malondialdehyde were significantly increased in parallel with the elevation of ADMA in untreated diabetic rats compared with control rats. These parameters were normalized after diabetic rats received insulin treatment for 8 weeks. These results provide the first evidence that an elevation in the concentration of ADMA in rats with streptozotocin-induced diabetes is closely related to metabolic control of the disease. PMID:12883321

Xiong, Yan; Fu, Yun-feng; Fu, Si-hai; Zhou, Hong-hao

2003-08-01

207

Angiotensin-converting enzyme activity is involved in the mechanism of increased endogenous nitric oxide synthase inhibitor in patients with type 2 diabetes mellitus.  

PubMed

The renin-angiotensin system plays an important role in the elevation of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, in hypertensive patients, so the present study was designed to examine whether angiotensin-converting enzyme (ACE) activity is also involved in the mechanism of ADMA elevation in type 2 diabetes mellitus (NIDDM). A crossover study was performed to determine if ACE inhibition with perindopril (4 mg/day) for 4 weeks decreases serum ADMA concentration and plasma von Willebrand factor (vWF) level (a marker of endothelial injury) in 11 patients with NIDDM. None of the patients was treated with insulin or oral hypoglycemic drugs, and none had major diabetic complications. Before the protocol began, serum ADMA and plasma vWF were significantly higher in the 11 NIDDM patients, when compared with 8 control subjects without diabetes. Perindopril did not affect blood pressure or glucose metabolism, but did significantly decrease serum ADMA and plasma vWF. These results suggest that endothelial injury associated with ADMA elevation may be present even in patients with non-complicated NIDDM, and that increased activity of ACE may be involved in such endothelial dysfunction. PMID:12224817

Ito, Akira; Egashira, Kensuke; Narishige, Takahiro; Muramatsu, Kouhei; Takeshita, Akira

2002-09-01

208

Application of nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester, on injured nerve attenuates neuropathy-induced thermal hyperalgesia in rats.  

PubMed

This study tested the ability of a nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME), to attenuate behavioral hyperalgesia in a rat model of neuropathic pain [Bennett, G.J. and Xie, Y.-K., Pain, 33 (1988) 87-107]. A mononeuropathy was produced by chronic constriction injury (CCI) of the sciatic nerve. Thermal hyperalgesia was assessed by a reduction of paw withdrawal latency to a noxious heat source. Following CCI, there was significant hyperalgesia in groups of rats treated with D-NAME (n = 7), an inactive isomer of L-NAME, saline (n = 7) or systemic L-NAME (n = 10). In contrast, when L-NAME was applied directly and continuously to the site of CCI (5.0 micrograms/microliter per h for up to 2 weeks) via an osmotic pump implanted at the time of the injury, no significant thermal hyperalgesia was observed (n = 8). The results suggest the involvement of nitric oxide in the development and maintenance of thermal hyperalgesia in a rat model of neuropathy. The blockade of nitric oxide production at the site of injury may provide a new approach for treatment of neuropathic pain. PMID:8783289

Thomas, D A; Ren, K; Besse, D; Ruda, M A; Dubner, R

1996-05-31

209

A nitric oxide synthase inhibitor reduces inflammation, down-regulates inflammatory cytokines and enhances interleukin-10 production in carrageenin-induced oedema in mice.  

PubMed Central

Mice injected in the footpad with carrageenin developed local inflammation which peaked at 48 hr. This delayed-type footpad swelling was significantly reduced in mice injected intraperitoneally (i.p.) with a specific nitric oxide (NO) synthase inhibitor, L-NGmonomethyl-arginine (L-NMMA). The draining lymph node (DLN) cells from mice injected 48 hr previously with carrageenin produced significantly higher levels of proliferation and interleukin-1 (IL-1), IL-2, IL-6 and interferon-gamma (IFN-gamma), but less IL-10, compared to cells from saline-injected controls, when stimulated with concanavalin A (Con A) in vitro. Treatment of the carrageenin-injected mice with L-NMMA had little effect on the proliferative response of the DLN cells, but significantly reduced the production of IL-1, IL-2, IL-6 and IFN-gamma, and increased the secretion of IL-10. These data demonstrate that NO plays a significant role in local inflammation and the pattern of cytokines induced in this model. Images Figure 2 PMID:7959870

Ianaro, A; O'Donnell, C A; Di Rosa, M; Liew, F Y

1994-01-01

210

Fabrication of optically transparent chitin nanocomposites  

NASA Astrophysics Data System (ADS)

This paper demonstrates the preparation of chitin nanofibers from crab shells using a simple mechanical treatment. The nanofibers are small enough to retain the transparency of neat acrylic resin. Possessing hydroxyl and amine/ N-acetyl functionalities, water suspension of chitin nanofibers was vacuum-filtered 9 times faster than cellulose nanofibers to prepare a nanofiber sheet of 90 mm in diameter. This is a prominent advantage of chitin nanofibers over cellulose nanofibers in terms of commercial application. Interestingly, chitin acrylic resin films exhibited much higher transparency than cellulose acrylic resin films owing to the close affinity between less hydrophilic chitin and hydrophobic resin. Furthermore, the incorporation of chitin nanofibers contributes to the significant improvement of the thermal expansion and mechanical properties of the neat acrylic resin. The properties of high light transmittance and low thermal expansion make chitin nanocomposites promising candidates for the substrate in a continuous roll-to-roll process in the manufacturing of various optoelectronic devices such as flat panel displays, bendable displays, and solar cells.

Shams, M. Iftekhar; Ifuku, Shinsuke; Nogi, Masaya; Oku, Takeshi; Yano, Hiroyuki

2011-02-01

211

An inhibitor-driven study for enhancing the selectivity of indirubin derivatives towards leishmanial Glycogen Synthase Kinase-3 over leishmanial cdc2-related protein kinase 3  

PubMed Central

Background In search of new antiparasitic agents for overcoming the limitations of current leishmaniasis chemotherapy, we have previously shown that 6-bromoindirubin-3'-oxime (6BIO) and several other 6-substituted analogues of indirubin, a naturally occurring bis-indole present in mollusks and plants, displayed reverse selectivity from the respective mammalian kinases, targeting more potently the leishmanial Cyclin-Dependent Kinase-1 (CDK1) homologue [cdc2-related protein kinase 3 (LCRK3)] over leishmanial Glycogen Synthase Kinase-3 (LGSK-3). This reversal of selectivity in Leishmania parasites compared to mammalian cells makes the design of specific indirubin-based LGSK-3 inhibitors difficult. In this context, the identification of compounds bearing specific substitutions that shift indirubin inhibition towards LGSK-3, previously found to be a potential drug target, over LCRK3 is imperative for antileishmanial targeted drug discovery. Methods A new in-house indirubin library, composed of 35 compounds, initially designed to target mammalian kinases (CDKs, GSK-3), was tested against Leishmania donovani promastigotes and intracellular amastigotes using the Alamar blue assay. Indirubins with antileishmanial activity were tested against LGSK-3 and LCRK3 kinases, purified from homologous expression systems. Flow cytometry (FACS) was used to measure the DNA content for cell-cycle analysis and the mode of cell death. Comparative structural analysis of the involved kinases was then performed using the Szmap algorithm. Results We have identified 7 new indirubin analogues that are selective inhibitors of LGSK-3 over LCRK3. These new inhibitors were also found to display potent antileishmanial activity with GI50 values of <1.5 ??. Surprisingly, all the compounds that displayed enhanced selectivity towards LGSK-3, were 6BIO analogues bearing an additional 3'-bulky amino substitution, namely a piperazine or pyrrolidine ring. A comparative structural analysis of the two aforementioned leishmanial kinases was subsequently undertaken to explain and rationalize the selectivity trend determined by the in vitro binding assays. Interestingly, the latter analysis showed that selectivity could be correlated with differences in kinase solvation thermo dynamics induced by minor sequence variations of the otherwise highly similar ATP binding pockets. Conclusions In conclusion, 3'-bulky amino substituted 6-BIO derivatives, which demonstrate enhanced specificity towards LGSK-3, represent a new scaffold for targeted drug development to treat leishmaniasis. PMID:24886176

2014-01-01

212

Suppressing the expression of a forkhead transcription factor disrupts the chitin biosynthesis pathway in Spodoptera exigua.  

PubMed

Forkhead (Fox) transcription factors display functional diversity and are involved in various metabolic and developmental processes. The Spodoptera exigua Fox (SeFox) encodes a protein of 353 amino acids with a theoretical molecular mass of approximately 38.99 kDa and an isoelectric point of 8.86. qPCR results revealed that SeFox was expressed mainly in the brain, fat body, epidermis, midgut, Malpighian tubules, and testis. SeFox was expressed, with some changes, throughout development in the fat body and whole body. Injection of dsSeFox (SeFox dsRNA) into larvae resulted in incidences of albino plus molting deformity (4.8%), molting deformity (26.2%), and albino phenotypes (69.1%). dsSeFox injection resulted in approximately 50% knockdown of transcript levels at 36 h. Compared with control groups, hexokinase (HK) expression was reduced to approximately 40% at 48 h postinjection. Chitin synthase A (CHSA) expression was reduced to two-thirds at 24 h, but increased at 72 h. Compared with untreated control and green fluorescent protein-treated groups, Chitin synthase B (CHSB) expression decreased to 33% following dsSeFox injection by 36 h. We infer from our results that forkhead transcription factors act in chitin synthesis in S. exigua. PMID:24464395

Zhao, Lina; Wei, Ping; Guo, Hongshuang; Wang, Shigui; Tang, Bin

2014-05-01

213

Dissolution of mechanically milled chitin in high temperature water.  

PubMed

Chitin is high in crystallinity in its natural form and does not dissolve into high temperature water (HTW), which often leads to decomposition reactions such as hydrolysis, deacetylation and dehydration when hydrothermally processed. In this work, we investigated the reactions of mechanically milled chitin in HTW. Mechanical milling pretreatment combined with HTW treatment improved the liquefaction of chitin giving a maximum water soluble fraction of 80%, where the untreated chitin was 55%. The reaction mechanism of the milled and raw chitin in HTW was shown to be different. For milled chitin, the dissolution of chitin occurred during the heating period to supercritical water conditions (400°C) at short reaction times (1 min). Extended reaction time (10 min) led to decomposition products and aromatic char formation. For raw chitin, the dissolution of chitin in HTW did not occur, due to its high crystallinity, so that liquefaction proceeded via decomposition reactions. PMID:24721066

Aida, Taku Michael; Oshima, Kenji; Abe, Chihiro; Maruta, Ryoma; Iguchi, Masayuki; Watanabe, Masaru; Smith, Richard L

2014-06-15

214

Cell cycle effects of CB30865, a lipophilic quinazoline-based analogue of the antifolate thymidylate synthase inhibitor ICI 198583 with an undefined mechanism of action.  

PubMed

CB30865 (p-[N-(7-bromo-3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl+ ++)-N-(prop-2-ynyl)amino]-N-(3-pyridylmethyl)benzamide) is a quinazoline-based pyridine-containing compound that emerged from a programme aimed at the development of thymidylate synthase (TS) inhibitors as anticancer agents. Its structure is based on the antifolate ICI 198583, but with a pyridine ring replacing the glutamate. Despite its structure, CB30865 does not act in vitro via inhibition of TS or, apparently, other known folate-dependent pathways, and extensive mechanistic studies suggest that it acts via a novel locus with respect to conventional antitumour agents. However, CB30865 is highly potent against a variety of human tumour cell lines (e.g., 50%-inhibitory concentration [IC50] values in the 1-10 nM range). Thus, the cell cycle effects of CB30865 were investigated. DNA histogram analysis of W1L2 human lymphoblastoid, L1210 murine leukaemia, and CH1 human ovarian cells (propidium iodide staining) has demonstrated that CB30865 does not cause a phase-specific arrest at concentrations that have been shown to inhibit colony formation. This is unexpected for an anticancer agent. In W1L2 cells, using bromodeoxyuridine (BrdUrd) labelling and bivariate Hoechst/ propidium iodide staining, it was revealed that 0.003-0.15 microM CB30865 (1-50 x 72 h IC50) caused cells to arrest in all phases of the cell cycle simultaneously after 20-24 h exposure. This effect was also observed in CH1 and L1210 cells, though the arrest was at slightly different times. Thus, using this technique, it has been demonstrated that CB30865 induces an unusual and delayed cell cycle arrest, which provides further evidence for a novel locus of action for this compound. PMID:9725559

Skelton, L A; Ormerod, M G; Titley, J C; Jackman, A L

1998-09-01

215

Celastrol, an inhibitor of heat shock protein 90? potently suppresses the expression of matrix metalloproteinases, inducible nitric oxide synthase and cyclooxygenase-2 in primary human osteoarthritic chondrocytes.  

PubMed

Overexpression of matrix metalloproteinases (MMPs), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) have long been suggested to play crucial roles in the progression of osteoarthritis. Studies have showed that selective MMPs, iNOS and COX-2 inhibitors possess great potential as chondroprotective agents for osteoarthritis. Therefore, there have been intensive efforts to develop novel natural compounds that target MMPs, iNOS and COX-2 activation. As interleukin-1? (IL-1?) is one of the key proinflammatory cytokines contributing to the progression in osteoarthritis, we investigated the effect of celastrol, a triterpenoid compound extracted from the Chinese herb Tript erygium wilfordii Hook F, in neutralizing the inflammatory effects of IL-1? on MMPs, iNOS and COX-2 expression as well as nitric oxide (NO) and prostaglandin E2 (PGE2) production. Protein expression was detected by Western blotting or by enzyme-linked immunosorbent assay (ELISA); messenger RNA (mRNA) expression was examined by real-time reverse transcription-polymerase chain reaction analysis and the involvement of signal pathway was assessed by transient transfection and luciferase activity assay. We found that treatment of primary human osteoarthritic chondrocytes with various concentrations of celastrol resulted in striking decrease in the expression of MMP-1, MMP-3, MMP-13, iNOS-2 and COX-2. In addition, celastrol treatment of cells also inhibited the activation of nuclear factor-kappa B (NF-kappaB). Taken together, we provide evidence that celastrol can protect human chondrocytes by downregulating the expression of MMPs, iNOS and COX-2. We suggest that celastrol could be a useful agent for prevention and treatment of osteoarthritis. PMID:23396231

Ding, Qian-Hai; Cheng, Ye; Chen, Wei-Ping; Zhong, Hui-Ming; Wang, Xiang-Hua

2013-05-15

216

Potencies of leflunomide and HR325 as inhibitors of prostaglandin endoperoxide H synthase-1 and -2: comparison with nonsteroidal anti-inflammatory drugs.  

PubMed

The relative anti-inflammatory activities of the immunomodulators HR325 and leflunomide, or its active metabolite A77 1726, were examined by determining potencies in vitro on prostaglandin endoperoxide H synthase (PGHS) and in vivo in rat air pouch inflammation. Nonsteroidal anti-inflammatory drugs (NSAIDs) were used as comparators. HR325 was more potent than A77 1726 as an inhibitor of PGHS in guinea pig polymorphonuclear leukocytes (IC50 = 415 and 4400 nM, respectively) and on isolated ovine PGHS-1 (IC50 = 64 and 742 microM) and PGHS-2 (IC50 = 100 and 2766 microM). In vivo, in rat carrageenan air pouch inflammation, HR325 but not leflunomide at 25 mg/kg inhibited accumulation of leukocytes (48%) and PGE2 (61%). HR325 was also more potent than A77 1726 against human peripheral blood mononuclear cell PGHS-1 [IC50 = 1.6 and 25.6 microM (thromboxane B2 production) or 1.1 and 8 microM (PGE2 production)] and lipopolysaccharide-induced PGHS-2 in human adherent peripheral blood mononuclear cells (IC50 = 435 nM and 9.5 microM) and peripheral blood polymorphonuclear leukocytes (IC50 = 91 nM and 3.2 microM). HR325 had low PGHS-2 selectivity in the human (2.5-12-fold) and was a more potent PGHS-2 inhibitor than naproxen, ibuprofen and piroxicam (28-fold). Assays using endogenous arachidonic acid as substrate yielded IC50 values for NSAIDs that were in general markedly lower than those published for assays using 10 microM substrate. With this approach, piroxicam had reasonable activity on human PGHS-2 (IC50 = 260-290 nM). Only NS398 and flufenamic acid were PGHS-2 selective in the human (90-330-fold and 37-60-fold, respectively); the other NSAIDs were either PGHS-1-selective (naproxen, ibuprofen, flurbiprofen and indomethacin) or nonselective (piroxicam and diclofenac). Inclusion of 10% human plasma reduced HR325 potency against PGHS-1 in human peripheral blood mononuclear cells approximately 32-fold (IC50 = 36 microM). Plasma protein binding further reduced HR325 potency (IC50 = 164 microM) and minimized the difference between HR325 and A77 1726 (IC50 = 292 microM) in a whole blood PGHS assay. Whether the greater activity against human PGHS-2 would allow HR325 to exhibit NSAID-like therapeutic effects in humans remains unclear. PMID:9223572

Curnock, A P; Robson, P A; Yea, C M; Moss, D; Gadher, S; Thomson, T A; Westwood, R; Ruuth, E; Williamson, R A

1997-07-01

217

Inhibitors of the fungal cell wall. Synthesis of 4-aryl-4- N-arylamine-1-butenes and related compounds with inhibitory activities on ?(1–3) glucan and chitin synthases  

Microsoft Academic Search

As part of our project devoted to the search for antifungal agents, which act via a selective mode of action, we synthesized a series of new 4-aryl- or 4-alkyl-N-arylamine-1-butenes and transformed some of them into 2-substituted 4-methyl-tetrahydroquinolines and quinolines by using a novel three-step synthesis. Results obtained in agar dilution assays have shown that 4-aryl homoallylamines not possessing halogen in

Juan M Urbina; Juan C. G Cortés; Alirio Palma; Silvia N López; Susana A Zacchino; Ricardo D Enriz; Juan C Ribas; Vladimir V Kouznetzov

2000-01-01

218

A Saccharomyces cerevisiae mutant with echinocandin-resistant 1,3-beta-D-glucan synthase.  

PubMed Central

A novel, potent, semisynthetic pneumocandin, L-733,560, was used to isolate a resistant mutant in Saccharomyces cerevisiae. This compound, like other pneumocandins and echinocandins, inhibits 1,3-beta-D-glucan synthase from Candida albicans (F.A. Bouffard, R.A. Zambias, J. F. Dropinski, J.M. Balkovec, M.L. Hammond, G.K. Abruzzo, K.F. Bartizal, J.A. Marrinan, M. B. Kurtz, D.C. McFadden, K.H. Nollstadt, M.A. Powles, and D.M. Schmatz, J. Med. Chem. 37:222-225, 1994). Glucan synthesis catalyzed by a crude membrane fraction prepared from the S. cerevisiae mutant R560-1C was resistant to inhibition by L-733,560. The nearly 50-fold increase in the 50% inhibitory concentration against glucan synthase was commensurate with the increase in whole-cell resistance. R560-1C was cross-resistant to other inhibitors of C. albicans 1,3-beta-D-glucan synthase (aculeacin A, dihydropapulacandin, and others) but not to compounds with different modes of action. Genetic analysis revealed that enzyme and whole-cell pneumocandin resistance was due to a single mutant gene, designated etg1-1 (echinocandin target gene 1), which was semidominant in heterozygous diploids. The etg1-1 mutation did not confer enhanced ability to metabolize L-733,560 and had no effect on the membrane-bound enzymes chitin synthase I and squalene synthase. Alkali-soluble beta-glucan synthesized by crude microsomes from R560-1C was indistinguishable from the wild-type product. 1,3-beta-D-Glucan synthase activity from R560-1C was fractionated with NaCl and Tergitol NP-40; reconstitution with fractions from wild-type membranes revealed that drug resistance is associated with the insoluble membrane fraction. We propose that the etg1-1 mutant gene encodes a subunit of the 1,3-beta-D-glucan synthase complex. PMID:8083161

Douglas, C M; Marrinan, J A; Li, W; Kurtz, M B

1994-01-01

219

Bacterial chitin degradation--mechanisms and ecophysiological strategies  

PubMed Central

Chitin is one the most abundant polymers in nature and interacts with both carbon and nitrogen cycles. Processes controlling chitin degradation are summarized in reviews published some 20 years ago, but the recent use of culture-independent molecular methods has led to a revised understanding of the ecology and biochemistry of this process and the organisms involved. This review summarizes different mechanisms and the principal steps involved in chitin degradation at a molecular level while also discussing the coupling of community composition to measured chitin hydrolysis activities and substrate uptake. Ecological consequences are then highlighted and discussed with a focus on the cross feeding associated with the different habitats that arise because of the need for extracellular hydrolysis of the chitin polymer prior to metabolic use. Principal environmental drivers of chitin degradation are identified which are likely to influence both community composition of chitin degrading bacteria and measured chitin hydrolysis activities. PMID:23785358

Beier, Sara; Bertilsson, Stefan

2013-01-01

220

Nitric oxide donors prevent while the nitric oxide synthase inhibitor L-NAME increases arachidonic acid plus CYP2E1-dependent toxicity  

SciTech Connect

Polyunsaturated fatty acids such as arachidonic acid (AA) play an important role in alcohol-induced liver injury. AA promotes toxicity in rat hepatocytes with high levels of cytochrome P4502E1 and in HepG2 E47 cells which express CYP2E1. Nitric oxide (NO) participates in the regulation of various cell activities as well as in cytotoxic events. NO may act as a protectant against cytotoxic stress or may enhance cytotoxicity when produced at elevated concentrations. The goal of the current study was to evaluate the effect of endogenously or exogenously produced NO on AA toxicity in liver cells with high expression of CYP2E1 and assess possible mechanisms for its actions. Pyrazole-induced rat hepatocytes or HepG2 cells expressing CYP2E1 were treated with AA in the presence or absence of an inhibitor of nitric oxide synthase L-N {sup G}-Nitroarginine Methylester (L-NAME) or the NO donors S-nitroso-N-acetylpenicillamine (SNAP), and (Z)-1-[-(2-aminoethyl)-N-(2-aminoethyl)]diazen-1-ium-1,2-diolate (DETA-NONO). AA decreased cell viability from 100% to 48 {+-} 6% after treatment for 48 h. In the presence of L-NAME, viability was further lowered to 23 {+-} 5%, while, SNAP or DETA-NONO increased viability to 66 {+-} 8 or 71 {+-} 6%. The L-NAME potentiated toxicity was primarily necrotic in nature. L-NAME did not affect CYP2E1 activity or CYP2E1 content. SNAP significantly lowered CYP2E1 activity but not protein. AA treatment increased lipid peroxidation and lowered GSH levels. L-NAME potentiated while SNAP prevented these changes. Thus, L-NAME increased, while NO donors decreased AA-induced oxidative stress. Antioxidants prevented the L-NAME potentiation of AA toxicity. Damage to mitochondria by AA was shown by a decline in the mitochondrial membrane potential (MMP). L-NAME potentiated this decline in MMP in association with its increase in AA-induced oxidative stress and toxicity. NO donors decreased this decline in MMP in association with their decrease in AA-induced oxidative stress and toxicity. These results indicate that NO can be hepatoprotective against CYP2E1-dependent toxicity, preventing AA-induced oxidative stress.

Wu Defeng [Department of Pharmacology and Biological Chemistry, Box 1603, One Gustave L. Levy Place, Mount Sinai School of Medicine, New York, NY 10029 (United States); Cederbaum, Arthur [Department of Pharmacology and Biological Chemistry, Box 1603, One Gustave L. Levy Place, Mount Sinai School of Medicine, New York, NY 10029 (United States)]. E-mail: arthur.cederbaum@mssm.edu

2006-10-15

221

Nanofibrillar chitin aerogels as renewable base catalysts.  

PubMed

We demonstrate the fabrication of chitin nanofibril aerogels and their successful application as base catalysts for the production of useful chemicals. Squid-pen chitin nanofibrils (ChNF) with primary C2-amine groups on their crystalline surfaces were fabricated into highly porous aerogels with high specific surface areas up to 289 m(2) g(-1) using freeze-drying or a supercritical drying process. The prepared ChNF aerogel was used in the aqueous Knoevenagel-condensation reaction and acted as a highly efficient base catalyst, suggesting that the combination of the nanofibrous aerogel structure and primary C2-amines exposed on the crystalline ChNF surface was effective for continuous flow catalysis. Because the ChNF aerogel can be easily prepared from abundant and renewable chitin present in nature, this strategy is a gateway to promoting and conducting green and sustainable chemistry. PMID:25285573

Tsutsumi, Yoshiyuki; Koga, Hirotaka; Qi, Zi-Dong; Saito, Tsuguyuki; Isogai, Akira

2014-11-10

222

Concurrent protein synthesis is required for in vivo chitin synthesis in postmolt blue crabs  

SciTech Connect

Chitin synthesis in crustaceans involves the deposition of a protein-polysaccharide complex at the apical surface of epithelial cells which secrete the cuticle or exoskeleton. The present study involves an examination of in vivo incorporation of radiolabeled amino acids and amino sugars into the cuticle of postmolt blue crabs, Callinectes sapidus. Rates of incorporation of both 3H leucine and 3H threonine were linear with respect to time of incubation. Incorporation of 3H threonine into the endocuticle was inhibited greater than 90% in the presence of the protein synthesis inhibitor, puromycin. Linear incorporation of 14C glucosamine into the cuticle was also demonstrated; a significant improvement of radiolabeling was achieved by using 14C-N-acetylglucosamine as the labeled precursor. Incorporation of 3H-N-acetylglucosamine into the cuticle of postmolt blue crabs was inhibited 89% by puromycin, indicating that concurrent protein synthesis is required for the deposition of chitin in the blue crab. Autoradiographic analysis of control vs. puromycin-treated crabs indicates that puromycin totally blocks labeling of the new endocuticle with 3H glucosamine. These results are consistent with the notion that crustacean chitin is synthesized as a protein-polysaccharide complex. Analysis of the postmolt and intermolt blue crab cuticle indicates that the exoskeleton contains about 60% protein and 40% chitin. The predominant amino acids are arginine, glutamic acid, alanine, aspartic acid, and threonine.

Horst, M.N. (Mercer Univ., Macon, GA (USA))

1990-12-01

223

Spermine synthase  

PubMed Central

Spermine is present in many organisms including animals, plants, some fungi, some archaea, and some bacteria. It is synthesized by spermine synthase, a highly specific aminopropyltransferase. This review describes spermine synthase structure, genetics, and function. Structural and biochemical studies reveal that human spermine synthase is an obligate dimer. Each monomer contains a C-terminal domain where the active site is located, a central linking domain that also forms the lid of the catalytic domain, and an N-terminal domain that is structurally very similar to S-adenosylmethionine decarboxylase. Gyro mice, which have an X-chromosomal deletion including the spermine synthase (SMS) gene, lack all spermine and have a greatly reduced size, sterility, deafness, neurological abnormalities, and a tendency to sudden death. Mutations in the human SMS lead to a rise in spermidine and reduction of spermine causing Snyder-Robinson syndrome, an X-linked recessive condition characterized by mental retardation, skeletal defects, hypotonia, and movement disorders. PMID:19859664

Michael, Anthony J.

2010-01-01

224

ICI D1694, a quinazoline antifolate thymidylate synthase inhibitor that is a potent inhibitor of L1210 tumor cell growth in vitro and in vivo: a new agent for clinical study.  

PubMed

N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N- methylamino]-2-thenoyl)-L-glutamic acid (ICI D1694) is a water-soluble, folate-based thymidylate synthase (TS) inhibitor designed to be a less toxic and more potent analogue of the clinically tested N10-propargyl-5,8-dideazafolic acid. Inhibition of isolated L1210 TS by ICI D1694 is mixed noncompetitive (although tending toward competitive), with a Ki of 62 nM (Kies = 960 nM). The synthetic gamma-polyglutamates are up to 2 orders of magnitude more potent as inhibitors of TS; e.g., the tetraglutamate (glu4) has a Ki of 1.0 nM (Kies = 15 nM). Although inhibitory activity of ICI D1694 toward rat liver dihydrofolate reductase was similar to that of TS (Ki = 92 nM; competitive inhibition) the polyglutamate derivatives did not show enhanced activity. ICI D1694 was also a very potent inhibitor of L1210 cell growth (50% inhibitory activity = 8 nM). L1210 growth inhibition was not observed in the presence of thymidine, consistent with TS being the locus of action. Folinic acid antagonized L1210 growth inhibition in a competitive fashion such that the highest folinic acid concentration used (25 microM) increased the 50% inhibitory activity 6000-fold. When given as a 4-h delayed "rescue", folinic acid was much less effective in antagonizing growth inhibition. These observations are consistent with folinic acid competing with ICI D1694 for uptake into the cell and/or intracellular polyglutamation. The L1210:1565 cell line, which has greatly impaired reduced-folate/methotrexate transport and thus is resistant to methotrexate, was significantly cross-resistant to ICI D1694 (121-fold), suggesting that ICI D1694 is dependent on this uptake mechanism for good cytotoxic potency in L1210 cells. L1210 cells that were incubated for 4 h with 0.1 microM 3H-ICI D1694 accumulated approximately 1.5 microM intracellular 3H, and the high performance liquid chromatography analysis of the cell extracts demonstrated that 96% of the 3H was associated with the ICI D1694 polyglutamate fractions (principally glu4). Upon resuspension in drug-free medium for 24 h, approximately 75% of the cellular 3H was retained, this being the higher polyglutamate pool (glu4-6). In mice, after a single bolus injection of 10 mg/kg of ICI D1694, TS was inhibited greater than 80% for 24 h in ascitic L1210:NCI cells (as measured by the rate of 3H release from [5-3H]deoxyuridine). ICI D1694 cured the L1210:ICR ascitic tumor in mice at 0.4 mg/kg daily for 5 days (maximum tolerated dose, approximately 50 mg/kg).(ABSTRACT TRUNCATED AT 400 WORDS) PMID:1913676

Jackman, A L; Taylor, G A; Gibson, W; Kimbell, R; Brown, M; Calvert, A H; Judson, I R; Hughes, L R

1991-10-15

225

Inhibitors of the Proteasome Pathway Interfere with Induction of Nitric Oxide Synthase in Macrophages by Blocking Activation of Transcription Factor NF-kappa B  

Microsoft Academic Search

The objective of this study was to elucidate the role of the proteasome pathway or multicatalytic proteinase complex in the induction of immunologic nitric oxide (NO) synthase (iNOS) in rat alveolar macrophages activated by lipopolysaccharide. Macrophages were incubated in the presence of lipopolysaccharide plus test agent for up to 24 hr. Culture media were analyzed for accumulation of stable oxidation

Jeanette M. Griscavage; Sherwin Wilk; Louis J. Ignarro

1996-01-01

226

Antimalarial drug targets: Screening for inhibitors of 2C-methyl- d-erythritol 4-phosphate synthase (IspC protein) in Mediterranean plants  

Microsoft Academic Search

The recently discovered non-mevalonate pathway of isoprenoid biosynthesis serves as the unique source of terpenoids in numerous pathogenic eubacteria and in apicoplast-type protozoa, most notably Plasmodium, but is absent in mammalian cells. It is therefore an attractive target for anti-infective chemotherapy. The first committed step of the non-mevalonate pathway is catalyzed by 2C-methyl-d-erythritol 4-phosphate synthase (IspC). Using photometric and NMR

J. Kaiser; M. Yassin; S. Prakash; N. Safi; M. Agami; S. Lauw; E. Ostrozhenkova; A. Bacher; F. Rohdich; W. Eisenreich; J. Safi; A. Golan-Goldhirsh

2007-01-01

227

Constitutive activation of glycogen synthase kinase-3? correlates with better prognosis and cyclin-dependent kinase inhibitors in human gastric cancer  

Microsoft Academic Search

BACKGROUND: Aberrant regulation of glycogen synthase kinase-3? (GSK-3?) has been implicated in several human cancers; however, it has not been reported in the gastric cancer tissues to date. The present study was performed to determine the expression status of active form of GSK-3? phosphorylated at Tyr216 (pGSK-3?) and its relationship with other tumor-associated proteins in human gastric cancers. METHODS: Immunohistochemistry

Yu Jin Cho; Ji Hun Kim; Jiyeon Yoon; Sung Jin Cho; Young San Ko; Jong-Wan Park; Hye Seung Lee; Hee Eun Lee; Woo Ho Kim; Byung Lan Lee

2010-01-01

228

Design, Synthesis, and Biological Evaluation of Classical and Nonclassical 2-Amino-4-oxo-5-substituted-6-methylpyrrolo[3,2-d]pyrimidines as Dual Thymidylate Synthase and Dihydrofolate Reductase Inhibitors  

PubMed Central

We designed and synthesized a classical antifolate N-{4-[(2-amino-6-methyl-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)methyl]benzoyl}-l-glutamic acid 4 and 11 nonclassical analogues 5–15 as potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors. The key intermediate in the synthesis was N-(4-chloro-6-methyl-5H-pyrrolo[3,2-d]pyrimidin-2-yl)-2,2-dimethylpropanamide, 29, to which various 5-benzyl substituents were attached. For the classical analogue 4, the ester obtained from the N-benzylation reaction was deprotected and coupled with diethyl l-glutamate followed by saponification. Compound 4 was a potent dual inhibitor of human TS (IC50 = 46 nM, about 206-fold more potent than pemetrexed) and DHFR (IC50 = 120 nM, about 55-fold more potent than pemetrexed). The nonclassical analogues were marginal inhibitors of human TS, but four analogues showed potent T. gondii DHFR inhibition along with >100-fold selectivity compared to human DHFR. PMID:18072727

Gangjee, Aleem; Li, Wei; Yang, Jie; Kisliuk, Roy L.

2013-01-01

229

6-Position optimization of tricyclic 4-quinolone-based inhibitors of glycogen synthase kinase-3?: discovery of nitrile derivatives with picomolar potency.  

PubMed

We previously disclosed tricylic, 6-carboxylic acid-bearing 4-quinolones as GSK-3? inhibitors. Herein we discuss the optimization of this series to yield a series of more potent 6-nitrile analogs with insignificant anti-microbial activity. Finally, kinase profiling indicated that members of this class were highly specific GSK-3 inhibitors. PMID:22202172

Li, Bei; Cociorva, Oana M; Nomanbhoy, Tyzoon; Li, Qiang; Nakamura, Kai; Nomura, Masahiro; Okada, Kyoko; Yumoto, Kazuhiro; Liyanage, Marek; Zhang, Melissa C; Aban, Arwin; Szardenings, Anna Katrin; Kozarich, John W; Kohno, Yasushi; Shreder, Kevin R

2012-01-15

230

Chitovibrin: a chitin-binding lectin from Vibrio parahemolyticus.  

PubMed

A novel 134 kDa, calcium-independent chitin-binding lectin, 'chitovibrin', is secreted by the marine bacterium Vibrio parahemolyticus, inducible with chitin or chitin-oligomers. Chitovibrin shows no apparent enzymatic activity but exhibits a strong affinity for chitin and chito-oligomers > dp9. The protein has an isoelectric pH of 3.6, shows thermal tolerance, binds chitin with an optimum at pH 6 and is active in 0-4 M NaCl. Chitovibrin appears to be completely different from other reported Vibrio lectins and may function to bind V. parahemolyticus to chitin substrates, or to capture or sequester chito-oligomers. It may be a member of a large group of recently described proteins in Vibrios related to a complex chitinoclastic (chitinivorous) system. PMID:7696854

Gildemeister, O S; Zhu, B C; Laine, R A

1994-12-01

231

4-methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic acid (C75), an inhibitor of fatty-acid synthase, suppresses the mitochondrial fatty acid synthesis pathway and impairs mitochondrial function.  

PubMed

4-Methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic acid (C75) is a synthetic fatty-acid synthase (FASN) inhibitor with potential therapeutic effects in several cancer models. Human mitochondrial ?-ketoacyl-acyl carrier protein synthase (HsmtKAS) is a key enzyme in the newly discovered mitochondrial fatty acid synthesis pathway that can produce the substrate for lipoic acid (LA) synthesis. HsmtKAS shares conserved catalytic domains with FASN, which are responsible for binding to C75. In our study, we explored the possible effect of C75 on HsmtKAS and mitochondrial function. C75 treatment decreased LA content, impaired mitochondrial function, increased reactive oxygen species content, and reduced cell viability. HsmtKAS but not FASN knockdown had an effect that was similar to C75 treatment. In addition, an LA supplement efficiently inhibited C75-induced mitochondrial dysfunction and oxidative stress. Overexpression of HsmtKAS showed cellular protection against low dose C75 addition, whereas there was no protective effect upon high dose C75 addition. In summary, the mitochondrial fatty acid synthesis pathway has a vital role in mitochondrial function. Besides FASN, C75 might also inhibit HsmtKAS, thereby reducing LA production, impairing mitochondrial function, and potentially having toxic effects. LA supplements sufficiently ameliorated the toxicity of C75, showing that a combination of C75 and LA may be a reliable cancer treatment. PMID:24784139

Chen, Cong; Han, Xiao; Zou, Xuan; Li, Yuan; Yang, Liang; Cao, Ke; Xu, Jie; Long, Jiangang; Liu, Jiankang; Feng, Zhihui

2014-06-13

232

Structure and function of enzymes acting on chitin and chitosan.  

PubMed

Enzymatic conversions of chitin and its soluble, partially deacetylated derivative chitosan are of great interest. Firstly, chitin metabolism is an important process in fungi, insects and crustaceans. Secondly, such enzymatic conversions may be used to transform an abundant biomass to useful products such as bioactive chito-oligosaccharides. Enzymes acting on chitin and chitosan are abundant in nature. Here we review current knowledge on the structure and function of enzymes involved in the conversion of these polymeric substrates: chitinases (glycoside hydrolase families 18 & 19), chitosanases (glycoside hydrolase families 8, 46, 75 & 80) and chitin deacetylases (carbohydrate esterase family 4). PMID:21415904

Eijsink, Vincent; Hoell, Ingunn; Vaaje-Kolstada, Gustav

2010-01-01

233

Isolation and characterization of chitin and chitosan from marine origin.  

PubMed

Nowadays, chitin and chitosan are produced from the shells of crabs and shrimps, and bone plate of squid in laboratory to industrial scale. Production of chitosan involved deproteinization, demineralization, and deacetylation. The characteristics of chitin and chitosan mainly depend on production processes and conditions. The characteristics of these biopolymers such as appearance of polymer, turbidity of polymer solution, degree of deacetylation, and molecular weight are of major importance on applications of these polymers. This chapter addresses the production processes and conditions to produce chitin, chitosan, and chito-oligosaccharide and methods for characterization of chitin, chitosan, and chito-oligosaccharide. PMID:25081074

Nwe, Nitar; Furuike, Tetsuya; Tamura, Hiroshi

2014-01-01

234

Immune suppression by recombinant interleukin (rIL)-12 involves interferon gamma induction of nitric oxide synthase 2 (iNOS) activity: inhibitors of NO generation reveal the extent of rIL-12 vaccine adjuvant effect.  

PubMed

Recombinant interleukin 12 (IL-12) can profoundly suppress cellular immune responses in mice. To define the underlying mechanism, recombinant murine (rm)IL-12 was given to C57BL/6 mice undergoing alloimmunization and found to transiently but profoundly suppress in vivo and in vitro allogeneic responses and in vitro splenocyte mitogenic responses. Use of neutralizing antibodies and genetically deficient mice showed that IFN-gamma (but not TNF-alpha) mediated rmIL-12-induced immune suppression. Splenocyte fractionation studies revealed that adherent cells from rmIL-12-treated mice suppressed the mitogenic response of normal nonadherent cells to concanavalin A and IL-2. Addition of an inhibitor of nitric oxide synthase (NOS) restored mitogenic responses, and inducible (i)NOS-/- mice were not immunosuppressed by rmIL-12. These results support the view that suppression of T cell responses is due to NO produced by macrophages responding to the high levels of IFN-gamma induced by rmIL-12. When a NOS inhibitor was given with rmIL-12 during vaccination of A/J mice with irradiated SCK tumor cells, immunosuppression was averted and the extent of rmIL-12's ability to enhance induction of protective antitumor immunity was revealed. This demonstrates that rmIL-12 is an effective vaccine adjuvant whose efficacy may be masked by its transient immunosuppressive effect. PMID:9802972

Koblish, H K; Hunter, C A; Wysocka, M; Trinchieri, G; Lee, W M

1998-11-01

235

Enhanced levan production using chitin-binding domain fused levansucrase immobilized on chitin beads.  

PubMed

Levan is a homopolymer of fructose which can be produced by the transfructosylation reaction of levansucrase (EC 2.4.1.10) from sucrose. In particular, levan synthesized by Zymomonas mobilis has found a wide and potential application in the food and pharmaceutical industry. In this study, the immobilization of Z. mobilis levansucrae (encoded by levU) was attempted for repeated production of levan. By fusion levU with the chitin-binding domain (ChBD), the hybrid protein was overproduced in a soluble form in Escherichia coli. After direct absorption of the protein mixture from E. coli onto chitin beads, levansucrase tagged with ChBD was found to specifically attach to the affinity matrix. Subsequent analysis indicated that the linkage between the enzyme and chitin beads was substantially stable. Furthermore, with 20% sucrose, the production of levan was enhanced by 60% to reach 83 g/l using the immobilized levansucrase as compared to that by the free counterpart. This production yield accounts for 41.5% conversion yield (g/g) on the basis of sucrose. After all, a total production of levan with 480 g/l was obtained by recycling of the immobilized enzyme for seven times. It is apparent that this approach offers a promising way for levan production by Z. mobilis levansucrase immobilized on chitin beads. PMID:19018526

Chiang, Chung-Jen; Wang, Jen-You; Chen, Po-Ting; Chao, Yun-Peng

2009-03-01

236

Lindane Adsorption-Desorption on Chitin in Sea Water  

Microsoft Academic Search

The adsorption and desorption processes by solid materials are important in determining the movement and fate of pesticide compounds in aquatic systems. Chitin is one of the constituents of natural organic matter and may serve as a model organic phase for studying the pesticide adsorption-desorption in marine systems. The lindane adsorption-desorption to chitin has been studied as a function of

M. Gonzalez-davila; J. Perez-Peña; J. M. Santana-casiano

1992-01-01

237

A gut-specific chitinase gene essential for regulation of chitin content of peritrophic matrix and growth of Ostrinia nubilalis larvae.  

PubMed

Chitinases belong to a large and diverse family of hydrolytic enzymes that break down glycosidic bonds of chitin. However, very little is known about the function of chitinase genes in regulating the chitin content in peritrophic matrix (PM) of the midgut in insects. We identified a cDNA putatively encoding a chitinase (OnCht) in European corn borer (ECB; Ostrinia nubilalis). The OnCht transcript was predominately found in larval midgut but undetectable in eggs, pupae, or adults. When the larvae were fed on an artificial diet, the OnCht transcript level increased by 4.4-fold but the transcript level of a gut-specific chitin synthase (OnCHS2) gene decreased by 2.5-fold as compared with those of unfed larvae. In contrast, when the larvae were fed with the food and then starved for 24h, the OnCht transcript level decreased by 1.8-fold but the transcript level of OnCHS2 increased by 1.8-fold. Furthermore, there was a negative relationship between OnCht transcript level and chitin content in the midgut. By using a feeding-based RNAi technique, we were able to reduce the OnCht transcript level by 63-64% in the larval midgut. Consequently, these larvae showed significantly increased chitin content (26%) in the PM but decreased larval body weight (54%) as compared with the control larvae fed on the diet containing GFP dsRNA. Therefore, for the first time, we provide strong evidence that OnCht plays an important role in regulating chitin content of the PM and subsequently affecting the growth and development of the ECB larvae. PMID:20542114

Khajuria, Chitvan; Buschman, Lawrent L; Chen, Ming-Shun; Muthukrishnan, Subbaratnam; Zhu, Kun Yan

2010-08-01

238

Detection and characterization of chitinases and other chitin-modifying enzymes  

Microsoft Academic Search

Multiple industrial and medical uses of chitin and its derivatives have been developed in recent years. The demand for enzymes with new or desirable properties continues to grow as additional uses of chitin, chitooligosaccharides, and chitosan become apparent. Microorganisms, the primary degraders of chitin in the environment, are a rich source of valuable chitin-modifying enzymes. This review summarizes many methods

Michael B. Howard; Nathan A. Ekborg; Ronald M. Weiner; Steven W. Hutcheson

2003-01-01

239

Crystal Structure and Binding Properties of the Serratia marcescens Chitin-binding Protein CBP21*S  

E-print Network

Crystal Structure and Binding Properties of the Serratia marcescens Chitin-binding Protein CBP21*S as a source of energy. CBP21 is a chitin- binding protein from Serratia marcescens, a Gram-neg- ative soil bacterium capable of efficient chitin degrada- tion. When grown on chitin, S. marcescens secretes large

van Aalten, Daan

240

Structural basis of chitin oligosaccharide deacetylation.  

PubMed

Cell signaling and other biological activities of chitooligosaccharides (COSs) seem to be dependent not only on the degree of polymerization, but markedly on the specific de-N-acetylation pattern. Chitin de-N-acetylases (CDAs) catalyze the hydrolysis of the acetamido group in GlcNAc residues of chitin, chitosan, and COS. A major challenge is to understand how CDAs specifically define the distribution of GlcNAc and GlcNH2 moieties in the oligomeric chain. We report the crystal structure of the Vibrio cholerae CDA in four relevant states of its catalytic cycle. The two enzyme complexes with chitobiose and chitotriose represent the first 3D structures of a CDA with its natural substrates in a productive mode for catalysis, thereby unraveling an induced-fit mechanism with a significant conformational change of a loop closing the active site. We propose that the deacetylation pattern exhibited by different CDAs is governed by critical loops that shape and differentially block accessible subsites in the binding cleft of CE4 enzymes. PMID:24810719

Andrés, Eduardo; Albesa-Jové, David; Biarnés, Xevi; Moerschbacher, Bruno M; Guerin, Marcelo E; Planas, Antoni

2014-07-01

241

Herbicide-resistant forms of Arabidopsis thaliana acetohydroxyacid synthase: characterization of the catalytic properties and sensitivity to inhibitors of four defined mutants.  

PubMed Central

Acetohydroxyacid synthase (AHAS) catalyses the first step in the synthesis of the branched-chain amino acids and is the target of several classes of herbicides. Four mutants (A122V, W574S, W574L and S653N) of the AHAS gene from Arabidopsis thaliana were constructed, expressed in Escherichia coli, and the enzymes were purified. Each mutant form and wild-type was characterized with respect to its catalytic properties and sensitivity to nine herbicides. Each enzyme had a pH optimum near 7.5. The specific activity varied from 13% (A122V) to 131% (W574L) of the wild-type and the Km for pyruvate of the mutants was similar to the wild-type, except for W574L where it was five-fold higher. The activation by cofactors (FAD, Mg2+ and thiamine diphosphate) was examined. A122V showed reduced affinity for all three cofactors, whereas S653N bound FAD more strongly than wild-type AHAS. Six sulphonylurea herbicides inhibited A122V to a similar degree as the wild-type but S653N showed a somewhat greater reduction in sensitivity to these compounds. In contrast, the W574 mutants were insensitive to these sulphonylureas, with increases in the Kiapp (apparent inhibition constant) of several hundred fold. All four mutants were resistant to three imidazolinone herbicides with decreases in sensitivity ranging from 100-fold to more than 1000-fold. PMID:9677339

Chang, A K; Duggleby, R G

1998-01-01

242

Vanillic acid: A potential inhibitor of cardiac and aortic wall remodeling in l-NAME induced hypertension through upregulation of endothelial nitric oxide synthase.  

PubMed

The objective of the present study is to investigate the effects of vanillic acid on blood pressure, cardiac marker enzymes, left ventricular function and endothelial nitric oxide synthase (eNOS) expression in N(?)-nitro-l-arginine methyl ester hydrochloride (l-NAME) induced hypertension in male albino Wistar rats. In hypertensive rats, mean arterial pressure (MAP), heart rate, cardiac marker enzymes and organ weight were increased. Impaired left ventricular function and decreased aortic eNOS expression was also observed in hypertensive rats. Moreover, treatment with vanillic acid exhibited beneficial effect on blood pressure, left ventricular function and cardiac marker enzymes. In addition, treatment with vanillic acid on hypertensive rats had upregulated eNOS expression and showed beneficial effects evidenced by histopathology and ultrastructural observations of aorta. In conclusion, vanillic acid has enough potential to normalize hypertension and left ventricular function in l-NAME induced hypertensive rats. With additional studies, vanillic acid might be used as a functional drug or as an adjuvant in the management of hypertension. PMID:25218092

Kumar, Subramanian; Prahalathan, Pichavaram; Raja, Boobalan

2014-09-01

243

Tetra- and Pentacyclic Triterpene Acids from the Ancient Anti-inflammatory Remedy Frankincense as Inhibitors of Microsomal Prostaglandin E2 Synthase-1  

PubMed Central

The microsomal prostaglandin E2 synthase (mPGES)-1 is the terminal enzyme in the biosynthesis of prostaglandin (PG)E2 from cyclooxygenase (COX)-derived PGH2. We previously found that mPGES-1 is inhibited by boswellic acids (IC50 = 3–30 ?M), which are bioactive triterpene acids present in the anti-inflammatory remedy frankincense. Here we show that besides boswellic acids, additional known triterpene acids (i.e., tircuallic, lupeolic, and roburic acids) isolated from frankincense suppress mPGES-1 with increased potencies. In particular, 3?-acetoxy-8,24-dienetirucallic acid (6) and 3?-acetoxy-7,24-dienetirucallic acid (10) inhibited mPGES-1 activity in a cell-free assay with IC50 = 0.4 ?M, each. Structure–activity relationship studies and docking simulations revealed concrete structure-related interactions with mPGES-1 and its cosubstrate glutathione. COX-1 and -2 were hardly affected by the triterpene acids (IC50 > 10 ?M). Given the crucial role of mPGES-1 in inflammation and the abundance of highly active triterpene acids in frankincence extracts, our findings provide further evidence of the anti-inflammatory potential of frankincense preparations and reveal novel, potent bioactivities of tirucallic acids, roburic acids, and lupeolic acids. PMID:24844534

2014-01-01

244

Tetra- and pentacyclic triterpene acids from the ancient anti-inflammatory remedy frankincense as inhibitors of microsomal prostaglandin E(2) synthase-1.  

PubMed

The microsomal prostaglandin E2 synthase (mPGES)-1 is the terminal enzyme in the biosynthesis of prostaglandin (PG)E2 from cyclooxygenase (COX)-derived PGH2. We previously found that mPGES-1 is inhibited by boswellic acids (IC50 = 3-30 ?M), which are bioactive triterpene acids present in the anti-inflammatory remedy frankincense. Here we show that besides boswellic acids, additional known triterpene acids (i.e., tircuallic, lupeolic, and roburic acids) isolated from frankincense suppress mPGES-1 with increased potencies. In particular, 3?-acetoxy-8,24-dienetirucallic acid (6) and 3?-acetoxy-7,24-dienetirucallic acid (10) inhibited mPGES-1 activity in a cell-free assay with IC50 = 0.4 ?M, each. Structure-activity relationship studies and docking simulations revealed concrete structure-related interactions with mPGES-1 and its cosubstrate glutathione. COX-1 and -2 were hardly affected by the triterpene acids (IC50 > 10 ?M). Given the crucial role of mPGES-1 in inflammation and the abundance of highly active triterpene acids in frankincence extracts, our findings provide further evidence of the anti-inflammatory potential of frankincense preparations and reveal novel, potent bioactivities of tirucallic acids, roburic acids, and lupeolic acids. PMID:24844534

Verhoff, Moritz; Seitz, Stefanie; Paul, Michael; Noha, Stefan M; Jauch, Johann; Schuster, Daniela; Werz, Oliver

2014-06-27

245

Salmonella Biofilm Formation on Aspergillus niger Involves Cellulose - Chitin Interactions  

PubMed Central

Salmonella cycles between host and nonhost environments, where it can become an active member of complex microbial communities. The role of fungi in the environmental adaptation of enteric pathogens remains relatively unexplored. We have discovered that S. enterica Typhimurium rapidly attaches to and forms biofilms on the hyphae of the common fungus, Aspergillus niger. Several Salmonella enterica serovars displayed a similar interaction, whereas other bacterial species were unable to bind to the fungus. Bacterial attachment to chitin, a major constituent of fungal cell walls, mirrored this specificity. Pre-incubation of S. Typhimurium with N-acetylglucosamine, the monomeric component of chitin, reduced binding to chitin beads by as much as 727-fold and inhibited attachment to A. niger hyphae considerably. A cellulose-deficient mutant of S. Typhimurium failed to attach to chitin beads and to the fungus. Complementation of this mutant with the cellulose operon restored binding to chitin beads to 79% of that of the parental strain and allowed for attachment and biofilm formation on A. niger, indicating that cellulose is involved in bacterial attachment to the fungus via the chitin component of its cell wall. In contrast to cellulose, S. Typhimurium curli fimbriae were not required for attachment and biofilm development on the hyphae but were critical for its stability. Our results suggest that cellulose–chitin interactions are required for the production of mixed Salmonella-A. niger biofilms, and support the hypothesis that encounters with chitinaceous alternate hosts may contribute to the ecological success of human pathogens. PMID:22003399

Brandl, Maria T.; Carter, Michelle Q.; Parker, Craig T.; Chapman, Matthew R.; Huynh, Steven; Zhou, Yaguang

2011-01-01

246

Timing of administration of dexamethasone or the nitric oxide synthase inhibitor, nitro-L-arginine methyl ester, is critical for effective treatment of ischaemia-reperfusion injury to rat skeletal muscle.  

PubMed

1. The effects of the nitric oxide synthase (NOS) inhibitors, NG-nitro-L-arginine-methyl ester (L-NAME), nitroiminoethyl-L-ornithine and S. methylisothiourea on skeletal muscle survival following 2 h of tourniquet ischaemia and 24 h of reperfusion were compared with those of the anti-inflammatory steroid, dexamethasone. 2. Administration of each of the NOS inhibitors or dexamethasone 30 min before reperfusion reduced the degree of skeletal muscle necrosis 24 h after reperfusion. 3. The influence of timing of drug administration was investigated. L-NAME administered 30 min before reperfusion, at 3 h after reperfusion, but not thereafter, significantly improved muscle survival compared with saline-treated controls. Dexamethasone administered 30 min before, or at 3 or 8 h after reperfusion, but not at 16 h, significantly improved muscle survival, but neither agent had protective effects when administered before ischaemia. 4. After 8 h of reperfusion of ischaemic skeletal muscle, cell-free homogenates contained Ca(2+)-independent (inducible) NOS activity which was reduced in dexamethasone-treated (2.5 mg/kg) rats. Furthermore, inducible NOS mRNA levels, as detected by reverse transcriptase-PCR, were increased after 8 h of reperfusion in saline, but not in dexamethasone-treated rats. 5. These data suggest a significant deleterious effect of endogenous NO which may be restricted to the first 3 h of the reperfusion phase of ischaemia-reperfusion injury, and raise the possibility of effective treatment of incipient reperfusion injury, even after several hours of reperfusion. PMID:9301432

Zhang, B; Knight, K R; Dowsing, B; Guida, E; Phan, L H; Hickey, M J; Morrison, W A; Stewart, A G

1997-08-01

247

Nature of isomerism of solid isothiourea salts, inhibitors of nitric oxide synthases, as studied by 1H-14N nuclear quadrupole double resonance, X-ray, and density functional theory/quantum theory of atoms in molecules.  

PubMed

Isothioureas, inhibitors of nitric oxide synthases, have been studied experimentally in solid state by nuclear quadrupole double resonance (NQDR) and X-ray methods and theoretically by the quantum theory of atoms in molecules/density functional theory. Resonance frequencies on (14)N have been detected and assigned to particular nitrogen sites in each molecule. The crystal packings of (S)-3,4-dichlorobenzyl-N-methylisothiouronium chloride with the disordered chlorine positions in benzene ring and (S)-butyloisothiouronium bromide have been resolved in X-ray diffraction studies. (14)N NQDR spectra have been found good indicators of isomer type and strength of intra- or intermolecular N-H···X (X = Cl, Br) interactions. From among all salts studied, only for (S)-2,3,4,5,6-pentabromobenzylisothiouronium chloride are both nitrogen sites equivalent, which has been explained by the slow exchange. This unique structural feature can be a key factor in the high biological activity of (S)-2,3,4,5,6-pentabromobenzylisothiouronium salts. PMID:22283980

Latosi?ska, J N; Latosi?ska, M; Seliger, J; Žagar, V; Maurin, J K; Kazimierczuk, Z

2012-02-01

248

Design, synthesis, biological evaluation and X-ray crystal structure of novel classical 6,5,6-tricyclic benzo[4,5]thieno[2,3-d]pyrimidines as dual thymidylate synthase and dihydrofolate reductase inhibitors  

PubMed Central

Classical antifolates (4-7) with a tricyclic benzo[4,5]thieno[2,3-d]pyrimidine scaffold and a flexible and rigid benzoylglutamate were synthesized as dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors. Oxidative aromatization of ethyl 2-amino-4-methyl-4,5,6,7-tetrahydro-1-benzothiophene-3-carboxylate (±)-9 to ethyl 2-amino-4-methyl-1-benzothiophene-3-carboxylate 10 with 10% Pd/C was a key synthetic step. Compounds with 2-CH3 substituents inhibited human (h) TS (IC50 = 0.26-0.8 ?M), but not hDHFR. Substitution of the 2-CH3 with a 2-NH2 increases hTS inhibition by more than 10-fold and also affords excellent hDHFR inhibition (IC50 = 0.09-0.1 ?M). This study shows that the tricyclic benzo[4,5]thieno[2,3-d]pyrimidine scaffold is highly conducive to single hTS or dual hTS-hDHFR inhibition depending on the 2-position substituents. The X-ray crystal structures of 6 and 7 with hDHFR reveal, for the first time, that tricyclics 6 and 7 bind with the benzo[4,5]thieno[2,3-d]pyrimidine ring in the folate binding mode with the thieno S mimicking the 4-amino of methotrexate. PMID:21550809

Zhang, Xin; Zhou, Xilin; L.Kisliuk, Roy; Piraino, Jennifer; Cody, Vivian

2011-01-01

249

Molecular modeling studies and synthesis of novel methyl 2-(2-(4-oxo-3-aryl-3,4-dihydroquinazolin-2-ylthio)acetamido)alkanoates with potential anti-cancer activity as inhibitors for methionine synthase.  

PubMed

Cobalamin-dependant cytosolic enzyme methionine synthase (MetS) catalyses the transfer of a methyl group from the methyltetrahydrofolate (MTHF) to homocysteine (Hcy) to produce methionine and tetrahydrofolate (THF). MetS is over-expressed in the cytosol of certain breast and prostate tumour cells. Methionine used as a source of one carbon atom for the building of the DNA of the tumour cells, structural protein and enzymes. In this study, we designed, synthesized and evaluated the cytotoxic activity of a series of substituted methyl 2-(2-(4-oxo-3-aryl-3,4-dihydroquinazolin-2-ylthio)acetamido)acetate and dipeptide that mimic the substructure of MTHF. These inhibitors were docked in to the MTHF binding domain in such the same way as MTHF in its binding domain. The free energies of the binding were calculated and compared to the IC50 values. This series has been developed by dicyclohexylcarbodiimide (DCC) and azide coupling methods of amino acid esters with carboxylic acid derivatives, respectively. Compound methyl 3-hydroxy-2-(2-(3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-ylthio)acetamido)propanoate exhibited the highest IC50 value 20?µg/mL against PC-3 cell line and scored the lowest free energy of the binding (-207.19?kJ/mol). PMID:24990505

Elfekki, Ismail Mahmoud; Hassan, Walid Fathalla Mohamed; Elshihawy, Hosam Eldin Abd Elhamed; Ali, Ibrahim Ahmed Ibrahim; Eltamany, Elsayed Hussein Mostafa

2014-01-01

250

4-Hydroxy-3-(naphthalen-1-ylmethyl)thiophen-2(5H)-one as inhibitors of tyrosyl-tRNA synthase: Synthesis, molecular docking and antibacterial evaluation  

NASA Astrophysics Data System (ADS)

A series of novel 4-hydroxy-3-(naphthalen-1-ylmethyl)thiophen-2(5H)-ones as tyrosyl-tRNA synthetase inhibitors were synthesized. Of these compounds, 4-(naphthalen-1-ylmethyl)-5-oxo-2,5-dihydrothiophen-3-yl-2-(4-hydroxyphenyl)acetate (29) was the most potent. The binding model and structure-activity relationship indicate that replacement of phenyl acetate in the side chain of 29 with a substituent containing more hydrophilic groups would be more suitable for further modification. Antibacterial assay revealed that the synthetic compounds are effective against growth of Gram-positive organisms, and 29 is the most potent agent against Staphylococcus aureus ATCC 25923 with MIC50 value of 0.21 ?g/mL.

Sun, Juan; Liu, Jia-Jia; Zhou, Wei; Guo, Feng-Jiao; Wang, Xin-Yi; Zhu, Hai-Liang

2014-01-01

251

Effect of Thymidylate Synthase Inhibitors on dUTP and TTP Pool Levels and the Activities of DNA Repair Glycosylases on Uracil and 5-Fluorouracil in DNA‡  

PubMed Central

5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-dUrd) and raltitrixed (RTX), are anticancer agents that target thymidylate synthase (TS) thereby blocking the conversion of dUMP into dTMP. In budding yeast, 5-FU promotes a large increase in the dUMP/dTMP ratio leading to massive polymerase-catalyzed incorporation of uracil (U) into genomic DNA, and to a lesser extent 5-FU, which are both excised by yeast uracil DNA glycosylase (UNG) leading to DNA fragmentation and cell death. In contrast, the toxicity of 5-FU and RTX in human and mouse cell lines does not involve UNG, but instead, other DNA glycosylases that can excise uracil derivatives. To elucidate the basis for these divergent findings in yeast and human cells, we have investigated the how these drugs perturb cellular dUTP and TTP pool levels and the relative abilities of three human DNA glycosylases (hUNG2, hSMUG1 and hTDG) to excise various TS drug-induced lesions in DNA. We found that 5-dUrd only modestly increases the dUTP/dTTP pool levels in asynchronous MEF, HeLa, and HT-29 human cell lines when growth is in standard culture media. In contrast, treatment of chicken DT40 B cells with 5-dUrd or RTX resulted in large increases in the dUTP/TTP ratio. Surprisingly, even though UNG is the only DNA glycosylase in DT40 cells that can act on U/A base pairs derived from dUTP incorporation, an isogenic ung?/? DT40 cell line showed little change its sensitivity to RTX as compared to control cells. In vitro kinetic analyses of the purified human enzymes show that hUNG2 is the most powerful catalyst for excision of 5-FU and U regardless of whether it is found in base pairs with A, G or present in ssDNA. Fully consistent with the in vitro activity assays, nuclear extracts isolated from human and chicken cell cultures show that hUNG2 is the overwhelming activity for removal of both U and 5-FU, despite its bystander status with respect to drug toxicity in these cell lines. The diverse outcomes of TS inhibition with respect to nucleotide pool levels, nature of the resulting DNA lesion, and the DNA repair response are discussed. PMID:21222484

Grogan, Breeana C.; Parker, Jared B.; Guminski, Amy F.; Stivers, James T.

2011-01-01

252

Chitin and Chitosan: Functional Biopolymers from Marine Crustaceans  

Microsoft Academic Search

Chitin and chitosan, typical marine polysaccharides as well as abundant biomass resources, are attracting a great deal of\\u000a attention because of their distinctive biological and physicochemical characteristics. To fully explore the high potential\\u000a of these specialty biopolymers, basic and application researches are being made extensively. This review deals with the fundamental\\u000a aspects of chitin and chitosan such as the preparation

Keisuke Kurita

2006-01-01

253

The Importance of Chitin in the Marine Environment  

Microsoft Academic Search

Chitin is the most abundant renewable polymer in the oceans and is an important source of carbon and nitrogen for marine organisms.\\u000a The process of chitin degradation is a key step in the cycling of nutrients in the oceans and chitinolytic bacteria play a\\u000a significant role in this process. These bacteria are autochthonous to both marine and freshwater ecosystems and

Claudiana P. Souza; Bianca C. Almeida; Rita R. Colwell; Irma N. G. Rivera

254

Comparison of extraction methods of chitin from Ganoderma lucidum mushroom obtained in submerged culture.  

PubMed

The chitin was isolated from the Ganoderma lucidum submerged cultures mycelium as potential source of chitin under biotechnological processes. The extraction of chitin was carried out through 5 different assays which involved mainly three phases: pulverization of the mushroom, deproteinization of the mycelia with NaOH solution, and a process of decolorization with potassium permanganate and oxalic acid. The chitin contents extracted from 9-day mycelia were 413, 339, 87, 78, and 144 mg/g(-1) (milligrams of chitin/grams of dry biomass) for A1, A2, A3, A4, and A5, respectively. Obtained chitin was characterized by X-Ray Diffraction (XRD), by Fourier transform infrared spectroscopy (FTIR), and by thermal analysis (TGA). The results showed that Ganoderma lucidum chitin has similar characteristic of chitin from different fonts. The advantage of the biotechnological processes and the fact that Ganoderma lucidum fungus may be used as a potential raw material for chitin production were demonstrated. PMID:24551839

Ospina Álvarez, Sandra Patricia; Ramírez Cadavid, David Alexander; Escobar Sierra, Diana Marcela; Ossa Orozco, Claudia Patricia; Rojas Vahos, Diego Fernando; Zapata Ocampo, Paola; Atehortúa, Lucía

2014-01-01

255

Comparison of Extraction Methods of Chitin from Ganoderma lucidum Mushroom Obtained in Submerged Culture  

PubMed Central

The chitin was isolated from the Ganoderma lucidum submerged cultures mycelium as potential source of chitin under biotechnological processes. The extraction of chitin was carried out through 5 different assays which involved mainly three phases: pulverization of the mushroom, deproteinization of the mycelia with NaOH solution, and a process of decolorization with potassium permanganate and oxalic acid. The chitin contents extracted from 9-day mycelia were 413, 339, 87, 78, and 144?mg/g?1 (milligrams of chitin/grams of dry biomass) for A1, A2, A3, A4, and A5, respectively. Obtained chitin was characterized by X-Ray Diffraction (XRD), by Fourier transform infrared spectroscopy (FTIR), and by thermal analysis (TGA). The results showed that Ganoderma lucidum chitin has similar characteristic of chitin from different fonts. The advantage of the biotechnological processes and the fact that Ganoderma lucidum fungus may be used as a potential raw material for chitin production were demonstrated. PMID:24551839

Ospina Alvarez, Sandra Patricia; Ramirez Cadavid, David Alexander; Ossa Orozco, Claudia Patricia; Zapata Ocampo, Paola; Atehortua, Lucia

2014-01-01

256

Bacterial Chitin Hydrolysis in Two Lakes with Contrasting Trophic Statuses  

PubMed Central

Chitin, which is a biopolymer of the amino sugar glucosamine (GlcN), is highly abundant in aquatic ecosystems, and its degradation is assigned a key role in the recycling of carbon and nitrogen. In order to study the significance of chitin decomposition in two temperate freshwater lakes with contrasting trophic and redox conditions, we measured the turnover rate of the chitin analog methylumbelliferyl-N,N?-diacetylchitobioside (MUF-DC) and the presence of chitinase (chiA) genes in zooplankton, water, and sediment samples. In contrast to the eutrophic and partially anoxic lake, chiA gene fragments were detectable throughout the oligotrophic water column and chiA copy numbers per ml of water were up to 15 times higher than in the eutrophic waters. For both lakes, the highest chiA abundance was found in the euphotic zone—the main habitat of zooplankton, but also the site of production of easily degradable algal chitin. The bulk of chitinase activity was measured in zooplankton samples and the sediments, where recalcitrant chitin is deposited. Both, chiA abundance and chitinase activity correlated well with organic carbon, nitrogen, and concentrations of particulate GlcN. Our findings show that chitin, although its overall contribution to the total organic carbon is small (?0.01 to 0.1%), constitutes an important microbial growth substrate in these temperate freshwater lakes, particularly where other easily degradable carbon sources are scarce. PMID:22101058

Carstens, Dorte; Keller, Esther; Vazquez, Francisco; Schubert, Carsten J.; Zeyer, Josef; Burgmann, Helmut

2012-01-01

257

Chitin membranes containing silver nanoparticles for wound dressing application.  

PubMed

Silver nanoparticles are gaining importance as an antimicrobial agent in wound dressings. Chitin is a biopolymer envisioned to promote rapid dermal regeneration and accelerate wound healing. This study was focused on the evaluation of chitin membranes containing silver nanoparticles for use as an antimicrobial wound dressing. Silver nanoparticles were synthesised by gamma irradiation at doses of 50 kGy in the presence of sodium alginate as stabiliser. The UV-Vis absorption spectra of nanoparticles exhibited an absorption band at 415-420 nm, which is the typical plasmon resonance band of silver nanoparticles. The peaks in the X-ray diffraction (XRD) pattern are in agreement with the standard values of the face-centred cubic silver. Transmission electron microscopy (TEM) images indicate silver nanoparticles with spherical morphology and small particle size in the range of 3-13 nm. In vitro antimicrobial tests were performed using Pseudomonas aeruginosa and Staphylococcus aureus to determine the antimicrobial efficiency of the chitin membranes containing 30, 50, 70 and 100 ppm nanosilver. No viable counts for P. aeruginosa were detected with 70 ppm silver nanoparticles dressing after 1-hour exposure. A 2-log reduction in viable cell count was observed for S. aureus after 1 hour and a 4-log reduction after 6 hours with 100 ppm nanosilver chitin membranes. This study demonstrates the antimicrobial capability of chitin membranes containing silver nanoparticles. The chitin membranes with 100 ppm nanosilver showed promising antimicrobial activity against common wound pathogens. PMID:22958740

Singh, Rita; Singh, Durgeshwer

2014-06-01

258

The Non-catalytic Chitin-binding Protein CBP21 from Serratia marcescens Is Essential for Chitin Degradation  

Microsoft Academic Search

The Gram-negative soil bacterium Serratia marc- escens uses three different family 18 chitinases to de- grade chitin, an abundant insoluble carbohydrate polymer composed of (1,4)-linked units of N-acetylglu- cosamine. We show that efficient chitin degradation additionally depends on the action of a small non- catalytic protein, CBP21, which binds to the insoluble crystalline substrate, leading to structural changes in the

G. Vaaje-Kolstad

2005-01-01

259

Characterization of pyrazofurin-resistant HeLa cells with amplification of UMP synthase gene  

Microsoft Academic Search

Three different phenotypes have been characterized in HeLa cells that have been selected for resistance to pyrazofurin, a potent inhibitor of the de novo pyrimidine biosynthetic enzyme UMP synthase. All of the resistant cell lines had a coordinate increase in UMP synthase activity, UMP synthase-specific mRNA, and UMP synthase gene sequences. In one of the resistant cell lines, the amplification

John J. Kanalas; John J. Hutton; D. Parker Suttle

1985-01-01

260

Structural differences between chitin and chitosan extracted from three different marine sources.  

PubMed

Three marine sources of chitin from Tunisia were investigated. Structural differences between ?-chitin from shrimp (Penaeus kerathurus) waste, crab (Carcinus mediterraneus) shells, and ?-chitin from cuttlefish (Sepia officinalis) bones were studied by the (13)C NMR, FTIR, and XRD diffractograms. The (13)C NMR analysis showed a splitting of the C3 and C5 carbon signals for ?-chitin, while that of ?-chitin was merged into a single resonance. The bands contour of deconvoluted and curve-fit FTIR spectra showed a more detailed structure of ?-chitin in the region of O-H, N-H and CO stretching regions. IR and (13)C NMR were used to determine the chitin degree of acetylation (DA). XRD analysis indicated that ?-chitins were more crystalline polymorph than ?-chitin. Shrimp chitin was obtained with a good yield (20% on raw material dry weight) and no residual protein and salts. Chitosans, with a DA lower than 20% and relatively low molecular masses were prepared from the wet chitins in the same experimental conditions. They were perfectly soluble in acidic medium. Nevertheless, chitin and chitosan characteristics were depending upon the chitin source. PMID:24468048

Hajji, Sawssen; Younes, Islem; Ghorbel-Bellaaj, Olfa; Hajji, Rachid; Rinaudo, Marguerite; Nasri, Moncef; Jellouli, Kemel

2014-04-01

261

The adsorption of copper to chitin in seawater  

NASA Astrophysics Data System (ADS)

The interaction of metal ions with particulate matter is important in limiting their concentration in seawater. Chitin is one of the constituents of the natural particulate organic matter than can interact with metal ions and therefore may serve as a reasonable model for natural organic solids. The adsorption of Cu 2+ on the chitin surface has been studied in seawater as a function of pH, temperature, and salinity. The amphoteric properties of the surface of chitin were characterized in 0.7 M NaCl in terms of a two-protic acid-base system ( pHPZC = 5.4) with acidity constants p ?K a1s = 4.4 ± 0.2 and p ?K a2s = 6.4 ± 0.2 . The maximum proton exchange capacity of chitin was found to be 2.3 ± 0.3 mol kg -1, broadly similar to other solids. The rates of the adsorption were quite rapid ( t 1/2 = 8 min ) and not strongly affected by the presence of other metals such as Cd 2+ and Pb 2+. The adsorption equilibrium data have been found to correlate well with surface complex-formation equilibria or the mathematically equivalent Langmuir- type adsorption equilibria. The value for the stability constant of Cu 2+ on chitin was found to be log ?K Hs = 8.95 ± 0.01 , and the complexing capacity of chitin was found to be 6.9 and 5.9 ?mol g -1, respectively, in the absence and in the presence of Cd 2+ and Pb 2+. An increase in the salinity and a decrease in the temperature result in greater adsorption of Cu 2+ to chitin

Gonzalez-Davila, Melchor; Millero, Frank J.

1990-03-01

262

Chitosan-sheath and chitin-core nanowhiskers.  

PubMed

Chitosan-sheath and ?-chitin-core nanowhiskers (CsNWs) have been successfully generated by surface deacetylation of chitin nanowhiskers (CtNWs) in the never-dried state. Acid hydrolysis (3N HCl, 30 mL/g, 104°C) of pure chitin derived from crab shell yielded 65% 4-10nm thick, 16 nm wide and 214 nm long chitin whiskers (CtNWs) that were 86% crystalline and 81% acetylated. Surface deacetylation of CtNWs was robust in their never-dried state in 50% NaOH at a moderate 50°C for 6h, yielding 92% CsNWs. All deacetylated CsNWs retain the same ?-chitin crystalline core at reduced 50% crystallinity and similar dimensions (4-12 nm thick, 15 nm wide, 247 long) as CtNWs, but reduced 60% acetylation reflecting the deacetylated surface layers. Progressive surface deacetylation was evident by the increased IP as well as increased positive charges under acidic pH and reduced negative charges at alkaline pH with increasing reaction time. PMID:24702931

Pereira, Antonio G B; Muniz, Edvani C; Hsieh, You-Lo

2014-07-17

263

Chitin in the Silk Gland Ducts of the Spider Nephila edulis and the Silkworm Bombyx mori  

PubMed Central

Here we report the detection and localisation of chitin in the cuticle of the spinning ducts of both the spider Nephila edulis and the silkworm Bombyx mori. Our observations demonstrate that the duct walls of both animals contain chitin notwithstanding totally independent evolutionary pathways of the systems. We conclude that chitin may well be an essential component for the construction of spinning ducts; we further conclude that in both species chitin may indicate the evolutionary origin of the spinning ducts. PMID:24015298

Davies, Gwilym J. G.; Knight, David P.; Vollrath, Fritz

2013-01-01

264

EGESTION OF CHITIN IN PELLETS OF AMERICAN KESTRELS AND EASTERN SCREECH OWLS  

Microsoft Academic Search

of the total amount of ingested chitin was found in excreta. The percent of chitin egested as pellets as compared to the amount ingested showed a negative correlation (r = -0.76, P (0.001). Our results suggest that the lower gastrointestinal tract contributes to total chitin digestion in American Kestrels.

CHIKAKO AKAKI; GARY E. DUI

265

Ultrasonication and steam-explosion as chitin pretreatments for chitin oligosaccharide production by chitinases of Lecanicillium lecanii.  

PubMed

In this study, chitin oligosaccharides have been successfully produced using chitinases from submerged fermentation of Lecanicillium lecanii. The highest Hex, Chit and Prot production was 0.14, 0.26 and 2.05 U/mg of protein, respectively, which were attained varying pH from 5 to 8 after 96 h. Culture conditions conducted at constant pH of 6 resulted in significantly lower enzyme production. The crude enzyme was partially purified by salting out with (NH4)2SO4 followed by size exclusion chromatography to isolate the chitinase mixture for further chitin hydrolysis assays. In this regard, chitin substrates were pretreated with sonication and steam explosion prior to enzymatic reaction. Structural changes were observed with steam explosion with 11.28% reduction of the crystallinity index attained with the lowest chitin/water ratio (0.1g/mL). Pretreated chitins reached the highest production of reducing sugars (0.37 mg/mL) and GlcNAc (0.59 mg/mL) in 23.6% yield. PMID:23993287

Villa-Lerma, Guadalupe; González-Márquez, Humberto; Gimeno, Miquel; López-Luna, Alberto; Bárzana, Eduardo; Shirai, Keiko

2013-10-01

266

Chitin nanocrystals for pickering high internal phase emulsions.  

PubMed

Chitin is a natural polymer of glucosamine bearing N-acetyl groups. Chitin nanocrystals (ChiNCs), resulting from the acid hydrolysis of amorphous regions of chitin, are crystalline positively charged rod-like particles. ChiNCs show some interfacial properties and very efficiently stabilize oil/water interfaces, leading to the so-called Pickering emulsions. In accordance with the irreversible adsorption of particles, these Pickering emulsions proved stable over time, with constant emulsion volume for several months, even though natural creaming may occur. The emulsions produced are not clearly susceptible to ionic strength or pH in terms of average droplet diameter. However, when mixed with a large amount of oil, high internal phase emulsions (HIPE) containing up to 96% of internal phase are formed as a gel with a texture that can be modulated from soft to solid gel by adjusting concentration, pH, and ionic strength. PMID:25180643

Perrin, Emilie; Bizot, Hervé; Cathala, Bernard; Capron, Isabelle

2014-10-13

267

Structure and inhibition of tuberculosinol synthase and decaprenyl diphosphate synthase from Mycobacterium tuberculosis.  

PubMed

We have obtained the structure of the bacterial diterpene synthase, tuberculosinol/iso-tuberculosinol synthase (Rv3378c) from Mycobacterium tuberculosis , a target for anti-infective therapies that block virulence factor formation. This phosphatase adopts the same fold as found in the Z- or cis-prenyltransferases. We also obtained structures containing the tuberculosinyl diphosphate substrate together with one bisphosphonate inhibitor-bound structure. These structures together with the results of site-directed mutagenesis suggest an unusual mechanism of action involving two Tyr residues. Given the similarity in local and global structure between Rv3378c and the M. tuberculosis cis-decaprenyl diphosphate synthase (DPPS; Rv2361c), the possibility exists for the development of inhibitors that target not only virulence but also cell wall biosynthesis, based in part on the structures reported here. PMID:24475925

Chan, Hsiu-Chien; Feng, Xinxin; Ko, Tzu-Ping; Huang, Chun-Hsiang; Hu, Yumei; Zheng, Yingying; Bogue, Shannon; Nakano, Chiaki; Hoshino, Tsutomu; Zhang, Lilan; Lv, Pin; Liu, Wenting; Crick, Dean C; Liang, Po-Huang; Wang, Andrew H-J; Oldfield, Eric; Guo, Rey-Ting

2014-02-19

268

Glycan-functionalized fluorescent chitin nanocrystals for biorecognition applications.  

PubMed

A new platform based on chitin nanocrystals has been developed for biorecognition applications. TEMPO-oxidized chitin nanocrystals (TCNs) were labeled with a fluorescent imidazoisoquinolinone dye, and simultaneously conjugated with carbohydrate ligands, resulting in dually functionalized TCNs. The biorecognition properties of the nanocrystals were probed with lectins and bacteria, resulting in selective interactions with their corresponding cognate carbohydrate-binding proteins, as visualized by optical, fluorescence, STEM, and TEM imaging. This represents a new approach to multifunctional nanomaterials based on naturally occurring polymers, holding high potential for biomedical applications. PMID:24625204

Zhou, Juan; Butchosa, Núria; Jayawardena, H Surangi N; Zhou, Qi; Yan, Mingdi; Ramström, Olof

2014-04-16

269

INHIBITOR STUDIES ON MYCOBACTERIUM TUBERCULOSIS MALATE SYNTHASE  

E-print Network

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis (Mtb) has intensified efforts to discover novel drugs for tuberculosis (TB) treatment. Targeting the persistent state of Mtb, a condition in which Mtb is resistant...

Owen, Joshua

2008-08-03

270

Preparation of chitin-silica composites by in vitro silicification of two-dimensional Ianthella basta demosponge chitinous scaffolds under modified Stöber conditions.  

PubMed

Chitin is a biopolymer found in cell walls of various fungi and skeletal structures of numerous invertebrates. The occurrence of chitin within calcium- and silica-containing biominerals has inspired development of chitin-based hybrids and composites in vitro with specific physico-chemical and material properties. We show here for the first time that the two-dimensional ?-chitin scaffolds isolated from the skeletons of marine demosponge Ianthella basta can be effectively silicified by the two-step method with the use of Stöber silica micro- and nanodispersions under Extreme Biomimetic conditions. The chitin-silica composites obtained at 120 °C were characterized by the presence of spherical SiO2 particles homogeneously distributed over the chitin fibers, which probably follows from the compatibility of Si-OH groups to the hydroxyl groups of chitin. The biocomposites obtained were characterized by various analytical techniques such as energy dispersive spectrometry, scanning electron microscopy, thermogravimetric/differential thermal analyses as well as X-ray photoelectron spectroscopy, Fourier transform infrared and Raman spectroscopy to determine possible interactions between silica and chitin molecule. The results presented proved that the character and course of the in vitro chitin silicification in Stöber dispersions depended considerably on the degree of hydrolysis of the SiO2 precursor. PMID:23910299

Wysokowski, Marcin; Behm, Thomas; Born, René; Bazhenov, Vasilii V; Meissner, Heike; Richter, Gert; Szwarc-Rzepka, Karolina; Makarova, Anna; Vyalikh, Denis; Schupp, Peter; Jesionowski, Teofil; Ehrlich, Hermann

2013-10-01

271

Dynamics of Gram-negative bacteria population density in a soil in the course of the succession initiated by chitin and cellulose  

NASA Astrophysics Data System (ADS)

The functions of actinomycetes in polymer destruction in soil traditionally considered as the dominant, compare to another groups of bacteria. Gram-positive bacteria also have ecological functions in destruction of soil organic matter. The role of Gram-negative bacteria has been researched in the microbial succession in terms of polymers destruction, which are widely spreads in soils: chitin and cellulose. The method with nalidixic acid as an inhibitor of DNA division of Gram-negative bacteria was modified. By modified method microbial succession of Gram-negative bacteria in the different horizons of a chernozem under aerobic and anaerobic conditions was researched. Chitin and cellulose as the source of nutrients with moistening was used in experiments. The introduction of chitin had no positive effect on the population density of Gram-negative bacteria in a chernozem, but it advanced the date of their appearance in microbial succession: the maximum of Gram-negative bacteria population density was registered on the 3rd- 7th day of the experiment with adding chitin. Compare to the control, which one was without any nutrient adding this dynamics registered much earlier. Consequently, the introduction of chitin as an additional source of nutrition promoted revealing of the Gram-negative bacteria in soil already at the early stages of the succession. In the course of the succession, when the fungal mycelium begins to die off, the actinomycetic mycelium increases in length, i.e., Gram-negative bacteria are replaced at this stage with Gram-positive ones, the leading role among which belongs to actinomycetes. The growth rate of Gram-negative bacteria is higher than that of actinomycetes, so they start chitin utilization at the early stages of the succession, whereas actinomycetes dominate at the late stages. The population density of Gram-negative bacteria was lower under the anaerobic conditions as compared with that in the aerobic ones. The population density of Gram-negative bacteria in the lower layer of the A horizon of the chernozem and in the B horizon was slightly higher only in the case of the chitin introduction. When cellulose was introduced into the soil under aerobic conditions, the population density of Gram-negative bacteria in all the layers of the A horizon of the chernozem was maximal from the 14th to the 22nd day of the experiment. Simultaneously, an increase in the length of the actinomycetal mycelium was observed, as these organisms also perform cellulose hydrolysis in soils. The Gram-negative bacteria began to develop at the stage of the fungal mycelium destruction, which indirectly confirmed the chitinolytic activity of these bacteria.

Konstantin, Ivanov; Lubov, Polyanskaya

2014-05-01

272

Development and Binding Mode Assessment of N-[4-[2-propyn-1-yl[(6S)-4,6,7,8-tetrahydro-2-(hydroxymethyl)-4-oxo-3H-cyclopenta[g]quinazolin-6-yl]amino]benzoyl]-L-?-glutamyl-D-glutamic acid (BGC 945), a Novel Thymidylate Synthase Inhibitor that Targets Tumor Cells  

PubMed Central

N-[4-[2-propyn-1-yl[(6S)-4,6,7,8-tetrahydro-2-(hydroxymethyl)-4-oxo-3H-cyclopenta[g]quinazolin-6-yl]amino]benzoyl]-L-?-glutamyl-D-glutamic acid 1 (BGC 945, now known as ONX 0801), is a small molecule thymidylate synthase (TS) inhibitor discovered at the Institute of Cancer Research in London. It is licensed by Onyx Pharmaceuticals and is in Phase 1 clinical studies. It is a novel antifolate drug resembling TS inhibitors plevitrexed and raltitrexed that combines enzymatic inhibition of thymidylate synthase with ?-folate receptor-mediated targeting of tumor cells. Thus, it has potential for efficacy with lower toxicity due to selective intracellular accumulation through ?-folate receptor (?-FR) transport. The ?-FR, a cell-surface receptor glycoprotein, which is over expressed mainly in ovarian and lung cancer tumors, has an affinity for 1 similar to that for its natural ligand, folic acid. This study describes a novel synthesis of 1, an X-ray crystal structure of its complex with Escherichia coli TS and 2’-deoxyuridine-5’-monophosphate, and a model for a similar complex with human TS. PMID:23710599

Tochowicz, Anna; Dalziel, Sean; Eidam, Oliv; O'Connell, Joseph D.; Griner, Sarah; Finer-Moore, Janet S.; Stroud, Robert M.

2013-01-01

273

Peptide induced crystallization of calcium carbonate on wrinkle patterned substrate: implications for chitin formation in molluscs.  

PubMed

We here present the nucleation and growth of calcium carbonate under the influence of synthetic peptides on topographically patterned poly(dimethylsiloxane) (PDMS) substrates, which have a controlled density of defects between the wrinkles. Experiments with two lysine-rich peptides derived from the extracellular conserved domain E22 of the mollusc chitin synthase Ar-CS1, AKKKKKAS (AS8) and EEKKKKKES (ES9) on these substrates showed their influence on the calcium carbonate morphology. A transition from polycrystalline composites to single crystalline phases was achieved with the peptide AS8 by changing the pH of the buffer solution. We analyzed three different pH values as previous experiments showed that E22 interacts with aragonite biominerals more strongly at pH 7.75 than at pH 9.0. At any given pH, crystals appeared in characteristic morphologies only on wrinkled substrates, and did not occur on the flat, wrinkle-free PDMS substrate. These results suggest that these wrinkled substrates could be useful for controlling the morphologies of other mineral/peptide and mineral/protein composites. In nature, these templates are formed enzymatically by glycosyltransferases containing pH-sensitive epitopes, similar to the peptides investigated here. Our in vitro test systems may be useful to gain understanding of the formation of distinct 3D morphologies in mollusc shells in response to local pH shifts during the mineralization of organic templates. PMID:23736692

Ghatak, Anindita Sengupta; Koch, Marcus; Guth, Christina; Weiss, Ingrid M

2013-01-01

274

Nikkomycin Z is an effective inhibitor of the chytrid fungus linked to global amphibian declines.  

PubMed

Fungal infections in humans, wildlife, and plants are a growing concern because of their devastating effects on human and ecosystem health. In recent years, populations of many amphibian species have declined, and some have become extinct due to chytridiomycosis caused by the fungal pathogen Batrachochytrium dendrobatidis. For some endangered amphibian species, captive colonies are the best intermediate solution towards eventual reintroduction, and effective antifungal treatments are needed to cure chytridiomycosis and limit the spread of this pathogen in such survival assurance colonies. Currently, the best accepted treatment for infected amphibians is itraconazole, but its toxic side effects reduce its usefulness for many species. Safer antifungal treatments are needed for disease control. Here, we show that nikkomycin Z, a chitin synthase inhibitor, dramatically alters the cell wall stability of B. dendrobatidis cells and completely inhibits growth of B. dendrobatidis at 250 ?M. Low doses of nikkomycin Z enhanced the effectiveness of natural antimicrobial skin peptide mixtures tested in vitro. These studies suggest that nikkomycin Z would be an effective treatment to significantly reduce the fungal burden in frogs infected by B. dendrobatidis. PMID:24433676

Holden, Whitney M; Fites, J Scott; Reinert, Laura K; Rollins-Smith, Louise A

2014-01-01

275

Functionality of chitin as a direct compression excipient: an acetaminophen comparative study.  

PubMed

The particle and tableting properties of chitin extracted from shrimp exoskeletons were evaluated and compared with common excipients used for the preparation of tablets. Chitin offered more benefits in terms of functionality than calcium diphosphate, lactose monohydrate and pregelatinized starch. Further, highly plastic deforming materials such as sorbitol and PVP K30 and microcrystalline cellulose showed the best compactibility and dilution potential, whereas brittle deforming materials such as lactose monohydrate and calcium diphosphate were poorly compactable. Chitin had better compactibility than pregelatinized starch, calcium diphosphate and lactose monohydrate. Further, along with calcium diphosphate, chitin was the least sensitive material to compaction speed due to a combination of a plastic and brittle behavior. Moreover, chitin was less sensitive to magnesium stearate and possessed better acetaminophen loading capacity than pregelatinized starch, calcium diphosphate and lactose monohydrate. Chitin showed potential for use as a direct compression excipient. PMID:24528710

Rojas, John; Ciro, Yhors; Correa, Luisa

2014-03-15

276

Electrospinning and characterization of chitin nanofibril/polycaprolactone nanocomposite fiber mats.  

PubMed

Nanocomposite fiber mats based on biodegradable polycaprolactone (PCL) and chitin nanofibril (n-chitin) were produced via electrospinning. The morphologies, thermal and mechanical properties as well as surface wettability of the fiber mats were studied by scanning electron microscopy, differential scanning calorimetry analysis, thermogravimetric analysis, dynamic mechanical analysis and static water-contact-angle analysis, respectively. The addition of chitin nanofibrils into PCL resulted in a small change in thermal behavior, but a significant improvement in mechanical properties. Moreover, the surface wettability of electrospun fiber mats transformed from hydrophobicity to hydrophilicity when the chitin nanofibril content was more than 25 wt%. In addition, in vitro cell culture results indicated that the addition of chitin nanofibrils can strongly improve the cellular infiltration and migration confirming that the chitin nanofibril was a good reinforcing as well as bioactive filler for PCL. PMID:24299750

Ji, Yali; Liang, Kai; Shen, Xinyuan; Bowlin, Gary L

2014-01-30

277

Effects of moisture on tablet compression of chitin  

Microsoft Academic Search

Direct compression of chitin was studied with special reference to the effects of moisture content on tablet formation and properties. Two cellulosic direct compression materials, microcrystalline cellulose (MCC) and spray-dried lactose-cellulose (SDLC, (Cellactose®) were used as reference materials. The compaction studies were carried out using an instrumented single-punch tablet machine. For physical material characterisation, water sorption isotherms were determined gravimetrically

Viviana García Mir; Jyrki Heinämäki; Osmo Antikainen; Antonio Iraizoz Colarte; Sari Airaksinen; Milja Karjalainen; Ofelia Bilbao Revoredo; Olga Maria Nieto; Jouko Yliruusi

2011-01-01

278

Characterization of a Novel, Antifungal, Chitin-Binding Protein from Streptomyces tendae T?901 That Interferes with Growth Polarity  

PubMed Central

The afp1 gene, which encodes the antifungal protein AFP1, was cloned from nikkomycin-producing Streptomyces tendae Tü901, using a nikkomycin-negative mutant as a host and screening transformants for antifungal activity against Paecilomyces variotii in agar diffusion assays. The 384-bp afp1 gene has a low G+C content (63%) and a transcription termination structure with a poly(T) region, unusual attributes for Streptomyces genes. AFP1 was purified from culture filtrate of S. tendae carrying the afp1 gene on the multicopy plasmid pIJ699. The purified protein had a molecular mass of 9,862 Da and lacked a 42-residue N-terminal peptide deduced from the nucleotide sequence. AFP1 was stable at extreme pH values and high temperatures and toward commercial proteinases. AFP1 had limited similarity to cellulose-binding domains of microbial plant cell wall hydrolases and bound to crab shell chitin, chitosan, and cell walls of P. variotii but showed no enzyme activity. The biological activity of AFP1, which represents the first chitin-binding protein from bacteria exhibiting antifungal activity, was directed against specific ascomycetes, and synergistic interaction with the chitin synthetase inhibitor nikkomycin inhibited growth of Aspergillus species. Microscopy studies revealed that fluorescein-labeled AFP1 strongly bound to the surface of germinated conidia and to tips of growing hyphae, causing severe alterations in cell morphogenesis that gave rise to large spherical conidia and/or swollen hyphae and to atypical branching. PMID:10601197

Bormann, Christiane; Baier, Daniel; Horr, Ingmar; Raps, Claudia; Berger, Jurgen; Jung, Gunther; Schwarz, Heinz

1999-01-01

279

Versatile carboxymethyl chitin and chitosan nanomaterials: a review.  

PubMed

Biocompatibility, biodegradability, and low cost of chitin and chitosan have drawn immense attention in many fields including medicine, bioinspired material science, pharmaceuticals, and agriculture. Their handling and processing are difficult owing to its insolubility in neutral aqueous solution or organic solvents. One of the methods used to improve the solubility characteristics of chitin and chitosan is chemical modification. Introducing a carboxymethyl group is the most advantageous method of increasing the solubility of chitosan at neutral and alkaline pH. Carboxymethyl chitin (CMC) and carboxymethyl chitosan (CMCS) are water soluble derivatives formed by introducing CH?COOH function into the polymer which endows it with better biological properties. The functional group makes CMC/CMCS nanoparticles (NPs) efficient vehicles for the delivery of DNA, proteins, and drugs. This review provides an overview of the characteristics of CMC/CMCS NPs as well as fulfills the task of describing and discussing its important roles primarily in cancer nanomedicine detailing the targeted drug delivery aspect. The application of these NPs in imaging, agriculture, and textiles has also been highlighted. The review also elaborates the advantages of using the CMC and CMCS NPs for drug and gene delivery. PMID:25266740

Narayanan, Deepa; Jayakumar, R; Chennazhi, K P

2014-01-01

280

Requirement for chitin biosynthesis in epithelial tube morphogenesis  

PubMed Central

Many organs are composed of branched networks of epithelial tubes that transport vital fluids or gases. The proper size and shape of tubes are crucial for their transport function, but the molecular processes that govern tube size and shape are not well understood. Here we show that three genes required for tracheal tube morphogenesis in Drosophila melanogaster encode proteins involved in the synthesis and accumulation of chitin, a polymer of N-acetyl-?-d-glucosamine that serves as a scaffold in the rigid extracellular matrix of insect cuticle. In all three mutants, developing tracheal tubes bud and extend normally, but the epithelial walls of the tubes do not expand uniformly, and the resultant tubes are grossly misshapen, with constricted and distended regions all along their lengths. The genes are expressed in tracheal cells during the expansion process, and chitin accumulates in the lumen of tubes, forming an expanding cylinder that we propose coordinates the behavior of the surrounding tracheal cells and stabilizes the expanding epithelium. These findings show that chitin regulates epithelial tube morphogenesis, in addition to its classical role protecting mature epithelia. PMID:16287975

Devine, W. Patrick; Lubarsky, Barry; Shaw, Ken; Luschnig, Stefan; Messina, Lisa; Krasnow, Mark A.

2005-01-01

281

Facile nanofibrillation of chitin derivatives by gas bubbling and ultrasonic treatments in water.  

PubMed

In this paper, we report that nanofiber network structures were constructed from chitin derivatives by gas bubbling and ultrasonic treatments in water. When chitin was first subjected to N2 gas bubbling with ultrasonication in water, the SEM images of the product showed nanofiber network morphology. However, nanofiber network was not re-constructed by the same N2 gas bubbling and ultrasonic treatments after agglomeration. We then have paid attention to an amidine group to provide the agglomeration-nanofibrillation behavior of chitin derivatives. An amidinated chitin was synthesized by the reaction of the amino groups in a partially deacetylated chitin with N,N-dimethylacetamide dimethyl acetal, which was subjected to CO2 gas bubbling and ultrasonic treatments in water to convert into an amidinium chitin by protonation. The SEM images of the product clearly showed nanofiber network morphology. We further examined re-nanofibrillation of the agglomerated material, which was obtained by mixing the nanofibrillated amidinium chitin with water, followed by drying under reduced pressure. Consequently, the material was re-nanofibrillated by N2 gas bubbling with ultrasonication in water owing to electrostatic repulsion between the amidinium groups. Furthermore, deprotonation of the amidinium chitin and re-protonation of the resulting amidinated chitin were conducted by alkaline treatment and CO2 gas bubbling-ultrasonic treatments, respectively. The material showed the agglomeration-nanofibrillation behavior during the processes. PMID:25238127

Tanaka, Kohei; Yamamoto, Kazuya; Kadokawa, Jun-Ichi

2014-10-29

282

A chalcone synthase/stilbene synthase DNA probe for conifers.  

PubMed

A probe for chalcone synthase (CHS) was generated by PCR using chalcone synthase conserved sequences. The cloned PCR product has high similarity to both chalcone synthase and stilbene synthase sequences. The probe was used to examine the organization of chalcone synthase and stilbene synthase genes in Abies procera, Pinus lambertiana, P. monticola, Picea glauca, P. sitchensis, Pseudostuga menziesii, Taxus brevifolia, and Thuja plicata. A large number of hybridizing bands were found in all species except T. plicata which did not cross hybridize. The hybridization patterns are highly polymorphic between the species and are also polymorphic within several of them. PMID:24166547

Baker, S M; White, E E

1996-05-01

283

Increase in chitin as an essential response to defects in assembly of cell wall polymers in the ggp1delta mutant of Saccharomyces cerevisiae.  

PubMed Central

The GGP1/GAS1 gene codes for a glycosylphosphatidylinositol-anchored plasma membrane glycoprotein of Saccharomyces cerevisiae. The ggp1delta mutant shows morphogenetic defects which suggest changes in the cell wall matrix. In this work, we have investigated cell wall glucan levels and the increase of chitin in ggp1delta mutant cells. In these cells, the level of alkali-insoluble 1,6-beta-D-glucan was found to be 50% of that of wild-type cells and was responsible for the observed decrease in the total alkali-insoluble glucan. Moreover, the ratio of alkali-soluble to alkali-insoluble glucan almost doubled, suggesting a change in glucan solubility. The increase of chitin in ggp1delta cells was found to be essential since the chs3delta ggp1delta mutations determined a severe reduction in the growth rate and in cell viability. Electron microscopy analysis showed the loss of the typical structure of yeast cell walls. Furthermore, in the chs3delta ggp1delta cells, the level of alkali-insoluble glucan was 57% of that of wild-type cells and the alkali-soluble/alkali-insoluble glucan ratio was doubled. We tested the effect of inhibition of chitin synthesis also by a different approach. The ggp1delta cells were treated with nikkomycin Z, a well-known inhibitor of chitin synthesis, and showed a hypersensitivity to this drug. In addition, studies of genetic interactions with genes related to the construction of the cell wall indicate a synthetic lethal effect of the ggp1delta kre6delta and the ggp1delta pkc1delta combined mutations. Our data point to an involvement of the GGP1 gene product in the cross-links between cell wall glucans (1,3-beta-D-glucans with 1,6-beta-D-glucans and with chitin). Chitin is essential to compensate for the defects due to the lack of Ggp1p. Moreover, the activities of Ggp1p and Chs3p are essential to the formation of the organized structure of the cell wall in vegetative cells. PMID:8990299

Popolo, L; Gilardelli, D; Bonfante, P; Vai, M

1997-01-01

284

Development of 2-aryl substituted quinazolin-4(3H)-one, pyrido[4,3-d]pyrimidin-4(3H)-one and pyrido[2,3-d]pyrimidin-4(3H)-one derivatives as microsomal prostaglandin E2 synthase-1 inhibitors.  

PubMed

mPGES-1 is inducible terminal synthase acting downstream of COX enzymes in arachidonic acid pathway, regulates the biosynthesis of pro-inflammatory prostaglandin PGE2. Cardiovascular side effect of coxibs and NSAIDs, selective for COX-2 inhibition, stimulated interest in mPGES-1, a therapeutic target with potential to deliver safe and effective anti-inflammatory drugs. The synthesis and structure activity relationship of a series of compounds from 2-aryl substituted quinazolin-4(3H)-one, pyrido[4,3-d]pyrimidin-4(3H)-one and pyrido[2,3-d]pyrimidin-4(3H)-one scaffolds as mPGES-1 inhibitor are discussed. A set of analogs (28, 48, 49) were identified with <10nM potencies in the recombinant human mPGES-1 enzyme and in the A549 cellular assays. These analogs were also found to be potent in the human whole blood assay (<400nM). Furthermore, the representative compound 48 was shown to be selective with other prostanoid synthases and was able to effectively regulate PGE2 biosynthesis in clinically relevant inflammatory settings, in comparison with celecoxib. PMID:25260492

Banerjee, Abhisek; Pawar, Mahesh Y; Patil, Sandip; Yadav, Pravin S; Kadam, Pradip A; Kattige, Vidya G; Deshpande, Durga S; Pednekar, Pallavi V; Pisat, Monali K; Gharat, Laxmikant A

2014-10-15

285

Soil Bacterial Community Shifts after Chitin Enrichment: An Integrative Metagenomic Approach  

PubMed Central

Chitin is the second most produced biopolymer on Earth after cellulose. Chitin degrading enzymes are promising but untapped sources for developing novel industrial biocatalysts. Hidden amongst uncultivated micro-organisms, new bacterial enzymes can be discovered and exploited by metagenomic approaches through extensive cloning and screening. Enrichment is also a well-known strategy, as it allows selection of organisms adapted to feed on a specific compound. In this study, we investigated how the soil bacterial community responded to chitin enrichment in a microcosm experiment. An integrative metagenomic approach coupling phylochips and high throughput shotgun pyrosequencing was established in order to assess the taxonomical and functional changes in the soil bacterial community. Results indicate that chitin enrichment leads to an increase of Actinobacteria, ?-proteobacteria and ?-proteobacteria suggesting specific selection of chitin degrading bacteria belonging to these classes. Part of enriched bacterial genera were not yet reported to be involved in chitin degradation, like the members from the Micrococcineae sub-order (Actinobacteria). An increase of the observed bacterial diversity was noticed, with detection of specific genera only in chitin treated conditions. The relative proportion of metagenomic sequences related to chitin degradation was significantly increased, even if it represents only a tiny fraction of the sequence diversity found in a soil metagenome. PMID:24278158

Jacquiod, Samuel; Franqueville, Laure; Cecillon, Sebastien; M. Vogel, Timothy; Simonet, Pascal

2013-01-01

286

The quick extraction of chitin from an epizoic crustacean species (Chelonibia patula).  

PubMed

Chitin was isolated from the shells of Chelonibia patula (barnacle, Crustacea), which lives on blue crab epizoically, following a 10-min demineralisation process through HCl and a 20-min deproteinisation process through NaOH. Due to the low-crystalline structure, and mineral-rich and low-protein content of the shells, chitin isolation was convenient. It was observed that the shell structure of C. patula contains 3.11% chitin per its dry weight. Following characterisation of the isolated chitin by using Fourier transform infrared spectroscopy, thermogravimetric analysis, X-ray diffractometry, elemental analysis and scanning electron microscopy, it was determined that there was close similarity with the ?-chitin isolated from crabs, shrimps and insects in various studies. It was observed that chitin was composed of nanofibres with a width of 10-20 nm. It was concluded that this was an economically advantageous chitin resource compared with crustaceans such as shrimp, crayfish and crab, because it is possible to isolate chitin in a significantly shorter time. PMID:24933023

Kaya, Murat; Karaarslan, Muhsin; Baran, Talat; Can, Esra; Ekemen, Gulcin; Bitim, Betul; Duman, Fatih

2014-12-01

287

Formation of ceramophilic chitin and biohybrid materials enabled by a genetically engineered bifunctional protein.  

PubMed

A bifunctional protein composed of a highly negatively charged oyster shell protein and a chitin-binding domain enabled the formation of biohybrid materials through non-covalent surface modification of chitin nanofibres. The results demonstrate that specific biomolecular interactions offer a route for the formation of biosynthetic materials. PMID:24871427

Malho, Jani-Markus; Heinonen, Hanna; Kontro, Inkeri; Mushi, Ngesa E; Serimaa, Ritva; Hentze, Hans-Peter; Linder, Markus B; Szilvay, Géza R

2014-07-14

288

Fungal chitin dampens inflammation through IL-10 induction mediated by NOD2 and TLR9 activation.  

PubMed

Chitin is an essential structural polysaccharide of fungal pathogens and parasites, but its role in human immune responses remains largely unknown. It is the second most abundant polysaccharide in nature after cellulose and its derivatives today are widely used for medical and industrial purposes. We analysed the immunological properties of purified chitin particles derived from the opportunistic human fungal pathogen Candida albicans, which led to the selective secretion of the anti-inflammatory cytokine IL-10. We identified NOD2, TLR9 and the mannose receptor as essential fungal chitin-recognition receptors for the induction of this response. Chitin reduced LPS-induced inflammation in vivo and may therefore contribute to the resolution of the immune response once the pathogen has been defeated. Fungal chitin also induced eosinophilia in vivo, underpinning its ability to induce asthma. Polymorphisms in the identified chitin receptors, NOD2 and TLR9, predispose individuals to inflammatory conditions and dysregulated expression of chitinases and chitinase-like binding proteins, whose activity is essential to generate IL-10-inducing fungal chitin particles in vitro, have also been linked to inflammatory conditions and asthma. Chitin recognition is therefore critical for immune homeostasis and is likely to have a significant role in infectious and allergic disease. PMID:24722226

Wagener, Jeanette; Malireddi, R K Subbarao; Lenardon, Megan D; Köberle, Martin; Vautier, Simon; MacCallum, Donna M; Biedermann, Tilo; Schaller, Martin; Netea, Mihai G; Kanneganti, Thirumala-Devi; Brown, Gordon D; Brown, Alistair J P; Gow, Neil A R

2014-04-01

289

Jak zskala Chlorella chitin??? We presented evidence suggesting that Chlorella could have acquired  

E-print Network

Jak získala Chlorella chitin??? We presented evidence suggesting that Chlorella could have acquired or a microorganism. In the case of Chlorella, the acquisition of a chitinous cell wall may have conferred required to penetrate and/or escape the algal cell. This might have increased the fitness of Chlorella

290

Screening of chitin deacetylase from Mucoralean strains (Zygomycetes) and its relationship to cell growth rate  

Microsoft Academic Search

Chitin deacetylase (CDA) is an enzyme that catalyzes the hydrolysis of acetamine groups of N-acetyl-d-glucosamine in chitin, converting it to chitosan in fungal cell walls. In the present study, the activity in batch culture of CDA from six Mucoralean strains, two of them wild type, isolated from dung of herbivores of Northeast Brazil, was screened. Among the strains tested, Cunninghamella

R. V. S. Amorim; W. M. Ledingham; K. Fukushima; G. M. Campos-Takaki

2005-01-01

291

Removal of copper, chromium, and arsenic from CCA-treated wood onto chitin and chitosan  

Microsoft Academic Search

Chitin and chitosan are naturally abundant biopolymers which are of interest to research concerning the sorption of metal ions since the amine and hydroxyl groups on their chemical structures act as chelation sites for metal ions. This study evaluates the removal of copper, chromium, and arsenic elements from chromated copper arsenate (CCA)-treated wood via biosorption by chitin and chitosan. Exposing

S. Nami Kartal; Yuji Imamura

2005-01-01

292

Hybrid of chitin and humic acid as high performance sorbent for Ni(II)  

NASA Astrophysics Data System (ADS)

Hybrid of humic acid (HA) and chitin has been synthesized and the hybrid material (chitin-HA) was then applied as sorbent to adsorb Ni(II). The HA was extracted from peat soil of Gambut District, South Kalimantan, Indonesia, according to the procedure recommended by IHSS (International Humic Substances Society). The chitin was isolated from crab shell waste of sea food restaurants through deproteination using NaOH 3.5% (w/v) and followed by removal of inorganic impurities using HCl 1 M. The synthesis of chitin-HA was performed by reacting gelatinous chitin solution in HCl 0.5 M and HA solution in NaOH 0.5 M. Parameters investigated in this work consists of effect of medium acidity on the sorption, sorption rate ( ks) and desorption rate ( kd) constants, Langmuir (monolayer) and Freundlich (multilayer) sorption capacities, and energy ( E) of sorption. The ks and kd were determined according to a kinetic model of first order sorption reaching equilibrium, monolayer sorption capacity ( b) and energy ( E) were determined according to the Langmuir isotherm model, and multilayer sorption capacity ( B) was determined based on the Freundlich isotherm model. Sorption of Ni(II) on both chitin and chitin-HA was maximum at pH 8.0. The kinetic expression resulted from the proposed kinetic model has been shown to be more applicable than the commonly known Lagergren equation obtained from the pseudo-first order sorption model. The application of the proposed model revealed that the presence of HA increased the ks from 0.018 min -1 for chitin to 0.031 min -1 for chitin-HA. As for ks, the value of b was also bigger in the presence of HA, i.e. 7.42 × 10 -5 mol/g for chitin and 9.93 × 10 -5 mol/g for the chitin-HA. Unlike ks and b, the value of E slightly decreased from 23.23 to 21.51 kJ/mol for the absence and presence of HA, respectively. It can also be deduced that the presence of HA on chitin contributed more to the additional layer of Ni(II) sorbed on sorbent. Without HA, B for chitin was only 6.17 times higher than b, while with the presence of HA, the enhancement of the sorption capacity from the multilayer ( B) to the monolayer ( b) was 19.40. The increase of ks, b, B, and the decrease of E would be very benefit in the real application of chitin-HA for the recovery of Ni(II) from aqueous samples.

Santosa, Sri Juari; Siswanta, Dwi; Kurniawan, Agusta; Rahmanto, Wasino H.

2007-11-01

293

Novel chitin/nanosilica composite scaffolds for bone tissue engineering applications.  

PubMed

Biopolymers like chitin are widely investigated as scaffolds in bone tissue engineering. Its properties like biocompatibility, biodegradability, non-toxicity, wound healing ability, antibacterial activity, hemostatic property, etc., are widely known. However, these materials are not much bioactive. Addition of material like silica can improve the bioactivity and biocompatibility of chitin. In this work, chitin composite scaffolds containing nanosilica were prepared using chitin hydrogel and their bioactivity, swelling ability and cytotoxicity was analyzed in vitro. These scaffolds were found to be bioactive in simulated body fluid (SBF) and biocompatible when tested with MG 63 cell line. These results suggest that chitin/nanosilica composite scaffolds can be useful for bone tissue engineering applications. PMID:19549539

Madhumathi, K; Sudheesh Kumar, P T; Kavya, K C; Furuike, T; Tamura, H; Nair, S V; Jayakumar, R

2009-10-01

294

Geranyl diphosphate synthase from mint  

DOEpatents

A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate. 5 figs.

Croteau, R.B.; Wildung, M.R.; Burke, C.C.; Gershenzon, J.

1999-03-02

295

Geranyl diphosphate synthase from mint  

DOEpatents

A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

Croteau, Rodney Bruce (Pullman, WA); Wildung, Mark Raymond (Colfax, WA); Burke, Charles Cullen (Moscow, ID); Gershenzon, Jonathan (Jena, DE)

1999-01-01

296

Dimers of mitochondrial ATP synthase form the permeability transition pore  

PubMed Central

Here we define the molecular nature of the mitochondrial permeability transition pore (PTP), a key effector of cell death. The PTP is regulated by matrix cyclophilin D (CyPD), which also binds the lateral stalk of the FOF1 ATP synthase. We show that CyPD binds the oligomycin sensitivity-conferring protein subunit of the enzyme at the same site as the ATP synthase inhibitor benzodiazepine 423 (Bz-423), that Bz-423 sensitizes the PTP to Ca2+ like CyPD itself, and that decreasing oligomycin sensitivity-conferring protein expression by RNAi increases the sensitivity of the PTP to Ca2+. Purified dimers of the ATP synthase, which did not contain voltage-dependent anion channel or adenine nucleotide translocator, were reconstituted into lipid bilayers. In the presence of Ca2+, addition of Bz-423 triggered opening of a channel with currents that were typical of the mitochondrial megachannel, which is the PTP electrophysiological equivalent. Channel openings were inhibited by the ATP synthase inhibitor AMP-PNP (?-imino ATP, a nonhydrolyzable ATP analog) and Mg2+/ADP. These results indicate that the PTP forms from dimers of the ATP synthase. PMID:23530243

Giorgio, Valentina; von Stockum, Sophia; Antoniel, Manuela; Fabbro, Astrid; Fogolari, Federico; Forte, Michael; Glick, Gary D.; Petronilli, Valeria; Zoratti, Mario; Szabo, Ildiko; Lippe, Giovanna; Bernardi, Paolo

2013-01-01

297

Design, synthesis, and biological evaluation of 1-phenylpyrazolo[3,4-e]pyrrolo[3,4-g]indolizine-4,6(1H,5H)-diones as new glycogen synthase kinase-3? inhibitors.  

PubMed

Compound 5 was selected from our in-house library as a suitable starting point for the rational design of new GSK-3? inhibitors. MC/FEP calculations of 5 led to the identification of a structural class of new GSK-3? inhibitors. Compound 18 inhibited GSK-3? with an IC50 of 0.24 ?M and inhibited tau phosphorylation in a cell-based assay. It proved to be a selective inhibitor of GSK-3 against a panel of 17 kinases and showed >10-fold selectivity against CDK2. Calculated physicochemical properties and Volsurf predictions suggested that compound 18 has the potential to diffuse passively across the blood-brain barrier. PMID:24295046

La Pietra, Valeria; La Regina, Giuseppe; Coluccia, Antonio; Famiglini, Valeria; Pelliccia, Sveva; Plotkin, Batya; Eldar-Finkelman, Hagit; Brancale, Andrea; Ballatore, Carlo; Crowe, Alex; Brunden, Kurt R; Marinelli, Luciana; Novellino, Ettore; Silvestri, Romano

2013-12-27

298

Fungal Chitin Dampens Inflammation through IL-10 Induction Mediated by NOD2 and TLR9 Activation  

PubMed Central

Chitin is an essential structural polysaccharide of fungal pathogens and parasites, but its role in human immune responses remains largely unknown. It is the second most abundant polysaccharide in nature after cellulose and its derivatives today are widely used for medical and industrial purposes. We analysed the immunological properties of purified chitin particles derived from the opportunistic human fungal pathogen Candida albicans, which led to the selective secretion of the anti-inflammatory cytokine IL-10. We identified NOD2, TLR9 and the mannose receptor as essential fungal chitin-recognition receptors for the induction of this response. Chitin reduced LPS-induced inflammation in vivo and may therefore contribute to the resolution of the immune response once the pathogen has been defeated. Fungal chitin also induced eosinophilia in vivo, underpinning its ability to induce asthma. Polymorphisms in the identified chitin receptors, NOD2 and TLR9, predispose individuals to inflammatory conditions and dysregulated expression of chitinases and chitinase-like binding proteins, whose activity is essential to generate IL-10-inducing fungal chitin particles in vitro, have also been linked to inflammatory conditions and asthma. Chitin recognition is therefore critical for immune homeostasis and is likely to have a significant role in infectious and allergic disease. Authors Summary Chitin is the second most abundant polysaccharide in nature after cellulose and an essential component of the cell wall of all fungal pathogens. The discovery of human chitinases and chitinase-like binding proteins indicates that fungal chitin is recognised by cells of the human immune system, shaping the immune response towards the invading pathogen. We show that three immune cell receptors– the mannose receptor, NOD2 and TLR9 recognise chitin and act together to mediate an anti-inflammatory response via secretion of the cytokine IL-10. This mechanism may prevent inflammation-based damage during fungal infection and restore immune balance after an infection has been cleared. By increasing the chitin content in the cell wall pathogenic fungi may influence the immune system in their favour, by down-regulating protective inflammatory immune responses. Furthermore, gene mutations and dysregulated enzyme activity in the described chitin recognition pathway are implicated in inflammatory conditions such as Crohn's Disease and asthma, highlighting the importance of the discovered mechanism in human health. PMID:24722226

Wagener, Jeanette; Malireddi, R. K. Subbarao; Lenardon, Megan D.; Koberle, Martin; Vautier, Simon; MacCallum, Donna M.; Biedermann, Tilo; Schaller, Martin; Netea, Mihai G.; Kanneganti, Thirumala-Devi; Brown, Gordon D.; Brown, Alistair J. P.; Gow, Neil A. R.

2014-01-01

299

Characterization of domain interfaces in monomeric and dimeric ATP synthase.  

PubMed

We disassembled monomeric and dimeric yeast ATP synthase under mild conditions to identify labile proteins and transiently stable subcomplexes that had not been observed before. Specific removal of subunits alpha, beta, oligomycin sensitivity conferring protein (OSCP), and h disrupted the ATP synthase at the gamma-alpha(3)beta(3) rotor-stator interface. Loss of two F(1)-parts from dimeric ATP synthase led to the isolation of a dimeric subcomplex containing membrane and peripheral stalk proteins thus identifying the membrane/peripheral stalk sectors immediately as the dimerizing parts of ATP synthase. Almost all subunit a was found associated with a ring of 10 c-subunits in two-dimensional blue native/SDS gels. We therefore postulate that c10a1-complex is a stable structure in resting ATP synthase until the entry of protons induces a breaking of interactions and stepwise rotation of the c-ring relative to the a-subunit in the catalytic mechanism. Dimeric subunit a was identified in SDS gels in association with two c10-rings suggesting that a c10a2c10-complex may constitute an important part of the monomer-monomer interface in dimeric ATP synthase that seems to be further tightened by subunits b, i, e, g, and h. In contrast to the monomer-monomer interface, the interface between dimers in higher oligomeric structures remains largely unknown. However, we could show that the natural inhibitor protein Inh1 is not required for oligomerization. PMID:18245802

Wittig, Ilka; Velours, Jean; Stuart, Rosemary; Schägger, Hermann

2008-05-01

300

Synthesis and Antibacterial Activity of Cinnamaldehyde Acylhydrazone with a 1,4-Benzodioxan Fragment as a Novel Class of Potent ?-Ketoacyl-Acyl Carrier Protein Synthase III (FabH) Inhibitor.  

PubMed

Fatty acid biosynthesis is essential for bacterial survival. ?-Ketoacyl-acyl carrier protein (ACP) synthase III (FabH), is a particularly attractive antibacterial target, since it is central to the initiation of fatty acid biosynthesis. Three series of 21 cinnamaldehyde acylhydrazone derivatives, A3-9, B3-9, and C3-9, were synthesized and evaluated for FabH-inhibitory activity. Compound B6 showed the most potent biological activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis (minimum inhibitory concentrations (MICs) values: 1.56-3.13?µg/mL) and was comparable with the positive control. Docking simulation by positioning compound B6 in the FabH structure active site was performed to explore the possible binding model. PMID:25196128

Song, Xiaoda; Yang, Yushun; Zhao, Jing; Chen, Yangjian

2014-11-01

301

Construction of chitin/PVA composite hydrogels with jellyfish gel-like structure and their biocompatibility.  

PubMed

High strength chitin/poly(vinyl alcohol) (PVA) composite hydrogels (RCP) were constructed by adding PVA into chitin dissolved in a NaOH/urea aqueous solution, and then by cross-linking with epichlorohydrin (ECH) and freezing-thawing process. The RCP hydrogels were characterized by field emission scanning electron microscopy, FTIR, differential scanning calorimetry, solid-state (13)C NMR, wide-angle X-ray diffraction, and compressive test. The results revealed that the repeated freezing/thawing cycles induced the bicrosslinked networks consisted of chitin and PVA crystals in the composite gels. Interestingly, a jellyfish gel-like structure occurred in the RCP75 gel with 25 wt % PVA content in which the amorphous and crystalline PVA were immobilized tightly in the chitin matrix through hydrogen bonding interaction. The freezing/thawing cycles played an important role in the formation of the layered porous PVA networks and the tight combining of PVA with the pore wall of chitin. The mechanical properties of RCP75 were much higher than the other RCP gels, and the compressive strength was 20× higher than that of pure chitin gels, as a result of broadly dispersing stress caused by the orderly multilayered networks. Furthermore, the cell culture tests indicated that the chitin/PVA composite hydrogels exhibited excellent biocompatibility and safety, showing potential applications in the field of tissue engineering. PMID:25077674

He, Meng; Wang, Zhenggang; Cao, Yan; Zhao, Yanteng; Duan, Bo; Chen, Yun; Xu, Min; Zhang, Lina

2014-09-01

302

Green conversion of agroindustrial wastes into chitin and chitosan by Rhizopus arrhizus and Cunninghamella elegans strains.  

PubMed

This article sets out a method for producing chitin and chitosan by Cunninghamella elegans and Rhizopus arrhizus strains using a green metabolic conversion of agroindustrial wastes (corn steep liquor and molasses). The physicochemical characteristics of the biopolymers and antimicrobial activity are described. Chitin and chitosan were extracted by alkali-acid treatment, and characterized by infrared spectroscopy, viscosity and X-ray diffraction. The effectiveness of chitosan from C. elegans and R. arrhizus in inhibiting the growth of Listeria monocytogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enterica, Escherichia coli and Yersinia enterocolitica were evaluated by determining the minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC). The highest production of biomass (24.60 g/L), chitin (83.20 mg/g) and chitosan (49.31 mg/g) was obtained by R. arrhizus. Chitin and chitosan from both fungi showed a similar degree of deacetylation, respectively of 25% and 82%, crystallinity indices of 33.80% and 32.80% for chitin, and 20.30% and 17.80% for chitosan. Both chitin and chitosan presented similar viscosimetry of 3.79-3.40 cP and low molecular weight of 5.08×10³ and 4.68×10³ g/mol. They both showed identical MIC and MBC for all bacteria assayed. These results suggest that: agricultural wastes can be produced in an environmentally friendly way; chitin and chitosan can be produced economically; and that chitosan has antimicrobial potential against pathogenic bacteria. PMID:24853288

Berger, Lúcia Raquel Ramos; Stamford, Thayza Christina Montenegro; Stamford-Arnaud, Thatiana Montenegro; de Alcântara, Sergio Roberto Cabral; da Silva, Antonio Cardoso; da Silva, Adamares Marques; do Nascimento, Aline Elesbão; de Campos-Takaki, Galba Maria

2014-01-01

303

The effect of chitin size, shape, source and purification method on immune recognition.  

PubMed

The animal immune response to chitin is not well understood and needs to be investigated further. However, this is a challenging topic to study because of the technical difficulties in purifying chitin, and because this material usually comes associated with contaminating components that can activate the immune system. In this study, improvements to previously described purification protocols were investigated for chitin obtained from different sources, including commercial shellfish, Candida albicans yeast and hyphal cell walls, as well as cell walls of the filamentous fungi Aspergillus fumigatus and Mucor circinelloides. The immune response to these different chitin preparations was tested using human peripheral blood mononuclear cells. In agreement with previous literature, small chitin particles of an average size of 0.2 µm were not immunogenic. On the other hand, bigger chitin particles induced in some cases a pro-inflammatory response. The results of this work suggest that not only the purity and size of the chitin particles, but also their shape can influence immune recognition. PMID:24727416

Alvarez, Francisco J

2014-01-01

304

Discovery of 505-million-year old chitin in the basal demosponge Vauxia gracilenta.  

PubMed

Sponges are probably the earliest branching animals, and their fossil record dates back to the Precambrian. Identifying their skeletal structure and composition is thus a crucial step in improving our understanding of the early evolution of metazoans. Here, we present the discovery of 505-million-year-old chitin, found in exceptionally well preserved Vauxia gracilenta sponges from the Middle Cambrian Burgess Shale. Our new findings indicate that, given the right fossilization conditions, chitin is stable for much longer than previously suspected. The preservation of chitin in these fossils opens new avenues for research into other ancient fossil groups. PMID:24336573

Ehrlich, H; Rigby, J Keith; Botting, J P; Tsurkan, M V; Werner, C; Schwille, P; Petrášek, Z; Pisera, A; Simon, P; Sivkov, V N; Vyalikh, D V; Molodtsov, S L; Kurek, D; Kammer, M; Hunoldt, S; Born, R; Stawski, D; Steinhof, A; Bazhenov, V V; Geisler, T

2013-01-01

305

Discovery of 505-million-year old chitin in the basal demosponge Vauxia gracilenta  

PubMed Central

Sponges are probably the earliest branching animals, and their fossil record dates back to the Precambrian. Identifying their skeletal structure and composition is thus a crucial step in improving our understanding of the early evolution of metazoans. Here, we present the discovery of 505–million-year-old chitin, found in exceptionally well preserved Vauxia gracilenta sponges from the Middle Cambrian Burgess Shale. Our new findings indicate that, given the right fossilization conditions, chitin is stable for much longer than previously suspected. The preservation of chitin in these fossils opens new avenues for research into other ancient fossil groups. PMID:24336573

Ehrlich, H.; Rigby, J. Keith; Botting, J. P.; Tsurkan, M. V.; Werner, C.; Schwille, P.; Petrasek, Z.; Pisera, A.; Simon, P.; Sivkov, V. N.; Vyalikh, D. V.; Molodtsov, S. L.; Kurek, D.; Kammer, M.; Hunoldt, S.; Born, R.; Stawski, D.; Steinhof, A.; Bazhenov, V. V.; Geisler, T.

2013-01-01

306

Multifaceted chitin/poly(lactic-co-glycolic) acid composite nanogels.  

PubMed

Cyto-compatible, 80nm sized chitin/PLGA composite nanogels (chit/PLGA-comp NGs) were prepared by regeneration method and characterized. The multifaceted chit/PLGA-comp NGs were surface modified with Au, Fe3O4, CdTe/ZnTe-QDs and umbelliferone, respectively. 185nm sized Au-chit/PLGA-comp NGs, 170nm sized QD-chit/PLGA-comp-NGs and 160nm sized Fe3O4-chit/PLGA-comp-NGs showed RF heating. The QD-chit/PLGA-comp-NGs and 180nm sized umb-chit/PLGA-comp-NGs were well uptaken by Escherichia coli, Staphylococcus aureus and Candida albicans. The chit/PLGA-comp NGs could be useful for microbial monitoring and RF application for cancer therapy. The preliminary data showed that multifaceted chit/PLGA-comp-NGs could be useful for hyperthermia for cancer treatment and microbial labelling and imaging. PMID:24685461

Rejinold, N Sanoj; Biswas, Raja; Chellan, Gopi; Jayakumar, R

2014-06-01

307

Isolation and identification of chitin in the black coral Parantipathes larix (Anthozoa: Cnidaria).  

PubMed

Until now, there is a lack of knowledge about the presence of chitin in numerous representatives of corals (Cnidaria). However, investigations concerning the chitin-based skeletal organization in different coral taxa are significant from biochemical, structural, developmental, ecological and evolutionary points of view. In this paper, we present a thorough screening for the presence of chitin within the skeletal formations of a poorly investigated Mediterranean black coral, Parantipathes larix (Esper, 1792), as a typical representative of the Schizopathidae family. Using a wide array variety of techniques ((13)C solid state NMR, Fourier transform infrared (FTIR), Raman, NEXAFS, Morgan-Elson assay and Calcofluor White Staining), we unambiguously show for the first time that chitin is an important component within the skeletal stalks as well as pinnules of this coral. PMID:22546360

Bo, Marzia; Bavestrello, Giorgio; Kurek, Denis; Paasch, Silvia; Brunner, Eike; Born, René; Galli, Roberta; Stelling, Allison L; Sivkov, Viktor N; Petrova, Olga V; Vyalikh, Denis; Kummer, Kurt; Molodtsov, Serguei L; Nowak, Dorota; Nowak, Jakub; Ehrlich, Hermann

2012-01-01

308

Application of Spectroscopic Methods for Structural Analysis of Chitin and Chitosan  

PubMed Central

Chitin, the second most important natural polymer in the world, and its N-deacetylated derivative chitosan, have been identified as versatile biopolymers for a broad range of applications in medicine, agriculture and the food industry. Two of the main reasons for this are firstly the unique chemical, physicochemical and biological properties of chitin and chitosan, and secondly the unlimited supply of raw materials for their production. These polymers exhibit widely differing physicochemical properties depending on the chitin source and the conditions of chitosan production. The presence of reactive functional groups as well as the polysaccharide nature of these biopolymers enables them to undergo diverse chemical modifications. A complete chemical and physicochemical characterization of chitin, chitosan and their derivatives is not possible without using spectroscopic techniques. This review focuses on the application of spectroscopic methods for the structural analysis of these compounds. PMID:20559489

Kumirska, Jolanta; Czerwicka, Ma?gorzata; Kaczy?ski, Zbigniew; Bychowska, Anna; Brzozowski, Krzysztof; Thöming, Jorg; Stepnowski, Piotr

2010-01-01

309

Chitin Microneedles for an Easy-to-Use Tuberculosis Skin Test.  

PubMed

An easy-to-use tuberculosis skin test is developed with chitin microneedles that deliver purified protein derivative at the correct skin depth and result in a positive test in BCG-immunized guinea pigs. PMID:23983170

Jin, Jungho; Reese, Valerie; Coler, Rhea; Carter, Darrick; Rolandi, Marco

2014-03-01

310

Analysis of a change in bacterial community in different environments with addition of chitin or chitosan  

Microsoft Academic Search

The temporal changes of a bacterial community in soil with chitin or chitosan added were analyzed by PCR-denaturing gradient gel electrophoresis (DGGE) targeting the 16S rRNA gene using total DNAs prepared from the community. Band patterns of PCR-DGGE confirmed that 31 species become predominant after the addition of chitin or chitosan. The determination of the nucleotide sequences of the bands

Kazuaki Sato; Yasuhito Azama; Masahiro Nogawa; Goro Taguchi; Makoto Shimosaka

2010-01-01

311

Chitin, Chitosan, and Glycated Chitosan Regulate Immune Responses: The Novel Adjuvants for Cancer Vaccine  

PubMed Central

With the development of cancer immunotherapy, cancer vaccine has become a novel modality for cancer treatment, and the important role of adjuvant has been realized recently. Chitin, chitosan, and their derivatives have shown their advantages as adjuvants for cancer vaccine. In this paper, the adjuvant properties of chitin and chitosan were discussed, and some detailed information about glycated chitosan and chitosan nanoparticles was also presented to illustrate the trend for future development. PMID:23533454

Li, Xiaosong; Min, Min; Du, Nan; Gu, Ying; Hode, Tomas; Naylor, Mark; Chen, Dianjun; Nordquist, Robert E.; Chen, Wei R.

2013-01-01

312

Use of colloid chitin and diatomaceous earth in continuous cake-filtration fermentation to produce creatinase  

Microsoft Academic Search

A recombinant Escherichia coli M15(pQE3208) producing creatinase was cultured in a cake-filtration fermentor containing colloid chitin and diatomaceous earth. The filter medium of this cake filtration was a hollow cylinder made of a 20 ?m stainless steel sieve located in the centre of the fermentor. During filtration, colloid chitin, diatomaceous earth, and E. coli cells formed a film of filter

Shiue-Cheng Tang; Ming-Chung Chang; Chu-Yuan Cheng

1998-01-01

313

Methyl jasmonate induces expression of a novel Brassica juncea chitinase with two chitin-binding domains  

Microsoft Academic Search

We have cloned a 1.3 kb Brassica juncea cDNA encoding BjCHI1, a novel acidic chitinase with two chitin-binding domains that shows 62% identity to Nicotiana tabacum Chia1 chitinase. BjCHI1 is structurally unlike Chia1 that has one chitin-binding domain, but resembles Chia5 chitinase UDA1, the precursor of Urtica dioica agglutinin; however there is only 36.9% identity between them. We propose that

Kai-Jun Zhao; Mee-Len Chye

1999-01-01

314

Chitin Binding Proteins Act Synergistically with Chitinases in Serratia proteamaculans 568  

PubMed Central

Genome sequence of Serratia proteamaculans 568 revealed the presence of three family 33 chitin binding proteins (CBPs). The three Sp CBPs (Sp CBP21, Sp CBP28 and Sp CBP50) were heterologously expressed and purified. Sp CBP21 and Sp CBP50 showed binding preference to ?-chitin, while Sp CBP28 did not bind to chitin and cellulose substrates. Both Sp CBP21 and Sp CBP50 were synergistic with four chitinases from S. proteamaculans 568 (Sp ChiA, Sp ChiB, Sp ChiC and Sp ChiD) in degradation of ?- and ?-chitin, especially in the presence of external electron donor (reduced glutathione). Sp ChiD benefited most from Sp CBP21 or Sp CBP50 on ?-chitin, while Sp ChiB and Sp ChiD had major advantage with these Sp CBPs on ?-chitin. Dose responsive studies indicated that both the Sp CBPs exhibit synergism ?0.2 µM. The addition of both Sp CBP21 and Sp CBP50 in different ratios to a synergistic mixture did not significantly increase the activity. Highly conserved polar residues, important in binding and activity of CBP21 from S. marcescens (Sm CBP21), were present in Sp CBP21 and Sp CBP50, while Sp CBP28 had only one such polar residue. The inability of Sp CBP28 to bind to the test substrates could be attributed to the absence of important polar residues. PMID:22590591

Purushotham, Pallinti; Arun, P. V. Parvati Sai; Prakash, Jogadhenu S. S.; Podile, Appa Rao

2012-01-01

315

Pharyngeal polysaccharide deacetylases affect development in the nematode C. elegans and deacetylate chitin in vitro.  

PubMed

Chitin (?-1,4-linked-N-acetylglucosamine) provides structural integrity to the nematode eggshell and pharyngeal lining. Chitin is synthesized in nematodes, but not in plants and vertebrates, which are often hosts to parasitic roundworms; hence, the chitin metabolism pathway is considered a potential target for selective interventions. Polysaccharide deacetylases (PDAs), including those that convert chitin to chitosan, have been previously demonstrated in protists, fungi and insects. We show that genes encoding PDAs are distributed throughout the phylum Nematoda, with the two paralogs F48E3.8 and C54G7.3 found in C. elegans. We confirm that the genes are somatically expressed and show that RNAi knockdown of these genes retards C. elegans development. Additionally, we show that proteins from the nematode deacetylate chitin in vitro, we quantify the substrate available in vivo as targets of these enzymes, and we show that Eosin Y (which specifically stains chitosan in fungal cells walls) stains the C. elegans pharynx. Our results suggest that one function of PDAs in nematodes may be deacetylation of the chitinous pharyngeal lining. PMID:22808160

Heustis, Ronald J; Ng, Hong K; Brand, Kenneth J; Rogers, Meredith C; Le, Linda T; Specht, Charles A; Fuhrman, Juliet A

2012-01-01

316

Squid-derived chitin oligosaccharides are a chemotactic signal during colonization by Vibrio fischeri.  

PubMed

Chitin, a polymer of N-acetylglucosamine (GlcNAc), is noted as the second most abundant biopolymer in nature. Chitin serves many functions for marine bacteria in the family Vibrionaceae ("vibrios"), in some instances providing a physical attachment site, inducing natural genetic competence, and serving as an attractant for chemotaxis. The marine luminous bacterium Vibrio fischeri is the specific symbiont in the light-emitting organ of the Hawaiian bobtail squid, Euprymna scolopes. The bacterium provides the squid with luminescence that the animal uses in an antipredatory defense, while the squid supports the symbiont's nutritional requirements. V. fischeri cells are harvested from seawater during each host generation, and V. fischeri is the only species that can complete this process in nature. Furthermore, chitin is located in squid hemocytes and plays a nutritional role in the symbiosis. We demonstrate here that chitin oligosaccharides produced by the squid host serve as a chemotactic signal for colonizing bacteria. V. fischeri uses the gradient of host chitin to enter the squid light organ duct and colonize the animal. We provide evidence that chitin serves a novel function in an animal-bacterial mutualism, as an animal-produced bacterium-attracting synomone. PMID:22522684

Mandel, Mark J; Schaefer, Amy L; Brennan, Caitlin A; Heath-Heckman, Elizabeth A C; Deloney-Marino, Cindy R; McFall-Ngai, Margaret J; Ruby, Edward G

2012-07-01

317

Squid-Derived Chitin Oligosaccharides Are a Chemotactic Signal during Colonization by Vibrio fischeri  

PubMed Central

Chitin, a polymer of N-acetylglucosamine (GlcNAc), is noted as the second most abundant biopolymer in nature. Chitin serves many functions for marine bacteria in the family Vibrionaceae (“vibrios”), in some instances providing a physical attachment site, inducing natural genetic competence, and serving as an attractant for chemotaxis. The marine luminous bacterium Vibrio fischeri is the specific symbiont in the light-emitting organ of the Hawaiian bobtail squid, Euprymna scolopes. The bacterium provides the squid with luminescence that the animal uses in an antipredatory defense, while the squid supports the symbiont's nutritional requirements. V. fischeri cells are harvested from seawater during each host generation, and V. fischeri is the only species that can complete this process in nature. Furthermore, chitin is located in squid hemocytes and plays a nutritional role in the symbiosis. We demonstrate here that chitin oligosaccharides produced by the squid host serve as a chemotactic signal for colonizing bacteria. V. fischeri uses the gradient of host chitin to enter the squid light organ duct and colonize the animal. We provide evidence that chitin serves a novel function in an animal-bacterial mutualism, as an animal-produced bacterium-attracting synomone. PMID:22522684

Schaefer, Amy L.; Brennan, Caitlin A.; Heath-Heckman, Elizabeth A. C.; DeLoney-Marino, Cindy R.; McFall-Ngai, Margaret J.

2012-01-01

318

Removal of copper, chromium, and arsenic from CCA-treated wood onto chitin and chitosan.  

PubMed

Chitin and chitosan are naturally abundant biopolymers which are of interest to research concerning the sorption of metal ions since the amine and hydroxyl groups on their chemical structures act as chelation sites for metal ions. This study evaluates the removal of copper, chromium, and arsenic elements from chromated copper arsenate (CCA)-treated wood via biosorption by chitin and chitosan. Exposing CCA-treated sawdust to various amounts of chitin and chitosan for 1, 5, and 10 days enhanced removal of CCA components compared to remediation by deionized water only. Remediation with a solution containing 2.5 g chitin for 10 days removed 74% copper, 62% chromium, and 63% arsenic from treated sawdust. Remediation of treated sawdust samples using the same amount of chitosan as chitin resulted in 57% copper, 43% chromium, and 30% arsenic removal. The results suggest that chitin and chitosan have a potential to remove copper element from CCA-treated wood. Thus, these more abundant natural amino polysaccharides could be important in the remediation of waste wood treated with the newest formulations of organometallic copper compounds and other water-borne wood preservatives containing copper. PMID:15474943

Kartal, S Nami; Imamura, Yuji

2005-02-01

319

Mammalian Ceramide Synthases  

PubMed Central

Summary In mammals, ceramide, a key intermediate in sphingolipid metabolism and an important signaling molecule, is synthesized by a family of six ceramide synthases (CerS), each of which synthesizes ceramides with distinct acyl chain lengths. There are a number of common biochemical features between the CerS, such as their catalytic mechanism, and their stucture and intracellular localization. Different CerS also display remarkable differences in their biological properties, with each of them playing distinct roles in processes as diverse as cancer and tumor suppression, in the response to chemotherapeutic drugs, in apoptosis, and in neurodegenerative diseases. PMID:20222015

Levy, Michal; Futerman, Anthony H.

2010-01-01

320

Synthesis and utilization of chitin humic acid hybrid as sorbent for Cr(III)  

NASA Astrophysics Data System (ADS)

New types of hybrid material have been synthesized by using four different methods of immobilization of humic acid (HA) on chitin. The most stable hybrid material toward the change of medium acidity was then utilized as sorbent for Cr(III). The HA was extracted from peat soil of Gambut District, South Kalimantan, Indonesia, using the recommended procedure of International Humic Substances Society (IHSS), while the chitin was isolated from crab shell waste through deproteination using 3.5% (w/v) NaOH and followed by removal of inorganic impurities using 1 M HCl. The four methods of immobilization of HA on chitin were (i) Method A: chitin powder (4 g) was gently poured into the stirred solution of 0.4 g HA in 40 mL of 0.01 M NaOH. After overnight stirring, the solid was separated, washed with water, and dried in oven at 70 °C. (ii) Method B: gelatinous chitin (40 g) in 250 mL of 0.5 M HCl was reacted with HA (4 g) in 500 mL of 0.5 M NaOH and aged for 24 h. The product was washed with water and dried. (iii) Method C: HA powder (0.5 g) was mixed with the stirred gel of chitin (2.5 g) in 60 mL of CaCl 2 saturated methanol and the mixture was then washed with the mixed solution of 25 mL of 2 M sodium citrate and ethylene glycol 1:1. The solid was separated, washed with water, and dried. (iv) Method D: the solution of HA (0.056 g) in 10 mL of 0.01 M NaOH was reacted with the gel of chitin (0.2 g) in 10 mL of CaCl 2 saturated methanol. After 24 h stirring, the solid was separated from the reaction medium, washed with the mixed solution of 2 M sodium citrate and ethylene glycol 1:1, and followed by washing with water and drying. Parameters investigated in this study consisted of the stability test of the immobilized HA, as well as the rate constant ( k1), capacity ( b), and energy ( E) of sorption as well as the rate constant of desorption ( k-1). The k1 and k-1 were determined according to a kinetic model of first order sorption reaching equilibrium, while the b and E were determined according to the Langmuir isotherm model. Compared to HA, Methods, A, C, and D; Method B produced the most stable immobilization of HA on chitin. The hybrid material (Chitin-HA) synthesized through Method B was stable in the acidity range that equivalent to pH 2.0-11.0. At the acidity giving maximum sorption, i.e. pH 5, the presence of immobilized HA on the Chitin-HA enhanced more than three times the k1 and k-1, i.e. from 0.057 min -1 and 8.51 × 10 -4 (min -1) (mol/L) for chitin to 0.183 min -1 and 3.27 × 10 -3 (min -1) (mol/L) for the Chitin-HA. On the contrary, the presence of HA on Chitin-HA only gave small increase on b and small decrease on E. The values of b and E for Cr(III) on chitin were 1.45 × 10 -2 mol/g and 23.12 kJ/mol, respectively, while those on Chitin-HA were 1.78 × 10 -2 mol/g and 19.95 kJ/mol, respectively.

Santosa, Sri Juari; Siswanta, Dwi; Sudiono, Sri; Sehol, Muhamad

2007-11-01

321

Effect of hydrolysis on heat capacity, thermodynamic functions, and the relaxation transition of crab chitin and chitosan  

NASA Astrophysics Data System (ADS)

The heat capacity of crab chitin and chitosan is measured in a vacuum adiabatic calorimeter at 10-330 K. The thermodynamic characteristics (enthalpy, entropy, and Gibbs function) are calculated at T ? 0 K to 330 K. Differential thermal analysis is used to calculate the relaxation transitions and thermal degradation of chitin and chitosan at 80-600 K. Acid hydrolysis is performed and its effect on the physicochemical properties and thermodynamic functions of chitin and chitosan is studied.

Kashtanov, E. A.; Uryash, V. F.; Kokurina, N. Yu.; Larina, V. N.

2014-02-01

322

A physicochemical characterization of fully acetylated chitin structure isolated from two spider species: with new surface morphology.  

PubMed

Spiders are a huge group which includes more than 44,000 species. But there has been no study of the chitin structure of spiders. In this study we physicochemically characterized chitin structure of two common spider species (Geolycosa vultuosa and Hogna radiata). Chitin content was determined as 8-8.5% for G. vultuosa and 6.5-7% for H. radiata. FTIR, TGA and XRD results showed that the chitin structures are in ?-form. Environmental scanning electron microscopy (ESEM) revealed that the surface morphology of each species is different. Chitin yielded from G. vultuosa has two different pore structures. The type one pore is rarely sequenced and its size ranges between 190 and 240 nm, while the type two pore is tightly sequenced and its size ranges between 11 and 32 nm. There is no information in previous studies about the chitin structure with two different pore morphologies. A new chitin surface morphology has been determined in G. vultuosa. The chitin isolated from H. radiata, has classic morphology: nanofibre structures (10-17 nm) and 195-260 nm sized pores. Acetylation degree of the chitin samples was calculated as 97% for G. vultuosa and 99% for H. radiata in accordance with elemental analysis results. PMID:24530368

Kaya, Murat; Seyyar, Osman; Baran, Talat; Erdo?an, Sevil; Kar, Musa

2014-04-01

323

Chitin Amendment Increases Soil Suppressiveness toward Plant Pathogens and Modulates the Actinobacterial and Oxalobacteraceal Communities in an Experimental Agricultural Field  

PubMed Central

A long-term experiment on the effect of chitin addition to soil on the suppression of soilborne pathogens was set up and monitored for 8 years in an experimental field, Vredepeel, The Netherlands. Chitinous matter obtained from shrimps was added to soil top layers on two different occasions, and the suppressiveness of soil toward Verticillium dahliae, as well as plant-pathogenic nematodes, was assessed, in addition to analyses of the abundances and community structures of members of the soil microbiota. The data revealed that chitin amendment had raised the suppressiveness of soil, in particular toward Verticillium dahliae, 9 months after the (second) treatment, extending to 2 years following treatment. Moreover, major effects of the added chitin on the soil microbial communities were detected. First, shifts in both the abundances and structures of the chitin-treated soil microbial communities, both of total soil bacteria and fungi, were found. In addition, the abundances and structures of soil actinobacteria and the Oxalobacteraceae were affected by chitin. At the functional gene level, the abundance of specific (family-18 glycoside hydrolase) chitinase genes carried by the soil bacteria also revealed upshifts as a result of the added chitin. The effects of chitin noted for the Oxalobacteraceae were specifically related to significant upshifts in the abundances of the species Duganella violaceinigra and Massilia plicata. These effects of chitin persisted over the time of the experiment. PMID:23811512

Cretoiu, Mariana Silvia; Korthals, Gerard W.; Visser, Johnny H. M.

2013-01-01

324

EFECTO DE LA CALIDAD DEL AGUA Y TAMAÑO DE PARTÍCULA EN LA PRODUCCIÓN DE QUITOSANO A PARTIR DE ?-QUITINA EXTRAÍDA DE DESPERDICIOS DE CALAMAR GIGANTE (Dosidicus gigas) EFFECT OF WATER QUALITY AND PARTICLE SIZE ON THE PRODUCTION OF CHITOSAN FROM ?-CHITIN ISOLATED FROM JUMBO SQUID PROCESSING WASTES (Dosidicus gigas)  

Microsoft Academic Search

Jumbo squid wastes represent an important source of ?-chitin, which recently has been studied for its properties and potential applications. ?-chitin is characterized for chains in parallel fashion with weaker intermolecular interactions than ?-chitin. Therefore ?-chitin is more soluble in common solvents as well as it display higher reactivity for deacetylation and chemical modification than ?-chitin. Preparation of ?-chitin was

Z. Rocha-Pino; K. Shirai; L. Arias; H. Vázquez-Torres

2008-01-01

325

Laboratory toxicity evaluation of Diflubenzuron, a chitin-synthesis inhibitor, against Anopheles darlingi (Diptera, Culicidae)  

Microsoft Academic Search

We evaluated Diflubenzuron toxicity against larvae and pupae of Anopheles darlingi. A series of bioassays were developed to assess the lethal concentrations LC50 and LC90 to larvae after two exposure periods (24 and 48 hours), and to evaluate its toxicity to 20-minute- and 24-hour-old pupae. The LC50 and LC90 obtained were 0.006 and 0.013 ppm, respectively. For concentrations of 0.01

Costa FM

2011-01-01

326

Interfacing whispering gallery mode optical microresonator biosensors with the plant defense elicitor chitin.  

PubMed

The biomaterial class of chitooligosaccharides (chitin), commonly found in insects and fungi, is one of the most abundant on earth. Substantial evidence implicates chitin in mediating a diverse array of plant cellular signaling events, including the induction of plant defense mechanisms against invading pests. However, these recognition and mediation mechanisms, including the binding kinetics between chitin and their plant recognition receptors, are not fully understood. Therefore, the creation of a platform capable of both interfacing with chitin and plant cell receptors, and monitoring their interactions, would significantly advance our understanding of this plant defense elicitor. Recently, a label-free, highly sensitive biosensor platform, based on Whispering Gallery Mode optical microresonators, has been developed to study such biomolecular interactions. Here, we demonstrate how this unique platform can be interfaced with chitin using simple carbohydrate chemistry. The surface chemistry is demonstrated using X-ray photoelectron spectroscopy, fluorescence microscopy, optical profilometry, ellipsometry, and contact angle measurements. The resulting surface is uniform, with an average surface roughness of 1.25nm, and is active toward chitin recognition elements. Optical loss measurements using standard quantitative cavity analysis techniques demonstrate that the bioconjugated platforms maintain the high performance (Q>10(6)) required to track binding interactions in this system. The platform is able to detect lectin, which binds COs, at 10?g/mL concentration. This biosensor platform's unique capabilities for label-free, high sensitivity biodetection, when properly interfaced with the biomaterials of interest, could provide the basis for a robust analytical technique to probe the binding dynamics of chitin-plant cell receptors. PMID:25051306

Dahmen, Jeremy L; Yang, Yongqiang; Greenlief, C Michael; Stacey, Gary; Hunt, Heather K

2014-10-01

327

The lysin motif receptor-like kinase (LysM-RLK) CERK1 is a major chitin-binding protein in Arabidopsis thaliana and subject to chitin-induced phosphorylation.  

PubMed

Plants detect potential pathogens by sensing microbe-associated molecular patterns via pattern recognition receptors. In the dicot model plant Arabidopsis, the lysin motif (LysM)-containing chitin elicitor receptor kinase 1 (CERK1) has been shown to be essential for perception of the fungal cell wall component chitin and for resistance to fungal pathogens. Recent in vitro studies with CERK1 protein expressed heterologously in yeast suggested direct chitin binding activity. Here we show in an affinity purification approach that CERK1 is a major chitin-binding protein of Arabidopsis cells, along with several known and putative chitinases. The ectodomain of CERK1 harbors three distinct LysM domains with potential ligand binding capacity. We demonstrate that the CERK1 ectodomain binds chitin and partially deacetylated chitosan directly without any requirement for interacting proteins and that all three LysM domains are necessary for chitin binding. Ligand-induced phosphorylation events are a general feature of animal and plant signal transduction pathways. Our studies show that chitin, chitin oligomers, and chitosan rapidly induce in vivo phosphorylation of CERK1 at multiple residues in the juxtamembrane and kinase domain. Functional analyses with a kinase dead variant provide evidence that kinase activity of CERK1 is required for its chitin-dependent in vivo phosphorylation, as well as for early defense responses and downstream signaling. Collectively, our data suggest that in Arabidopsis, CERK1 is a major chitin, chitosan, and chito-oligomer binding component and that chitin signaling depends on CERK1 post-translational modification and kinase activity. PMID:20610395

Petutschnig, Elena K; Jones, Alexandra M E; Serazetdinova, Liliya; Lipka, Ulrike; Lipka, Volker

2010-09-10

328

Characterization of potential drug targets farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase in Schistosoma mansoni.  

PubMed

Schistosomiasis affects over 200 million people worldwide, with over 200,000 deaths annually. Currently, praziquantel is the only drug available against schistosomiasis. We report here that Schistosoma mansoni farnesyl diphosphate synthase (SmFPPS) and geranylgeranyl diphosphate synthase (SmGGPPS) are potential drug targets for the treatment of schistosomiasis. We expressed active, recombinant SmFPPS and SmGGPPS for subsequent kinetic characterization and testing against a variety of bisphosphonate inhibitors. Recombinant SmFPPS was found to be a soluble 44.2-kDa protein, while SmGGPPS was a soluble 38.3-kDa protein. Characterization of the substrate utilization of the two enzymes indicates that they have overlapping substrate specificities. Against SmFPPS, several bisphosphonates had 50% inhibitory concentrations (IC50s) in the low micromolar to nanomolar range; these inhibitors had significantly less activity against SmGGPPS. Several lipophilic bisphosphonates were active against ex vivo adult worms, with worm death occurring over 4 to 6 days. These results indicate that FPPS and GGPPS could be of interest in the context of the emerging resistance to praziquantel in schistosomiasis therapy. PMID:24041901

Ziniel, Peter D; Desai, Janish; Cass, Cynthia L; Gatto, Craig; Oldfield, Eric; Williams, David L

2013-12-01

329

Design, synthesis and biological evaluation of N-alkyl or aryl substituted isoindigo derivatives as potential dual cyclin-dependent kinase 2 (CDK2)/glycogen synthase kinase 3? (GSK-3?) phosphorylation inhibitors.  

PubMed

A series of N-alkyl or aryl substituted isoindigo derivatives have been synthesized and their anti-proliferative activity was evaluated by Sulforhodamine B (SRB) assay. Some of the target compounds exhibited significant antitumor activity, including compounds 6h and 6k (against K562 cells), 6i (against HeLa cells) and 6j (against A549 cells). N-(p-methoxy-phenyl)-isoindigo (6k) exhibited a high and selective anti-proliferative activity against K562 cells (IC50 7.8 ?M) and induced the apoptosis of K562 cells in a dose-dependent manner. Compound 6k arrested the cell cycle at S phase in K562 cells by decreasing the expression of cyclin A and CDK2, which played critical roles in DNA replication and passage through G2 phase. Moreover, compound 6k down-regulated the expression of p-GSK-3? (Ser9), ?-catenin and c-myc proteins, up-regulated the expression of GSK-3?, consequently, suppressed Wnt/?-catenin signaling pathway and induced the apoptosis of K562 cells. The binding mode of compound 6k with GSK-3? was simulated using molecular docking tools. All of these studies gave a better understanding to the molecular mechanisms of this class of agents and clues to develop dual CDK2/GSK-3? (Ser9) phosphorylation inhibitors applied in cancer chemotherapy. PMID:25151579

Zhao, Ping; Li, Yanzhong; Gao, Guangwei; Wang, Shuai; Yan, Yun; Zhan, Xiaoping; Liu, Zenglu; Mao, Zhenmin; Chen, Shaoxiong; Wang, Liqun

2014-10-30

330

Chitin extraction from shrimp shell waste using Bacillus bacteria.  

PubMed

The ability of six protease-producing Bacillus species (Bacillus pumilus A1, Bacillus mojavencis A21, Bacillus licheniformis RP1, Bacillus cereus SV1, Bacillus amyloliquefaciens An6 and Bacillus subtilis A26) to ferment media containing only shrimp shell waste, for chitin extraction, was investigated. More than 80% deproteinization was attained by all the strains tested. However, demineralization rates not exceeding 67% were registered. Cultures conducted in media containing shrimp shell waste supplemented with 5% (w/v) glucose were found to remarkably promote demineralization efficiency, without affecting deproteinization rates. The antioxidant activities of hydrolysates, at different concentrations, produced during fermentation in medium supplemented with glucose, were determined using different tests: 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging method, reducing power assay and chelating activity. All hydrolysates showed varying degrees of antioxidant activity. Hydrolysate produced by B. pumilus A1 exhibited the highest DPPH radical scavenging activity, with an IC(50) value of 0.3 mg/ml. Highest reducing power (DO 700 nm=1.55 at 1.5 mg/ml) and metal chelating activity (98% at 5mg/ml) were obtained with B. pumilus A1 and B. licheniformis RP1 hydrolysates, respectively. PMID:22981824

Ghorbel-Bellaaj, Olfa; Younes, Islem; Maâlej, Hana; Hajji, Sawssen; Nasri, Moncef

2012-12-01

331

Multifunctional chitin nanogels for simultaneous drug delivery, bioimaging, and biosensing.  

PubMed

In this work, we developed biodegradable chitin nanogels (CNGs) by controlled regeneration method. For multifunctionalization, we have conjugated CNGs with MPA-capped-CdTe-QDs (QD-CNGs) for the in vitro cellular localization studies. In addition, the Bovine Serum Albumin (BSA) was loaded on to QD-CNGs (BSA-QD-CNGs). The CNGs, QD-CNGs, and BSA-QD-CNGs were well-characterized by SEM and AFM, which shows that the nanogels are in the range of <100 nm. These were further characterized by FT-IR and Cyclic Voltametry. The cytocompatibility assay showed that the nanogels are nontoxic to L929, NIH-3T3, KB, MCF-7, PC3, and VERO cells. The cell uptake studies of the QD-CNGs were analyzed, which showed retention of these nanogels inside the cells (L929, PC3, and VERO). In addition, the protein loading efficiency of the nano gels has also been analyzed. Our preliminary studies reveal that these multifunctionalized nanogels could be useful for drug delivery with simultaneous imaging and biosensing. PMID:21863797

Rejinold N, Sanoj; Chennazhi, Krishna Prasad; Tamura, Hiroshi; Nair, Shantikumar V; Rangasamy, Jayakumar

2011-09-01

332

Depletion of cellular cholesterol enhances macrophage MAPK activation by chitin microparticles but not by heat-killed Mycobacterium bovis BCG.  

PubMed

When macrophages phagocytose chitin (N-acetyl-d-glucosamine polymer) microparticles, mitogen-activated protein kinases (MAPK) are immediately activated, followed by the release of Th1 cytokines, but not IL-10. To determine whether phagocytosis and macrophage activation in response to chitin microparticles are dependent on membrane cholesterol, RAW264.7 macrophages were treated with methyl-beta-cytodextrin (MBCD) and stimulated with chitin. These results were compared with the corresponding effects of bacterial components including heat-killed (HK) Mycobacterium bovis bacillus Calmette-Guèrin (BCG) and an oligodeoxynucleotide (ODN) of bacterial DNA (CpG-ODN). The MBCD treatment did not alter chitin binding or the phagocytosis of chitin particles 20 min after stimulation. At the same time, however, chitin-induced phosphorylation of cellular MAPK was accelerated and enhanced in an MBCD dose-dependent manner. The increased phosphorylation was also observed for chitin phagosome-associated p38 and ERK1/2. In contrast, CpG-ODN and HK-BCG induced activation of MAPK in MBCD-treated cells at levels comparable to, or only slightly more than, those of control cells. We also found that MBCD treatment enhanced the production of tumor necrosis factor-alpha (TNF-alpha) and the expression of cyclooxygenase-2 (COX-2) in response to chitin microparticles. In neither MBCD- nor saline-treated macrophages, did chitin particles induce detectable IL-10 mRNA synthesis. CpG-ODN induced TNF-alpha production, and COX-2 expression were less sensitive to MBCD treatment. Among the agonists studied, our results indicate that macrophage activation by chitin microparticles was most sensitive to cholesterol depletion, suggesting that membrane structures integrated by cholesterol are important for physiological regulation of chitin microparticle-induced cellular activation. PMID:18524942

Nishiyama, Akihito; Shinohara, Tsutomu; Pantuso, Traci; Tsuji, Shoutaro; Yamashita, Makiko; Shinohara, Shizuka; Myrvik, Quentin N; Henriksen, Ruth Ann; Shibata, Yoshimi

2008-08-01

333

Structure of dihydrodipicolinate synthase from Methanocaldococcus jannaschii  

PubMed Central

In bacteria and plants, dihydrodipicolinate synthase (DHDPS) plays a key role in the (S)-lysine biosynthesis pathway. DHDPS catalyzes the first step of the condensation of (S)-aspartate-?-semialdehyde and pyruvate to form an unstable compound, (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. The activity of DHDPS is allosterically regulated by (S)-lysine, a feedback inhibitor. The crystal structure of DHDPS from Methanocaldococcus jannaschii (MjDHDPS) was solved by the molecular-replacement method and was refined to 2.2?Å resolution. The structure revealed that MjDHDPS forms a functional homo­tetramer, as also observed in Escherichia coli DHDPS, Thermotoga maritima DHDPS and Bacillus anthracis DHDPS. The binding-site region of MjDHDPS is essentially similar to those found in other known DHDPS structures. PMID:20054116

Padmanabhan, Balasundaram; Strange, Richard W.; Antonyuk, Svetlana V.; Ellis, Mark J.; Hasnain, S. Samar; Iino, Hitoshi; Agari, Yoshihiro; Bessho, Yoshitaka; Yokoyama, Shigeyuki

2009-01-01

334

Modification of Chitin with Kraft Lignin and Development of New Biosorbents for Removal of Cadmium(II) and Nickel(II) Ions  

PubMed Central

Novel, functional materials based on chitin of marine origin and lignin were prepared. The synthesized materials were subjected to physicochemical, dispersive-morphological and electrokinetic analysis. The results confirm the effectiveness of the proposed method of synthesis of functional chitin/lignin materials. Mechanism of chitin modification by lignin is based on formation of hydrogen bonds between chitin and lignin. Additionally, the chitin/lignin materials were studied from the perspective of waste water treatment. The synthetic method presented in this work shows an attractive and facile route for producing low-cost chitin/lignin biosorbents with high efficiency of nickel and cadmium adsorption (88.0% and 98.4%, respectively). The discovery of this facile method of synthesis of functional chitin/lignin materials will also have a significant impact on the problematic issue of the utilization of chitinous waste from the seafood industry, as well as lignin by-products from the pulp and paper industry. PMID:24727394

Wysokowski, Marcin; Klapiszewski, Lukasz; Moszynski, Dariusz; Bartczak, Przemyslaw; Szatkowski, Tomasz; Majchrzak, Izabela; Siwinska-Stefanska, Katarzyna; Bazhenov, Vasilii V.; Jesionowski, Teofil

2014-01-01

335

Modification of chitin with kraft lignin and development of new biosorbents for removal of cadmium(II) and nickel(II) ions.  

PubMed

Novel, functional materials based on chitin of marine origin and lignin were prepared. The synthesized materials were subjected to physicochemical, dispersive-morphological and electrokinetic analysis. The results confirm the effectiveness of the proposed method of synthesis of functional chitin/lignin materials. Mechanism of chitin modification by lignin is based on formation of hydrogen bonds between chitin and lignin. Additionally, the chitin/lignin materials were studied from the perspective of waste water treatment. The synthetic method presented in this work shows an attractive and facile route for producing low-cost chitin/lignin biosorbents with high efficiency of nickel and cadmium adsorption (88.0% and 98.4%, respectively). The discovery of this facile method of synthesis of functional chitin/lignin materials will also have a significant impact on the problematic issue of the utilization of chitinous waste from the seafood industry, as well as lignin by-products from the pulp and paper industry. PMID:24727394

Wysokowski, Marcin; Klapiszewski, ?ukasz; Moszy?ski, Dariusz; Bartczak, Przemys?aw; Szatkowski, Tomasz; Majchrzak, Izabela; Siwi?ska-Stefa?ska, Katarzyna; Bazhenov, Vasilii V; Jesionowski, Teofil

2014-04-01

336

Analysis of a change in bacterial community in different environments with addition of chitin or chitosan.  

PubMed

The temporal changes of a bacterial community in soil with chitin or chitosan added were analyzed by PCR-denaturing gradient gel electrophoresis (DGGE) targeting the 16S rRNA gene using total DNAs prepared from the community. Band patterns of PCR-DGGE confirmed that 31 species become predominant after the addition of chitin or chitosan. The determination of the nucleotide sequences of the bands of the 31 species indicated that 20 species belonged to the division Proteobacteria, and that the genus Cellvibrio was apparently predominant among them (7/20). The 16S rRNA sequences of the 16 deduced species (16/31) showed less than 98% similarities to those of previously identified bacteria, indicating that the species were derived from unidentified bacteria. The total community DNAs extracted from bacterial cells adsorbed on the surface of flakes of chitin and chitosan placed in a river, a moat, or soil were subjected to PCR-DGGE to examine the extent of diversity of chitinolytic bacteria among different environments. The predominant species significantly differed between the chitin and chitosan placed in the river and moat, but not so much between those placed in the soil. The large difference between the diversities of the three bacterial communities indicated that a wide variety of bacteria including unidentified ones are involved in the degradation of chitin and chitosan in the above-mentioned natural environments. PMID:20347770

Sato, Kazuaki; Azama, Yasuhito; Nogawa, Masahiro; Taguchi, Goro; Shimosaka, Makoto

2010-05-01

337

Thin-film Nanofibrous Composite Membranes Containing Cellulose or Chitin Barrier Layers Fabricated by Ionic Liquids  

SciTech Connect

The barrier layer of high-flux ultrafiltration (UF) thin-film nanofibrous composite (TFNC) membranes for purification of wastewater (e.g., bilge water) have been prepared by using cellulose, chitin, and a cellulose-chitin blend, regenerated from an ionic liquid. The structures and properties of regenerated cellulose, chitin, and a cellulose-chitin blend were analyzed with thermogravimetric analysis (TGA) and wide-angle X-ray diffraction (WAXD). The surface morphology, pore size and pore size distribution of TFNC membranes were determined by SEM images and molecular weight cut-off (MWCO) methods. An oil/water emulsion, a model of bilge water, was used as the feed solution, and the permeation flux and rejection ratio of the membranes were investigated. TFNC membranes based on the cellulose-chitin blend exhibited 10 times higher permeation flux when compared with a commercial UF membrane (PAN10, Sepro) with a similar rejection ratio after filtration over a time period of up to 100 h, implying the practical feasibility of such membranes for UF applications.

H Ma; B Hsiao; B Chu

2011-12-31

338

Proteomic analysis of insect molting fluid with a focus on enzymes involved in chitin degradation.  

PubMed

Cuticular chitin degradation is extremely important for insect growth and development, which has not been fully understood thus far. One obstacle to understanding this mechanism is the lack of a systematic analysis of the chitinolytic enzymes involved in cuticular chitin degradation. In this study, we used the silkmoth Bombyx mori as a model organism and compared proteomic analyses for larval-pupal (L-P) and pupal-adult (P-A) molting fluids using tandem mass tag quantitative mass spectrometry. There were 195 proteins identified from both L-P and P-A molting fluids. A total of 170 out of 195 proteins were deduced to be secretory and were enriched for GO terms associated with chitin metabolism and proteolysis by using AgriGO. Although the chitinolytic enzymes are encoded by many insect genes, the proteomics analysis unexpectedly showed that only four chitinolytic enzymes with the combination "211" were abundant in both molting fluids, namely, two insect GH18 Chitinase family members (ChtI and ChtII), one bacterial-type GH18 Chitinase (Chi-h), and one insect GH20 hexosaminidase (Hex1). A tissue-specific and stage-specific gene expression pattern verified that the "211" enzymes are involved in cuticular chitin degradation. This work first demonstrates that specific enzymes ChtI, ChtII, Chi-h, and Hex1 can be assigned to cuticular chitin degradation. PMID:24779478

Qu, Mingbo; Ma, Li; Chen, Peng; Yang, Qing

2014-06-01

339

Characterization of a Chitin-Binding Protein from Bacillus thuringiensis HD-1  

PubMed Central

Strains of Bacillus thuringiensis produce insecticidal proteins. These strains have been isolated from diverse ecological niches, such as soil, phylloplane, insect cadavers and grain dust. To effectively propagate, these strains produce a range of molecules that facilitate its multiplication in a competing environment. In this report, we have examined synthesis of a chitin-binding protein and evaluated its effect on fungi encountered in environment and its interaction with insecticidal proteins synthesized by B. thuringiensis. The gene encoding chitin-binding protein has been cloned and expressed. The purified protein has been demonstrated to interact with Cry insecticidal protein, Cry1Ac by Circular Dichrosim spectroscopy (CD) and in vitro pull down assays. The chitin-binding protein potentiates insecticidal activity of bacillar insecticidal protein, Cry1Ac. Further, chitin-binding protein was fungistatic against several soil fungi. The chitin binding protein is expressed in spore mother cell and deposited along with insecticidal protein, Cry1Ac. It interacts with Cry1Ac to potentiate its insecticidal activity and facilitate propagation of Bacillus strain in environment by inhibiting growth of certain fungi. PMID:23824872

Gupta, Rani; Vimala, Y.; Bhatnagar, Raj K.

2013-01-01

340

Chitin-Induced Gene Expression in Secondary Metabolic Pathways of Streptomyces coelicolor A3(2) Grown in Soil  

PubMed Central

Microarray analyses revealed that the expression of genes for secondary metabolism together with that of primary metabolic genes was induced by chitin in autoclaved soil cultures of Streptomyces coelicolor A3(2). The data also indicated that DasR was involved in the regulation of gene expression for chitin catabolism, secondary metabolism, and stress responses. PMID:23124229

Nazari, Behnam; Kobayashi, Michihiko; Saito, Akihiro; Hassaninasab, Azam; Miyashita, Kiyotaka

2013-01-01

341

Applying Molecular Dynamics Simulations to Identify Rarely Sampled Ligand-bound Conformational States of Undecaprenyl Pyrophosphate Synthase, an Antibacterial Target  

SciTech Connect

Undecaprenyl pyrophosphate synthase is a cis-prenyltransferase enzyme, which is required for cell wall biosynthesis in bacteria. Undecaprenyl pyrophosphate synthase is an attractive target for antimicrobial therapy. We performed long molecular dynamics simulations and docking studies on undecaprenyl pyrophosphate synthase to investigate its dynamic behavior and the influence of protein flexibility on the design of undecaprenyl pyrophosphate synthase inhibitors. We also describe the first X-ray crystallographic structure of Escherichia coli apo-undecaprenyl pyrophosphate synthase. The molecular dynamics simulations indicate that undecaprenyl pyrophosphate synthase is a highly flexible protein, with mobile binding pockets in the active site. By carrying out docking studies with experimentally validated undecaprenyl pyrophosphate synthase inhibitors using high- and low-populated conformational states extracted from the molecular dynamics simulations, we show that structurally dissimilar compounds can bind preferentially to different and rarely sampled conformational states. By performing structural analyses on the newly obtained apo-undecaprenyl pyrophosphate synthase and other crystal structures previously published, we show that the changes observed during the molecular dynamics simulation are very similar to those seen in the crystal structures obtained in the presence or absence of ligands. We believe that this is the first time that a rare 'expanded pocket' state, key to drug design and verified by crystallography, has been extracted from a molecular dynamics simulation.

Sinko, William; de Oliveira, César; Williams, Sarah; Van Wynsberghe, Adam; Durrant, Jacob D.; Cao, Rong; Oldfield, Eric; McCammon, J. Andrew (UIUC); (UCSD); (Hamilton)

2012-04-30

342

Expression and characterization of Bacillus licheniformis chitinase (ChiA), suitable for bioconversion of chitin waste.  

PubMed

Chitinase (EC 3.2.1.14) is an enzyme with multiple industrial applications. These include bioconversion of chitin waste, a highly resistant and abundant biopolymer from crustacean food industry, into glucosamine and chito-oligosaccharide value-added products. This paper reports on the expression of endochitinase (ChiA) from Bacillus licheniformis strain DSM8785 in E. coli and characterization of the recombinant enzyme. Recombinant ChiA could efficiently convert colloidal chitin to N-acetyl glucosamine and chitobiose at pH 4.0, 6.0 and 9.0 at 50 degrees C and retained its activity up to 3days under these conditions, suggesting that this enzyme is suitable for bioconversion of chitin waste. PMID:20133129

Songsiriritthigul, Chomphunuch; Lapboonrueng, Sasithorn; Pechsrichuang, Phornsiri; Pesatcha, Puntarika; Yamabhai, Montarop

2010-06-01

343

Evaluation of the effects of chitin nanofibrils on skin function using skin models.  

PubMed

Chitins are highly crystalline structures that are predominantly found in crustacean shells. Alpha-chitin is composed of microfibers, which are made up of nanofibrils that are 2-5 nm in diameter and 30 nm in length and embedded in a protein matrix. Crystalline nanofibrils can also be prepared by acid treatment. We verified the effect of chitin nanofibrils (NF) and nanocrystals (NC) on skin using a three-dimensional skin culture model and Franz cells. The application of NF and NC to skin improved the epithelial granular layer and increased granular density. Furthermore, NF and NC application to the skin resulted in a lower production of TGF-? compared to that of the control group. NF and NC might have protective effects to skin. Therefore, their potential use as components of skin-protective formulations merits consideration. PMID:24299799

Ito, Ikuko; Osaki, Tomohiro; Ifuku, Shinsuke; Saimoto, Hiroyuki; Takamori, Yoshimori; Kurozumi, Seiji; Imagawa, Tomohiro; Azuma, Kazuo; Tsuka, Takeshi; Okamoto, Yoshiharu; Minami, Saburo

2014-01-30

344

Facile production of chitin from crab shells using ionic liquid and citric acid.  

PubMed

Facile production of chitin from crab shells was performed by direct extraction using an ionic liquid, 1-allyl-3-methylimidazolium bromide (AMIMBr), followed by demineralization using citric acid. First, dried crab shells were treated with AMIMBr at elevated temperatures to extract chitin. Supernatants separated by centrifugation were then subjected to a chelating treatment with an aqueous solution of citric acid to achieve demineralization. The precipitated extracts were filtered and dried. The isolated material was subjected to X-ray diffraction, IR, (1)H NMR, and energy-dispersive X-ray spectroscopy, and thermal gravimetric analysis; the results indicated the structure of chitin. On the basis of the IR spectra, the degree of deacetylation in the samples obtained was calculated to be <7%. Furthermore, the protein content was <0.1% and the M(w) values were 0.7-2.2×10(5). PMID:22108289

Setoguchi, Tatsuya; Kato, Takeshi; Yamamoto, Kazuya; Kadokawa, Jun-ichi

2012-04-01

345

Bridging peripheral nerves using a deacetyl chitin conduit combined with short-term electrical stimulation.  

PubMed

Previous studies have demonstrated that deacetyl chitin conduit nerve bridging or electrical stimulation can effectively promote the regeneration of the injured peripheral nerve. We hypothesized that the combination of these two approaches could result in enhanced regeneration. Rats with right sciatic nerve injury were subjected to deacetyl chitin conduit bridging combined with electrical stimulation (0.1 ms, 3 V, 20 Hz, for 1 hour). At 6 and 12 weeks after treatment, nerve conduction velocity, myelinated axon number, fiber diameter, axon diameter and the thickness of the myelin sheath in the stimulation group were better than in the non-stimulation group. The results indicate that deacetyl chitin conduit bridging combined with temporary electrical stimulation can promote peripheral nerve repair. PMID:25206762

Zhang, Zhongli; Li, Xin; Zuo, Songjie; Xin, Jie; Zhang, Peixun

2014-05-15

346

Cytotoxicity and mechanical behavior of chitin-bentonite clay based polyurethane bio-nanocomposites.  

PubMed

Chitin based polyurethane bio-nanocomposites (PUBNC) were prepared using chitin, Delite HPS bentonite nanoclay enriched in montmorillonite (MMT), 4,4'-diphenylmethane diisocyanate (MDI) and polycaprolactone polyol CAPA 231 (3000 g/mol(-1)). The prepolymers having different concentration of Delite HPS bentonite nanoclay were extended with 2 moles of chitin. The structures of the resulted polymers were determined by FT-IR technique. The effect of nanoclay contents on mechanical properties and in vitro biocompatibility was investigated. The mechanical properties of the synthesized materials were improved with increase in the Delite HPS bentonite nanoclay contents. Optimum mechanical properties were obtained from the PU bio-nanocomposite samples having 4% Delite HPS bentonite nanoclay. The results revealed that the final PU bio-nanocomposite having 2% Delite HPS bentonite nanoclay contents is ideal contenders for surgical threads with on going investigations into their in vitro biocompatibility, non-toxicity, and mechanical properties. PMID:21945787

Zia, Khalid Mahmood; Zuber, Mohammad; Barikani, Mehdi; Hussain, Rizwan; Jamil, Tahir; Anjum, Sohail

2011-12-01

347

Transforming nanostructured chitin from crustacean waste into beneficial health products: a must for our society  

PubMed Central

Chitin, obtained principally from crustacean waste, is a sugar-like polymer that is available at low cost. It has been shown to be bio- and ecocompatible, and has a very low level of toxicity. Recently, it has become possible to industrially produce pure chitin crystals, named “chitin nanofibrils” (CN) for their needle-like shape and nanostructured average size (240 × 5 × 7 nm). Due to their specific chemical and physical characteristics, CN may have a range of industrial applications, from its use in biomedical products and biomimetic cosmetics, to biotextiles and health foods. At present, world offshore disposal of this natural waste material is around 250 billion tons per year. It is an underutilized resource and has the potential to supply a wide range of useful products if suitably recycled, thus contributing to sustainable growth and a greener economy. PMID:24198491

Morganti, P; Morganti, G; Morganti, A

2011-01-01

348

ARF6 Regulates Neuron Differentiation through Glucosylceramide Synthase  

PubMed Central

The small GTPase ADP ribosylation factor 6 (ARF6) mediates endocytosis and has in addition been shown to regulate neuron differentiation. Here we investigated whether ARF6 promotes differentiation of Neuro-2a neuronal cells by modifying the cellular lipid composition. We showed that knockdown of ARF6 by siRNA in Neuro-2a cells increased neuronal outgrowth as expected. ARF6 knockdown also resulted in increased glucosylceramide levels and decreased sphingomyelin levels, but did not affect the levels of ceramide or phospholipids. We speculated that the ARF6 knockdown-induced increase in glucosylceramide was caused by an effect on glucosylceramide synthase and, in agreement, showed that ARF6 knockdown increased the mRNA levels and activity of glucosylceramide synthase. Finally, we showed that incubation of Neuro-2a cells with the glucosylceramide synthase inhibitor D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) normalized the increased neuronal outgrowth induced by ARF6 knockdown. Our results thus show that ARF6 regulates neuronal differentiation through an effect on glucosylceramide synthase and glucosylceramide levels. PMID:23555901

Li, Lu; Ståhlman, Marcus; Rutberg, Mikael; Håversen, Liliana; Fogelstrand, Per; Andersson, Linda; Levin, Malin; Borén, Jan

2013-01-01

349

Elicitation of Diterpene Biosynthesis in Rice (Oryza sativa L.) by Chitin 1  

PubMed Central

Cell-free extracts of UV-irradiated rice (Oryza sativa L.) leaves have a much greater capacity for the synthesis from geranylgeranyl pyrophosphate of diterpene hydrocarbons, including the putative precursors of rice phytoalexins, than extracts of unstressed leaves (KA Wickham, CA West [1992] Arch Biochem Biophys 293: 320-332). An elicitor bioassay was developed on the basis of these observations in which 6-day-old rice cell suspension cultures were incubated for 40 hours with the substance to be tested, and an enzyme extract of the treated cells was assayed for its diterpene hydrocarbon synthesis activity as a measure of the response to elicitor. Four types of cell wall polysaccharides and oligosaccharide fragments that have elicitor activity for other plants were tested. Of these, polymeric chitin was the most active; a suspension concentration of approximately 7 micrograms per milliliter gave 50% of the maximum response in the bioassay. Chitosan and a branched ?-1,3-glucan fraction from Phytophthora megasperma f. sp. glycinea cell walls were only weakly active, and a mixture of oligogalacturonides was only slightly active. A crude mycelial cell wall preparation from the rice pathogen, Fusarium moniliforme, gave a response comparable to that of chitin, and this activity was sensitive to predigestion of the cell wall material with chitinase before the elicitor assay. N-Acetylglucosamine, chitobiose, chitotriose, and chitotetrose were inactive as elicitors, whereas a mixture of chitin fragments solubilized from insoluble chitin by partial acid hydrolysis was highly active. Constitutive chitinase activity was detected in the culture filtrate and enzyme extract of cells from a 6-day-old rice cell culture; the amount of chitinase activity increased markedly in both the culture filtrate and cell extracts after treatment of the culture with chitin. We propose on the basis of these results that soluble chitin fragments released from fungal cell walls through the action of constitutive rice chitinases serve as biotic elicitors of defense-related responses in rice. PMID:16668985

Ren, Yue-Ying; West, Charles A.

1992-01-01

350

Study of Fourier transform infrared spectra of cockroach nervous tissue and chitin  

NASA Astrophysics Data System (ADS)

Fourier Transform Infrared Spectroscopy (FTIR) is a very sensitive tool which is capable of providing strong insight on structural and functional changes in lipids and proteins induced by laser radiation. In the present work cockroach nervous tissue and chitin from tibia region are irradiated with Nd: YAG laser (?= 1064 nm, Power =150mW) via fiber optics (Numerical aperture=0.22, diameter = 8 ?). Nd: YAG laser exposure time is varied from 10 sec to 50 sec for nervous tissue and chitin. FTIR (Fourier Transform Infra Red spectra) of cockroach nervous tissue and chitin are compared before and after laser irradiation. The FTIR spectrum of non irradiated cockroach nervous tissue shows clearly the peaks due to O-H (Carboxylic acid), C=O (Amide I), C=C (Aromatic), N=0 (Nitro), C-H (Alkenes), CH (Aromatics). FTIR Spectra of non irradiated cockroach chitin clearly shows O-H (Carboxylic acid), C=O (Carbonyl stretch), C=C (Aromatic), N=O (Nitro), C-O, (anhydrides), C-H (Alkenes stretch) group. FTIR spectra of laser radiated nervous tissue from cockroach tibia and chitin shows significant changes in transmittance for O-H, C=O, C=C, C-H, N=O, C-O and C-H groups. The percentage transmittance increases for O-H, C=C group for exposure time 10sec, 40sec and 50 sec for nervous tissue. The percentage transmittance increases for O-H, C=C group for exposure time 10sec, 20sec, 30sec and 40 sec for chitin. The study shows clearly that FTIR spectroscopy of nervous tissue can reveal the interactions between infrared laser light and nervous tissue.

Ghadage, Vijay H.; Kulkarni, Gauri R.; Bhoraskar, Sudha V.

2012-03-01

351

Micro-CT Imaging of Denatured Chitin by Silver to Explore Honey Bee and Insect Pathologies  

PubMed Central

Background Chitin and cuticle coatings are important to the environmental and immune defense of honey bees and insect pollinators. Pesticides or environmental effects may target the biochemistry of insect chitin and cuticle coating. Denaturing of chitin involves a combination of deacetylation, intercalation, oxidation, Schweiger-peeling, and the formation of amine hydrochloride salt. The term “denatured chitin” calls attention to structural and property changes to the internal membranes and external carapace of organisms so that some properties affecting biological activities are diminished. Methodology/Principal Findings A case study was performed on honey bees using silver staining and microscopic computer-tomographic x-ray radiography (micro-CT). Silver nitrate formed counter-ion complexes with labile ammonium cations and reacted with amine hydrochloride. Silver was concentrated in the peritrophic membrane, on the abdomen, in the glossa, at intersegmental joints (tarsi), at wing attachments, and in tracheal air sacs. Imaged mono-esters and fatty acids from cuticle coating on external surfaces were apparently reduced by an alcohol pretreatment. Conclusions/Significance The technique provides 3-dimensional and sectional images of individual honey bees consistent with the chemistries of silver reaction and complex formation with denatured chitin. Environmental exposures and influences such as gaseous nitric oxide intercalant, trace oxidants such as ozone gas, oligosachharide salt conversion, exposure to acid rain, and chemical or biochemical denaturing by pesticides may be studied using this technique. Peritrophic membranes, which protect against food abrasion, microorganisms, and permit efficient digestion, were imaged. Apparent surface damage to the corneal lenses of compound eyes by dilute acid exposure consistent with chitin amine hydrochloride formation was imaged. The technique can contribute to existing insect pathology research, and may provide an additional tool for research on CCD. PMID:22110654

Butzloff, Peter R.

2011-01-01

352

The ion channel of F-ATP synthase is the target of toxic organotin compounds  

PubMed Central

ATP is the universal energy currency of living cells, and the majority of it is synthesized by the F1F0 ATP synthase. Inhibitors of this enzyme are therefore potentially detrimental for all life forms. Tributyltin chloride (TBT-Cl) inhibits ATP hydrolysis by the Na+-translocating ATP synthase of Ilyobacter tartaricus or the H+-translocating counterpart of Escherichia coli with apparent Ki of 200 nM. To target the site of this inhibition, we synthesized a tritium-labeled derivative of TBT-Cl in which one of the butyl groups was replaced by a photoactivatable aryldiazirine residue. Upon illumination, subunit a of the ATP synthase becomes specifically modified, and this labeling is suppressed in the presence of the original inhibitor. In case of the Na+ ATP synthase, labeling is also suppressed in the presence of Na+ ions, suggesting an interference in Na+ or TBT-Cl binding to subunit a. This interference is corroborated by the protection of ATP hydrolysis from TBT-Cl inhibition by 105 mM Na+. TBT-Cl strongly inhibits Na+ exchange by the reconstituted I. tartaricus ATP synthase. Taken together these results indicate that the subunit a ion channel is the target site for ATPase inhibition by toxic organotin compounds. An inhibitor interacting specifically with this site has not been reported previously. PMID:15277681

von Ballmoos, Christoph; Brunner, Josef; Dimroth, Peter

2004-01-01

353

Mechanism of Action and Inhibition of dehydrosqualene Synthase  

SciTech Connect

'Head-to-head' terpene synthases catalyze the first committed steps in sterol and carotenoid biosynthesis: the condensation of two isoprenoid diphosphates to form cyclopropylcarbinyl diphosphates, followed by ring opening. Here, we report the structures of Staphylococcus aureus dehydrosqualene synthase (CrtM) complexed with its reaction intermediate, presqualene diphosphate (PSPP), the dehydrosqualene (DHS) product, as well as a series of inhibitors. The results indicate that, on initial diphosphate loss, the primary carbocation so formed bends down into the interior of the protein to react with C2,3 double bond in the prenyl acceptor to form PSPP, with the lower two-thirds of both PSPP chains occupying essentially the same positions as found in the two farnesyl chains in the substrates. The second-half reaction is then initiated by the PSPP diphosphate returning back to the Mg{sup 2+} cluster for ionization, with the resultant DHS so formed being trapped in a surface pocket. This mechanism is supported by the observation that cationic inhibitors (of interest as antiinfectives) bind with their positive charge located in the same region as the cyclopropyl carbinyl group; that S-thiolo-diphosphates only inhibit when in the allylic site; activity results on 11 mutants show that both DXXXD conserved domains are essential for PSPP ionization; and the observation that head-to-tail isoprenoid synthases as well as terpene cyclases have ionization and alkene-donor sites which spatially overlap those found in CrtM.

F Lin; C Liu; Y Liu; Y Zhang; K Wang; W Jeng; T Ko; R Cao; A Wang; E Oldfield

2011-12-31

354

Statistical optimization for production of chitin deacetylase from Rhodococcus erythropolis HG05.  

PubMed

A strain producing chitin deacetylase (CDA) was isolated and identified as Rhodococcus erythropolis by morphological characteristics and 16S rDNA analysis, named as R. erythropolis HG05. By Plackett-Burman and central composite design, CDA production from R. erythropolis HG05 was increased from 58.00 U/mL to 238.89 U/mL. With the crude enzyme from R. erythropolis HG05, the hydrolysate components from colloid chitin were chito-oligosaccharides with polymerization number larger than hexaose. PMID:24507331

Sun, Yuying; Zhang, Jiquan; Wu, Shengjun; Wang, Shujun

2014-02-15

355

Isolation and identification of chitin in three-dimensional skeleton of Aplysina fistularis marine sponge.  

PubMed

The recent discovery of chitin within skeletons of numerous marine and freshwater sponges (Porifera) stimulates further experiments to identify this structural aminopolysaccharide in new species of these aquatical animals. Aplysina fistularis (Verongida: Demospongiae: Porifera) is well known to produce biologically active bromotyrosines. Here, we present a detailed study of the structural and physico-chemical properties of the three-dimensional skeletal scaffolds of this sponge. Calcofluor white staining, Raman and IR spectroscopy, ESI-MS as well as chitinase digestion test were applied in order to unequivocally prove the first discovery of ?-chitin in skeleton of A. fistularis. PMID:23994783

Wysokowski, Marcin; Bazhenov, Vasilii V; Tsurkan, Mikhail V; Galli, Roberta; Stelling, Allison L; Stöcker, Hartmut; Kaiser, Sabine; Niederschlag, Elke; Gärtner, Günter; Behm, Thomas; Ilan, Micha; Petrenko, Alexander Y; Jesionowski, Teofil; Ehrlich, Hermann

2013-11-01

356

Mimicking chitin: chemical synthesis, conformational analysis, and molecular recognition of the beta(1-->3) N-acetylchitopentaose analogue.  

PubMed

Mimicking Nature by using synthetic molecules that resemble natural products may open avenues to key knowledge that is difficult to access by using substances from natural sources. In this context, a novel N-acetylchitooligosaccharide analogue, beta-1,3-N-acetamido-gluco-pentasaccharide, has been designed and synthesized by using aminoglucose as the starting material. A phthalic group has been employed as the protecting group of the amine moiety, whereas a thioalkyl was used as the leaving group on the reducing end. The conformational properties of this new molecule have been explored and compared to those of the its chito analogue, with the beta-1,3 linkages, by a combined NMR spectroscopic/molecular modeling approach. Furthermore, the study of its molecular recognition properties towards two proteins, a lectin (wheat germ agglutinin) and one enzyme (a chitinase) have also been performed by using NMR spectroscopy and docking protocols. There are subtle differences in the conformational behavior of the mimetic versus the natural chitooligosaccharide, whereas this mimetic is still recognized by these two proteins and can act as a moderate inhibitor of chitin hydrolysis. PMID:20229531

Morando, Maria; Yao, Yanping; Martín-Santamaría, Sonsoles; Zhu, Zhenyuan; Xu, Tong; Cañada, F Javier; Zhang, Yongmin; Jiménez-Barbero, Jesús

2010-04-12

357

Nitric oxide synthase inhibition can initiate or prevent gut inflammation: Role of enzyme source  

Microsoft Academic Search

The role of nitric oxide in gut inflammation was evaluated by comparing the effects of selective or nonselective inhibitors of nitric oxide synthase (NOS). Aminoguanidine, a selective inducible NOS (iNOS) inhibitor, or NG-nitro-l-arginine methyl ester (l-NAME) were administered via the drinking water to normal guinea pigs or following induction of ileitis with trinitrobenzene sulfonic acid (TNBS 30 mg\\/kg). Aminoguanidine had

M. J. S. Miller; D. A. Clark

1994-01-01

358

Virtual Screening, Selection and Development of a Benzindolone Structural Scaffold for Inhibition of Lumazine Synthase  

PubMed Central

Virtual screening of a library of commercially available compounds vs. the structure of Mycobacterium tuberculosis lumazine synthase identified 2-(2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamido)acetic acid (9) as a possible lead compound. Compound 9 proved to be an effective inhibitor of M. tuberculosis lumazine synthase with a Ki of 70 ?M. Lead optimization through replacement of the carboxymethylsulfonamide sidechain with sulfonamides substituted with alkyl phosphates led to a four-carbon phosphate 38 that displayed a moderate increase in enzyme inhibitory activity (Ki 38 ?M). Molecular modeling based on known lumazine synthase/inhibitor crystal structures suggests that the main forces stabilizing the present benzindolone/enzyme complexes involve ?–? stacking interactions with Trp27 and hydrogen bonding of the phosphates with Arg128, the backbone nitrogens of Gly85 and Gln86, and the side chain hydroxyl of Thr87. PMID:20430628

Talukdar, Arindam; Morgunova, Ekaterina; Duan, Jianxin; Meining, Winfried; Foloppe, Nicolas; Nilsson, Lennart; Bacher, Adelbert; Illarionov, Boris; Fischer, Markus; Ladenstein, Rudolf; Cushman, Mark

2010-01-01

359

Binding and inhibition of human spermidine synthase by decarboxylated S-adenosylhomocysteine  

SciTech Connect

Aminopropyltransferases are essential enzymes that form polyamines in eukaryotic and most prokaryotic cells. Spermidine synthase (SpdS) is one of the most well-studied enzymes in this biosynthetic pathway. The enzyme uses decarboxylated S-adenosylmethionine and a short-chain polyamine (putrescine) to make a medium-chain polyamine (spermidine) and 5'-deoxy-5'-methylthioadenosine as a byproduct. Here, we report a new spermidine synthase inhibitor, decarboxylated S-adenosylhomocysteine (dcSAH). The inhibitor was synthesized, and dose-dependent inhibition of human, Thermatoga maritima, and Plasmodium falciparum spermidine synthases, as well as functionally homologous human spermine synthase, was determined. The human SpdS/dcSAH complex structure was determined by X-ray crystallography at 2.0 {angstrom} resolution and showed consistent active site positioning and coordination with previously known structures. Isothermal calorimetry binding assays confirmed inhibitor binding to human SpdS with K{sub d} of 1.1 {+-} 0.3 {mu}M in the absence of putrescine and 3.2 {+-} 0.1 {mu}M in the presence of putrescine. These results indicate a potential for further inhibitor development based on the dcSAH scaffold.

Š e; #269; kut; #279; , Jolita; McCloskey, Diane E.; Thomas, H. Jeanette; Secrist III, John A.; Pegg, Anthony E.; Ealick, Steven E. (Cornell); (Southern Research); (UPENN-MED)

2011-11-17

360

Prostaglandin H Synthase 2 is Expressed Abnormally in Human Colon Cancer: Evidence for a Transcriptional Effect  

Microsoft Academic Search

Evidence from epidemiological studies, clinical trials, and animal experiments indicates that inhibitors of prostaglandin synthesis lower the risk of colon cancer. We tested the hypothesis that abnormal expression of prostaglandin H synthase 2 (PHS-2), which can be induced by oncogenes and tumor promoters, occurs during colon carcinogenesis by examining its level in colon tumors. Human colon cancers were found to

William Kutchera; David A. Jones; Norisada Matsunami; Joanna Groden; Thomas M. McIntyre; Guy A. Zimmerman; Raymond L. White; Stephen M. Prescott

1996-01-01

361

Colloid chitin azure is a dispersible, low-cost substrate for chitinase measurements in a sensitive, fast, reproducible assay.  

PubMed

Chitin and its derivatives are widely used as biomedical materials because of their versatility and biocompatibility. Chitinases are enzymes that produce chito-oligosaccharides from chitin. The assay of chitinase activity is difficult because few appropriate substrates are available. In this study, the authors developed an efficient and low-cost chitinase assay using colloidal chitin azure. The assay feasibility is evaluated and compared with traditional assays employing colloidal chitin and chitin azure. The authors found that the optimum pH for determining chitinase activity using colloid chitin azure was pH 5 or 8. The method was sensitive, and the assay was complete within 30 min. When the assay was used to measure chitinase activities produced by 2 strains of chitinolytic bacteria, BCTS (an Escherichia coli BL21 [DE3] expressing a secretory recombinant chitinase) and AS1 (a chitinolytic bacterium with low levels of chitinase), it was shown that cultivation in Bushnell-Haas selection medium caused AS1 to secrete a higher level of chitinase than was secreted when the bacterium grew in other media. In summary, colloid chitin azure is a sensitive, feasible, reproducible, and low-cost substrate for the assay of chitinase activity. PMID:20042532

Shen, Chia-Rui; Chen, Yu-Sheng; Yang, Ching-Jen; Chen, Jeen-Kuan; Liu, Chao-Lin

2010-02-01

362

Suppression of Meloidogyne hapla and Its Damage to Lettuce Grown in a Mineral Soil Amended with Chitin and Biocontrol Organisms.  

PubMed

Chitin was used as soil amendment in fiberglass field microplots, alone or with one or a combination of two to three species of Hirsutella rhossiliensis, Paecilomyces marquandii, Verticillium chlamydosporium, Bacillus thuringiensis, and Streptomyces costaricanus. Sudangrass and rapeseed were planted as cover crops and incorporated into soil as green manure amendments. Chitin amendment alone increased the marketable yield of lettuce in 1995 and reduced root-galling ratings and the reproduction of Meloidogyne hapla in both 1995 and 1996. Green manure amendments of sudangrass and rapeseed increased total and marketable yields of lettuce, and decreased root-galling ratings and the reproduction of M. hapla in 1996. Hirsutella rhossiliensis in combination with chitin increased total yield of lettuce over the chitin amendment alone in 1995. The combination of B. thuringiensis, S. costaricanus, and chitin either with or without P. marquandii increased total yield of lettuce over the chitin amendment alone in 1996. In most cases, however, the nematode-antagonistic organisms did not improve lettuce yield or further suppression of M. hapla compared to the chitin amendment alone. The introduced fungi were recoverable from the infested soil. The rifampicin-resistant mutant of B. thuringiensis was not isolated at the end of the season. PMID:19270942

Chen, J; Abawi, G S; Zuckerman, B M

1999-12-01

363

Suppression of Meloidogyne hapla and Its Damage to Lettuce Grown in a Mineral Soil Amended with Chitin and Biocontrol Organisms  

PubMed Central

Chitin was used as soil amendment in fiberglass field microplots, alone or with one or a combination of two to three species of Hirsutella rhossiliensis, Paecilomyces marquandii, Verticillium chlamydosporium, Bacillus thuringiensis, and Streptomyces costaricanus. Sudangrass and rapeseed were planted as cover crops and incorporated into soil as green manure amendments. Chitin amendment alone increased the marketable yield of lettuce in 1995 and reduced root-galling ratings and the reproduction of Meloidogyne hapla in both 1995 and 1996. Green manure amendments of sudangrass and rapeseed increased total and marketable yields of lettuce, and decreased root-galling ratings and the reproduction of M. hapla in 1996. Hirsutella rhossiliensis in combination with chitin increased total yield of lettuce over the chitin amendment alone in 1995. The combination of B. thuringiensis, S. costaricanus, and chitin either with or without P. marquandii increased total yield of lettuce over the chitin amendment alone in 1996. In most cases, however, the nematode-antagonistic organisms did not improve lettuce yield or further suppression of M. hapla compared to the chitin amendment alone. The introduced fungi were recoverable from the infested soil. The rifampicin-resistant mutant of B. thuringiensis was not isolated at the end of the season. PMID:19270942

Chen, J.; Abawi, G. S.; Zuckerman, B. M.

1999-01-01

364

Beta-D-glycan synthases and the CesA gene family: lessons to be learned from the mixed-linkage (1-->3),(1-->4)beta-D-glucan synthase.  

PubMed

Cellulose synthase genes (CesAs) encode a broad range of processive glycosyltransferases that synthesize (1-->4)beta-D-glycosyl units. The proteins predicted to be encoded by these genes contain up to eight membrane-spanning domains and four 'U-motifs' with conserved aspartate residues and a QxxRW motif that are essential for substrate binding and catalysis. In higher plants, the domain structure includes two plant-specific regions, one that is relatively conserved and a second, so-called 'hypervariable region' (HVR). Analysis of the phylogenetic relationships among members of the CesA multi-gene families from two grass species, Oryza sativa and Zea mays, with Arabidopsis thaliana and other dicotyledonous species reveals that the CesA genes cluster into several distinct sub-classes. Whereas some sub-classes are populated by CesAs from all species, two sub-classes are populated solely by CesAs from grass species. The sub-class identity is primarily defined by the HVR, and the sequence in this region does not vary substantially among members of the same sub-class. Hence, we suggest that the region is more aptly termed a 'class-specific region' (CSR). Several motifs containing cysteine, basic, acidic and aromatic residues indicate that the CSR may function in substrate binding specificity and catalysis. Similar motifs are conserved in bacterial cellulose synthases, the Dictyostelium discoideum cellulose synthase, and other processive glycosyltransferases involved in the synthesis of non-cellulosic polymers with (1-->4)beta-linked backbones, including chitin, heparan, and hyaluronan. These analyses re-open the question whether all the CesA genes encode cellulose synthases or whether some of the sub-class members may encode other non-cellulosic (1-->4)beta-glycan synthases in plants. For example, the mixed-linkage (1-->3)(1-->4)beta-D-glucan synthase is found specifically in grasses and possesses many features more similar to those of cellulose synthase than to those of other beta-linked cross-linking glycans. In this respect, the enzymatic properties of the mixed-linkage beta-glucan synthases not only provide special insight into the mechanisms of (1-->4)beta-glycan synthesis but may also uncover the genes that encode the synthases themselves. PMID:11554469

Vergara, C E; Carpita, N C

2001-09-01

365

Characteristics of deacetylation and depolymerization of ?-chitin from jumbo squid ( Dosidicus gigas) pens  

Microsoft Academic Search

This study evaluated the deacetylation characteristics of ?-chitin from jumbo squid (Dosidicus gigas) pens by using strongly alkaline solutions of NaOH or KOH. Taguchi design was employed to investigate the effect of reagent concentration, temperature, time, and treatment step on molecular mass (MM) and degree of deacetylation (DDA) of the chitosan obtained. The optimal treatment conditions for achieving high MM

Jooyeoun Jung; Yanyun Zhao

2011-01-01

366

Study on radiation-induced grafting of styrene onto chitin and chitosan  

NASA Astrophysics Data System (ADS)

Radiation-induced grafting of styrene onto chitin and chitosan powder was performed at room temperature. The effect of various conditions such as absorbed dose, solvent and oxygen on grafting was investigated. The grafting yield increased with the increase in absorbed dose. At the same dose, the grafting yield of styrene on chitosan was higher than that on chitin. The grafting reaction was promoted in the presence of methanol, and oxygen delayed the grafting reaction but did not inhibit it completely. In order to study the mechanism of grafting reaction and analyze the grafted samples, the grafted products were extracted first by benzene, then they were hydrolyzed in the presence of acid and separated by thin layer chromatography (TLC). Three different kinds of polystyrene (PS), i.e. PS grafted onto chitin, PS embedded in chitin and PS in grafting solution were gained. The variation of their molecular weight (MW) and width index of molecular weight distribution ( Mw/ Mn) determined by gel permeation chromatography (GPC) at different grafting conditions is discussed.

Pengfei, Liu; Maolin, Zhai; Jilan, Wu

2001-05-01

367

A New Type I Peritrophic Membrane Protein from Larval Holotrichia oblita (Coleoptera: Melolonthidae) Binds to Chitin  

PubMed Central

Peritrophic membranes (PMs) are composed of chitin and protein. Chitin and protein play important roles in the structural formation and function of the PM. A new type I PM protein, HoCBP76, was identified from the Holotrichia oblita. HoCBP76 was shown as a 62.3 kDa protein by SDS-PAGE analysis and appeard to be associated with the PM throughout its entire length. In H. oblita larvae, the midgut is the only tissue where HoCBP76 could be detected during the feeding period of the larvae. The predicted amino acid sequence indicates that it contains seven tandem chitin binding domains belonging to the peritrophin-A family. HoCBP76 has chitin binding activity and is strongly associated with the PM. The HoCBP76 was not a mucin-like glycoprotein, and the consensus of conserved cysteines appeared to be CX13–17CX5CX9CX12CX7C. Western blot analysis showed that the abundance of HoCBP76 in the anterior, middle and posterior regions of the midgut was similar, indicating that HoCBP76 was secreted by the whole midgut epithelium, and confirmed the H. oblita PM belonged to the Type I PM. Immunolocalization analysis showed that HoCBP76 was mainly localized in the PM. The HoCBP76 is the first PM protein found in the H. oblita; however, its biochemical and physiological functions require further investigation. PMID:24758927

Liu, Xiaomin; Li, Jie; Guo, Wei; Li, Ruijun; Zhao, Dan; Li, Xinna

2014-01-01

368

Functional Monomers and Polymers. 90 Radiation-Induced Graft Polymerization of Styrene onto Chitin and Chitosan  

Microsoft Academic Search

The graft polymerization of styrene onto chitin powder, and chitosan powder and film initiated by ?-ray irradiation was carried out at 30°C. The graft polymerization was found to proceed predominantly in the presence of water, and the degree of grafting increased with an increase in the radiation dose. From ESR spectroscopy it was noted that the free radicals generated both

Yasuhiro Shigeno; Koichi Kondo; Kiichi Takemoto

1982-01-01

369

Serratia marcescens is one of the most effective bacteria for degradation of chitin, a  

E-print Network

in a variety of organisms, varying from prokaryotes to man (reviewed in 1 - 7). In chitin-containing organisms, whereas plants produce chitinases as part of their defence against fungal pathogens. Many bacteria) peptide Reference 57 - 58 chiA (ChiA) extracellular yes 27 - 29, 32 52 - 54 chiB (ChiB) periplasm / no 27

van Aalten, Daan

370

A new type I peritrophic membrane protein from larval Holotrichia oblita (Coleoptera: Melolonthidae) binds to chitin.  

PubMed

Peritrophic membranes (PMs) are composed of chitin and protein. Chitin and protein play important roles in the structural formation and function of the PM. A new type I PM protein, HoCBP76, was identified from the Holotrichia oblita. HoCBP76 was shown as a 62.3 kDa protein by SDS-PAGE analysis and appeard to be associated with the PM throughout its entire length. In H. oblita larvae, the midgut is the only tissue where HoCBP76 could be detected during the feeding period of the larvae. The predicted amino acid sequence indicates that it contains seven tandem chitin binding domains belonging to the peritrophin-A family. HoCBP76 has chitin binding activity and is strongly associated with the PM. The HoCBP76 was not a mucin-like glycoprotein, and the consensus of conserved cysteines appeared to be CX13-17CX5CX9CX12CX7C. Western blot analysis showed that the abundance of HoCBP76 in the anterior, middle and posterior regions of the midgut was similar, indicating that HoCBP76 was secreted by the whole midgut epithelium, and confirmed the H. oblita PM belonged to the Type I PM. Immunolocalization analysis showed that HoCBP76 was mainly localized in the PM. The HoCBP76 is the first PM protein found in the H. oblita; however, its biochemical and physiological functions require further investigation. PMID:24758927

Liu, Xiaomin; Li, Jie; Guo, Wei; Li, Ruijun; Zhao, Dan; Li, Xinna

2014-01-01

371

Chitin deacetylase family genes in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae).  

PubMed

Chitin deacetylases (CDAs) are enzymes required for one of the pathways of chitin degradation, in which chitosan is produced by the deacetylation of chitin. Bioinformatic investigations with genomic and transcriptomic databases identified four genes encoding CDAs in Nilaparvata lugens (NlCDAs). Phylogenetic analysis showed that insect CDAs were clustered into five major groups. Group I, III and IV CDAs are found in all insect species, whereas the pupa-specific group II and gut-specific group V CDAs are not found in the plant-sap/blood-sucking hemimetabolous species from Hemiptera and Anoplura. The developmental and tissue-specific expression patterns of four NlCDAs revealed that NlCDA3 was a gut-specific CDA, with high expression at all developmental stages; NlCDA1, NlCDA2 and NlCDA4 were highly expressed in the integument and peaked periodically during every moulting, which suggests their roles in chitin turnover of the insect old cuticle. Lethal phenotypes of cuticle shedding failure and high mortality after the injection of double-stranded RNAs (dsRNAs) for NlCDA1, NlCDA2 and NlCDA4 provide further evidence for their functions associated with moulting. No observable morphological and internal structural abnormality was obtained in insects treated with dsRNA for gut-specific NlCDA3. PMID:24989071

Xi, Y; Pan, P-L; Ye, Y-X; Yu, B; Zhang, C-X

2014-12-01

372

Genetics Home Reference: GM3 synthase deficiency  

MedlinePLUS

... GM3 synthase deficiency is characterized by recurrent seizures (epilepsy) and problems with brain development. Within the first ... synthase deficiency and may include treatment providers. American Epilepsy Society: Find a Doctor Clinic for Special Children ( ...

373

Are rod outer segment ATP-ase and ATP-synthase activity expression of the same protein?  

PubMed

Vertebrate retinal rod outer segments (OS) consist of a stack of disks surrounded by the plasma membrane, where phototransduction takes place. Energetic metabolism in rod OS remains obscure. Literature described a so-called Mg(2+)-dependent ATPase activity, while our previous results demonstrated the presence of oxidative phosphorylation (OXPHOS) in OS, sustained by an ATP synthetic activity. Here we propose that the OS ATPase and ATP synthase are the expression of the same protein, i.e., of F1Fo-ATP synthase. Imaging on bovine retinal sections showed that some OXPHOS proteins are expressed in the OS. Biochemical data on bovine purified rod OS, characterized for purity, show an ATP synthase activity, inhibited by classical F1Fo-ATP synthase inhibitors. Moreover, OS possess a pH-dependent ATP hydrolysis, inhibited by pH values below 7, suggestive of the functioning of the inhibitor of F1 (IF1) protein. WB confirmed the presence of IF1 in OS, substantiating the expression of F1Fo ATP synthase in OS. Data suggest that the OS F1Fo ATP synthase is able to hydrolyze or synthesize ATP, depending on in vitro or in vivo conditions and that the role of IF1 would be pivotal in the prevention of the reversal of ATP synthase in OS, for example during hypoxia, granting photoreceptor survival. PMID:23568658

Calzia, Daniela; Candiani, Simona; Garbarino, Greta; Caicci, Federico; Ravera, Silvia; Bruschi, Maurizio; Manni, Lucia; Morelli, Alessandro; Traverso, Carlo Enrico; Candiano, Giovanni; Tacchetti, Carlo; Panfoli, Isabella

2013-07-01

374

[The composition of the chitinolytic microbial complex and its effect on chitin decomposition at various humidity levels].  

PubMed

The dynamics of assimilation of chitin by soil microorganisms (primarily prokaryotes) as a source of carbon and nitrogen has been determined by gas chromatography and fluorescence microscopy. The highest rates of chitin decomposition in chernozem were detected at humidity levels corresponding to the pressure of soil moisture (P) of -1.4 atm. The rate of microbial consumption of chitin is three times higher than that of the carbon of soil organic matter. Fluorescence microscopy revealed that an increase in the pressure of soil moisture from P = -10 atm to P = -0.7 atm resulted in a considerable increase in the proportion of the specific surface of mycelial bacteria (actinomycetes). PMID:18069323

Vorob'ev, A V; Manucharova, N A; Iaroslavtsev, A M; Belova, E V; Zviagintsev, D G; Sudnitsyn, I I

2007-01-01

375

An insecticidal GroEL protein with chitin binding activity from Xenorhabdus nematophila.  

PubMed

Xenorhabdus nematophila secretes insecticidal proteins to kill its larval prey. We have isolated an approximately 58-kDa GroEL homolog, secreted in the culture medium through outer membrane vesicles. The protein was orally insecticidal to the major crop pest Helicoverpa armigera with an LC50 of approximately 3.6 microg/g diet. For optimal insecticidal activity all three domains of the protein, apical, intermediate, and equatorial, were necessary. The apical domain alone was able to bind to the larval gut membranes and manifest low level insecticidal activity. At equimolar concentrations, the apical domain contained approximately one-third and the apical-intermediate domain approximately one-half bioactivity of that of the full-length protein. Interaction of the protein with the larval gut membrane was specifically inhibited by N-acetylglucosamine and chito-oligosaccharides. Treatment of the larval gut membranes with chitinase abolished protein binding. Based on the three-dimensional structural model, mutational analysis demonstrated that surface-exposed residues Thr-347 and Ser-356 in the apical domain were crucial for both binding to the gut epithelium and insecticidal activity. Double mutant T347A,S356A was 80% less toxic (p < 0.001) than the wild type protein. The GroEL homolog showed alpha-chitin binding activity with Kd approximately 0.64 microm and Bmax approximately 4.68 micromol/g chitin. The variation in chitin binding activity of the mutant proteins was in good agreement with membrane binding characteristics and insecticidal activity. The less toxic double mutant XnGroEL showed an approximately 8-fold increase of Kd in chitin binding assay. Our results demonstrate that X. nematophila secretes an insecticidal GroEL protein with chitin binding activity. PMID:18667427

Joshi, Mohan Chandra; Sharma, Animesh; Kant, Sashi; Birah, Ajanta; Gupta, Gorakh Prasad; Khan, Sharik R; Bhatnagar, Rakesh; Banerjee, Nirupama

2008-10-17

376

An Insecticidal GroEL Protein with Chitin Binding Activity from Xenorhabdus nematophila*  

PubMed Central

Xenorhabdus nematophila secretes insecticidal proteins to kill its larval prey. We have isolated an ?58-kDa GroEL homolog, secreted in the culture medium through outer membrane vesicles. The protein was orally insecticidal to the major crop pest Helicoverpa armigera with an LC50 of ?3.6 ?g/g diet. For optimal insecticidal activity all three domains of the protein, apical, intermediate, and equatorial, were necessary. The apical domain alone was able to bind to the larval gut membranes and manifest low level insecticidal activity. At equimolar concentrations, the apical domain contained approximately one-third and the apical-intermediate domain approximately one-half bioactivity of that of the full-length protein. Interaction of the protein with the larval gut membrane was specifically inhibited by N-acetylglucosamine and chito-oligosaccharides. Treatment of the larval gut membranes with chitinase abolished protein binding. Based on the three-dimensional structural model, mutational analysis demonstrated that surface-exposed residues Thr-347 and Ser-356 in the apical domain were crucial for both binding to the gut epithelium and insecticidal activity. Double mutant T347A,S356A was 80% less toxic (p < 0.001) than the wild type protein. The GroEL homolog showed ?-chitin binding activity with Kd ? 0.64 ?m and Bmax ? 4.68 ?mol/g chitin. The variation in chitin binding activity of the mutant proteins was in good agreement with membrane binding characteristics and insecticidal activity. The less toxic double mutant XnGroEL showed an ?8-fold increase of Kd in chitin binding assay. Our results demonstrate that X. nematophila secretes an insecticidal GroEL protein with chitin binding activity. PMID:18667427

Joshi, Mohan Chandra; Sharma, Animesh; Kant, Sashi; Birah, Ajanta; Gupta, Gorakh Prasad; Khan, Sharik R.; Bhatnagar, Rakesh; Banerjee, Nirupama

2008-01-01

377

Biphenyl synthase, a novel type III polyketide synthase.  

PubMed

Biphenyls and dibenzofurans are the phytoalexins of the Maloideae, a subfamily of the economically important Rosaceae. The carbon skeleton of the two classes of antimicrobial secondary metabolites is formed by biphenyl synthase (BIS). A cDNA encoding this key enzyme was cloned from yeast-extract-treated cell cultures of Sorbus aucuparia. BIS is a novel type III polyketide synthase (PKS) that shares about 60% amino acid sequence identity with other members of the enzyme superfamily. Its preferred starter substrate is benzoyl-CoA that undergoes iterative condensation with three molecules of malonyl-CoA to give 3,5-dihydroxybiphenyl via intramolecular aldol condensation. BIS did not accept CoA-linked cinnamic acids such as 4-coumaroyl-CoA. This substrate, however, was the preferential starter molecule for chalcone synthase (CHS) that was also cloned from S. aucuparia cell cultures. While BIS expression was rapidly, strongly and transiently induced by yeast extract treatment, CHS expression was not. In a phylogenetic tree, BIS grouped together closely with benzophenone synthase (BPS) that also uses benzoyl-CoA as starter molecule but cyclizes the common intermediate via intramolecular Claisen condensation. The molecular characterization of BIS thus contributes to the understanding of the functional diversity and evolution of type III PKSs. PMID:17109150

Liu, B; Raeth, T; Beuerle, T; Beerhues, L

2007-05-01

378

Discovery and development of neuronal nitric oxide synthase inhibitors  

Microsoft Academic Search

The role of neuronally derived nitric oxide (NO) in neurotransmission and neural injury remains an area of active investigation. NO generation has been postulated to be involved in the deleterious events surrounding ischemia\\/reperfusion injury either directly or via the production of more reactive oxidants such as peroxynitrite. In our search for novel therapeutics for the treatment of a variety of

David W Reif; Dennis J McCarthy; Ed Cregan; James E Macdonald

2000-01-01

379

Induction of 1-aminocyclopropane-1-carboxylate synthase mRNA by auxin in mung bean hypocotyls and cultured apple shoots.  

PubMed

Auxin is known to promote ethylene production in vegetative tissues by increasing the activity of 1-aminocyclopropane-1-carboxylate (ACC) synthase; therefore, we have studied the effect of auxins on ACC synthase mRNA expression. Total RNA was isolated from auxin-incubated cultured apple (Malus sylvestris Mill.) shoots or mung bean (Vigna radiata L.) hypocotyls. These RNAs and a set of oligonucleotide primers corresponding to two conserved amino acid sequences (SNPLGTT and MSSFGLV) found in ACC synthases isolated from other species were used for polymerase chain reaction-based amplification of DNA fragments encoding the ACC synthase-active site domain. We obtained and sequenced a 290-base pair cDNA fragment (pAA1) from cultured apple shoots and a 328-base pair cDNA clone (pMBA1) from mung bean hypocotyls. Comparisons of their deduced amino acid sequences with those of previously characterized ACC synthase cDNAs indicate that both fragments are, indeed, closely related to ACC synthase cDNA. Northern blot analyses further showed that the expression of these transcripts is regulated by auxin treatment. These data indicate that auxin induces ethylene production transcriptionally by increasing the ACC synthase transcripts. The pAA1 shares 46% amino acid sequence homology with ripening-regulated apple fruit ACC synthase, indicating that ripening-regulated and auxin-regulated ACC synthases are encoded by different genes. In mung bean hypocotyls, aminooxyacetic acid, a potent inhibitor of ACC synthase activity, promoted the expression of auxin-induced ACC synthase mRNA, but cycloheximide inhibited this induction. PMID:16668663

Kim, W T; Silverstone, A; Yip, W K; Dong, J G; Yang, S F

1992-02-01

380

Alkali- or acid-induced changes in structure, moisture absorption ability and deacetylating reaction of ?-chitin extracted from jumbo squid (Dosidicus gigas) pens.  

PubMed

Alkali- or acid-induced structural modifications in ?-chitin from squid (Dosidicus gigas, d'Orbigny, 1835) pens and their moisture absorption ability (MAA) and deacetylating reaction were investigated and compared with ?-chitin from shrimp shells. ?-Chitin was converted into the ?-form after 3h in 40% NaOH or 1-3 h in 40% HCl solution, and ?-chitin obtained from NaOH treatment had higher MAA than had native ?-chitin, due to polymorphic destructions. In contrast, induced ?-chitin from acid treatment of ?-chitin had few polymorphic modifications, showing no significant change (P>0.05) in MAA. ?-Chitin was more susceptible to alkali deacetylation than was ?-chitin, and required a lower concentration of NaOH and shorter reaction time. These results demonstrate that alkali- or acid-treated ?-chitin retained high susceptibility toward solvents, which in turn resulted in good biological activity of ?-chitosan for use as a natural antioxidant and antimicrobial substance or application as edible coatings and films for various food applications. PMID:24444948

Jung, Jooyeoun; Zhao, Yanyun

2014-01-01

381

A chitinolytic endochitinase and ?-N-acetylglucosaminidase-based system from Hevea latex in generating N-acetylglucosamine from chitin.  

PubMed

An endochitinase and ?-N-acetylglucosaminidase (NAGase) were purified and characterised from fresh rubber latex serum. These enzymes were used in a total enzyme-based system to produce pure N-acetylglucosamine (NAG) from chitin. The N-terminal amino acid sequences of both purified endochitinase (KEESRRRRHR) and NAGase (AAVDSDTLEI) lacked homology with other known chitinases, including hevamine from rubber latex lutoids. The apparent kinetic parameters, Km and Vmax, for the endochitinase using 4-MU-?-(NAG)3 as a substrate were 99.73 ?M and 29.49 pkat mg(-1), respectively. For NAGase, using 4-MU-?-NAG as a substrate, the corresponding Km and Vmax values were 20.4 ?M and 25.82 pkat mg(-1). When an enzyme incubation mixture containing a 1:1 (pkat/pkat) activity mixed ratio of endochitinase: NAGase was employed, the maximum yield of N-acetylglucosamine (NAG) obtained was 98% from ?-chitin and 20% from ?-chitin. These yields were obtained after 4 days of hydrolysis of equal amounts of ?-chitin and ?-chitin in the mixture. Thus, ?-chitin was the preferred substrate compared to ?-chitin by a ratio of nearly five to one. Mass spectroscopic analysis, using electrospray ionisation mass spectrometry (ESI-MS), of the product obtained from ?-chitin after digestion (for 24h) depicted one distinct major molecular ion peak m/z 260.1, a small minor ion peak m/z 481.2, a potassium adduct of NAG and a potassium adduct of two NAG molecules. Furthermore, experiments to establish the commercial production of NAG using crude enzymes of Hevea latex serum are currently in progress. PMID:24833032

Sukprasirt, Pannawich; Wititsuwannakul, Rapepun

2014-08-01

382

Functional analyses of the chitin-binding domains and the catalytic domain of Brassica juncea chitinase BjCHI1  

Microsoft Academic Search

We previously isolated a Brassica juncea cDNA encoding BjCHI1, a novel chitinase with two chitin-binding domains. Synthesis of its mRNA is induced by wounding, methyl jasmonate treatment, Aspergillus niger infection and caterpillar Pieris rapae feeding, suggesting that the protein has a role in defense. In that it possesses two chitin-binding domains, BjCHI1 resembles the precursor of Urtica dioica agglutinin but

Ce Mun Tang; Mee-Len Chye; Sathishkumar Ramalingam; Shi-Wen Ouyang; Kai-Jun Zhao; Wimal Ubhayasekera; Sherry L. Mowbray

2004-01-01

383

Measurement of the extraradical mycelium of a vesicular-arbuscular mycorrhizal fungus in soil by chitin determination  

Microsoft Academic Search

Summary  Development of a vesicular-arbuscular mycorrhizal (VAM) fungus in association with soybean was determined in a greenhouse\\u000a soil mix by chitin assay. Samples were sieved to eliminate hexosamine-containing contaminants. This preparation reduced the\\u000a interference caused by extraneous soil substances and permitted quantitative measurement of extraradical VAM fungal mycelium\\u000a in the soil mix by colorimetric assay. Recovery of added chitin, used as

R. S. Pacovsky; G. J. Bethlenfalvay

1982-01-01

384

Resistance Phenotypes Mediated by Aminoacyl-Phosphatidylglycerol Synthases  

PubMed Central

The specific aminoacylation of the phospholipid phosphatidylglycerol (PG) with alanine or with lysine catalyzed by aminoacyl-phosphatidylglycerol synthases (aaPGS) was shown to render various organisms less susceptible to antibacterial agents. This study makes use of Pseudomonas aeruginosa chimeric mutant strains producing lysyl-phosphatidylglycerol (L-PG) instead of the naturally occurring alanyl-phosphatidylglycerol (A-PG) to study the resulting impact on bacterial resistance. Consequences of such artificial phospholipid composition were studied in the presence of an overall of seven antimicrobials (?-lactams, a lipopeptide antibiotic, cationic antimicrobial peptides [CAMPs]) to quantitatively assess the effect of A-PG substitution (with L-PG, L-PG and A-PG, increased A-PG levels). For the employed Gram-negative P. aeruginosa model system, an exclusive charge repulsion mechanism does not explain the attenuated antimicrobial susceptibility due to PG modification. Additionally, the specificity of nine orthologous aaPGS enzymes was experimentally determined. The newly characterized protein sequences allowed for the establishment of a significant group of A-PG synthase sequences which were bioinformatically compared to the related group of L-PG synthesizing enzymes. The analysis revealed a diverse origin for the evolution of A-PG and L-PG synthases, as