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Selected AbstractsCopper-containing nitrite reductase from Pseudomonas chlororaphis DSM 50135FEBS JOURNAL, Issue 12 2004Evidence for modulation of the rate of intramolecular electron transfer through nitrite binding to the type 2 copper center The nitrite reductase (Nir) isolated from Pseudomonas chlororaphis DSM 50135 is a blue enzyme, with type 1 and type 2 copper centers, as in all copper-containing Nirs described so far. For the first time, a direct determination of the reduction potentials of both copper centers in a Cu-Nir was performed: type 2 copper (T2Cu), 172 mV and type 1 copper (T1Cu), 298 mV at pH 7.6. Although the obtained values seem to be inconsistent with the established electron-transfer mechanism, EPR data indicate that the binding of nitrite to the T2Cu center increases its potential, favoring the electron-transfer process. Analysis of the EPR spectrum of the turnover form of the enzyme also suggests that the electron-transfer process between T1Cu and T2Cu is the fastest of the three redox processes involved in the catalysis: (a) reduction of T1Cu; (b) oxidation of T1Cu by T2Cu; and (c) reoxidation of T2Cu by NO2,. Electrochemical experiments show that azurin from the same organism can donate electrons to this enzyme. [source] 2-Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulansFEBS JOURNAL, Issue 12 2001Characterization, comparison of both enzymes In Escherichia coli and Aspergillus nidulans, propionate is oxidized to pyruvate via the methylcitrate cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate is catalysed by 2-methylisocitrate lyase. The enzymes from both organisms were assayed with chemically synthesized threo -2-methylisocitrate; the erythro -diastereomer was not active. 2-Methylisocitrate lyase from E. coli corresponds to the PrpB protein of the prp operon involved in propionate oxidation. The purified enzyme has a molecular mass of approximately 32 kDa per subunit, which is lower than those of isocitrate lyases from bacterial sources (, 48 kDa). 2-Methylisocitrate lyase from A. nidulans shows an apparent molecular mass of 66 kDa per subunit, almost equal to that of isocitrate lyase of the same organism. Both 2-methylisocitrate lyases have a native homotetrameric structure as identified by size-exclusion chromatography. The enzymes show no measurable activity with isocitrate. Starting from 250 mm pyruvate, 150 mm succinate and 10 µm PrpB, the enzymatically active stereoisomer could be synthesized in 1% yield. As revealed by chiral HPLC, the product consisted of a single enantiomer. This isomer is cleaved by 2-methylisocitrate lyases from A. nidulans and E. coli. The PrpB protein reacted with stoichiometric amounts of 3-bromopyruvate whereby the activity was lost and one amino-acid residue per subunit became modified, most likely a cysteine as shown for isocitrate lyase of E. coli. PrpB exhibits 34% sequence identity with carboxyphosphoenolpyruvate phosphonomutase from Streptomyces hygroscopicus, in which the essential cysteine residue is conserved. [source] Aragonite Formation in the Chiton (Mollusca) GirdleHELVETICA CHIMICA ACTA, Issue 4 2003Keren Treves In the chitons (Polyplacophora, Mollusca), the body is not entirely protected by the shell. Mineralized spicules or scales often, but not always, decorate the exposed part of the girdle. Here, we report a study on the composition and ultrastructural organization of these mineralized skeletal parts in four different chiton species. In all specimens, the mineral component (97,98,wt-%) is aragonite, and the organic matrix (2,3,wt-%) consists of highly glycosylated proteins. X-Ray diffraction and scanning electron microscopy show that the organic matrix fibers are aligned, morphologically and crystallographically, with the prismatic aragonite crystals. Matrix and mineral are thus clearly related. The matrix,mineral composite bundles are, however, assembled in the various skeletal parts examined with widely different degrees of alignment and order. In the same organism, the crystals are aligned within a range of ±15° in one type of spicule, while they are randomly oriented in another type. The wide heterogeneity in shape, density, and ultrastructure suggests that the girdle mineralized tissues do not fulfill a fundamental role necessary for the survival of the organism. This, together with the lack of chitin in the organic matrix, supports the hypothesis that they evolved separately from the other chiton mineralized tissues, namely the shell plates and teeth. [source] Multiple strategies for O2 transport: from simplicity to complexityIUBMB LIFE, Issue 8-9 2007Paolo Ascenzi Abstract O2carriers (extracellular and intracellular as well as monomeric and multimeric) have evolved over the last billion of years, displaying iron and copper reactive centers; very different O2carriers may co-exist in the same organism. Circulating O2carriers, faced to the external environment, are responsible for maintaining an adequate delivery of O2to tissues and organs almost independently of the environmental O2partial pressure. Then, intracellular globins facilitate O2transfer to mitochondria sustaining cellular respiration. Here, molecular aspects of multiple strategies evolved for O2transport and delivery are examined, from the simplest myoglobin to the most complex giant O2carriers and the red blood cell, mostly focusing on the aspects which have been mainly addressed by the so called 'Rome Group'. [source] Modular changes of cis-regulatory elements from two functional Pit1 genes in the duplicated genome of Cyprinus carpioJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2006G. Kausel Abstract The pituitary-specific transcription factor Pit1 is involved in its own regulation and in a network of transcriptional regulation of hypothalamo-hypophyseal factors including prolactin (PRL) and growth hormone (GH). In the ectotherm teleost Cyprinus carpio, Pit1 plays an important role in regulation of the adaptive response to seasonal environmental changes. Two Pit1 genes exist in carp, a tetraploid vertebrate and transcripts of both genes were detected by RT-PCR analysis. Powerful comparative analyses of the 5,-flanking regions revealed copy specific changes comprising modular functional units in the naturally evolved promoters. These include the precise replacement of four nucleotides around the transcription start site embedded in completely conserved regions extending upstream of the TATA-box, an additional transcription factor binding site in the 5,-UTR of gene-I and, instead, duplication of a 9 bp element in gene-II. Binding of nuclear factors was assessed by electro mobility shift assays using extracts from rat pituitary cells and carp pituitary. Binding was confirmed at one conserved Pit1, one conserved CREB and one consensus MTF1. Interestingly, two functional Pit1 sites and one putative MTF1 binding site are unique to the Pit1 gene-I. In situ hybridization experiments revealed that the expression of gene-I in winter carp was significantly stronger than that of gene-II. Our data suggest that the specific control elements identified in the proximal regulatory region are physiologically relevant for the function of the duplicated Pit1 genes in carp and highlight modular changes in the architecture of two Pit1 genes that evolved for at least 12 MYA in the same organism. J. Cell. Biochem. 99: 905,921, 2006. © 2006 Wiley-Liss, Inc. [source] Synthesis and Biological Evaluation of Novel Pyrazoles and Pyrazolo[3,4- d]pyrimidines Incorporating a Benzenesulfonamide MoietyARCHIV DER PHARMAZIE, Issue 4 2009Hayam M. A. Ashour Abstract Synthesis and biological evaluation of novel pyrazoles and pyrazolo[3,4- d]pyrimidines are reported. Fourteen compounds were selected by the NCI and tested for their preliminary in-vitro anticancer activity, whereas all the synthesized compounds were evaluated for their in-vitro antimicrobial activity. Compound 12a was proven to possess the highest anticancer activity with a broad spectrum profile. It showed particular effectiveness towards leukemia HL-60 (TB), K-562, non-small cell lung cancer NCI-H23, and colon cancer HT 29, KM 12 cell lines (GI50 = 6.59, 4.44, 1.37, 3.33, and 9.63 ,M, respectively). Out of the synthesized compounds, thirteen derivatives were found to display pronounced antimicrobial activity especially against P. aeruginosa. Compounds 2c, 5b, 10, 11b, 17b, 18b, and 19 were proven to be the most active with a broad spectrum of activity. Compound 19 was found to be equipotent to ampicillin against B. subtilis, whereas compounds 11b and 19 were four times superior to ampicillin against P. aeruginosa, while compounds 5b and 18b were equipotent to ampicillin against the same organism. Moreover, compounds 2c, 10, and 11b were nearly equipotent to ampicillin against E. coli. On the other hand, compounds 2c, 5b, 10, 11a, 17b, and 18b exerted nearly half the activity of clotrimazole against C. albicans. [source] Identification and comparison of aerobic and denitrifying polyphosphate-accumulating organismsBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2003Raymond J. Zeng Abstract Two laboratory-scale sequencing batch reactors (SBRs) were operated for enhanced biological phosphorus removal (EBPR) in alternating anaerobic,aerobic or alternating anaerobic,anoxic modes, respectively. Polyphosphate-accumulating organisms (PAOs) were enriched in the anaerobic,aerobic SBR and denitrifying PAOs (DPAOs) were enriched in the anaerobic,aerobic SBR. Fluorescence in situ hybridization (FISH) demonstrated that the well-known PAO, "Candidatus Accumulibacter phosphatis" was abundant in both SBRs, and post-FISH chemical staining with 4,6-diamidino-2-phenylindol (DAPI) confirmed that they accumulated polyphosphate. When the anaerobic,anoxic SBR enriched for DPAOs was converted to anaerobic,aerobic operation, aerobic uptake of phosphorus by the resident microbial community occurred immediately. However, when the anaerobic,aerobic SBR enriched for PAOs was exposed to one cycle with anoxic rather than aerobic conditions, a 5-h lag period elapsed before phosphorus uptake proceeded. This anoxic phosphorus-uptake lag phase was not observed in the subsequent anaerobic,aerobic cycle. These results demonstrate that the PAOs that dominated the anaerobic,aerobic SBR biomass were the same organisms as the DPAOs enriched under anaerobic,anoxic conditions. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 140,148, 2003. [source] | |||||||||||||