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101.
Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a binuclear metallohydrolase that catalyzes the breakdown of a broad range of phosphate ester substrates, and it is of interest for its potential application in the destruction of organophosphate nerve agents and pesticides. The reaction mechanism of GpdQ has been proposed to involve a nucleophilic attack by a terminally bound hydroxide molecule. The hydroxide species bridging the two metal ions is suggested to activate the nucleophile, thus favoring a sequential rather than a processive mechanism of action. Here, the hydrolysis of the two ester bonds in the substrate bis(para-nitrophenyl) phosphate (bpNPP) is probed using 31P NMR. The kinetic rates measured compare well with those determined spectrophotometrically. Furthermore, the data indicate that the diester bonds are cleaved in two separate (non-processive) reactions, indicating that only a single nucleophile (the terminal hydroxide molecule) is likely to be employed as a nucleophile for GpdQ. 相似文献
102.
Quantum-chemical study of structures, energies, and effective partial charge distribution for several models of the Rieske protein redox center is performed in terms of the B3LYP density functional method in combination with the broken symmetry approach using three different atomic basis sets. The structure of the redox complex optimized in vacuum differs markedly from that inside the protein. This means that the protein matrix imposes some stress on the active site resulting in distortion of its structure. The redox potentials calculated for the real active site structure are in a substantially better agreement with the experiment than those calculated for the idealized structure. This shows an important role of the active site distortion in tuning its redox potential. The reference absolute electrode potential of the standard hydrogen electrode is used that accounts for the correction caused by the water surface potential. Electrostatic calculations are performed in the framework of the polarizable solute model. Two dielectric permittivities of the protein are employed: the optical permittivity for calculation of the intraprotein electric field, and the static permittivity for calculation of the dielectric response energy. Only this approach results in a reasonable agreement of the calculated and experimental redox potentials. 相似文献
103.
Kyoung-Ho Pyo Bong-Kwang Jung Jong-Yil Chai Eun-Hee Shin 《The Korean journal of parasitology》2010,48(2):171-174
The anti-tumorigenic effects of Toxoplasma gondii (RH) antigens were studied in a murine sarcoma-180 tumor model. To determine the anti-tumor effects, the reduction in tumor size and expression of CD31 (an angiogenesis marker in the tumor tissue) were examined after injection of BALB/c mice with T. gondii lysate antigen (TLA) or formalin-fixed, proliferation-inhibited, T. gondii tachyzoites. Tumors were successfully produced by an intradermal injection of sarcoma-180 cells with plain Matrigel in the mid-backs of mice. After injection with TLA or formalin-fixed T. gondii tachyzoites, the increase in tumor size and weight nearly stopped while tumor growth continued in control mice that were injected with PBS. CD31 expression in TLA-treated or formalin-fixed T. gondii-injected mice was lower than the control mice. Accordingly, the present study shows that the treatment of mice with formalin-fixed T. gondii or TLA in the murine sarcoma-180 tumor model results in a decrease of both tumor size and CD31 expression. 相似文献
104.
105.
Human eosinophil-derived neurotoxin (EDN) or RNase 2, found in the non-core matrix of eosinophils is a ribonuclease belonging
to the Ribonuclease A superfamily. EDN manifests a number of bioactions including neurotoxic and antiviral activities, which
are dependent on its ribonuclease activity. The core of the catalytic site of EDN contains various base and phosphate-binding
subsites. Unlike many members of the RNase A superfamily, EDN contains an additional non-catalytic phosphate-binding subsite,
P−1. Although RNase A also contains a P−1 subsite, the composition of the site in EDN and RNase A is different. In the current study we have generated site-specific
mutants to study the role of P−1 subsite residues Arg36, Asn39, and Gln40 of EDN in its catalytic activity. The individual mutation of Arg36, Asn 39, and Gln40 resulted in a reduction in the catalytic activity of EDN on poly(U) and poly(C). However, there was no change in the activities
on yeast tRNA and dinucleotide substrates. The study shows that the P−1 subsite is crucial for the ribonucleolytic activity of EDN on polymeric RNA substrates.
Deepa Sikriwal and Divya Seth contributed equally to this work. 相似文献
106.
Soil Organic Phosphorus Transformations During Pedogenesis 总被引:2,自引:0,他引:2
Benjamin L. Turner Leo M. Condron Sarah J. Richardson Duane A. Peltzer Victoria J. Allison 《Ecosystems》2007,10(7):1166-1181
Abstract
Long-term changes in soil phosphorus influence ecosystem development and lead to a decline in the productivity of forests
in undisturbed landscapes. Much of the soil phosphorus occurs in a series of organic compounds that differ in their availability
to organisms, but changes in the relative abundance of these compounds during pedogenesis remain unknown. We used alkaline
extraction and solution phosphorus-31 nuclear magnetic resonance spectroscopy to assess the chemical nature of soil organic
phosphorus along a 120,000-year post-glacial chronosequence at Franz Josef, New Zealand. Inositol phosphates, DNA, phospholipids,
and phosphonates accumulated rapidly during the first 500 years of soil development characterized by nitrogen limitation of
biological productivity, but then declined slowly to low concentrations in older soils characterized by intense phosphorus
limitation. However, the relative contribution of the various compounds to the total organic phosphorus varied along the sequence
in dramatic and surprising ways. The proportion of inositol hexakisphosphate, conventionally considered to be relatively recalcitrant
in the environment, declined markedly in older soils, apparently due to a corresponding decline in amorphous metal oxides,
which weather to crystalline forms during pedogenesis. In contrast, the proportion of DNA, considered relatively bioavailable
in soil, increased continually throughout the sequence, due apparently to incorporation within organic structures that provide
protection from biological attack. The changes in soil organic phosphorus coincided with marked shifts in plant and microbial
communities, suggesting that differences in the forms and bioavailability of soil organic phosphorus have ecological significance.
Overall, the results strengthen our understanding of phosphorus transformations during pedogenesis and provide important insight
into factors regulating the composition of soil organic phosphorus. 相似文献
107.
Lucy Cherbas Jennifer Hackney Lei Gong Claire Salzer Eric Mauser Dayu Zhang Peter Cherbas 《Genetics》2015,201(4):1307-1318
We describe an adaptation of φC31 integrase–mediated targeted cassette exchange for use in Drosophila cell lines. Single copies of an attP-bounded docking platform carrying a GFP-expression marker, with or without insulator elements flanking the attP sites, were inserted by P-element transformation into the Kc167 and Sg4 cell lines; each of the resulting docking-site lines carries a single mapped copy of one of the docking platforms. Vectors for targeted substitution contain a cloning cassette flanked by attB sites. Targeted substitution occurs by integrase-mediated substitution between the attP sites (integrated) and the attB sites (vector). We describe procedures for isolating cells carrying the substitutions and for eliminating the products of secondary off-target events. We demonstrate the technology by integrating a cassette containing a Cu2+-inducible mCherry marker, and we report the expression properties of those lines. When compared with clonal lines made by traditional transformation methods, which lead to the illegitimate insertion of tandem arrays, targeted insertion lines give more uniform expression, lower basal expression, and higher induction ratios. Targeted substitution, though intricate, affords results that should greatly improve comparative expression assays—a major emphasis of cell-based studies. 相似文献
108.
Takashi Okumura Takeshi Sasamura Momoko Inatomi Shunya Hozumi Mitsutoshi Nakamura Ryo Hatori Kiichiro Taniguchi Naotaka Nakazawa Emiko Suzuki Reo Maeda Tomoko Yamakawa Kenji Matsuno 《Genetics》2015,199(4):1183-1199
The class I myosin genes are conserved in diverse organisms, and their gene products are involved in actin dynamics, endocytosis, and signal transduction. Drosophila melanogaster has three class I myosin genes, Myosin 31DF (Myo31DF), Myosin 61F (Myo61F), and Myosin 95E (Myo95E). Myo31DF, Myo61F, and Myo95E belong to the Myosin ID, Myosin IC, and Myosin IB families, respectively. Previous loss-of-function analyses of Myo31DF and Myo61F revealed important roles in left–right (LR) asymmetric development and enterocyte maintenance, respectively. However, it was difficult to elucidate their roles in vivo, because of potential redundant activities. Here we generated class I myosin double and triple mutants to address this issue. We found that the triple mutant was viable and fertile, indicating that all three class I myosins were dispensable for survival. A loss-of-function analysis revealed further that Myo31DF and Myo61F, but not Myo95E, had redundant functions in promoting the dextral LR asymmetric development of the male genitalia. Myo61F overexpression is known to antagonize the dextral activity of Myo31DF in various Drosophila organs. Thus, the LR-reversing activity of overexpressed Myo61F may not reflect its physiological function. The endogenous activity of Myo61F in promoting dextral LR asymmetric development was observed in the male genitalia, but not the embryonic gut, another LR asymmetric organ. Thus, Myo61F and Myo31DF, but not Myo95E, play tissue-specific, redundant roles in LR asymmetric development. Our studies also revealed differential colocalization of the class I myosins with filamentous (F)-actin in the brush border of intestinal enterocytes. 相似文献
109.
Transgenesis in numerous eukaryotes has been facilitated by the use of site-specific integrases to stably insert transgenes at predefined genomic positions (landing sites). However, the utility of integrase-mediated transgenesis in any system is constrained by the limited number and variable expression properties of available landing sites. By exploiting the nonstandard recombination activity exhibited by a phiC31 integrase mutant, we developed a rapid and inexpensive method for isolating landing sites that exhibit desired expression properties. Additionally, we devised a simple technique for constructing arrays of transgenes at a single landing site, thereby extending the utility of previously characterized landing sites. Using the fruit fly Drosophila melanogaster, we demonstrate the feasibility of these approaches by isolating new landing sites optimized to express transgenes in the nervous system and by building fluorescent reporter arrays at several landing sites. Because these strategies require the activity of only a single exogenous protein, we anticipate that they will be portable to species such as nonmodel organisms, in which genetic manipulation is more challenging, expediting the development of genetic resources in these systems. 相似文献
110.
α‐ l‐iduronidase gene‐based therapy using the phiC31 system to treat mucopolysaccharidose type I mice
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Roberta Sessa Stilhano Priscila Keiko Matsumoto Martin Suely Maymone de Melo Vivian Yochiko Samoto Giovani Bravin Peres Yara Maria Correa da Silva Michelacci Flavia Helena da Silva Vanessa Gonçalves Pereira Vania D'Almeida Adriana Taveira da Cruz Miriam Galvonas Jasiulionis Sang Won Han 《The journal of gene medicine》2015,17(1-2):1-13