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51.
Phosphoribosyl pyrophosphate (PRPP) synthetase catalyzes the transfer of the pyrophosphate group from ATP to ribose-5-phosphate (R5P) yielding PRPP and AMP. PRPP is an essential metabolite that plays a central role in cellular metabolism. The enzyme from a thermophilic archaeon Thermoplasma volcanium (Tv) was expressed in Escherichia coli, crystallized, and its X-ray molecular structure was determined in a complex with its substrate R5P and with substrate analogs β,γ-methylene ATP and ADP in two monoclinic crystal forms, P21. The β,γ-methylene ATP- and the ADP-bound binary structures were determined from crystals grown from ammonium sulfate solutions; these crystals diffracted to 1.8 Å and 1.5 Å resolutions, respectively. Crystals of the ternary complex with ADP-Mg2+ and R5P were grown from a polyethylene glycol solution in the absence of sulfate ions, and they diffracted to 1.8 Å resolution; the unit cell is approximately double the size of the unit cell of the crystals grown in the presence of sulfate. The Tv PRPP synthetase adopts two conformations, open and closed, at different stages in the catalytic cycle. The binding of substrates, R5P and ATP, occurs with PRPP synthetase in the open conformation, whereas catalysis presumably takes place with PRPP synthetase in the closed conformation. The Tv PRPP synthetase forms a biological dimer in contrast to the tetrameric or hexameric quaternary structures of the Methanocaldococcus jannaschii and Bacillus subtilis PRPP synthetases, respectively. 相似文献
52.
Carsten Kintscher 《Journal of molecular biology》2009,387(2):270-5145
Cdc42, a member of the Ras superfamily of small guanine nucleotide binding proteins, plays an important role in regulating the actin cytoskeleton, intracellular trafficking, and cell polarity. Its activation is controlled by guanine nucleotide exchange factors (GEFs), which stimulate the dissociation of bound guanosine-5′-diphosphate (GDP) to allow guanosine-5′-triphosphate (GTP) binding. Here, we investigate the exchange factor activity of the Dbl-homology domain containing constructs of the adaptor protein Intersectin1L (ITSN1L), which is a specific GEF for Cdc42. A detailed kinetic characterisation comparing ITSN1L-mediated nucleotide exchange on Cdc42 in its GTP- versus GDP-bound state reveals a kinetic discrimination for GEF-stimulated dissociation of GTP: The maximum acceleration of the intrinsic mGDP [2′/3′-O-(N-methyl-anthraniloyl)-GDP] release from Cdc42 by ITSN1L is accelerated at least 68,000-fold, whereas the exchange of mGTP [2′/3′-O-(N-methyl-anthraniloyl)-GTP] is stimulated only up to 6000-fold at the same GEF concentration. The selectivity in nucleotide exchange kinetics for GDP over GTP is even more pronounced when a Cdc42 mutant, F28L, is used, which is characterised by fast intrinsic dissociation of nucleotides. We furthermore show that both GTP and Mg2+ ions are required for the interaction with effectors. We suggest a novel model for selective nucleotide exchange residing on a conformational change of Cdc42 upon binding of GTP, which enables effector binding to the Cdc42 · GTP complex but, at the same time, excludes efficient modulation by the GEF. The higher exchange activity of ITSN1L towards the GDP-bound conformation of Cdc42 could represent an evolutionary adaptation of this GEF that ensures nucleotide exchange towards the formation of the signalling-active GTP-bound form of Cdc42 and avoids dissociation of the active complex. 相似文献
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55.
Sandrine Théoleyre Damien Masson Marc G. Denis 《Biochemical and biophysical research communications》2010,394(3):453-458
Peroxisome proliferator-activated receptor gamma (PPARγ) ligands have been shown to possess anti-proliferative effects in many types of cancer. In clear cell renal cell carcinoma (CCRCC), the targets involved in these effects are not known. In this study, we demonstrated that, in CCRCC cell lines, the endogenous PPARγ ligand 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) induces the expression, both at the mRNA and the protein levels, of the HtrA3 gene. This gene belongs to the High-Temperature Requirement Factor A family of serine proteases that repress signaling by TGF-β family members and inhibit cell migration. Rosiglitazone or ciglitazone, synthetic PPARγ agonists, did not induce HtrA3 expression, and the PPARγ antagonist GW9662 did not prevent 15dPGJ2 induction, suggesting that the up-regulation of HtrA3 by 15dPGJ2 is independent of PPARγ. The MEK/ERK inhibitor PD98059 dramatically repressed HtrA3 induction. Altogether, these data indicate that 15dPGJ2 is able to stimulate the expression of HtrA3 through an indirect mechanism involving the MEK/ERK pathway but independent of PPARγ. Our results provide a better understanding of the mechanisms involved in the regulation of HtrA3, a potential tumor suppressor gene. 相似文献
56.
The aims of current study are to describe the immunogenicity and protective efficacy of prime boost vaccine using C-terminal extension (CTE) of cysteine proteinase type I of Leishmania infantum in BALB/c mice. Group I as vaccinated group primed with 100 microg of pcDNA-CTE and 3 weeks later boosted with combination of 30 microg rCTE, 50 microg of CpG and Montanide 720. Groups II and III were served as control groups. Although, this vaccination regimen did not protect mice against the infectious challenge but it was highly immunogenic. IgG2a has been raised strongly against rCTE in contrast to IgG1 and remains high at every time point under study. By analysis of CTE synthetic peptides (CTE100) before challenge, both IgG1 and IgG2a were produced and for all overlapping synthetic peptides (CTE 1-8) IgG1 raised significantly. This statue is changed at 7 weeks after challenge and only CTE2 and CTE3 have shown to induce considerable amount of IgG1. In all groups, the level of IL-5 started to increase with high concentration shortly passing only 3 weeks after infectious challenge. In compare with two control groups, the vaccinated group produced significantly higher level of IL-5 at 7 weeks post-infection. The parasite burden of all groups is similar at 4 weeks post-challenge in both liver and spleen. In contrast, at 8 weeks post challenge, the spleen of the vaccinated group showed significantly higher level of parasite load in compare with two control groups. This study demonstrated that immunization with CTE display both type 1 and 2 immune signatures in experimental murine model of L. infantum infection. 相似文献
57.
沙眼衣原体(Chlamydia trachomatis,Ct)具有广泛致病谱,是引起感染性致盲的首要病因,也是性传播疾病的主要病原体。γ干扰素在抗Ct感染中起重要作用。自噬是维持细胞内环境自稳的一种自我保护机制,与γ干扰素介导的抗Ct感染作用关系密切。就γ干扰素与自噬抗Ct感染的作用进行综述。 相似文献
58.
The humoral and cellular responses to DNA vaccination of BALB/c mice with a novel antigen from the Fasciola hepatica saposin-like protein family (FhSAP-2) have been investigated. Two constructs were produced containing the FhSAP-2 DNA sequence, one intended for extracellular secretion of FhSAP-2 protein, and one expressing FhSAP-2 in the cytoplasm of a transfected cell. The constructs were tested in HEK 293T cells, with the secretory construct producing less detectable FhSAP-2 relative to cytoplasmic construct when observed by fluorescence. The size of expressed protein was confirmed by Western blot of cell lysate, but FhSAP-2 was undetectable in cell supernatants. Both, secretory and cytoplasmic constructs as well as FhSAP-2 recombinant protein were tested in mice. The antibody response elicited in mice vaccinated with the rFhSAP-2 induced high levels of IgG(1), IgG(2), and IgE as well as high levels of IL-10 and IFNgamma indicating a mixed Th1/Th2 response. Vaccination of mice intramuscularly with the cytoplasmic FhSAP-2 construct resulted in a dominant IgG(2a) isotype antibody as well as a dominant IFNgamma cytokine, with significant IgE, IgG(1), and IL-10 responses also present, suggesting a mixed Th1/Th2 profile. Isotype and cytokine profiles elicited by the FhSAP-2 secretory construct were similar to those obtained with the cytoplasmic construct but at levels that were significantly lower. The results demonstrate that FhSAP-2 can be delivered as a DNA vaccine construct and induces a stronger Th1 response than the recombinant protein alone. This could result in an improvement in the immunoprophylactic potential of this candidate vaccine against F. hepatica. 相似文献
59.
Hajime Yoshida Yoshitake Tanaka Kiyoshi Nakayama 《Bioscience, biotechnology, and biochemistry》2013,77(9):2121-2126
Six strains of bacteria belonging to Vibrio and Pseudomonas were selected as good producers of L-DOPA from L-tyrosine out of various bacteria. The condition for the formation of L-DOPA by Vibrio tyrosinaticus ATCC 19378 was examined and the following results were obtained. (1) Intermittent addition of L-tyrosine in small portions gave higher titer of L-DOPA than single addition of L-tyrosine. (2) Higher amount of L-DOPA was produced in stationary phase of growth than in logarithmic phase. (3) Addition of antioxidant, chelating agent or reductant such as L-ascorbic acid, araboascorbic acid, hydrazine, citric acid and 5-ketofructose increased the amount of L-DOPA formed. (4) L-Tyrosine derivatives such as N-acetyl-L-tyrosine amide, N-acetyl-L-tyrosine, L-tyrosine amide, L-tyrosine methyl ester and L-tyrosine benzyl ester were converted to the corresponding L-DOPA derivatives.In the selected condition about 4 mg/ml of L-DOPA was produced from 4.3 mg/ml of L-tyrosine. 相似文献
60.
The possibility that the GABA-receptor agonists isoguvacine and THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) might be taken up into brain cells via the high affinity GABA transport system was tested by incubation of cultured neurons and astrocytes in media containing either [3H]GABA, [3H]isoguvacine or [3H]THIP at different concentrations. While GABA was actively taken up into both cell types via high affinity transport mechanisms, no high affinity transport could be demonstrated for isoguvacine or THIP. Both compounds did, however, penetrate into the cells. It is concluded that isoguvacine and THIP interact with the high affinity GABA-carrier neither in neurons nor in astrocytes. 相似文献