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1.
Expression of human asparagine synthetase in Escherichia coli 总被引:4,自引:0,他引:4
Human asparagine synthetase was expressed in Escherichia coli. Synthesis of the enzyme was demonstrated by immunoblotting and by complementation of asparagine auxotrophy in E. coli. The recombinant enzyme was shown to have both the ammonia- and glutamine-dependent asparagine synthetase activity in vitro. Compared to asparagine synthetase isolated from beef pancreas, the one expressed in E. coli migrated at a slightly slower rate on a denaturing protein gel. In contrast with previous reports, the data obtained here strongly suggest that the active enzyme is a homodimer. The production of soluble and active enzyme was shown to be highly temperature-dependent. Expression at 37 degrees C yielded no soluble enzyme, whereas growth at 30 and 21 degrees C favored the production of soluble asparagine synthetase. The incubation temperature was also important for complementation of asparagine auxotrophy in E. coli, as growth in the absence of asparagine occurred at 30 degrees C and not at 37 degrees C. 相似文献
2.
Paulo FP Pimenta Alessandra S Orfano Ana C Bahia Ana PM Duarte Claudia M Ríos-Velásquez Fabrício F Melo Felipe AC Pessoa Giselle A Oliveira Keillen MM Campos Luis Martínez Villegas Nilton Barnabé Rodrigues Rafael Nacif-Pimenta Rejane C Sim?es Wuelton M Monteiro Rogerio Amino Yara M Traub-Cseko José BP Lima Maria GV Barbosa Marcus VG Lacerda Wanderli P Tadei Nágila FC Secundino 《Memórias do Instituto Oswaldo Cruz》2015,110(1):23-47
In the Americas, areas with a high risk of malaria transmission are mainly located in
the Amazon Forest, which extends across nine countries. One keystone step to
understanding the Plasmodium life cycle in Anopheles species from the Amazon Region
is to obtain experimentally infected mosquito vectors. Several attempts to colonise
Ano- pheles species have been conducted, but with only short-lived success or no
success at all. In this review, we review the literature on malaria transmission from
the perspective of its Amazon vectors. Currently, it is possible to develop
experimental Plasmodium vivax infection of the colonised and field-captured vectors
in laboratories located close to Amazonian endemic areas. We are also reviewing
studies related to the immune response to P. vivax infection of Anopheles aquasalis,
a coastal mosquito species. Finally, we discuss the importance of the modulation of
Plasmodium infection by the vector microbiota and also consider the anopheline
genomes. The establishment of experimental mosquito infections with Plasmodium
falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide
interesting models for studying malaria in the Amazonian scenario is important.
Understanding the molecular mechanisms involved in the development of the parasites
in New World vectors is crucial in order to better determine the interaction process
and vectorial competence. 相似文献
3.
Structure based virtual screening of ligands to identify cysteinyl leukotriene receptor 1 antagonist
Srinivas Bandaru Vijaya Kumar Marri Priyadarshani Kasera Purnima Kovuri Amandeep Girdhar Deepti Raj Mittal Sabeen Ikram Ravi GV Anuraj Nayarisseri 《Bioinformation》2014,10(10):652-657
Montelukast and Zafirlukast are known leukotriene receptor antagonists prescribed in asthma treatment. However, these fall short
as mono therapy and are frequently used in combination with inhaled glucocorticosteroids with or without long acting beta 2
agonists. Therefore, it is of interest to apply ligand and structure based virtual screening strategies to identify compounds akin to
lead compounds Montelukast and Zafirlukast. Hence, compounds with structures having 95% similarity to these compounds were
retrieved from NCBI׳s PubChem database. Compounds similar to lead were grouped and docked at the antagonist binding site of
cysteinyl leukotriene receptor 1. This exercise identified compounds UNII 70RV86E50Q (Pub Cid 71587778) and Sure CN 9587085
(Pub Cid 19793614) with higher predicted binding compared to Montelukast and Zafirlukast. It is shown that the compound Sure
CN 9587085 showed appreciable ligand receptor interaction compared to UNII 70RV86E50Q. Thus, the compound Sure CN
9587085 is selected as a potent antagonist to cysteinyl leukotriene receptor 1 for further consideration in vitro and in vivo validation. 相似文献
4.
5.
Ott TL Van Heeke G Hostetler CE Schalue TK Olmsted JJ Johnson HM Bazer FW 《Theriogenology》1993,40(4):757-769
The ability of recombinant ovine interferon-tau (roIFNtau) to extend the interestrous interval (IEI) in sheep was studied. Ewes were fitted with bilateral uterine catheters 7 or 8 days post estrus and were assigned to receive either 10 or 20 million antiviral (AV) units/day i.u. ( approximately 100 or 200 ug) of roIFNtau or ovine conceptus secretory proteins containing equivalent AV units of native oIFNtau (noIFNtau; 4 ewes/treatment). Four control ewes received ovine serum proteins (SP). Total protein injected was 6 mg per day, half at 0700 hours and half at 1730 hours. The treatments were administered from Day 11.5 (estrus=Day 0) to Day 16. Blood samples were collected by jugular vienipuncture daily from Day 11 until ewes returned to estrus. Concentrations of progesterone (P) in plasma were determined by RIA. Treatment with either noIFNtau or roIFNtau extended IEI beyond that of SP-treated ewes (19.1 vs 31.2+/-3.4 days P<0.03). Of the ewes receiving 100 mug/day of oIFNtau, 2 of 4 receiving noIFNtau (23.6+/-5.2 days) and 3 of 4 receiving roIFNtau (34.2+/-5.2 days) had an extended IEI. All ewes receiving 200 mug/day of noIFNtau or roIFNtau had an extended IEI (28.8 and 38.5+/-5.2 days. respectively). Ewes receiving roIFNtau had a longer IEI than those receiving noIFNtau (36.7 vs 26.2+/-3.4 days; P=0.07). Ewes with an extended IEI had functional corpora lutea, as assessed by P production. The results demonstrate that 10 or 20 million AV units ( approximately 100 or 200 ug) of roIFNtau extends the IEI and that the length of the IEI is longer for ewes receiving roIFNtau than noIFNtau following injection of equivalent AV units. 相似文献
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7.
The N-terminal cysteine of human asparagine synthetase is essential for glutamine-dependent activity 总被引:2,自引:0,他引:2
Site-specific mutagenesis was used to replace the N-terminal cysteine in human asparagine synthetase by an alanine. The mutant enzyme was expressed in the yeast Saccharomyces cerevisiae, and the asparagine synthetase activity was analyzed in vitro. The mutation resulted in the loss of the glutamine-dependent asparagine synthetase activity, while the ammonia-dependent activity remained unaffected. These results confirm the existence of a glutamine amidotransfer domain with an N-terminal cysteine essential for the glutamine-dependent asparagine synthetase activity. 相似文献
8.
Human asparagine synthetase was expressed in the yeast Saccharomyces cerevisiae. The identity of the expressed protein was confirmed by immunoblotting and in vitro enzymatic activity. The recombinant enzyme was shown to have both the ammonia- and glutamine-dependent asparagine synthetase activity in vitro. In contrast to overproduction in Escherichia coli, the expressed protein was found to be soluble in the yeast cell. Furthermore, expression in yeast made it possible to isolate non-degraded human asparagine synthetase which had also the N-terminal methionine correctly processed. The yeast expression plasmid was constructed for optimal production of the recombinant enzyme. In addition, unique restriction enzyme sites that bracket the first five codons of the human asparagine synthetase gene were introduced. This will allow the use of oligonucleotide cassette mutagenesis to investigate the role of the N-terminal amino acids in asparagine synthetase enzymatic activity. 相似文献
9.
McCahill A McSorley T Huston E Hill EV Lynch MJ Gall I Keryer G Lygren B Tasken K van Heeke G Houslay MD 《Cellular signalling》2005,17(9):1158-1173
We employ a novel, dominant negative approach to identify a key role for certain tethered cyclic AMP specific phosphodiesterase-4 (PDE4) isoforms in regulating cyclic AMP dependent protein kinase A (PKA) sub-populations in resting COS1 cells. A fraction of PKA is clearly active in resting COS1 cells and this activity increases when cells are treated with the selective PDE4 inhibitor, rolipram. Point mutation of a critical, conserved aspartate residue in the catalytic site of long PDE4A4, PDE4B1, PDE4C2 and PDE4D3 isoforms renders them catalytically inactive. Overexpressed in resting COS1 cells, catalytically inactive forms of PDE4C2 and PDE4D3, but not PDE4A4 and PDE4B1, are constitutively PKA phosphorylated while overexpressed active versions of all these isoforms are not. Inactive and active versions of all these isoforms are PKA phosphorylated in cells where protein kinase A is maximally activated with forskolin and IBMX. By contrast, rolipram challenge of COS1 cells selectively triggers the PKA phosphorylation of recombinant, active PDE4D3 and PDE4C2 but not recombinant, active PDE4A4 and PDE4B1. Purified, recombinant PDE4D3 and PDE4A4 show a similar dose-dependency for in vitro phosphorylation by PKA. Disruption of the tethering of PKA type-II to PKA anchor proteins (AKAPs), achieved using the peptide Ht31, prevents inactive forms of PDE4C2 and PDE4D3 being constitutively PKA phosphorylated in resting cells as does siRNA-mediated knockdown of PKA-RII, but not PKA-RI. PDE4C2 and PDE4D3 co-immunoprecipitate from COS1 cell lysates with 250 kDa and 450 kDa AKAPs that tether PKA type-II and not PKA type-I. PKA type-II co-localises with AKAP450 in the centrosomal region of COS1 cells. The perinuclear distribution of recombinant, inactive PDE4D3, but not inactive PDE4A4, overlaps with AKAP450 and PKA type-II. The distribution of PKA phosphorylated inactive PDE4D3 also overlaps with that of AKAP450 in the centrosomal region of COS1 cells. We propose that a novel role for PDE4D3 and PDE4C2 is to gate the activation of AKAP450-tethered PKA type-II localised in the perinuclear region under conditions of basal cAMP generation in resting cells. 相似文献
10.
Lynch MJ Baillie GS Mohamed A Li X Maisonneuve C Klussmann E van Heeke G Houslay MD 《The Journal of biological chemistry》2005,280(39):33178-33189
PDE4B and PDE4D provide >90% of PDE4 cAMP phosphodiesterase activity in human embryonic kidney (HEK293B2) cells. Their selective small interference RNA (siRNA)-mediated knockdown potentiates isoprenaline-stimulated protein kinase A (PKA) activation. Whereas endogenous PDE4D co-immunoprecipitates with beta arrestin, endogenous PDE4B does not, even upon PDE4D knockdown. Ectopic overexpression of PDE4B2 confers co-immunoprecipitation with beta arrestin. Knockdown of PDE4D, but not PDE4B, amplifies isoprenaline-stimulated phosphorylation of the beta2-adrenergic receptor (beta2-AR) by PKA and activation of extracellular signal-regulated kinase (ERK) through G(i). Isoform-selective knockdown identifies PDE4D5 as the functionally important species regulating isoprenaline stimulation of both these processes. Ht31-mediated disruption of the tethering of PKA to AKAP scaffold proteins attenuates isoprenaline activation of ERK, even upon PDE4D knockdown. Selective siRNA-mediated knockdown identifies AKAP79, which is constitutively associated with the beta2-AR, rather than isoprenaline-recruited gravin, as being the functionally relevant AKAP in this process. Isoprenaline-stimulated membrane recruitment of PDE4D is ablated upon beta arrestin knockdown. A mutation that compromises interactions with beta arrestin prevents catalytically inactive PDE4D5 from performing a dominant negative role in potentiating isoprenaline-stimulated ERK activation. Beta arrestin-recruited PDE4D5 desensitizes isoprenaline-stimulated PKA phosphorylation of the beta2-AR and the consequential switching of its signaling to ERK. The ability to observe a cellular phenotype upon PDE4D5 knockdown demonstrates that other PDE4 isoforms, expressed at endogenous levels, are unable to afford rescue in HEK293B2 cells. 相似文献