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1.
Expression of the recombinant antibacterial peptide sarcotoxin IA in Escherichia coli cells 总被引:5,自引:0,他引:5
Skosyrev VS Kulesskiy EA Yakhnin AV Temirov YV Vinokurov LM 《Protein expression and purification》2003,28(2):350-356
Sarcotoxin IA is an antibacterial peptide that is secreted by a meat-fly Sarcophaga peregrina larva in response to a hypodermic injury or bacterial infection. This peptide is highly toxic against a broad spectrum of both Gram-positive and Gram-negative bacteria and lethal to microbes even at nanomolar concentrations. However, research needs as well as its potential use in medicine require substantial amounts of highly purified sarcotoxin. Because heterologous expression systems proved to be inefficient due to sarcotoxin sensitivity to intracellular proteases, here we propose the biosynthesis of sarcotoxin precursors in Escherichia coli cells that are highly sensitive to the mature peptide. To optimize its biosynthesis, sarcotoxin was translationally fused with proteins highly expressed in E. coli. A fusion partner and the position of sarcotoxin in the chimeric polypeptide were crucial for protecting the sarcotoxin portion of the fusion protein from proteolysis. Released after chemical cleavage of the fusion protein and purified to homogeneity, sarcotoxin displayed antibacterial activity comparable to that previously reported for the natural peptide. 相似文献
2.
Dysregulation of signaling pathways is believed to contribute to Parkinson's disease pathology and l-DOPA-induced motor complications. Long-lived dopamine (DA) agonists are less likely to cause motor complications by virtue of continuous stimulation of DA receptors. In this study, we compared the effects of the unilateral 6-hydroxydopamine lesion and subsequent treatment with l-DOPA and DA agonist pergolide on signaling pathways in rats. Pergolide caused less pronounced behavioral sensitization than l-DOPA (25 mg/kg, i.p., 10 days), particularly at lower dose (0.5 and 0.25 mg/kg, i.p.). Pergolide, but not l-DOPA, reversed lesion-induced up-regulation of preproenkephalin and did not up-regulate preprodynorphine or DA D3 receptor in the lesioned hemisphere. Pergolide was as effective as l-DOPA in reversing the lesion-induced elevation of ERK2 phosphorylation in response to acute apomorphine administration (0.05 mg/kg, s.c.). Chronic l-DOPA significantly elevated the level of Akt phosphorylation at both Thr(308) and Ser(473) and concentration of phosphorylated GSK3alpha, whereas pergolide suppressed the lesion- and/or challenge-induced supersensitive Akt responses. The data indicate that l-DOPA, unlike pergolide, exacerbates imbalances in the Akt pathway caused by the loss of DA. The results support the hypothesis that the Akt pathway is involved in long-term actions of l-DOPA and may be linked to l-DOPA-induced dyskinesia. 相似文献
3.
Gram-negative rod shaped bacterium Myxococcus xanthus DK1622 produces a smooth-type LPS. The structure of the polysaccharide O-chain and the core-lipid A region of the LPS has been determined by chemical and spectroscopic methods. The O-chain was built up of disaccharide repeating units having the following structure: -->6)-alpha-D-Glcp-(1-->4)-alpha-D-GalpNAc6oMe*-(1--> with partially methylated GalNAc residue. The core region consisted of a phosphorylated hexasaccharide, containing one Kdo residue, unsubstituted at O-4, and no heptose residues. The lipid A component consisted of beta-GlcN-(1-->6)-alpha-GlcN1P disaccharide, N-acylated with 13-methyl-C14-3OH (iso-C15-3OH), C16-3OH, and 15-methyl-C16-3OH (iso-C17-3OH) acids. The lipid portion contained O-linked iso-C16 acid. 相似文献
4.
5.
Cdk1 and okadaic acid-sensitive phosphatases control assembly of nuclear pore complexes in Drosophila embryos 下载免费PDF全文
Onischenko EA Gubanova NV Kiseleva EV Hallberg E 《Molecular biology of the cell》2005,16(11):5152-5162
Disassembly and reassembly of the nuclear pore complexes (NPCs) is one of the major events during open mitosis in higher eukaryotes. However, how this process is controlled by the mitotic machinery is not clear. To investigate this we developed a novel in vivo model system based on syncytial Drosophila embryos. We microinjected different mitotic effectors into the embryonic cytoplasm and monitored the dynamics of disassembly/reassembly of NPCs in live embryos using fluorescently labeled wheat germ agglutinin (WGA) or in fixed embryos using electron microscopy and immunostaining techniques. We found that in live embryos Cdk1 activity was necessary and sufficient to induce disassembly of NPCs as well as their cytoplasmic mimics: annulate lamellae pore complexes (ALPCs). Cdk1 activity was also required for keeping NPCs and ALPCs disassembled during mitosis. In agreement recombinant Cdk1/cyclin B was able to induce phosphorylation and dissociation of nucleoporins from the NPCs in vitro. Conversely, reassembly of NPCs and ALPCs was dependent on the activity of protein phosphatases, sensitive to okadaic acid (OA). Our findings suggest a model where mitotic disassembly/reassembly of the NPCs is regulated by a dynamic equilibrium of Cdk1 and OA-sensitive phosphatase activities and provide evidence that mitotic phosphorylation mediates disassembly of the NPC. 相似文献
6.
Kobrinsky E Tiwari S Maltsev VA Harry JB Lakatta E Abernethy DR Soldatov NM 《The Journal of biological chemistry》2005,280(13):12474-12485
Voltage-gated Ca(v)1.2 channels are composed of the pore-forming alpha1C and auxiliary beta and alpha2delta subunits. Voltage-dependent conformational rearrangements of the alpha1C subunit C-tail have been implicated in Ca2+ signal transduction. In contrast, the alpha1C N-tail demonstrates limited voltage-gated mobility. We have asked whether these properties are critical for the channel function. Here we report that transient anchoring of the alpha1C subunit C-tail in the plasma membrane inhibits Ca2+-dependent and slow voltage-dependent inactivation. Both alpha2delta and beta subunits remain essential for the functional channel. In contrast, if alpha1C subunits with are expressed alpha2delta but in the absence of a beta subunit, plasma membrane anchoring of the alpha1C N terminus or its deletion inhibit both voltage- and Ca2+-dependent inactivation of the current. The following findings all corroborate the importance of the alpha1C N-tail/beta interaction: (i) co-expression of beta restores inactivation properties, (ii) release of the alpha1C N terminus inhibits the beta-deficient channel, and (iii) voltage-gated mobility of the alpha1C N-tail vis a vis the plasma membrane is increased in the beta-deficient (silent) channel. Together, these data argue that both the alpha1C N- and C-tails have important but different roles in the voltage- and Ca2+-dependent inactivation, as well as beta subunit modulation of the channel. The alpha1C N-tail may have a role in the channel trafficking and is a target of the beta subunit modulation. The beta subunit facilitates voltage gating by competing with the N-tail and constraining its voltage-dependent rearrangements. Thus, cross-talk between the alpha1C C and N termini, beta subunit, and the cytoplasmic pore region confers the multifactorial regulation of Ca(v)1.2 channels. 相似文献
7.
Mesozooplankton surveys were conducted in April/May for fourconsecutive years (19961999) in the vicinity of the PrinceEdward Islands (PEIs), Southern Ocean. The PEIs are locatedin the Polar Frontal Zone, directly in the path of the east-flowingAntarctic Circumpolar Current. Zooplankton were collected byoblique tows using a Bongo net fitted with 300 µm mesh.The abundance, biomass and average size of the mesozooplanktonin the upstream (USR), inter-island (IIR) and downstream (DSR)regions indicated that some groups and species were significantlyaffected by their interaction with the shallow shelf watersof the PEIs. Total mesozooplankton abundance and biomass weretypically highest in the DSR, but no consistent pattern wasevident in the USR and IIR. Copepods, euphausiids and fish weregenerally of a low average size in the IIR. This small sizewas largely attributed to the reduced abundance, or completeabsence, of mesopelagic species from the shelf region. Of totalbiomass, the mesopelagic species Euphausia longirostris, Euphausiasimilis, Pleuromamma abdominalis, Paraeuchaeta biloba and Oncaeaantarctica together contributed an average of 16% to the USR,2% to the IIR and 15% to the DSR. Conversely, epipelagic speciesshowed no consistent pattern of abundance and biomass distributionbetween regions. The low incidence of mesopelagic species overthe island shelf was attributed mainly to reduced advectionof deep water into the shelf region (average depth = 200 m),rather than predation, particularly during the through-flowmode between the islands. This resulted in substantial regionaldifferences in euphausiid community structure. The epipelagicspecies Euphausia vallentini and Thysanoessa vicina completelydominated the IIR, comprising on average 89% of total euphausiidbiomass in this region. However, predation may be importantduring the water-trapping mode between the islands. Advectionof zooplankton into the IIR appeared to be affected by the proximityof the Subantarctic Front (SAF). In 1996, when the SAF was farnorth of the PEIs, reduced current velocities resulted in somedegree of water retention over the shelf and an increased predationimpact. Conversely, when the SAF was close to the PEIs in 1999,more large plankton were transported over the island shelf.High current velocities and productivity associated with theSAF appear to increase the biomass and size of allochthonouszooplankton/nekton advected into the IIR, and consequently mayhave increased the availability of prey to land-based predators.The long-term southward movement of the SAF recently observedin the vicinity of the PEIs may therefore have important implicationsfor the ecosystem of these islands. 相似文献
8.
Eugene G. Maksimov Nikolai N. Sluchanko Kirill S. Mironov Evgeny A. Shirshin Konstantin E. Klementiev Georgy V. Tsoraev Marcus Moldenhauer Thomas Friedrich Dmitry A. Los Suleyman I. Allakhverdiev Vladimir Z. Paschenko Andrew B. Rubin 《Biophysical journal》2017,112(1):46-56
Orange carotenoid protein (OCP), responsible for the photoprotection of the cyanobacterial photosynthetic apparatus under excessive light conditions, undergoes significant rearrangements upon photoconversion and transits from the stable orange to the signaling red state. This is thought to involve a 12-Å translocation of the carotenoid cofactor and separation of the N- and C-terminal protein domains. Despite clear recent progress, the detailed mechanism of the OCP photoconversion and associated photoprotection remains elusive. Here, we labeled the OCP of Synechocystis with tetramethylrhodamine-maleimide (TMR) and obtained a photoactive OCP-TMR complex, the fluorescence of which was highly sensitive to the protein state, showing unprecedented contrast between the orange and red states and reflecting changes in protein conformation and the distances from TMR to the carotenoid throughout the photocycle. The OCP-TMR complex was sensitive to the light intensity, temperature, and viscosity of the solvent. Based on the observed Förster resonance energy transfer, we determined that upon photoconversion, the distance between TMR (donor) bound to a cysteine in the C-terminal domain and the carotenoid (acceptor) increased by 18 Å, with simultaneous translocation of the carotenoid into the N-terminal domain. Time-resolved fluorescence anisotropy revealed a significant decrease of the OCP rotation rate in the red state, indicating that the light-triggered conversion of the protein is accompanied by an increase of its hydrodynamic radius. Thus, our results support the idea of significant structural rearrangements of OCP, providing, to our knowledge, new insights into the structural rearrangements of OCP throughout the photocycle and a completely novel approach to the study of its photocycle and non-photochemical quenching. We suggest that this approach can be generally applied to other photoactive proteins. 相似文献
9.
Igor N. Stadnichuk Evgeny P. Lukashev Irina V. Elanskaya 《Photosynthesis research》2009,99(3):227-241
The features of the two types of short-term light-adaptations of photosynthetic apparatus, State 1/State 2 transitions, and
non-photochemical fluorescence quenching of phycobilisomes (PBS) by orange carotene-protein (OCP) were compared in the cyanobacterium
Synechocystis sp. PCC 6803 wild type, CK pigment mutant lacking phycocyanin, and PAL mutant totally devoid of phycobiliproteins. The permanent
presence of PBS-specific peaks in the in situ action spectra of photosystem I (PSI) and photosystem II (PSII), as well as
in the 77 K fluorescence excitation spectra for chlorophyll emission at 690 nm (PSII) and 725 nm (PSI) showed that PBS are
constitutive antenna complexes of both photosystems. The mutant strains compensated the lack of phycobiliproteins by higher
PSII content and by intensification of photosynthetic linear electron transfer. The detectable changes of energy migration
from PBS to the PSI and PSII in the Synechocystis wild type and the CK mutant in State 1 and State 2 according to the fluorescence excitation spectra measurements were not
registered. The constant level of fluorescence emission of PSI during State 1/State 2 transitions and simultaneous increase
of chlorophyll fluorescence emission of PSII in State 1 in Synechocystis PAL mutant allowed to propose that spillover is an unlikely mechanism of state transitions. Blue–green light absorbed by
OCP diminished the rout of energy from PBS to PSI while energy migration from PBS to PSII was less influenced. Therefore,
the main role of OCP-induced quenching of PBS is the limitation of PSI activity and cyclic electron transport under relatively
high light conditions. 相似文献
10.
Evelina L Zdorovenko Evgeny Vinogradov Galina M Zdorovenko Buko Lindner Olga V Bystrova Alexander S Shashkov Klaus Rudolph Ulrich Z?hringer Yuriy A Knirel 《European journal of biochemistry》2004,271(23-24):4968-4977
The core structure of the lipopolysaccharide (LPS) isolated from a rough strain of the phytopathogenic bacterium Pseudomonas syringae pv. phaseolicola, GSPB 711, was investigated by sugar and methylation analyses, Fourier transform ion-cyclotron resonance ESI MS, and one- and two-dimensional 1H-, 13C- and 31P-NMR spectroscopy. Strong alkaline deacylation of the LPS resulted in two core-lipid A backbone undecasaccharide pentakisphosphates in the ratio approximately 2.5 : 1, which corresponded to outer core glycoforms 1 and 2 terminated with either L-rhamnose or 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), respectively. Mild acid degradation of the LPS gave the major glycoform 1 core octasaccharide and a minor truncated glycoform 2 core heptasaccharide, which resulted from the cleavage of the terminal Kdo residues. The inner core of P. syringae is distinguished by a high degree of phosphorylation of L-glycero-D-manno-heptose residues with phosphate, diphosphate and ethanolamine diphosphate groups. The glycoform 1 core is structurally similar but not identical to one of the core glycoforms of the human pathogenic bacterium Pseudomonas aeruginosa. The outer core composition and structure may be useful as a chemotaxonomic marker for the P. syringae group of bacteria, whereas a more conserved inner core structure appears to be representative for the whole genus Pseudomonas. 相似文献