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1.
The effects of hematopoietic stem/progenitor cells (HSPCs) expanded in the two step coculture with human bone marrow mesenchymal
stem cells (hMSCs) on the hematopoietic reconstruction of irradiated NOD/SCID mice were studied. Mononuclear cells (MNCs)
were isolated from human umbilical cord blood (UCB) and cultured in the non-coculture scheme of rhSCF + rhG-CSF + rhMDGF combination
and the coculture scheme of rhSCF + rhG-CSF + rhMDGF + hMSCs. Sublethally-irradiated NOD/SCID mice were transplanted with
ex vivo expanded HSPCs with the dose of 8.5 × 106 cells per mouse. After transplantation, the dynamics of WBC in the transplanted mice was measured periodically, and the Alu
sequence fragment special for human in the transplanted mice was inspected by PCR. Results showed that the coculture scheme
increased proliferation of UCB-derived HSPCs. After transplantation with expanded HSPCs, the population of WBC in the transplanted
mice increased in 12 d and reached the first peak in 25 d, then showed the second increasing of WBC in 45∼55 d. Expanded cells
from the coculture scheme appeared to be favorable for the second increasing of WBC in the transplanted mice. After 85 d,
the Alu sequence fragment was detected in the probability of 87.5% (7/8) for the non-coculture scheme and 88.9% (8/9) for
the coculture scheme.
__________
Translated from Journal of Zhejiang University (Science Edition), 2005, 32 (4) [译自: 浙江大学学报 (理学版), 2005, 32 (4)] 相似文献
2.
Luiz Eduardo Maia Nery Marcelo Alves da Silva Lars Josefsson Ana Maria Lauro Castrucci 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1997,167(8):570-575
The cellular system responsible for the transduction of the pigment-concentrating hormone (PCH) signal was investigated in
erythrophores of the freshwater shrimp, Macrobrachium potiuna. Dose-response curves to the hormone were determined in the absence and in the presence of several drugs that affect sequential
steps of the Ca2+-dependent signalling pathway. Additionally, the ability of forskolin to induce pigment dispersion was evaluated. Neomycin
sulphate (10−4 and 10−3 mol · l−1), trifluoperazine (10−5 and 10−4 mol · l−1), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (10−7 and 10−5 mol · l−1) and okadaic acid (10−7 mol · l−1) significantly (P<0.05) decreased the responses to PCH. However, okadaic acid at low concentration (10−9 mol · l−1) and cyclosporin A (10−6 and 10−5 mol · l−1) did not significantly (P>0.05) affect PCH activity. Forskolin (10−4 mol · l−1) was able to half-maximally reverse the hormone-induced aggregation. Our results suggest that the pigment-concentrating hormone
induces pigment aggregation through a Ca2+-dependent pathway with a posteriori phosphatase activation, probably the serine/threonine phosphatase 1.
Accepted: 30 June 1997 相似文献
3.
Solubilization of enzymes in water-in-oil microemulsions and their rapid and efficient release through use of a pH-degradable surfactant 总被引:3,自引:0,他引:3
α-Chymotrypsin and lysozyme were solubilized in a water/O-[(2-tridecyl, 2-ethyl-1,3-dioxolan-4-yl)methoxy]–O′-methoxy poly(ethylene glycol) (CK-2,13 surfactant)/isooctane water-in-oil microemulsion solution at 1.5–2 and 10 g l−1 for 0.15 and 1.2 M CK-2,13, respectively. Upon contact with an equal volume of 0.1 M NaH2PO4/Na2HPO4 buffer, pH 5, a three-phase system (Winsor-III system) was formed, consisting of a surfactant-rich middle phase and aqueous
and isooctane-rich “excess” phases. Both enzymes were rapidly released into the aqueous excess phase, with 70% recovery of
each in 30 and 60 min for microemulsion solutions containing 0.15 and 1.2 M surfactant, respectively. The recovered enzymes retained >90% of their original specific activity. 相似文献
4.
The role of 3′,5′-cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), protein kinase C (PKC) and phosphatases
in the regulation of the taurine influx via the β-system in Ehrlich ascites tumor cells has been investigated. The taurine
uptake by the β-system in Ehrlich cells is inhibited when PKC is activated by phorbol 12-myristate 13-acetate (PMA) and when
protein phosphatases are inhibited by calyculin A (CLA). On the other hand, taurine uptake by the β-system is stimulated by
an increased level of cAMP or following addition of N6,2′-O-dibutyryl-3′,5′-cyclic adenosine monophosphate (dbcAMP). The effect of dbcAMP is partially blocked by addition of the
protein kinase inhibitor H-89, and suppressed in the presence of CLA. It is proposed that the β-system in the Ehrlich cells
exists in three states of activity: State I, where a PKC phosphorylation site on the transporter or on a regulator is phosphorylated and transport activity is low. State II, where the PKC phosphorylation site is dephosphorylated and transport activity is normal. State III, representing a state with high transport activity, induced by an elevated cellular cAMP level. Apparently, cAMP preferentially
stimulates taurine transport when the β-system is in State II.
Received: 8 September/Revised: 9 November 1995 相似文献
5.
Amalia Muñoz Frank Laib David H. Petering C. F. Shaw III 《Journal of biological inorganic chemistry》1999,4(4):495-507
The synthetic peptide fragment containing residues 49–61 of rabbit liver metallothionein II (MT-II) (Ac-Ile-Cys-Lys-Gly-Ala-Ser-Asp-Lys-Cys-Ser-Cys-Cys-Ala-COOH),
which includes the only sequential four cysteines bound to the same metal ion in Cd7MT, forms a stable, monomeric Cd-peptide complex with 1 : 1 stoichiometry (Cd:peptide) via Cd-thiolate interactions. This
represents the first synthesis of a single metal-binding site of MT independent of the domains. The 111Cd NMR chemical shift at 716 ppm indicates that the 111Cd2+ in the metal site is terminally coordinated to four side-chain thiolates of the cysteine residues. The pH of half dissociation
for this Cd-peptide derivative, ∼3.3, demonstrates an affinity similar to that for Cd7MT. Molecular mechanics calculations show that the thermodynamically most stable folding for this isolated Cd2+ center has the same counterclockwise chirality (Λ or S) observed in the native holo-protein. These properties are consistent with its proposed role as a nucleation center for cadmium-induced
protein folding. However, the kinetic reactivity of the CdS4 structure toward 5,5′-dithiobis(5-nitrobenzoate) (DTNB) and EDTA is greatly increased compared to the complete cluster (α-domain
or holo-protein). The rate law for the reaction with DTNB is rate=(k
uf +k
1,f +k
2,f [DTNB])[peptide], where k
uf=0.15 s–1, k
1,f=2.59×10–3 s–1, and k
2,f=0.88 M–1 s–1. The ultrafast step (uf), observable only by stopped-flow measurement, is unprecedented for mammalian (M7MT) and crustacean (M6MT) holo-proteins or the isolated domains. The accommodation of other metal ions by the peptide indicates a rich coordination
chemistry, including stoichiometries of M-peptide for Hg2+, Cd2+, and Zn2+, M2-peptide for Hg2+ and Au+, and (Et3PAu)2-peptide.
Received: 9 December 1998 / Accepted: 20 May 1999 相似文献
6.
7.
Quantification of arbuscular mycorrhizal fungi activity by the glomalin concentration on hyphal traps 总被引:9,自引:0,他引:9
Strips of horticultural film (16–32 cm2) were used to trap extraradical hyphae emanating from roots of sudangrass [Sorghum sudanense (Piper) Staph] enclosed in 40-μm mesh bags and colonized by Gigaspora rosea FL 224-1, Glomus intraradices EY 113/114, or Glomus caledonium UK 301-1. Strips of film were placed at opposite sides of 17–21 replicate sand culture pots for each isolate and were removed
after 12–14 weeks of plant growth. To extract glomalin, a strip was cut into small pieces and submerged in 2 ml of 20 mM citrate,
pH 7.0 and then autoclaved for 60 min. A quantitative enzyme-linked immunosorbent assay (ELISA) detected 0.005–0.04 μg glomalin
in the volume of extract tested. The Bradford protein assay detected 1.25–5 μg of protein in the volume of extract tested.
Both assays gave results ranging from 5–40 μg glomalin/cm2 of film. Protein assay values were correlated with ELISA values (r=0.6091, P≤0.001, n=118). Analysis of variance indicated that isolates differed in Bradford protein values (P=0.001), but not ELISA values (P=0.154). Spatial variability of glomalin deposition ca. 7 cm from roots on opposite sides of pots was indicated by significant
paired T tests (P<0.05) for protein values for each of the three isolates and ELISA for two isolates. These results indicate that hyphal traps,
Bradford protein assay and ELISA are useful to assess hyphal activity over a growing season.
Accepted: 11 October 1998 相似文献
8.
Background
Stem cell factor (SCF) can stimulate hematopoietic stem cell (HSC) expansion; however, the specific structural region(s) of SCF protein that are critical for this function are still unknown. A novel monoclonal antibody (named 23C8) against recombinant human SCF (rhSCF) was previously found to inhibit the ability of rhSCF to enhance HSC expansion, making it possible to identify the relevant active region to HSC.Methods
Eleven polypeptides were synthesized, which were designed to cover the full-length of rhSCF, with overlaps that are at least 3 amino acids long. ELISA was used to identify the polypeptide(s) that specifically react with the anti-SCF. The effects of the synthetic polypeptides on human HSC expansion, or on the ability of the full-length rhSCF to stimulate cell proliferation, were evaluated ex vivo. Total cell number was monitored using hemocytometer whereas CD34+ cell number was calculated based on the proportion determined via flow cytometry on day 6 of culture.Results
Of all polypeptides analyzed, only one, named P0, corresponding to the SCF protein sequence at residues 39–56, was recognized by 23C8 mAb during ELISA. P0 stimulated the expansion of CD34+ cells derived from human umbilical cord blood (UCB). Addition of P0 increased the numbers of total mononucleated cells and CD34+ cells, by ~2 fold on day 6. P0 also showed partial competition against full-length rhSCF in the ex vivo cell expansion assay.Conclusion
Residues 39–56 of rhSCF comprise a critical functional region for its ability to enhance expansion of human UCB CD34+ cells. The peptide P0 is a potential candidate for further development as a synthetic substitute for rhSCF in laboratory and clinical applications. 相似文献9.
We previously described a dominant negative secY
-d
1 allele in Escherichia coli, whose product interferes with protein export, presumably by sequestering SecE, the stabilizing partner of SecY. Syd is the
product of a multicopy suppressor of the secY
-d
1 phenotype, and its overproduction preferentially stabilizes the wild-type SecY protein. In contrast, overproduction of Syd
is toxic to the secY24 mutant, which shows a partial defect in SecY-SecE interaction. We isolated Syd-resistant revertants from the secY24 mutant. Pseudo-reversions mapped to sites at or near the secY24 mutation site (Gly240→Asp). The secY249 mutation (Ala249→Val) intragenically suppressed Syd sensitivity, but not the temperature-sensitive Sec phenotype of the secY24 mutation. The SecY249 mutant protein shows a reduced capacity to be stabilized by Syd, suggesting that the mutation weakens
the SecY-Syd interaction. The other two mutations changed residue 240 (the site of the secY24 alteration) to Asn (secY245) or Ala (secY241) and restored the ability of the cell to export protein. Although the secY245 mutant retained some sensitivity␣to Syd overproduction, the secY241 mutant was completely Syd-resistant. Furthermore, the secY241 mutation seemed to represent a “hyper reversion” with respect to the SecY-SecE interaction. Protein export in this mutant
was no longer sensitive to SecY-d1. When the secY
-d
1 mutation was combined intragenically with secY241, the resulting double mutant gene (secY
-d
1–241) showed an increased ability to interfere with protein export. On the basis of our model for SecY-d1, these results suggest that the secY241 alteration enhances SecY-SecE interaction. These results indicate that residue 240 of SecY is crucial for the interaction
between the cytosolic domains of SecY and SecE required for the establishment of the translocase complex.
Received: 20 October 1997 / Accepted: 1 December 1997 相似文献
10.
11.
The effect of ethylene and cytokinin on guanosine 5′-triphosphate binding and protein phosphorylation in leaves of Arabidopsis thaliana 总被引:2,自引:0,他引:2
Binding of [α-32P]guanosine 5′-triphosphate ([α-32P]GTP) has been demonstrated in a Triton X-100-solubilised membrane fraction from leaves of Arabidopsis thaliana (L.) Heynh. Binding was stimulated by 1 h pre-treatment of leaves with ethylene and this effect was antagonised by the inclusion
of N6-benzyladenine in the medium used for homogenisation. The ethylene-insensitive mutants eti5 and etr showed contrasting responses. In eti5 the constitutive level of GTP binding was higher than in the wild type whereas in etr the level was much lower. Neither ethylene nor cytokinin affected GTP binding in the mutants. The GTP-binding activity was
localised in two bands at 22 and 25 kDa, both of which were immunoprecipitated by anti-pan-Ras antibodies, indicating that
the activity is due to small GTP-binding proteins. In a similar membrane fraction, ethylene was shown to increase protein
phosphorylation and benzyladenine antagonised this effect. In eti5 the constitutive level of protein phosphorylation was higher than in the wild type, but benzyladenine increased activity
substantially while ethylene was without effect. In etr, protein phosphorylation was lower than in the wild type, ethylene was without effect, but cytokinin increased activity.
A protein of Mr 17 kDa was detected on gels using antibodies to nucleoside diphosphate kinase. Phosphorylation of this protein was upregulated
by ethylene but nucleoside diphosphate kinase activity was unaffected. The results are compared with the effect of the two
hormones on the senescence of detached leaves and discussed in relation to pathways proposed for ethylene signal transduction.
Received: 23 November 1998 / Accepted: 10 December 1998 相似文献
12.
Giuseppe Digilio Chiara Bracco Laura Vergani Mauro Botta Domenico Osella Aldo Viarengo 《Journal of biological inorganic chemistry》2009,14(2):167-178
The metal–thiolate connectivity of recombinant Cd7-MT10 metallothionein from the sea mussel Mytilus galloprovincialis has been investigated for the first time by means of multinuclear, multidimensional NMR spectroscopy. The internal backbone
dynamics of the protein have been assessed by the analysis of 15N T
1 and T
2 relaxation times and steady state {1H}–15N heteronuclear NOEs. The 113Cd NMR spectrum of mussel MT10 shows unique features, with a remarkably wide dispersion (210 ppm) of 113Cd NMR signals. The complete assignment of cysteine Hα and Hβ proton resonances and the analysis of 2D 113Cd–113Cd COSY and 1H–113Cd HMQC type spectra allowed us to identify a four metal–thiolate cluster (α-domain) and a three metal–thiolate cluster (β-domain),
located at the N-terminal and the C-terminal, respectively. With respect to vertebrate MTs, the mussel MT10 displays an inversion
of the α and β domains inside the chain, similar to what observed in the echinoderm MT-A. Moreover, unlike the MTs characterized
so far, the α-domain of mussel Cd7-MT10 is of the form M4S12 instead of M4S11, and has a novel topology. The β-domain has a metal–thiolate binding pattern similar to other vertebrate MTs, but it is conformationally
more rigid. This feature is quite unusual for MTs, in which the β-domain displays a more disordered conformation than the
α-domain. It is concluded that in mussel Cd7-MT10, the spacing of cysteine residues and the plasticity of the protein backbone (due to the high number of glycine residues)
increase the adaptability of the protein backbone towards enfolding around the metal–thiolate clusters, resulting in minimal
alterations of the ideal tetrahedral geometry around the metal centres. 相似文献
13.
Tissue density and growth response of ectomycorrhizal fungi to nitrogen source and concentration 总被引:4,自引:0,他引:4
Amanita rubescens Pers., Lactarius affinis Pk., Leccinum aurantiacum (Fr.) S.F. Gray, Tylopilus felleus (Bull. ex Fe.) Karsten, and two isolates of Suillus intermedius (Smith & Thiers) Smith & Thiers collected from an approximately 55-year-old Pinus resinosa Ait. plantation, and Pisolithus tinctorius (Pers.) Coker & Couch obtained from another source, were tested for their abilities to grow with protein as the primary source
of nitrogen. Protein plates contained 63 mg l–1 N as bovine serum albumen and 7 mg l–1 N as arginine. Control plates contained only 7 mg l–1 N as arginine. All isolates except Leccinum aurantiacum and one isolate of S. intermedius attained greater dry weight with protein as the primary source of N. Lactarius affinis, Leccinum aurantiacum, P. tinctorius, and both isolates of S. intermedius had higher tissue densities on protein medium. Amanita rubescens had lower tissue density. To determine if increase in tissue density was an effect of total N concentration or an effect
of N source (protein versus arginine), we performed a second experiment in which arginine concentration was increased (7 mg
l–1 N versus 70 mg l–1 N). The second experiment also included Cenococcum geophilum Fr. but excluded T. felleus. Higher tissue densities with increased nutrients were found in C. geophilum, Lactarius affinis, Leccinum aurantiacum, and both isolates of S. intermedius. Only A. rubescens and P. tinctorius did not have increased densities. The results suggest that these ectomycorrhizal fungi alter their growth forms according
to N concentration. At low N concentrations, a growth form likely to promote exploitation of a large volume of medium for
a given biomass is produced. At high concentrations, a growth form likely to promote exploitation of a rich source of N is
produced. Whether ectomycorrhizal fungi growing in association with roots would act in a similar fashion is not known.
Accepted: 30 July 1998 相似文献
14.
Jose Neptuno Rodriguez-Lopez Andrew T. Smith R. N. F. Thorneley 《Journal of biological inorganic chemistry》1996,1(2):136-142
Horseradish peroxidase isoenzyme C (HRPC) mutants were constructed in order to understand the role of two key distal haem
cavity residues, histidine 42 and arginine 38, in the formation of compound I and in substrate binding. The role of these
residues as general acid-base catalysts, originally proposed for cytochrome c peroxidase by Poulos and Kraut in 1980 was assessed for HRPC. Replacement of histidine 42 by leucine [(H42L)HRPC*] decreased
the apparent bimolecular rate constant for the reaction with hydrogen peroxide by five orders of magnitude (k
1 = 1.4×102 M–1s–1) compared with both native-glycosylated and recombinant forms of HRPC (k
1 = 1.7×107 M–1s–1). The first-order rate constant for the heterolytic cleavage of the oxygen-oxygen bond to form compound I was estimated to
be four orders of magnitude slower for this variant. Replacement of arginine 38 by leucine [(R38L)HRPC*] decreased the observed
pseudo-first-order rate constant for the reaction with hydrogen peroxide by three orders of magnitude (k
1 = 1.1×104 M–1s–1), while the observed rate constant of oxygen bond scission was decreased sixfold (k
2 = 142 s–1). These rate constants are consistent with arginine 38 having two roles in catalysing compound I formation: firstly, promotion
of proton transfer to the imidazole group of histidine 42 to facilitate peroxide anion binding to the haem, and secondly,
stabilisation of the transition state for the heterolytic cleavage of the oxygen-oxygen bond. These roles for arginine 38
explain, in part, why dioxygen-binding globins, which do not have an arginine in the distal cavity, are poor peroxidases.
Binding studies of benzhydroxamic acid to (H42L)HRPC* and (R38L)HRPC* indicate that both histidine 42 and arginine 38 are
involved in the modulation of substrate affinity.
Received: 21 July 1995 / Accepted: 27 November 1995 相似文献
15.
Irma Rosas Alma Yela Eva Salinas Roberto Arreguin Adela Rodriguez-Romero 《Aerobiologia》1995,11(2):81-86
The protein associated with airborne particles was measured during 1991 as an indicator of airborne biological material in
different outdoor urban environments. Fifty air samples were collected simultaneously at three sampling sites, located in
the north, south and downtown Mexico City, using a PM10 high-volume sampler (particles<10 μm). The air filters were weighed
and protein extracted using a phosphate buffer. Protein concentrations were determined by Lowry assay. The extracts were also
analysed by SDS electrophoresis and IEF using a Phastsystem. High concentrations of airborne particles were recorded at the
sampling sites with a geometric mean of 70.2 μg/m3 in the south (residential area), 95.5 μg/m3 in the center (urban-commercial area), and with the highest value of 108.9 μg/m3 in the north (urban-industrial area). No statistically significant difference (P>0.05) was observed among the protein concentrations from the sampling sites and the concentrations ranged from non-detectable
to 2.54 μg/m3. However, the protein concentrations presented significant difference (P<0.05) with respect to rainy and dry seasons. The Spearman correlation coefficient between protein concentration and airborne
particles concentration was statistically significant (r=0.50). The molecular weights (MW) and isoelectric points (pI) for the proteins present in some of the extracts were determined.
The values ranged from approximately 8000 to 106 000 Da and the pI values from nearly 4.0 to 5.85. This is important because
the major allergens from inhalants are mostly acidic proteins with molecular weights in the range of 20 000–40 000 Da. 相似文献
16.
Sari Tähtiharju Veena Sangwan Antonio F. Monroy Rajinder S. Dhindsa Marianne Borg 《Planta》1997,203(4):442-447
The involvement of calcium signaling during cold-induction of the kin genes of Arabidopsis thaliana (L.) Heynh. was examined. Treatments with chemicals which either chelate extracellular calcium (EGTA) or block the plasma-membrane
calcium channels (La3+, Gd3+) inhibited cold acclimation as well as kin gene expression. Ruthenium red, an inhibitor of calcium release from intracellular stores partially inhibited kin gene expression and development of freezing tolerance. An inhibitor of calcium-dependent protein kinases (CDPKs) and calmodulin
prevented cold acclimation as well as the cold induction of kin genes. Using restriction fragment length polymorphism-coupled domain-directed differential display, five CDPK clones were
identified which showed differential regulation by cold. The amplified fragments showed homology to known plant CDPKs. The
involvement of calcium and calcium-binding proteins in cold acclimation of A. thaliana is discussed.
Received: 28 November 1996 / Accepted: 5 May 1997 相似文献
17.
M. Muddassar F. A. Pasha M. M. Neaz Y. Saleem S. J. Cho 《Journal of molecular modeling》2009,15(2):183-192
Protein kinase B (PKB; also known as Akt kinase) is located downstream in the PI-3 kinase pathway. Overexpression and constitutive
activation of PKB/Akt leads to human prostate, breast and ovarian carcinomas. A series of 69 PKB/Akt inhibitors were examined
to explore their binding modes using FlexX, and three-dimensional quantitative structure–activity relationship (3D-QSAR) studies
based on comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were
performed to provide structural insights into these compounds. CoMFA produced statistically significant results, with cross-validated
q
2
and non-cross validated correlation r
2
coefficients of 0.53 and 0.95, respectively. For CoMSIA, steric, hydrophobic and hydrogen bond acceptor fields jointly yielded
‘leave one out’ q
2
= 0.51 and r
2
= 0.84. The predictive power of CoMFA and CoMSIA was determined using a test set of 13 molecules, which gave correlation
coefficients, of 0.58 and 0.62, respectively. Molecular docking revealed that the binding modes of these molecules in the ATP binding sites
of the Akt kinase domain were very similar to those of the co-crystallized ligand. The information obtained from 3D contour
maps will allow the design of more potent and selective Akt kinase inhibitors. 相似文献
18.
Maize glutathione-dependent formaldehyde dehydrogenase: protein sequence and catalytic properties 总被引:2,自引:0,他引:2
Glutathione-dependent formaldehyde dehydrogenase (FDH; EC 1.2.1.1) has been purified 3900-fold from maize cell-suspension
cultures to a specific activity of 4.68 μmol (mg protein)−1 min−1. The homogeneous enzyme consisted of two identical subunits with a molecular mass of 42 kDa, and an isoelectric point of
5.8. Eight tryptic peptides were sequenced and gave a perfect fit to the protein sequence derived from maize Fdh cDNA (J. Fliegmann and H. Sandermann, 1997, Plant Mol Biol 34: 843–854). There was 62% identity with the eucaryotic FDH consensus
sequence. Michaelis constants of approx. 20 μm (formaldehyde), approx. 50 μm (glutathione) and approx. 31 μm (NAD+) were determined for the maize enzyme as well as for FDH partially purified from dog lung. Besides S-hydroxymethylglutathione, pentanol-1, octanol-1, and ω-hydroxyfatty acids served as substrates for both FDH preparations.
The unusual substrate specificity indicates that FDH may be involved in the detoxification of long-chain lipid peroxidation
products.
Received: 1 April 1998 / Accepted: 18 November 1998 相似文献
19.
Jiang Yunbo Fang Liurong Xiao Shaobo Zhang Hui Chen Huanchun 《Frontiers of Biology in China》2007,2(1):85-91
Pseudorabies virus (PRV), an alpha-herpesvirus, has been developed as a live viral vector for animal vaccines. However, the
PRV recombinant virus TK−/gE−/GP5+ expressing GP5 of porcine reproductive and respiratory syndrome virus (PRRSV), based on the PRV genetically depleted vaccine
strain TK−/gE−/LacZ+, scarcely stimulated the vaccinated animals to produce neutralizing antibodies against PRRSV. To develop a booster-specific
immune response of such PRV recombinants, the ORF5m gene (the modified ORF5 gene having better immune responses) was substituted for the ORF5 gene and introduced into PRV TK−/gE−/LacZ+, resulting in a PRV recombinant named TK−/gE−/GP5m+, which expressed the modified GP5m protein. The recombinant virus was confirmed using PCR, Southern blotting and Western
blotting. TK−/gE−/GP5m+ and TK−/gE−/GP5+ expressing the authentic GP5 protein were inoculated into Balb/c mice to evaluate their immune responses. The results indicated
that the protecting neutralization antibodies (the 3/6 vaccinated mice obtained 1:16) and cell immune responses induced by
TK−/gE−/GP5m+ against PRRSV were higher than that induced by TK−/gE−/GP5+. Thus, the development of the new PRV recombinant expressing the modified GP5m protein as a candidate vaccine established
the basis for the study of bivalent genetic engineering vaccines against PRRSV and PRV.
Translated from Journal of Biotechnology, 2005, 21(6): 858–864 [译自: 生物工程学报] 相似文献
20.
α-Synuclein (α-syn) is the major protein component of the insoluble fibrils that make up Lewy bodies, the hallmark lesions of Parkinson’s
disease. Its C-terminal region contains motifs of charged amino acids that potentially bind metal ions, as well as several
identified phosphorylation sites. We have investigated the metal-binding properties of synthetic model peptides and phosphopeptides
that correspond to residues 119–132 of the C-terminal, polyacidic stretch of human α-syn, with the sequence Ac-Asp-Pro-Asp-Asn-Glu-Ala-Tyr-Glu-Met-Pro-Ser-Glu-Glu-Gly (α-syn119–132). The peptide pY125 replaces tyrosine with phosphotyrosine, whereas pS129 replaces serine with phosphoserine.
By using Tb3+ as a luminescent probe of metal binding, we find a marked selectivity of pY125 for Tb3+ compared with pS129 and α-syn119–132, a result confirmed by isothermal titration calorimetry. Truncated or alanine-substituted peptides show that the
phosphoester group on tyrosine provides a metal-binding anchor that is supplemented by carboxylic acid groups at positions
119, 121, and 126 to establish a multidentate ligand, while two glutamic acid residues at positions 130 and 131 contribute
to binding additional Tb3+ ions. The interaction of other metal ions was investigated by electrospray ionization mass spectrometry, which confirmed
that pY125 is selective for trivalent metal ions over divalent metal ions, and revealed that Fe3+ and Al3+ induce peptide dimerization through metal ion cross-links. Circular dichroism showed that Fe3+ can induce a partially folded structure for pY125, whereas no change was observed for pS129 or the unphosphorylated analog.
The results of this study show that the type and location of a phosphorylated amino acid influence a peptide’s metal-binding
specificity and affinity as well as its overall conformation.
Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. 相似文献