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341.
Characterization of humoral and CD4+ cellular responses after genetic immunization with retroviral vectors expressing different forms of the hepatitis B virus core and e antigens.
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M Sllberg K Townsend M Chen J O'Dea T Banks D J Jolly S M Chang W T Lee D R Milich 《Journal of virology》1997,71(7):5295-5303
The humoral and CD4+ cellular immune responses in mice following genetic immunization with three retroviral vectors encoding different forms of hepatitis B virus core antigen (HBcAg) and e antigen (HBeAg) were analyzed. The retroviral vectors induced expression of intracellular HBcAg (HBc[3A4]), secreted HBeAg (HBe[5A2]), or an intracellular HBcAg-neomycin phosphoryltransferase fusion protein (HBc-NEO[6A3]). Specific antibody levels and immunoglobulin G isotype restriction were highly dependent on both the host major histocompatibility complex and the transferred gene. Humoral and CD4+ cellular HBcAg and/or HBeAg (HBc/eAg)-specific immune responses following retroviral vector immunization were of a lower magnitude but followed the same characteristics compared with those after immunization with HBc/eAg in adjuvant. Two factors influenced the humoral responses. First, in vivo depletion of CD8+ cells in HBc-NEO[6A3]-immunized H-2k mice abrogated both HBcAg-specific antibodies and in vitro-detectable cytotoxic T lymphocytes. Second, priming of H-2b mice with an HBc/eAg-derived T-helper (Th) peptide in adjuvant prior to retroviral vector immunization greatly enhanced the HBc/eAg-specific humoral responses to all three vectors, suggesting that insufficient HBc/eAg-specific CD4+ Th-cell priming limits the humoral responses. In conclusion, direct injection of retroviral vectors seems to be effective in priming HBc/eAg-specific CD8+ but comparatively inefficient in priming CD4+ Th cells and subsequently specific antibodies. However, the limited HBc/eAg-specific CD4+ cell priming can effectively be circumvented by prior administration of a recombinant or synthetic form of HBc/eAg in adjuvant. 相似文献
342.
Long-lasting adenovirus transgene expression in mice through neonatal intrathymic tolerance induction without the use of immunosuppression. 总被引:6,自引:0,他引:6
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The major barrier to the clinical application of adenovirus gene therapy for diseases that require stable transgene expression is the immunogenicity of recombinant adenovirus, which ordinarily limits the duration of its effects to a period of about 2 weeks. We postulated that tolerance to adenovirus could be induced and transgene expression could be prolonged if T lymphocytes underwent thymic selection in the presence of adenovirus antigens. Mice were inoculated in the thymus with a recombinant adenovirus containing the lacZ marker gene during the neonatal period at a time before T-cell maturation had occurred. When the virus was administered intravenously to these mice in adulthood, they were found to have an impaired adenovirus-specific cytotoxic T-lymphocyte response which allowed prolonged hepatic lacZ expression, for up to 260 days. The ability to achieve unresponsiveness to a recombinant adenovirus after inoculation of the thymus in neonates extends the paradigm of intrathymic tolerance induction. Furthermore, this model will enable the study of stable adenovirus transgene expression in vivo without the use of immunosuppression and ultimately may have clinical utility. 相似文献
343.
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345.
Sequence variability in homologs of the aflatoxin pathway gene aflR distinguishes species in Aspergillus section Flavi. 总被引:3,自引:3,他引:0
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The Aspergillus parasiticus aflR gene, a gene that may be involved in the regulation of aflatoxin biosynthesis, encodes a putative zinc finger DNA-binding protein. PCR and sequencing were used to examine the presence of aflR homologs in other members of Aspergillus Section Flavi. The predicted amino acid sequences indicated that the same zinc finger domain, CTSCASSKVRCTKEKPACARCIERGLAC, was present in all of the Aspergillus sojae, Aspergillus flavus, and Aspergillus parasiticus isolates examined and in some of the Aspergillus oryzae isolates examined. Unique base substitutions and a specific base deletion were found in the 5' untranslated and zinc finger region; these differences provided distinct fingerprints. A. oryzae and A. flavus had the T-G-A-A-X-C fingerprint, whereas A. parasiticus and A sojae had the C-C-C-C-C-T fingerprint at the corresponding positions. Specific nucleotides at positions -90 (C or T) and -132 (G or A) further distinguished A. flavus from A. oryzae and A. parasiticus from A. sojae, respectively. A sojae ATCC 9362, which was previously designated A. oryzae NRRL 1988, was determined to be a A. sojae strain on the basis of the presence of the characteristic fingerprint, A-C-C-C-C-C-C-T. The DNAs of other members of Aspergillus Section Flavi, such as Aspergillus nomius and Aspergillus tamarii, and some isolates of A. oryzae appeared to exhibit low levels of similarity to the A. parasiticus aflR gene since low amounts of PCR products or no PCR products were obtained when DNAs from these strains were used. 相似文献
346.
Summary We have introduced the gene encoding luciferase from Photinus pyralis into pear and tobacco cells in order to judge the reaction of plant tissue to damaging conditions such as incubation at high temperature or inoculation with a pathogen. The constitutive expression of the luciferase gene via a strong promoter slowly decreased during propagation of the transformed pear cell line. After various stress treatments the resulting luciferase activity and the ATP content of the plant cells were determined by bioluminescence and found to correspond to each other. Inoculation of transformed pear cells with Erwinia amylovora resulted in a continuous decrease of luciferase activity in contrast to tobacco cells, where the enzyme activity was significantly higher in the first period after inoculation with bacteria compared to the untreated control cells. The pattern of the luciferase activity reflected the slow damage of the host-plant cells by E. amylovora and the elevated metabolism of the non-host cells after inoculation with the pathogen.Abbreviations 2,4-D
2,4-dichloro-phenoxyacetic acid
- CaMV
Cauliflower mosaic virus
- DTT
dithiothreitol
- EDTA
ethylenediaminetetraacetate
- FDA
fluorescein diacetate
- HEPES
(hydroxyethyl)piperazine(ethanesulfonic acid)
- HR
hypersensitive reaction
- Tris
tris (hydroxymethyl)amino-methane 相似文献
347.
Rabindranath Chakrabarti Joseph Y. Chang Kent L. Erickson 《Journal of cellular biochemistry》1995,58(3):344-359
In this study, we showed that cross-linking CD3 molecules on the T cell surface resulted in Ca2+ release from the intracellular stores followed by a sustained Ca2+ influx. Inhibition of release with TMB-8 did not block the influx. However, inhibition of phospholipase C activity suppressed both Ca2+ release and influx. Once activated, the influx pathway remained open in the absence of further hydrolysis of PIP2. Thapsigargin, a microsomal Ca2+ -ATPase inhibitor, stimulated Ca2+ entry into the cells by a mechanism other than emptying Ca2+ stores. In addition, Ca2+ entry into the Ca2+ -depleted cells was stimulated by low basal level of cytosolic Ca2+, not by the emptying of intracellular Ca2+ stores. Both the Ca2+ release and influx were dependent on high and low concentrations of extracellular Ca2+. At low concentrations, Mn2+ entered the cell through the Ca2+ influx pathway and quenched the sustained phase of fluorescence; whereas, at higher Mn2+ concentration both the transient and the sustained phases of fluorescence were quenched. Moreover, Ca2+ release was inhibited by low concentrations of Ni2+, La3+, and EGTA, while Ca2+ influx was inhibited by high concentrations. Thus, in T cells Ca2+ influx occurs independently of IP3-dependent Ca2+ release. However, some other PIP2 hydrolysis-dependent event was involved in prolonged activation of Ca2+ influx. Extracellular Ca2+ influenced Ca2+ release and influx through the action of two plasma membrane Ca2+ entry pathways with different pharmacological and biochemical properties. 相似文献
348.
Cheom Gil Cheong Soo Hyun Eom Changsoo Chang Dong Hae Shin Hyun Kyu Song Kyeongsik Min Jin Ho Moon Kyeong Kyu Kim Kwang Yeon Hwang Se Won Suh 《Proteins》1995,21(2):105-117
Sweet potato β-amylase is a tetramer of identical subunits, which are arranged to exhibit 222 molecular symmetry. Its subunit consists of 498 amino acid residues (Mr 55,880). It has been crystallized at room temperature using polyethylene glycol 1500 as precipitant. The crystals, growing to dimensions of 0.4 mm × 0.4 mm × 1.0 mm within 2 weeks, belong to the tetragonal space group P42212 with unit cell dimensions of a = b = 129.63 Å and c = 68.42 Å. The asymmetric unit contains 1 subunit of β-amylase, with a crystal volume per protein mass (VM) of 2.57 Å3/Da and a solvent content of 52% by volume. The three-dimensional structure of the tetrameric β-amylase from sweet potato has been determined by molecular replacement methods using the monomeric structure of soybean enzyme as the starting model. The refined subunit model contains 3,863 nonhydrogen protein atoms (488 amino acid residues) and 319 water oxygen atoms. The current R-value is 20.3% for data in the resolution range of 8–2.3 Å (with 2 σ cut-off) with good stereochemistry. The subunit structure of sweet potato β-amylase (crystallized in the absence of α-cyclodextrin) is very similar to that of soybean β-amylase (complexed with α-cyclodextrin). The root-mean-square (RMS) difference for 487 equivalent Cα atoms of the two β-amylases is 0.96 Å. Each subunit of sweet potato β-amylase is composed of a large (α/β)8 core domain, a small one made up of three long loops [L3 (residues 91–150), LA (residues 183–258), and L5 (residues 300–327)], and a long C-terminal loop formed by residues 445–493. Conserved Glu 187, believed to play an important role in catalysis, is located at the cleft between the (α/β)8 barrel core and a small domain made up of three long loops (L3, L4, and L5). Conserved Cys 96, important in the inactivation of enzyme activity by sulfhydryl reagents, is located at the entrance of the (α/β)8 barrel. © 1995 Wiley-Liss, Inc. 相似文献
349.
350.
Molecular dynamics simulations have been performed on three phenylimidazole inhibitor complexes ofP450
cam, utilizing the X-ray structures and the AMBER suite of programs. Compared to their corresponding optimized X-ray structures, very similar features were observed for the 1-phenylimidazole (1-PI) and 2-phenylimidazole (2-PI) complexes during a 100 ps MD simulation. The 1-PI inhibitor binds as a Type II complex with the imidazole nitrogen as a ligand of the heme iron. Analysis of the inhibitor-enzyme interctions during the MD simulations reveals that electrostatic interactions of the imidazole with the heme and van der Waals interactions of the phenyl ring with nearby hydrophobic residues are dominant. By contrast, 2-PI binds as a Type I inhibitor in the substrate binding pocket, but not as a ligand of the iron. The interactions of this inhibitor are qualitatively different from that of the Type II 1-PI, being mainly electrostatic/H-bonding interactions with a bound water and polar residues. Although the third compound, 4-PI, in common with 1-PI, also binds as a Type II inhibitor, with one nitrogen of the imidazole as a ligand to the iron, the MD average binding orientation deviates significantly from the X-ray structure. The most important changes observed include: (1) the rotation of the imidazole ring of this inhibitor by about 90° to enhance electrostatic interactions of the imidazole NH group with the carbonyl group of LEU244, and (2) the rotation of the carbonyl group of ASP251 to form a H-bond with VAL254. An analysis of the H-bonding network surrounding this substrate in the optimized crystal structure revealed that there is no H-bonding partner either for the free polar NH group in the imidazole ring of 4-phenylimidazole or for the polar carbonyl group of the nearby ASP251 residue. The deviation of the dynamically averaged inhibitor-enzyme structure of the 4-PI complex from the optimized crystal structure can therefore be rationalized as a consequence of the optimization of the electrostatic interactions among the polar groups. 相似文献