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101.
María del L. Yácono Inmaculada Farran Melina L. Becher Valeria Sander Vanesa R. Sánchez Valentina Martín Jon Veramendi Marina Clemente 《Plant biotechnology journal》2012,10(9):1136-1144
The parasitic protozoan Toxoplasma gondii, the causal agent of toxoplasmosis, can infect most mammals and birds. In human medicine, T. gondii can cause complications in pregnant women and immunodeficient individuals, while in veterinary medicine, T. gondii infection has economic importance due to abortion and neonatal loss in livestock. Thus, the development of an effective anti‐Toxoplasma vaccine would be of great value. In this study, we analysed the expression of T. gondii GRA4 antigen by chloroplast transformation (chlGRA4) in tobacco plants and evaluated the humoral and cellular responses and the grade of protection after oral administration of chlGRA4 in a murine model. The Western blot analysis revealed a specific 34‐kDa band mainly present in the insoluble fractions. The chlGRA4 accumulation levels were approximately 6 μg/g of fresh weight (equivalent to 0.2% of total protein). Oral immunization with chlGRA4 resulted in a decrease of 59% in the brain cyst load of mice compared to control mice. ChlGRA4 immunization elicited both a mucosal immune response characterized by the production of specific IgA, and IFN‐γ, IL‐4 and IL‐10 secretion by mesenteric lymph node cells, and a systemic response in terms of GRA4‐specific serum antibodies and secretion of IFN‐γ, IL‐4 and IL‐10 by splenocytes. Our results indicate that oral administration of chlGRA4 promotes the elicitation of both mucosal and systemic balanced Th1/Th2 responses that control Toxoplasma infection, reducing parasite loads. 相似文献
102.
The spinal muscular atrophy disease gene product, SMN: A link between snRNP biogenesis and the Cajal (coiled) body 总被引:1,自引:0,他引:1 下载免费PDF全文
Carvalho T Almeida F Calapez A Lafarga M Berciano MT Carmo-Fonseca M 《The Journal of cell biology》1999,147(4):715-728
The spliceosomal snRNAs U1, U2, U4, and U5 are synthesized in the nucleus, exported to the cytoplasm to assemble with Sm proteins, and reimported to the nucleus as ribonucleoprotein particles. Recently, two novel proteins involved in biogenesis of small nuclear ribonucleoproteins (snRNPs) were identified, the Spinal muscular atrophy disease gene product (SMN) and its associated protein SIP1. It was previously reported that in HeLa cells, SMN and SIP1 form discrete foci located next to Cajal (coiled) bodies, the so-called "gemini of coiled bodies" or "gems." An intriguing feature of gems is that they do not appear to contain snRNPs. Here we show that gems are present in a variable but small proportion of rapidly proliferating cells in culture. In the vast majority of cultured cells and in all primary neurons analyzed, SMN and SIP1 colocalize precisely with snRNPs in the Cajal body. The presence of SMN and SIP1 in Cajal bodies is confirmed by immunoelectron microscopy and by microinjection of antibodies that interfere with the integrity of the structure. The association of SMN with snRNPs and coilin persists during cell division, but at the end of mitosis there is a lag period between assembly of new Cajal bodies in the nucleus and detection of SMN in these structures, suggesting that SMN is targeted to preformed Cajal bodies. Finally, treatment of cells with leptomycin B (a drug that blocks export of U snRNAs to the cytoplasm and consequently import of new snRNPs into the nucleus) is shown to deplete snRNPs (but not SMN or SIP1) from the Cajal body. This suggests that snRNPs flow through the Cajal body during their biogenesis pathway. 相似文献
103.
Zylberman V Craig PO Klinke S Braden BC Cauerhff A Goldbaum FA 《The Journal of biological chemistry》2004,279(9):8093-8101
The penultimate step in the pathway of riboflavin biosynthesis is catalyzed by the enzyme lumazine synthase (LS). One of the most distinctive characteristics of this enzyme is the structural quaternary divergence found in different species. The protein exists as pentameric and icosahedral forms, built from practically the same structural monomeric unit. The pentameric structure is formed by five 18-kDa monomers, each extensively contacting neighboring monomers. The icosahedrical structure consists of 60 LS monomers arranged as 12 pentamers giving rise to a capsid exhibiting icosahedral 532 symmetry. In all lumazine synthases studied, the topologically equivalent active sites are located at the interfaces between adjacent subunits in the pentameric modules. The Brucella sp. lumazine synthase (BLS) sequence clearly diverges from pentameric and icosahedric enzymes. This unusual divergence prompted us to further investigate its quaternary arrangement. In the present work, we demonstrate by means of solution light scattering and x-ray structural analyses that BLS assembles as a very stable dimer of pentamers, representing a third category of quaternary assembly for lumazine synthases. We also describe by spectroscopic studies the thermodynamic stability of this oligomeric protein and postulate a mechanism for dissociation/unfolding of this macromolecular assembly. The higher molecular order of BLS increases its stability 20 degrees C compared with pentameric lumazine synthases. The decameric arrangement described in this work highlights the importance of quaternary interactions in the stabilization of proteins. 相似文献
104.
Influence of maternal environment on the number of transferable embryos obtained in response to superovulatory FSH treatments in ewes 总被引:3,自引:0,他引:3
González-Bulnes A García-García RM Castellanos V Santiago-Moreno J Ariznavarreta C Domínguez V López-Sebastián A Tresguerres JA Cocero MJ 《Reproduction, nutrition, development》2003,43(1):17-28
In a first experiment, embryo viability was estimated after recovery in the uterus or the oviduct of 70 Manchega ewes following a treatment of superovulation with decreasing doses of OVAGEN. Fewer viable embryos (5.6 +/- 0.9 vs. 8.3 +/- 0.8, P < 0.05) and more degenerative embryos (31.3% vs. 6.8%, P < 0.005) were obtained from the uterus than from the oviduct respectively. In a second experiment performed on 14 ewes, embryo viability was analyzed in relation to the follicular population estimated by ultrasonography (follicles > or = 2 mm) at the first FSH administration. Progesterone (P4) and oestradiol 17beta (E2) concentrations were also determined from the beginning of the superovulation treatment to the recovery of the embryos. The number of viable embryos (4.3 +/- 1.4) was positively correlated (r = 0.824) with of 2-4 mm diameter follicles (P < 0.05), and with E2 concentrations at -12 h (r = 0.891, P < 0.01) , 0 h (r = 0.943, P < 0.0001) and +24 h (r = 0.948, P < 0.05) from estrus detection. Prolonged high levels of E2 up to 72 h with low levels of P4 on days 3 and 4 after estrus had a negative (P < 0.05) effect on embryo viability. These results indicate that ovarian response to superovulatory protocols is related to the individual variations in the number of follicles of 2-4 mm at the start of FSH treatment, and that embryo viability is conditioned by the steroid patterns during the time spent in the genital tract of the super-ovulated ewes. 相似文献
105.
Caetano-da-Silva A Ferre I Collantes-Fernández E Navarro V Aduriz G Ugarte-Garagalza C Ortega-Mora LM 《Theriogenology》2004,62(7):1329-1336
Recently, the presence of Neospora caninum DNA in semen from naturally infected bulls was reported. In the present work, the presence and quantification of N. caninum by PCR techniques in frozen extended semen straws from naturally infected bulls was investigated. A total of 20 seropositive and five seronegative bulls raised for reproductive purposes in an AI centre were used. Ten extended semen straws from each bull obtained at different time-points during the previous 2 years were selected for Neospora testing. Eight of the seropositive bulls (40%) studied showed at least one positive straw to N. caninum DNA and 14 of their 180 semen straws examined (7.8%) were found to be positive. In all positive samples, N. caninum DNA was consistently detected in the cell fraction and not in the seminal plasma. However, the parasite number in each positive straw was under the detection level of real-time PCR. In parallel, 10 semen straws from each of the five seronegative bulls were also analyzed by the nested-PCR and no N. caninum DNA products were obtained. In order to check the consistent presence of N. caninum in a positive semen batch, three additional semen straws from the same batch of each positive straw from three seropositive bulls were analyzed but N. caninum DNA was only detected in one straw from one bull. In conclusion, we report the sporadic detection of N. caninum DNA in semen straws of naturally infected bulls but the low frequency of contaminated semen straws and the low parasite load observed indicate a minor chance of bovine neosporosis transmission by AI. 相似文献
106.
Among the eukaryotic members of the Hsp70 family, mitochondrial Hsp70 shows the highest degree of sequence identity with bacterial DnaK. Although they share a functional mechanism and homologous co-chaperones, they are highly specific and cannot be exchanged between Escherichia coli and yeast mitochondria. To provide a structural basis for this finding, we characterized both proteins, as well as two DnaK/mtHsp70 chimeras constructed by domain swapping, using biochemical and biophysical methods. Here, we show that DnaK and mtHsp70 display different conformational and biochemical properties. Replacing different regions of the DnaK peptide-binding domain with those of mtHsp70 results in chimeric proteins that: (a) are not able to support growth of an E. coli DnaK deletion strain at stress temperatures (e.g. 42 degrees C); (b) show increased accessibility and decreased thermal stability of the peptide-binding pocket; and (c) have reduced activation by bacterial, but not mitochondrial co-chaperones, as compared with DnaK. Importantly, swapping the C-terminal alpha-helical subdomain promotes a conformational change in the chimeras to an mtHsp70-like conformation. Thus, interaction with bacterial co-chaperones correlates well with the conformation that natural and chimeric Hsp70s adopt in solution. Our results support the hypothesis that a specific protein structure might regulate the interaction of Hsp70s with particular components of the cellular machinery, such as Tim44, so that they perform specific functions. 相似文献
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110.
Jens S?chtig Vanesa álvarez-Iglesias Ana Mosquera-Miguel Miguel Gelabert-Besada Alberto Gómez-Carballa Antonio Salas 《BMC genomics》2015,16(1)