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121.
Isabel Guerrero Montero Kirsty L. Richards Chillel Jawara Douglas F. Browning Amber R. Peswani Mickael Labrit Matthew Allen Cedric Aubry Emma Davé David P. Humphreys Stephen J. W. Busby Colin Robinson 《Biotechnology and bioengineering》2019,116(12):3282-3291
Escherichia coli is a heavily used platform for the production of biotherapeutic and other high-value proteins, and a favored strategy is to export the protein of interest to the periplasm to simplify downstream processing and facilitate disulfide bond formation. The Sec pathway is the standard means of transporting the target protein but it is unable to transport complex or rapidly folding proteins because the Sec system can only transport proteins in an unfolded state. The Tat system also operates to transport proteins to the periplasm, and it has significant potential as an alternative means of recombinant protein production because it transports fully folded proteins. Here, we have tested the Tat system's full potential for the production of biotherapeutics for the first time using fed-batch fermentation. We expressed human growth hormone (hGH) with a Tat signal peptide in E. coli W3110 “TatExpress” strains that contain elevated levels of the Tat apparatus. This construct contained four amino acids from TorA at the hGH N-terminus as well as the initiation methionine from hGH, which is removed in vivo. We show that the protein is efficiently exported to the periplasm during extended fed-batch fermentation, to the extent that it is by far the most abundant protein in the periplasm. The protein was shown to be homogeneous, disulfide bonded, and active. The bioassay showed that the yields of purified periplasmic hGH are 5.4 g/L culture whereas an enzyme-linked immunosorbent assay gave a figure of 2.39 g/L. Separate analysis of a TorA signal peptide linked to hGH construct lacking any additional amino acids likewise showed efficient export to the periplasm, although yields were approximately two-fold lower. 相似文献
122.
The chronology of post-larval development in S. chrysophrii, a polyopisthocotylean monogenean parasite of the gilthead seabream (Sparus aurata L.), was experimentally studied. It is compared with other species within the Microcotylidae and the Heteraxinidae, including an analysis of the changes in attachment and the growth rate. Gilthead seabreams infected by larvae of S. chrysophrii were killed periodically in order to collect the different developmental stages. Parasite total body length, haptor length, largest clamp width, and total number of clamps were recorded. Specimens of S. chrysophrii in culture conditions at 20°C became gravid after 26-30 days, with 37 pairs of clamps. The S. chrysophrii growth curve appears to be sigmoid with 3 growth periods (slow-fast-slow). The haptor of S. chrysophrii grows linearly with total body length, but the main contribution to total body length growth is that of the non-haptoral body. The relationship between number of clamps and total body length during development can be fitted to an exponential curve for all the reviewed species, i.e.: Microcotyle spinicirrus, Microcotyle donavini, Microcotyle gotoi, Microcotyle sebastis, Microcotyle hiatulae, Polylabroides multispinosus, Bivagina tai, Heteraxinoides xanthophilis, Heteraxine heterocerca, and Zeuxapta seriolae. The sequence of events was common for all of the species compared: terminal lappet is lost when about 15% of clamps were developed; primordia of testes at approximately 30% of clamps developed, and maturity (as first egg appearance) at about 65% of clamps developed. 相似文献
123.
Manoeli Lupatini Afnan Khalil Ahmad Suleiman Rodrigo Josemar Seminoti Jacques Zaida Inês Antoniolli Eiko Eurya Kuramae Flávio Anastácio de Oliveira Camargo Luiz Fernando Würdig Roesch 《PloS one》2013,8(10)
The Pampa biome is considered one of the main hotspots of the world’s biodiversity and it is estimated that half of its original vegetation was removed and converted to agricultural land and tree plantations. Although an increasing amount of knowledge is being assembled regarding the response of soil bacterial communities to land use change, to the associated plant community and to soil properties, our understanding about how these interactions affect the microbial community from the Brazilian Pampa is still poor and incomplete. In this study, we hypothesized that the same soil type from the same geographic region but under distinct land use present dissimilar soil bacterial communities. To test this hypothesis, we assessed the soil bacterial communities from four land-uses within the same soil type by 454-pyrosequencing of 16S rRNA gene and by soil microbial activity analyzes. We found that the same soil type under different land uses harbor similar (but not equal) bacterial communities and the differences were controlled by many microbial taxa. No differences regarding diversity and richness between natural areas and areas under anthropogenic disturbance were detected. However, the measures of microbial activity did not converge with the 16S rRNA data supporting the idea that the coupling between functioning and composition of bacterial communities is not necessarily correlated. 相似文献
124.
Lysosomal cathepsins in embryonic programmed cell death 总被引:1,自引:0,他引:1
Zuzarte-Luis V Montero JA Kawakami Y Izpisua-Belmonte JC Hurle JM 《Developmental biology》2007,301(1):205-217
During limb development, expression of cathepsin D and B genes prefigure the pattern of interdigital apoptosis including the differences between the chick and the webbed digits of the duck. Expression of cathepsin L is associated with advanced stages of degeneration. Analysis of Gremlin-/- and Dkk-/- mouse mutants and local treatments with BMP proteins reveal that the expression of cathepsin B and D genes is regulated by BMP signaling, a pathway responsible for triggering cell death. Further cathepsin D protein is upregulated in the preapoptotic mesenchyme before being released into the cytosol, and overexpression of cathepsin D induces cell death in embryonic tissues by a mechanism including mitochondrial permeabilization and nuclear translocation of AIF. Combined inhibition of cathepsin and caspases suggests a redundancy in the apoptotic molecular machinery, providing evidence for compensatory activation mechanisms in the cathepsin pathway when caspases are blocked. It is concluded that lysosomal enzymes are functionally implicated in embryonic programmed cell death. 相似文献
125.
González LM Daniel-Mwambete K Montero E Rosenzvit MC McManus DP Gárate T Cuesta-Bandera C 《Experimental parasitology》2002,102(1):46-56
We have designed two polymerase chain reaction (PCR) primer sets (PEg9F1-PEg9R1 and PEg16F1-PEg16R1) and two PCR protocols (Eg9-PCR and Eg16-PCR) for discrimination of Echinococcus granulosus genotypes. The oligonucleotide sequences originate from two E. granulosus DNA multiplex-PCR amplification fragments, previously reported, that allows species-specific discrimination between Taenia saginata, Taenia solium, and E. granulosus. The Eg9-PCR, Eg16-PCR, and Eg9-PCR linked restriction fragment length polymorphism (RFLP) analysis was used to characterize 53 E. granulosus isolates from the central region of Spain, highly endemic for echinococcosis. The analysis resulted in: (i) the discrimination of E. granulosus from Echinococcus multilocularis; (ii) the characterisation and discrimination of discrete E. granulosus strains from Spain; and (iii) the identification of two distinct genotypes within E. granulosus Spanish pig isolates. To further characterize the genetic variants in pigs, fragments of the NADH dehydrogenase I (ND1) and the cytochrome c oxidase subunit I (CO1) genes were amplified from parasite DNA and sequenced. The results again revealed the presence of two distinct genotypes: the G1 (sheep-dog strain) and G7 (pig-dog strain) genotypes. This observation could have important consequences for human health in Spain. Furthermore, the Eg9-PCR, Eg16-PCR, and Eg9-PCR-RFLP protocols can be used as additional methods to discriminate various E. granulosus genotypes. 相似文献
126.
Zaida Agüera Xandra Romero Jon Arcelus Isabel Sánchez Nadine Riesco Susana Jiménez-Murcia Jana González-Gómez Roser Granero Nuria Custal Monica Montserrat-Gil de Bernabé Salomé Tárrega Rosa M. Ba?os Cristina Botella Rafael de la Torre José C. Fernández-García José M. Fernández-Real Gema Frühbeck Javier Gómez-Ambrosi Francisco J. Tinahones Ana B. Crujeiras Felipe F. Casanueva José M. Menchón Fernando Fernández-Aranda 《PloS one》2015,10(11)
The restoration of body composition (BC) parameters is considered to be one of the most important goals in the treatment of patients with anorexia nervosa (AN). However, little is known about differences between AN diagnostic subtypes [restricting (AN-R) and binge/purging (AN-BP)] and weekly changes in BC during refeeding treatment. Therefore, the main objectives of our study were twofold: 1) to assess the changes in BC throughout nutritional treatment in an AN sample and 2) to analyze predictors of BC changes during treatment, as well as predictors of treatment outcome. The whole sample comprised 261 participants [118 adult females with AN (70 AN-R vs. 48 AN-BP), and 143 healthy controls]. BC was measured weekly during 15 weeks of day-hospital treatment using bioelectrical impedance analysis (BIA). Assessment measures also included the Eating Disorders Inventory-2, as well as a number of other clinical indices. Overall, the results showed that AN-R and AN-BP patients statistically differed in all BC measures at admission. However, no significant time×group interaction was found for almost all BC parameters. Significant time×group interactions were only found for basal metabolic rate (p = .041) and body mass index (BMI) (p = .035). Multiple regression models showed that the best predictors of pre-post changes in BC parameters (namely fat-free mass, muscular mass, total body water and BMI) were the baseline values of BC parameters. Stepwise predictive logistic regressions showed that only BMI and age were significantly associated with outcome, but not with the percentage of body fat. In conclusion, these data suggest that although AN patients tended to restore all BC parameters during nutritional treatment, only AN-BP patients obtained the same fat mass values as healthy controls. Put succinctly, the best predictors of changes in BC were baseline BC values, which did not, however, seem to influence treatment outcome. 相似文献
127.
Induction of intracellular and extracellular beta-galactosidase activity in Phycomyces blakesleeanus
The inductive effect of different sugars on beta-galactosidase synthesis in Phycomyces blakesleeanus has been studied. The enzyme was inducible by galactose and fructose. When grown on these sugars the enzyme level was 10-20 times greater than when grown on glucose. We have detected both intra- and extracellular beta-galactosidase activity when Phycomyces blakesleeanus was grown on galactose, but only extracellular beta-galactosidase activity when grown on fructose plus lactose. 相似文献
128.
129.
Carlos G. Montero Dr. Antonio Ventosa Joaquín J. Nieto Francisco Ruiz-Berraquero 《Current microbiology》1991,23(6):299-302
We report here the first available data on the genomic structure of an archaebacterial, extremely halophilic coccus. Thus, the physical map of a region of 257 kilobase-pairs (kbp) from the extremely halophilic aerobic archaebacteriumHalococcus saccharolyticus ATCC 49257 has been constructed. This DNA fragment could be ascribed to the FI fraction of the chromosome. Long repeated sequences were not detected, but, on the other hand, some losses of fragments in the recombinants were found. These data suggest that only a minor part of the chromosome ofH. saccharolyticus, which is not cccDNA, is accompanied by multiple rearrangements, while the other, which includes most of the chromosome, possesses more genetic stability. Our results indicate that there is a low degree of genomic variability in contrast to the high instability reported forHalobacterium salinarium. 相似文献
130.
J Sánchez Olavarría C Galindo M Montero Y Baquero J Victorica J Satrústegui 《Biochimica et biophysica acta》1988,935(3):322-332
(1) A method is presented for continuous and simultaneous monitoring of the 'in situ' mitochondrial membrane potential (delta psi m) and respiration rate of Ehrlich ascites tumor cells. The method involves permeabilization of the plasma membrane, achieved by treatment with low digitonin concentration, and the use of a TPP+ selective electrode attached to an oxygraph vessel. Binding of the probe inside the cells was analyzed assuming a proportional relationship between the amount of bound TPP+ and the free concentration of the lipophilic cation. (2) Evidence is reported that the addition of glucose to digitonin-permeabilized Ehrlich ascites tumor cells causes a decrease of mitochondrial membrane potential that coincided with a transient enhancement of the respiration rate and remained unchanged during the subsequent Crabtree effect. We have characterized the effect of glucose on delta psi m by determining its dependent on the glycolytic pathway and its sensitivity towards oligomycin. The mutual relationships between glucose and ADP effects on the mitochondrial membrane potential were also studied. A plausible mechanism underlying the depolarization of mitochondrial membrane induced by glucose is presented. 相似文献