We present the potential of inclusion bodies (IBs) as a protein delivery method for polymeric filamentous proteins. We used as cell factory a strain of E. coli, a conventional host organism, and keratin 14 (K14) as an example of a complex protein. Keratins build the intermediate filament cytoskeleton of all epithelial cells. In order to build filaments, monomeric K14 needs first to dimerize with its binding partner (keratin 5, K5), which is then followed by heterodimer assembly into filaments.
Results
K14 IBs were electroporated into SW13 cells grown in culture together with a ??reporter?? plasmid containing EYFP labeled keratin 5 (K5) cDNA. As SW13 cells do not normally express keratins, and keratin filaments are built exclusively of keratin heterodimers (i.e. K5/K14), the short filamentous structures we obtained in this study can only be the result of: a) if both IBs and plasmid DNA are transfected simultaneously into the cell(s); b) once inside the cells, K14 protein is being released from IBs; c) released K14 is functional, able to form heterodimers with EYFP-K5.
Conclusions
Soluble IBs may be also developed for complex cytoskeletal proteins and used as nanoparticles for their delivery into epithelial cells. 相似文献
Highlights? H. pylori is highly susceptible to the bactericidal activity of hBD3 ? hBD3 is initially induced by H. pylori via activation of EGFR/MAPK/JAK signaling ? H. pylori evades killing by downregulating hBD3 via CagA-mediated SHP-2 activation ? CagA virulence factor is a potent inhibitor of an immune effector mechanism 相似文献
The composition of the colonic microbiota of 91 northern Europeans was characterized by fluorescent in situ hybridization using 18 phylogenetic probes. On average 75% of the bacteria were identified, and large interindividual variations were observed. Clostridium coccoides and Clostridium leptum were the dominant groups (28.0% and 25.2%), followed by the Bacteroides (8.5%). According to principal component analysis, no significant grouping with respect to geographic origin, age, or gender was observed. 相似文献
In this study, we investigate correlation properties of fluctuations in heart interbeat (RR) time series in a broad range of physiological and pathological conditions. Using detrended fluctuation analysis (DFA) method we determined short-term (alpha (1)) and long-term (alpha (2)) scaling exponent. In addition, we calculated standard deviation of RR intervals (SDRR) as the simplest variability measure. We found that the difference between alpha (1) and alpha (2) is related to RR interval length. At the shortest RR intervals, which correspond to extreme physiological and pathological conditions, we found the highest reduction of variability and the biggest difference between scaling exponents. In this case, DFA reveals a white noise over short scales (alpha (1 )about 0.5) and strongly correlated noise over large scales (alpha (2) about 1.5). With an increase in RR interval, accompanied by increased variability (increase in parasympathetic control), the difference between alpha (1) and alpha (2) decreases. The difference between scaling exponents disappeared in a state of efficient autonomic control. We suggest that the complexity in heart rhythm is achieved through coupling between intrinsically controlled heart rhythm and autonomic control, and that the model of stochastic resonance mechanism could be applied to this system. 相似文献
The binding of mercury (Hg) to metallothioneins (MTs) and the relation between Hg and selenium in supernatants of hepatopancreas
and gill tissues of the common mussel Mytilus galloprovincialis (Lamarck, 1819) was investigated. The mussels were exposed to different Hg concentrations in laboratory conditions: 2.5 μgHg/L,
4 d exposure (short term) and 60 μgHg/L, 33 d exposure (long term). In addition, the results were compared to those found
for mussels from nature (polluted and unpolluted region). In control and short-term-exposed mussles, the level of Hg extraction
(cytosol) from hepatopancreas and gill cells was very low with respect to the total Hg concentrations in the corresponding
tissues, around 10% in control and around 20% after exposure. As expected, Hg exposure was followed by Se increase. For Se,
the levels of extraction were higher, around 20% in control and up to 50% (heaptopacrease) of 70% (gills) after exposure.
In order to study the distribution of Hg and Se in the cells of these organs, the total Hg and Secconcentrations were analyzed
in the subcellular fractions obtained after differential centrifugation. Although after exposure the concentrations of both
element increased in all subcellular fractions, their percentages in particular fractions were lower or higher. In this study,
the convincing binding of Hg to metallothionein-like proteins was perceived after long-term laboratory exposure (gills, heapatopancreas)
and in wild mussels collected near industrial port (hepatopancreas). In latter case, we also detected the traces of Se bound
to the MT fractions after size-exclusion chromatography. 相似文献
A facile synthesis of 7-epi-(-)-goniofufurone as well as the first synthesis of (-)-crassalactone C was achieved starting from D-xylose. A comparison of their in vitro antitumour activities with those observed for the corresponding naturally occurring enantiomers was provided. 相似文献
The metabolic syndrome, an assemblage of risk factors, viz., obesity, dyslipidemia, carbohydrate intolerance, and hypertension, associated with development of cardiovascular diseases and Type 2 diabetes, has become a major public health problem in the developed countries. However, data on its prevalence in worldwide populations, particularly in non-industrialized societies is sparse. We report the prevalence of metabolic syndrome in an island population of the eastern Adriatic coast of Croatia, a distinctly homogenous population living in relative isolation with a traditional way of life style pattern. The participants consist of 381 unrelated individuals (136 males, 245 females) from the island of Hvar, age 19 and above. Hvar is among the most populated Croatian islands with a total population of 11,459 individuals according to the 1991 census. Metabolic syndrome was assessed following the National Cholesterol Education Program (NCEP) criteria, with the exception of using body mass index and waist hip ratio as the predictors of obesity. Overall age-adjusted prevalence of metabolic syndrome is 26% (32% in males and 24% in females) with body mass index, and 42% (57% in males and 36% in females) with waist hip ratio as the measures of obesity. Pairwise correlations of the age and sex-adjusted individual components show that with the exception of fasting plasma glucose, the other components of metabolic syndrome are significantly associated with each other, suggesting their co-morbidity. In spite of adherence to a relatively traditional life-style pattern together with a "Mediterranean diet" and rural habitat, the prevalence of metabolic syndrome is substantially high in the population of Hvar. It is likely that factors other than nutritional practices, that might include genetic susceptibility, could potentially be important in predisposition to metabolic syndrome. 相似文献
Structural component of plant biomass, lignocellulose, is the most abundant renewable resource in nature. Lignin is the most recalcitrant natural aromatic polymer and its degradation presents great challenge. Nowadays, the special attention is given to biological delignification, the process where white-rot fungi take the crucial place owing to strong ligninolytic enzyme system. However, fungal species, even strains, differ in potential to produce high active ligninolytic enzymes and consequently to delignify plant biomass. Therefore, the goals of the study were characterization of Mn-oxidizing peroxidases and laccases of numerous mushrooms as well as determination of their potential to delignify wheat straw, the plant raw material that, according to annual yield, takes the first place in Europe and the second one in the world.
Results
During wheat straw fermentation, Lentinus edodes HAI 858 produced the most active Mn-dependent and Mn-independent peroxidases (1443.2 U L−1 and 1045.5 U L−1, respectively), while Pleurotus eryngii HAI 711 was the best laccase producer (7804.3 U L−1). Visualized bends on zymogram confirmed these activities and demonstrated that laccases were the dominant ligninolytic enzymes in the studied species. Ganoderma lucidum BEOFB 435 showed considerable ability to degrade lignin (58.5%) and especially hemicellulose (74.8%), while the cellulose remained almost intact (0.7%). Remarkable selectivity in lignocellulose degradation was also noted in Pleurotus pulmonarius HAI 573 where degraded amounts of lignin, hemicellulose and cellulose were in ratio of 50.4%:15.3%:3.8%.
Conclusions
According to the presented results, it can be concluded that white-rot fungi, due to ligninolytic enzymes features and degradation potential, could be important participants in various biotechnological processes including biotransformation of lignocellulose residues/wastes in food, feed, paper and biofuels.
New protocols for successful asymbiotic seed germination of two European orchid species, Himantoglossum jankae Somlyay, Kreutz & Óvári and Spiranthes spiralis (L.) Chevall., were established in this study. The influence of two basal media, organic supplements, illumination, and cytokinins on germination, protocorm formation, and seedling development was examined. A strong species-specific dependence on illumination conditions and nutrient medium composition was observed. Improved germination under continual darkness indicated that H. jankae had negatively photoblastic seeds, while S. spiralis seeds could germinate under both light conditions examined in this study, but slightly faster development was achieved under the 16-h photoperiod protocol. The H. jankae seeds cultured on Knudson C medium failed to germinate in any medium combination, irrespective of the organic supplement use. The highest germination rate of H. jankae was achieved on Malmgren medium supplemented with coconut water, peptone, and L-glutamine, indicating that germination of this species was amino acid–dependent. Although S. spiralis seeds germinated on all medium combinations, Knudson C basal medium (without supplements) yielded better results when compared with the Malmgren medium. The best S. spiralis seedling development was achieved on the medium supplemented with 6-(γ,γ-dimethylallylamino) purine, while the cytokinin 6-benzyladenine promoted H. jankae seedling development. With the implementation of these protocols, well-developed seedlings were acclimatized to greenhouse conditions after 7 mo in culture.
