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1.
Paloheimo M Mäntylä A Kallio J Puranen T Suominen P 《Applied and environmental microbiology》2007,73(10):3215-3224
We have previously shown that the Nonomuraea flexuosa Xyn11A polypeptides devoid of the carbohydrate binding module (CBM) have better thermostability than the full-length xylanase and are effective in bleaching of pulp. To produce an enzyme preparation useful for industrial applications requiring high temperature, the region encoding the CBM was deleted from the N. flexuosa xyn11A gene and the truncated gene was expressed in Trichoderma reesei. The xylanase sequence was fused to the T. reesei mannanase I (Man5A) signal sequence or 3' to a T. reesei carrier polypeptide, either the Man5A core/hinge or the cellulose binding domain (CBD) of cellobiohydrolase II (Cel6A, CBHII). The gene and fusion genes were expressed using the cellobiohydrolase 1 (cel7A, cbh1) promoter. Single-copy isogenic transformants in which the expression cassette replaced the cel7A gene were cultivated and analyzed. The transformants expressing the truncated N. flexuosa xyn11A produced clearly increased amounts of both the xylanase/fusion mRNA and xylanase activity compared to the corresponding strains expressing the full-length N. flexuosa xyn11A. The transformant expressing the cel6A CBD-truncated N. flexuosa xyn11A produced about 1.9 g liter-1 of the xylanase in laboratory-scale fermentations. The xylanase constituted about 25% of the secreted proteins. The production of the truncated xylanase did not induce the unfolded protein response (UPR) pathway. However, the UPR was induced when the full-length N. flexuosa xyn11A with an exact fusion to the cel7A terminator was expressed. We suggest that the T. reesei folding/secretion machinery is not able to cope properly with the bacterial CBM when the mRNA of the full-length N. flexuosa xyn11A is efficiently translated. 相似文献
2.
Hydrobiologia - Forestry-related land use can cause increasing instream sedimentation, burying and eradicating stream bryophytes, with severe ecological consequences. However, there is limited... 相似文献
3.
K. Rissanen T. Hölttä A. Vanhatalo J. Aalto E. Nikinmaa H. Rita J. Bäck 《Plant, cell & environment》2016,39(3):527-538
Coniferous tree stems contain large amounts of oleoresin under positive pressure in the resin ducts. Studies in North‐American pines indicated that the stem oleoresin exudation pressure (OEP) correlates negatively with transpiration rate and soil water content. However, it is not known how the OEP changes affect the emissions of volatile vapours from the trees. We measured the OEP, xylem diameter changes indicating changes in xylem water potential and monoterpene emissions under field conditions in mature Scots pine (Pinus sylvestris L.) trees in southern Finland. Contrary to earlier reports, the diurnal OEP changes were positively correlated with temperature and transpiration rate. OEP was lowest at the top part of the stem, where water potentials were also more negative, and often closely linked to ambient temperature and stem monoterpene emissions. However, occasionally OEP was affected by sudden changes in vapour pressure deficit (VPD), indicating the importance of xylem water potential on OEP as well. We conclude that the oleoresin storage pools in tree stems are in a dynamic relationship with ambient temperature and xylem water potential, and that the canopy monoterpene emission rates may therefore be also regulated by whole tree processes and not only by the conditions prevailing in the upper canopy. 相似文献
4.
Gamauf C Marchetti M Kallio J Puranen T Vehmaanperä J Allmaier G Kubicek CP Seiboth B 《The FEBS journal》2007,274(7):1691-1700
The extracellular bga1-encoded beta-galactosidase of Hypocrea jecorina (Trichoderma reesei) was overexpressed under the pyruvat kinase (pki1) promoter region and purified to apparent homogeneity. The monomeric enzyme is a glycoprotein with a molecular mass of 118.8 +/- 0.5 kDa (MALDI-MS) and an isoelectric point of 6.6. Bga1 is active with several disaccharides, e.g. lactose, lactulose and galactobiose, as well as with aryl- and alkyl-beta-D-galactosides. Based on the catalytic efficiencies, lactitol and lactobionic acid are the poorest substrates and o-nitrophenyl-beta-D-galactoside and lactulose are the best. The pH optimum for the hydrolysis of galactosides is approximately 5.0, and the optimum temperature was found to be 60 degrees C. Bga1 is also capable of releasing D-galactose from beta-galactans and is thus actually a galacto-beta-D-galactanase. beta-Galactosidase is inhibited by its reaction product D-galactose and the enzyme also shows a significant transferase activity which results in the formation of galacto-oligosaccharides. 相似文献
5.
Laasanen MS Saarakkala S Töyräs J Hirvonen J Rieppo J Korhonen RK Jurvelin JS 《Journal of biomechanics》2003,36(9):1259-1267
We have earlier developed a handheld ultrasound indentation instrument for the diagnosis of articular cartilage degeneration. In ultrasound indentation, cartilage is compressed with the ultrasound transducer. Tissue thickness and deformation are calculated from the A-mode ultrasound signal and the stress applied is registered with the strain gauges. In this study, the applicability of the ultrasound indentation instrument to quantify site-dependent variation in the mechano-acoustic properties of bovine knee cartilage was investigated. Osteochondral blocks (n=6 per site) were prepared from the femoral medial condyle (FMC), the lateral facet of the patello-femoral groove (LPG) and the medial tibial plateau (MTP). Cartilage stiffness (dynamic modulus, E(dyn)), as obtained with the ultrasound indentation instrument in situ, correlated highly linearly (r=0.913, p<0.01) with the values obtained using the reference material-testing device in vitro. Reproducibility (standardized coefficient of variation) of the ultrasound indentation measurements was 5.2%, 1.7% and 3.1% for E(dyn), ultrasound reflection coefficient of articular surface (R) and thickness, respectively. E(dyn) and R were site dependent (p<0.05, Kruskall-Wallis H test). E(dyn) was significantly higher (p<0.05, Kruskall-Wallis Post Hoc test) in LPG (mean+/-SD: 10.1+/-3.1MPa) than in MTP (2.9+/-1.4MPa). In FMC, E(dyn) was 4.6+/-1.3MPa. R was significantly (p<0.05) lower at MTP (2.0+/-0.7%) than at other sites (FMC: 4.2+/-0.9%; LPG: 4.4+/-0.8%). Cartilage glycosaminoglycan concentration, as quantified with the digital densitometry, correlated positively with E(dyn) (r=0.678, p<0.01) and especially with the equilibrium Young's modulus (reference device, r=0.874, p<0.01) but it was not associated with R (r=0.294, p=0.24). We conclude that manual measurements are reproducible and the instrument may be used for detection of cartilage quality in situ. Especially, combined measurement of thickness, E(dyn) and R provides valuable diagnostic information on cartilage status. 相似文献
6.
Paloheimo M Mäntylä A Kallio J Suominen P 《Applied and environmental microbiology》2003,69(12):7073-7082
A bacterial xylanase gene, Nonomuraea flexuosa xyn11A, was expressed in the filamentous fungus Trichoderma reesei from the strong cellobiohydrolase 1 promoter as fusions to a variety of carrier polypeptides. By using single-copy isogenic transformants, it was shown that production of this xylanase was clearly increased (up to 820 mg/liter) when it was produced as a fusion protein with a carrier polypeptide having an intact domain structure compared to the production (150 to 300 mg/liter) of fusions to the signal sequence alone or to carriers having incomplete domain structures. The carriers tested were the T. reesei mannanase I (Man5A, or MANI) core-hinge and a fragment thereof and the cellulose binding domain of T. reesei cellobiohydrolase II (Cel6A, or CBHII) with and without the hinge region(s) and a fragment thereof. The flexible hinge region was shown to have a positive effect on both the production of Xyn11A and the efficiency of cleavage of the fusion polypeptide. The recombinant Xyn11A produced had properties similar to those of the native xylanase. It constituted 6 to 10% of the total proteins secreted by the transformants. About three times more of the Man5A core-hinge carrier polypeptide than of the recombinant Xyn11A was observed. Even in the best Xyn11A producers, the levels of the fusion mRNAs were only approximately 10% of the level of cel7A (cbh1) mRNA in the untransformed host strain. 相似文献
7.
Niina Tohmola Jouni Ahtinen Juha-Pekka Pitkänen Ville Parviainen Sakari Joenväärä Mika Hautamäki Peter Lindroos Jarno Mäkinen Risto Renkonen 《Biotechnology and Bioprocess Engineering》2011,16(2):264-272
We constructed a bioprocess environment enabling automatic sampling from a bioreactor combined with a compact on-line high
performance liquid chromatography (HPLC) unit. This setup allowed us to measure extracellular glucose, ethanol, glycerol,
and acetate concentrations automatically at 5 min intervals during the cultivation. This environment also provides mechanical
measurement of the optical density (OD) of cells and enables us to collect and store (−35°C) samples for further off-line
analyses. Among the available devices, the performance of the sampling-analysis unit is by far the best with regard to speed
and number of analytes. Both the sampling and analysis phases are easily controlled by software; thus, providing a unique
environment to perform various bioprocess activity tasks, whether they would be cell line screening or optimisation of conditions
for growth and productivity. Complex research set-ups can be created and continuous automated measurements empower long-term
cultivations with a time series. We provide evidence for the applicability of this environment by performing three comparable
batch cultivations with Saccharomyces cerevisiae yeast and show that both the on-line sampling and analysis modes produce reliable data for further use in the monitoring
and controlling of bioprocesses. On-line data provided new insight into the dynamics of the diauxic shift during aerobic glucose
batch cultivation. When cell growth and carbon dioxide production ceased for the first time during the diauxic shift, acetate
accumulation and consumption of the remaining glucose below 0.15 g/L continued to occur for 1 h. At the same time, glycerol
and ethanol began to be consumed. Samples were also collected during cultivation for later analysis of intracellular metabolites
and to collect more valuable information about metabolism. 相似文献
8.
Saarakkala S Korhonen RK Laasanen MS Töyräs J Rieppo J Jurvelin JS 《Biorheology》2004,41(3-4):167-179
The compressive stiffness of an elastic material is traditionally characterized by its Young's modulus. Young's modulus of articular cartilage can be directly measured using unconfined compression geometry by assuming the cartilage to be homogeneous and isotropic. In isotropic materials, Young's modulus can also be determined acoustically by the measurement of sound speed and density of the material. In the present study, acoustic and mechanical techniques, feasible for in vivo measurements, were investigated to quantify the static and dynamic compressive stiffness of bovine articular cartilage in situ. Ultrasound reflection from the cartilage surface, as well as the dynamic modulus were determined with the recently developed ultrasound indentation instrument and compared with the reference mechanical and ultrasound speed measurements in unconfined compression (n=72). In addition, the applicability of manual creep measurements with the ultrasound indentation instrument was evaluated both experimentally and numerically. Our experimental results indicated that the sound speed could predict 47% and 53% of the variation in the Young's modulus and dynamic modulus of cartilage, respectively. The dynamic modulus, as determined manually with the ultrasound indentation instrument, showed significant linear correlations with the reference Young's modulus (r(2)=0.445, p<0.01, n=70) and dynamic modulus (r(2)=0.779, p<0.01, n=70) of the cartilage. Numerical analyses indicated that the creep measurements, conducted manually with the ultrasound indentation instrument, were sensitive to changes in Young's modulus and permeability of the tissue, and were significantly influenced by the tissue thickness. We conclude that acoustic parameters, i.e. ultrasound speed and reflection, are indicative to the intrinsic mechanical properties of the articular cartilage. Ultrasound indentation instrument, when further developed, provides an applicable tool for the in vivo detection of cartilage mechano-acoustic properties. These techniques could promote the diagnostics of osteoarthrosis. 相似文献
9.
Ajay Pradhan Hazem Khalaf Scott A. Ochsner Rajini Sreenivasan Jarno Koskinen Marie Karlsson Jesper Karlsson Neil J. McKenna L��szl�� Orb��n Per-Erik Olsson 《The Journal of biological chemistry》2012,287(45):37926-37938
Testis differentiation in zebrafish involves juvenile ovary to testis transformation initiated by an apoptotic wave. The molecular regulation of this transformation process is not fully understood. NF-κB is activated at an early stage of development and has been shown to interact with steroidogenic factor-1 in mammals, leading to the suppression of anti-Müllerian hormone (Amh) gene expression. Because steroidogenic factor-1 and Amh are important for proper testis development, NF-κB-mediated induction of anti-apoptotic genes could, therefore, also play a role in zebrafish gonad differentiation. The aim of this study was to examine the potential role of NF-κB in zebrafish gonad differentiation. Exposure of juvenile zebrafish to heat-killed Escherichia coli activated the NF-κB pathways and resulted in an increased ratio of females from 30 to 85%. Microarray and quantitative real-time-PCR analysis of gonads showed elevated expression of NF-κB-regulated genes. To confirm the involvement of NF-κB-induced anti-apoptotic effects, zebrafish were treated with sodium deoxycholate, a known inducer of NF-κB or NF-κB activation inhibitor (NAI). Sodium deoxycholate treatment mimicked the effect of heat-killed bacteria and resulted in an increased proportion of females from 25 to 45%, whereas the inhibition of NF-κB using NAI resulted in a decrease in females from 45 to 20%. This study provides proof for an essential role of NF-κB in gonadal differentiation of zebrafish and represents an important step toward the complete understanding of the complicated process of sex differentiation in this species and possibly other cyprinid teleosts as well. 相似文献
10.
Expression analysis of immune response genes in fish epithelial cells following ranavirus infection 总被引:1,自引:0,他引:1
Ranaviruses (family Iridoviridae) are a growing threat to fish and amphibian populations worldwide. The immune response to ranavirus infection has been studied in amphibians, but little is known about the responses elicited in piscine hosts. In this study, the immune response and apoptosis induced by ranaviruses were investigated in fish epithelial cells. Epithelioma papulosum cyprini (EPC) cells were infected with four different viral isolates: epizootic haematopoietic necrosis virus (EHNV), frog virus 3 (FV3), European catfish virus (ECV) and doctor fish virus (DFV). Quantitative real-time PCR (qPCR) assays were developed to measure the mRNA expression of immune response genes during ranavirus infection. The target genes included tumour necrosis factor α (TNF-α), interleukin-1β (IL-1β), β2-microglobulin (β2M), interleukin-10 (IL-10) and transforming growth factor β (TGF-β). All ranaviruses elicited changes in immune gene expression. EHNV and FV3 caused a strong pro-inflammatory response with an increase in the expression of both IL-1β and TNF-α, whereas ECV and DFV evoked transient up-regulation of regulatory cytokine TGF-β. Additionally, all viral isolates induced increased β2M expression as well as apoptosis in the EPC cells. Our results indicate that epithelial cells can serve as an in vitro model for studying the mechanisms of immune response in the piscine host in the first stages of ranavirus infection. 相似文献