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31.
32.
瑞氏木霉木糖醇脱氢酶基因的分离与鉴定 总被引:2,自引:0,他引:2
将在木聚糖上生长的瑞氏木霉(Trichoderma reesei)RutC-30的cDNA文库全部质粒转化已携带有毕赤氏酵(Pithia stipitis)木糖还原酶基因的重组酿酒酵母(Saccharomycescerevisiae)菌株H475,在H475中构建了瑞氏木霉的cDNA表达亚文库。在以木糖为唯一碳源的选择性酵母合成培养基上,从该亚文库中筛选到瑞氏木霉木糖醇脱氢酶cDNA基因.该基因片段长为1.3kb。Southern、Norhern印迹杂交分析和蛋白质凝胶电泳结果表明该基因确实来源于瑞氏木霉,所编码蛋白质分子量约为40kDa。携带有毕赤氏酵母木糖还原酶和瑞氏木霉木糖醇脱氢酶基因的重组酵母能够在以木糖为唯一碳源的培养基上生长,并能将90%以上的木糖转化为木糖醇、乙醇和其它副产品。 相似文献
33.
Merja Itävaara 《Physiologia plantarum》1990,80(1):36-42
Growth characteristics of a spontaneous mutant of shiitake Lentinula edodes (Berk.) Pegler were studied. The mutant was first detected as a result of changes in the growth habit of the normal strain in the liquid medium. Abundant formation of aerial hyphae was distinctive. In sawdust logs the mutant strain produced abnormal basidiocarps, lacking stipe, gill and spore formation.
Growth rates of the normal and the mutant strain were compared in two liquid media: malt-yeast extract and Leatham's medium. The increase in dry weight of the mutant's mycelium was much higher than that of the wild type in both media, which indicated better adaptation to liquid culture. In the sawdust, however, growth of the mutant was slower than that of the normal strain. The mutant's intracellular protein content was lower than that of the normal strain. The pH of the liquid cultures differed: the wild type decreased the pH during growth, while the mutant increased the pH. Comparison of the protein and esterase isoenzyme profiles of the vegetative hyphae of both strains indicated profound differences. One protein (pI 6.5, 39 kDa), which in earlier studies has been found to be typical of L. edodes species, was absent from the mutant's profile. Differences in the esterase profile were also clear. 相似文献
Growth rates of the normal and the mutant strain were compared in two liquid media: malt-yeast extract and Leatham's medium. The increase in dry weight of the mutant's mycelium was much higher than that of the wild type in both media, which indicated better adaptation to liquid culture. In the sawdust, however, growth of the mutant was slower than that of the normal strain. The mutant's intracellular protein content was lower than that of the normal strain. The pH of the liquid cultures differed: the wild type decreased the pH during growth, while the mutant increased the pH. Comparison of the protein and esterase isoenzyme profiles of the vegetative hyphae of both strains indicated profound differences. One protein (pI 6.5, 39 kDa), which in earlier studies has been found to be typical of L. edodes species, was absent from the mutant's profile. Differences in the esterase profile were also clear. 相似文献
34.
Jenni Lahtela Barun Pradhan Katja N?rhi Annabrita Hemmes Merja S?rkioja Panu E. Kovanen Arthur Brown Emmy W. Verschuren 《Disease models & mechanisms》2015,8(4):393-401
Treatment of non-small cell lung cancer (NSCLC) is based on histological analysis and molecular profiling of targetable driver oncogenes. Therapeutic responses are further defined by the landscape of passenger mutations, or loss of tumor suppressor genes. We report here a thorough study to address the physiological role of the putative lung cancer tumor suppressor EPH receptor A3 (EPHA3), a gene that is frequently mutated in human lung adenocarcinomas. Our data shows that homozygous or heterozygous loss of EphA3 does not alter the progression of murine adenocarcinomas that result from Kras mutation or loss of Trp53, and we detected negligible postnatal expression of EphA3 in adult wild-type lungs. Yet, EphA3 was expressed in the distal mesenchyme of developing mouse lungs, neighboring the epithelial expression of its Efna1 ligand; this is consistent with the known roles of EPH receptors in embryonic development. However, the partial loss of EphA3 leads only to subtle changes in epithelial Nkx2-1, endothelial Cd31 and mesenchymal Fgf10 RNA expression levels, and no macroscopic phenotypic effects on lung epithelial branching, mesenchymal cell proliferation, or abundance and localization of CD31-positive endothelia. The lack of a discernible lung phenotype in EphA3-null mice might indicate lack of an overt role for EPHA3 in the murine lung, or imply functional redundancy between EPHA receptors. Our study shows how biological complexity can challenge in vivo functional validation of mutations identified in sequencing efforts, and provides an incentive for the design of knock-in or conditional models to assign the role of EPHA3 mutation during lung tumorigenesis.KEY WORDS: EPHA3, EPH receptor A3, GEMM, Adenocarcinoma, Lung morphogenesis 相似文献
35.
Objectives
Physical activity–an important determinant of health and function in old age–may vary according to the life-space area reached. Our aim was to study how moving through greater life-space areas is associated with greater physical activity of community-dwelling older people. The association between objectively measured physical activity and life-space area reached on different days by the same individual was studied using one-week longitudinal data, to provide insight in causal relationships.Methods
One-week surveillance of objectively assessed physical activity of community-dwelling 70–90-year-old people in central Finland from the “Life-space mobility in old age” cohort substudy (N = 174). In spring 2012, participants wore an accelerometer for 7 days and completed a daily diary including the largest life-space area reached (inside home, outside home, neighborhood, town, and beyond town). The daily step count, and the time in moderate (incl. walking) and low activity and sedentary behavior were assessed. Differences in physical activity between days on which different life-space areas were reached were tested using Generalized Estimation Equation models (within-group comparison).Results
Participants’ mean age was 80.4±4.2 years and 63.5% were female. Participants had higher average step counts (p < .001) and greater moderate and low activity time (p < .001) on days when greater life-space areas were reached, from the home to the town area. Only low activity time continued to increase when moving beyond the town.Conclusion
Community-dwelling older people were more physically active on days when they moved through greater life-space areas. While it is unknown whether physical activity was a motivator to leave the home, intervention studies are needed to determine whether facilitation of daily outdoor mobility, regardless of the purpose, may be beneficial in terms of promoting physical activity. 相似文献36.
Eero Lauhkonen Petri Koponen Johanna Ter?sj?rvi Kirsi Gr?ndahl-Yli-Hannuksela Juho Vuononvirta Kirsi Nuolivirta Jyri O. Toikka Merja Helminen Qiushui He Matti Korppi 《PloS one》2015,10(10)
Aim
Interleukin-10 (IL-10) has been associated with wheezing and asthma in children and the genetic variation of the IL-10 cytokine production may be linked to post-bronchiolitis lung function. We used impulse oscillometry (IOS) to evaluate the associations of IL10 polymorphisms with lung function at a median age of 6.3 years in children hospitalised for bronchiolitis before six months of age.Methods
We performed baseline and post-exercise IOS on 103 former bronchiolitis patients. Data on single nucleotide polymorphisms (SNP) of IL10 rs1800896 (–1082G/A), rs1800871 (–819C/T), rs1800872 (–592C/A) were available for 99 children and of IL10 rs1800890 (–3575T/A) for 98 children.Results
IL10 rs1800896, rs1800871 and rs1800872 combined genotype AA+CT+CA and carriage of haplotype ATA, respectively, were associated with higher resistance and lower reactance in baseline IOS in adjusted analyses. At IL10 rs1800890, the A/A-genotype and carriers of A-allele were associated with lower reactance in baseline IOS. There were no significant associations between the studied SNPs and airway hyper-reactivity to exercise.Conclusion
Low-IL-10-producing polymorphisms in the IL-10 encoding gene were associated with obstructive lung function parameters, suggesting an important role for IL-10 in development of lung function deficit in early bronchiolitis patients. 相似文献37.
Christopher P. Landowski Anne Huuskonen Ramon Wahl Ann Westerholm-Parvinen Anne Kanerva Anna-Liisa H?nninen Noora Salovuori Merja Penttil? Jari Natunen Christian Ostermeier Bernhard Helk Juhani Saarinen Markku Saloheimo 《PloS one》2015,10(8)
The filamentous fungus Trichoderma reesei has tremendous capability to secrete proteins. Therefore, it would be an excellent host for producing high levels of therapeutic proteins at low cost. Developing a filamentous fungus to produce sensitive therapeutic proteins requires that protease secretion is drastically reduced. We have identified 13 major secreted proteases that are related to degradation of therapeutic antibodies, interferon alpha 2b, and insulin like growth factor. The major proteases observed were aspartic, glutamic, subtilisin-like, and trypsin-like proteases. The seven most problematic proteases were sequentially removed from a strain to develop it for producing therapeutic proteins. After this the protease activity in the supernatant was dramatically reduced down to 4% of the original level based upon a casein substrate. When antibody was incubated in the six protease deletion strain supernatant, the heavy chain remained fully intact and no degradation products were observed. Interferon alpha 2b and insulin like growth factor were less stable in the same supernatant, but full length proteins remained when incubated overnight, in contrast to the original strain. As additional benefits, the multiple protease deletions have led to faster strain growth and higher levels of total protein in the culture supernatant. 相似文献
38.
Mervi H. Toivari Laura Ruohonen Peter Richard Merja Penttilä Marilyn G. Wiebe 《Applied microbiology and biotechnology》2010,88(3):751-760
Saccharomyces cerevisiae was engineered to produce D-xylonate by introducing the Trichoderma reesei xyd1 gene, encoding a D-xylose dehydrogenase. D-xylonate was not toxic to S. cerevisiae, and the cells were able to export D-xylonate produced in the cytoplasm to the supernatant. Up to 3.8 g of D-xylonate per
litre, at rates of 25–36 mg of D-xylonate per litre per hour, was produced. Up to 4.8 g of xylitol per litre was also produced.
The yield of D-xylonate from D-xylose was approximately 0.4 g of D-xylonate per gramme of D-xylose consumed. Deletion of the
aldose reductase encoding gene GRE3 in S. cerevisiae strains expressing xyd1 reduced xylitol production by 67%, increasing the yield of D-xylonate from D-xylose. However, D-xylose uptake was reduced
compared to strains containing GRE3, and the total amount of D-xylonate produced was reduced. To determine whether the co-factor NADP+ was limiting for D-xylonate production the Escherichia coli transhydrogenase encoded by udhA, the Bacillus subtilis glyceraldehyde 3-phosphate dehydrogenase encoded by gapB or the S. cerevisiae glutamate dehydrogenase encoded by GDH2 was co-expressed with xyd1 in the parent and GRE3 deficient strains. Although each of these enzymes enhanced NADPH consumption on D-glucose, they did not enhance D-xylonate production, suggesting
that NADP+ was not the main limitation in the current D-xylonate producing strains. 相似文献
39.
Dominik Mojzita Marilyn Wiebe Satu Hilditch Harry Boer Merja Penttil? Peter Richard 《Applied and environmental microbiology》2010,76(1):169-175
d-Galacturonic acid can be obtained by hydrolyzing pectin, which is an abundant and low value raw material. By means of metabolic engineering, we constructed fungal strains for the conversion of d-galacturonate to meso-galactarate (mucate). Galactarate has applications in food, cosmetics, and pharmaceuticals and as a platform chemical. In fungi d-galacturonate is catabolized through a reductive pathway with a d-galacturonate reductase as the first enzyme. Deleting the corresponding gene in the fungi Hypocrea jecorina and Aspergillus niger resulted in strains unable to grow on d-galacturonate. The genes of the pathway for d-galacturonate catabolism were upregulated in the presence of d-galacturonate in A. niger, even when the gene for d-galacturonate reductase was deleted, indicating that d-galacturonate itself is an inducer for the pathway. A bacterial gene coding for a d-galacturonate dehydrogenase catalyzing the NAD-dependent oxidation of d-galacturonate to galactarate was introduced to both strains with disrupted d-galacturonate catabolism. Both strains converted d-galacturonate to galactarate. The resulting H. jecorina strain produced galactarate at high yield. The A. niger strain regained the ability to grow on d-galacturonate when the d-galacturonate dehydrogenase was introduced, suggesting that it has a pathway for galactarate catabolism.d-Galacturonate is the main component of pectin, an abundant and cheap raw material. Sugar beet pulp and citrus peel are both rich in pectin residues. At present, these residues are mainly used as cattle feed. However, since energy-consuming drying and pelletizing of the residues is required to prevent them from rotting, it is not always economical to process the residues, and it is desirable to find alternative uses.Various microbes which live on decaying plant material have the ability to catabolize d-galacturonate using various, completely different pathways (19). Eukaryotic microorganisms use a reductive pathway in which d-galacturonate is first reduced to l-galactonate by an NAD(P)H-dependent reductase (12, 17). In the following steps a dehydratase, aldolase, and reductase convert the l-galactonate to pyruvate and glycerol (9, 11, 14).In Hypocrea jecorina (anamorph Trichoderma reesei) the gar1 gene codes for a strictly NADPH-dependent d-galacturonate reductase. In Aspergillus niger a homologue gene sequence, gar2, exists; however, a different gene, gaaA, is upregulated during growth on d-galacturonate containing medium (16). The gaaA codes for a d-galacturonate reductase with different kinetic properties than the H. jecorina enzyme, having a higher affinity toward d-galacturonate and using either NADH or NADPH as cofactor. It is not known whether gar2 codes for an active protein.Some bacteria, such as Agrobacterium tumefaciens or Pseudomonas syringae, have an oxidative pathway for d-galacturonate catabolism. In this pathway d-galacturonate is first oxidized to meso-galactarate (mucate) by an NAD-utilizing d-galacturonate dehydrogenase. Galactarate is then converted in the following steps to α-ketoglutarate. This route is sometimes called the α-ketoglutarate pathway (20). Galactarate can also be catabolized through the glycerate pathway (20). The products of this pathway are pyruvate and d-glycerate. These pathways have been described in prokaryotes, and it is not certain whether similar pathways also exist in fungi, some of which are able to metabolize galactarate.d-Galacturonate dehydrogenase (EC 1.1.1.203) has been described in Agrobacterium tumefaciens and in Pseudomonas syringae, and the enzymes from these organisms have been purified and characterized (3, 6, 22). Recently, the corresponding genes were also identified (4, 24). Both enzymes are specific for NAD as a cofactor but are not specific for the substrate. They oxidize d-galacturonate and d-glucuronate to meso-galactarate (mucate) and d-glucarate (saccharate), respectively. The reaction product is probably the hexaro-lactone which spontaneously hydrolyzes. The reverse reaction can only be observed at acidic pH where some of the galactarate is in the lactone form (22).We describe here strains of filamentous fungi that have been genetically engineered to produce galactarate by disruption of d-galacturonate reductase and expression of d-galacturonate dehydrogenase (Fig. (Fig.1).1). Galactarate is currently commercially produced from d-galactose by oxidation with nitric acid (1) or from d-galacturonate by electrolytic oxidation (8). Oxidation with nitric acid is expensive and produces toxic wastes. Galactarate is used as a chelator and in skin care products. It was formerly used as a leavening agent in self-rising flour (2) and has potential applications in polymer synthesis (10) and as a platform chemical (for a review, see reference 13).Open in a separate windowFIG. 1.Engineering the d-galacturonic acid pathway in fungi. Deletion of the gene encoding d-galacturonate reductase resulted in strains unable to utilize d-galacturonic acid as a carbon source. The expression of a bacterial udh gene, encoding an NAD-dependent d-galacturonate dehydrogenase, resulted in fungal strains, which were able to oxidize d-galacturonic acid to meso-galactaric acid (mucic acid). d-Galacturonate dehydrogenase forms a galactaro-lactone which spontaneously hydrolyzes. 相似文献
40.
Impacts of nutrient enrichment and sediment on phytoplankton community structure in the northern Baltic Sea 总被引:2,自引:0,他引:2
A three-week mesocosm experiment was conducted in order to study the effects of bottom sediment and nutrient enrichment on
phytoplankton and zooplankton community structure in the Archipelago Sea, northern Baltic Sea. The transparent polyethylene
enclosures included the whole water column and varied in volume from 30 to 40 m3. There were two types of enclosures: some with natural sediment as a bottom and others with a plastic bottom. The experiment
was a 2 × 2 factorial design with presence of sediment and nutrient enrichment as treatment factors. Both the sediment presence
and nutrient enrichment significantly increased water nutrient concentrations and the rate of primary production. However,
external nutrient enrichment and the presence of sediment stimulated the growth of different phytoplankton groups, indicating
that the effect of sediment was not related to nutrient fluxes alone, but involved more complex interactions. External nutrient
enrichment was primarily channelled to picoplanktonic cyanobacteria, the biomass of which increased four- to fivefold due
to enrichment. The presence of sediment increased the biomass of cryptophytes, chrysophytes and prasinophytes, but decreased
the biomass of N2-fixing cyanobacteria. Zooplankton biomass increased during the experiment, but was not affected by the treatments. The study
shows that sediment plays a significant role in phytoplankton dynamics, underlining the importance of including sediment in
shallow-water mesocosm experiments.
Handling editor: J. Padisak 相似文献