Activated white cells use oxidants generated by the heme enzyme myeloperoxidase to kill invading pathogens. This enzyme utilizes H2O2 and Cl−, Br−, or SCN− to generate the oxidants HOCl, HOBr, and HOSCN, respectively. Whereas controlled production of these species is vital in maintaining good health, their uncontrolled or inappropriate formation (as occurs at sites of inflammation) can cause host tissue damage that has been associated with multiple inflammatory pathologies including cardiovascular diseases and cancer. Previous studies have reported that sulfur-containing species are major targets for HOCl but as the reactions are fast the only physiologically relevant kinetic data available have been extrapolated from data measured at high pH (>10). In this study these values have been determined at pH 7.4 using a newly developed competition kinetic approach that employs a fluorescently tagged methionine derivative as the competitive substrate (k(HOCl + Fmoc-Met), 1.5×108 M−1 s−1). This assay was validated using the known k(HOCl + NADH) value and has allowed revised k values for the reactions of HOCl with Cys, N-acetylcysteine, and glutathione to be determined as 3.6×108, 2.9×107, and 1.24×108 M−1 s−1, respectively. Similar experiments with methionine derivatives yielded k values of 3.4×107 M−1 s−1 for Met and 1.7×108 M−1 s−1 for N-acetylmethionine. The k values determined here for the reaction of HOCl with thiols are up to 10-fold higher than those previously determined and further emphasize the critical importance of reactions of HOCl with thiol targets in biological systems. 相似文献
Phenylethanoid glycosides (PeG) are a class of polyphenols found in some plants that have pharmaceutical effects as anti-inflammatories and anti-oxidants. The presence of PeG (acteoside) in the aerial parts of Scrophularia striata Boiss. has been demonstrated. Considerable progress has been made using plant cell cultures to stimulate formation and accumulation of secondary metabolites. The present study optimized phenylethanoid production from shake flasks to bioreactor using a cell culture of S. striata. The optimal conditions for production of cell biomass by scale-up to a bioreactor were determined to be a pH of 4.8, air flow rate of 0.5–1.5 l min−1, and mixing speed of 110–170 rpm at 25 ± 1 °C in darkness. Growth parameters and PeG production were measured and compared with the results from the shake flasks. The results showed that cell biomass was high in the bioreactor (15.64 g l−1 DW) and in the shake flasks (14.16 g l−1 DW). The acteoside content in the bioreactor was 1404.20 μg g−1 DW, which is threefold higher than in the shake flasks (459.71 μg g−1 DW). The echinacoside concentration in the bioreactor was 1449.39 μg g−1, 1.36-fold lower than in the shake flasks (1973.03 μg g−1 DW). This study established an efficient way for production of acteoside, the major PeG, in a bioreactor.
A process was developed for producing human menin from transformed Drosophila Schneider 2 cells. Protein expression was achieved after inducing the metallothionein promoter by adding copper sulfate to
cells growing in suspension in a stirred-tank reactor. Experiments in shake flasks showed that the production of menin was
improved when the induction was conducted late in the exponential phase of cell growth at a concentration of 1–2 × 107 cells ml-1, with a copper concentration of 0.2 mM for no more than 24 h. This observation was confirmed by experiments in bench-scale
fermentors. Subsequently, a pilot-scale fermentation yielded 1 mg l-1 culture of purified menin.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
The patterns of serum luteinizing hormone (LH), follicle stimulating hormone (FSH), progesterone and estradiol-17β during the estrous cycle of six crossbred (Alpine × Nubian × Native) and six native goats showing a 21 day estrous cycle in a semiarid zone of Venezuela are presented. In the crossbred goats, FSH had two significant peaks on Days 19 and 0 (33 ± 8.6 ng ml−1 and 25 ± 6 ng ml−1, respectively); in contrast, native goats only had one significant peak on the day of estrus (22 ± 2 ng ml−1), with the increase beginning on Day 17. During the follicular phase of crossbred goats, estradiol-17β and LH increased to 28 ± 6 pg ml−1 and 23 ± 6.9 ng ml−1, respectively, on Day 0. Prior to Day 0, LH increased to 10.0 ± 4.9 ng ml−1 on Day 18, decreasing to 1.5 ng ml−1 on Day 19, while estradiol-17β was increasing. This relationship between estradiol-17β and LH was not found to exist in native does, which presented a LH peak on Day 0 (30 ± 8 ng ml−1 and 35 ± 10 ng ml−1 in first and second estrus, respectively). LH basal levels were notably higher in native does. The highest concentrations of progesterone (10 and 12 ng ml−1) were detected on Days 12 and 15 in crossbred and native females, respectively. In conclusion, the relationship between estradiol-17β and gonadotropins during the follicular phase in crossbred goats suggests negative and positive feedback effects on both LH and FSH. Serum concentrations of LH were higher in native than in crossbred goats, whereas concentrations of FSH were higher in crossbred does. Thus, genetic factors need to be taken into account when comparing blood levels of gonadotropins in goats raised in tropical semiarid zones. 相似文献
Long-term and intensive use of synthetic insecticides to control the green peach aphid, Myzus persicae (Homoptera: Aphididae) in agricultural production in the world has resulted in pest resistance and environmental pollution. The aim of the study was to develop an environmentally-friendly and effective mycoinsecticide from a local fungal isolate against M. persicae. According to the results of the screening experiments using 15 isolates (6 × Metarhizium, 5 × Beauveria, 2 × Isaria, 2 × Lecanicillium) at 1 × 107 conidia ml?1 concentrations and the dose–response experiments at 1 × 105 –1 × 109 conidia ml?1 concentrations against M. persicae nymphs, Beauveria bassiana (KTU-24) was identified as the most promising isolate. The strain KTU-24 is also had tolerance to ecological conditions such as temperature, and UV-B. KTU-24 with advantageous and superior properties was used to develop a test mycoinsecticide. Mass production of spores by KTU-24 was conducted out by liquid-state fermentation using liquid medium. Spores harvested from the sporulated biomass were used to develop an oil-based mycoinsecticide, and the product was designated as AFIDISIDAL-OD Bbas-TR61. The product had lethal effect on M. persicae nymphs at a concentration of 1 × 108 conidia ml?1 in leaf-disc (82.5%) and pot (84.33%) experiments in a climate chamber. The oil-based mycoinsecticide developed in this study could be profitable when used in aphid-IPM prgrams by reducing crop loss and synthetic pesticides use. 相似文献
Progesterone production of granulosa cells cultured in vitro is stimulated and cell differentiation increased, by follicle-stimulating hormone (FSH). This study examined whether the increased progesterone production observed when bovine granulosa cells are cultured occurs because (1) progesterone production by undifferentiated and/or differentiated cells is increased or (2) the differentiation of granulosa cells is stimulated. Viable bovine granulosa cells (2−3×105) from follicles 5–8 mm in diameter were cultured in the presence of 0, 1, 10 and 100 μu FSH (1 μu ≡ 1 μg NIH-FSH-S1) for 6 days at 37°C in a humidified atmosphere of 5% CO2 in air in 1 ml of a 1:1 mixture of Dulbecco's modified Eagle medium: Ham's F10 medium supplemented with 365 μg ml−1 l-glutamine, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin. Progesterone production, total DNA and protein, and cell diameter were determined sequentially over the culture period. The increases in progesterone production (ng μg−1 DNA per 24 h), cytoplasmic:nuclear ratio (μg protein μg−1 DNA) and cell diameter (μm) over 6 days culture indicated that granulosa cells underwent differentiation in the presence of FSH. Progesterone production of undifferentiated granulosa cells (diameter 14 μm or less) was stimulated by FSH (P < 0.01) in a dose dependent manner (1.0±0.2, 2.9±0.3, 3.7±0.3 and 4.9±0.4 ng μg−1 DNA per 24 h for 0, 1, 10 and 100 μu ml−1 FSH respectively) but remained constant within dose (P > 0.05) during a 6 day culture period. FSH stimulated (P < 0.05) the rate of granulosa cell differentiation (10±3%, 53±13%, 74±21% and 82±10% differentiating cells per well for 0 μu, 1 μu, 10 μu and 100 μu ml−1 FSH respectively) but did not stimulate (P > 0.05) progesterone production by differentiating granulosa cells (8.7±0.5 ng μg−1 DNA per 24 h). In conclusion, the increase in progesterone production of FSH-stimulated granulosa cells cultured in vitro appears to be mainly due to an increase in the number of differentiating cells with a constant rather than an increasing progesterone production per cell. 相似文献
The kinetics of the formation of the thiomolybdate ions MoOS32− and MoS42− were determined spectroscopically from the addition of excess sulphide to MoO2S22− in pH buffered media (6–8) at 30 °C. The reverse (hydrolysis) reactions of MoO2S22− and MoOS32− were measured under the same conditions. The reaction rates measured are shown below: Values of the rate-constants (s−1) obtained at pH 7.0 were k10 2.4 × 10−3, k21 1.5 × 10−5, k30 2.1 × 10−5, k23 6.0 × 10−4, and k34 1.9 × 10−5; where the results are comparable they are in good agreement with those obtained by earlier workers, although different conditions were used. However, in this work it was found that certain reactions had to be mathematically treated as two consecutively occurring reactions. There is also a difference in interpretation of the mechanism of the hydrolysis reactions of the tri- and tetrathio ions. In general the lability towards further S replacement of O atoms, and the reverse reaction, decreased with increased S substitution. All reaction rates increased with increasing H+ ion concentration, mostly this was a linear relationship over the limited pH range examined. The effect of the H+ ion is interpreted in terms of protonation of the oxythiomolybdate ions at an O atom leading to increased lability. 相似文献
In the present work, we evaluated the feeding selectivity of starved Daphnia magna on two freshwater green algae Chlamydomonas sajao and Chlorellapyrenoidosa. Compared to C. pyrenoidosa, food quality of C. sajao are better in food palatability (cell size and digestibility), but poor in nutritional content (total carbon content). D. magna was starved for 0 and 8 d, and then was allowed to graze on a mixture of C. sajao and C. pyrenoidosa with following proportion: 5 × 104: 35 × 104 cells ml?1, 20 × 104: 20 × 104 cells ml?1 and 35 × 104: 5 × 104 cells ml?1. The results indicated that the ingestion rate and filtration rate of starved D. magna, comparing with satiated groups, on C. pyrenoidosa increased significantly, while, inverse trends was observed in C. sajao. Base on selectivity coefficient of D. magna, we observed that when D. magna was in satiation C. sajao will be preferred, while, C. pyrenoidosa will be selected when D. magna was in starvation, and moreover, these foraging behaviors were not influenced by the relative food abundance of each green alga. Therefore, a tradeoff between food palatability (physical makeup) and food nutritional content (chemical composition) can be hypothesized in the foraging behavior of D. magna, which is modified by the starvation of feeder. High valuable food is always selected by D. magna as predicted by optimal foraging theory. However, when D. magna is in satiation food diets with adequate size and easy digestibility will be preferred, while, those foods with relatively higher lipid or total carbon content will be selected when D. magna is in starvation. 相似文献
In the present work, we evaluated the feeding selectivity of starved Daphnia magna on two freshwater green algae Chlamydomonas sajao and Chlorellapyrenoidosa. Compared to C. pyrenoidosa, food quality of C. sajao are better in food palatability (cell size and digestibility), but poor in nutritional content (total carbon content). D. magna was starved for 0 and 8 d, and then was allowed to graze on a mixture of C. sajao and C. pyrenoidosa with following proportion: 5 × 104: 35 × 104 cells ml?1, 20 × 104: 20 × 104 cells ml?1 and 35 × 104: 5 × 104 cells ml?1. The results indicated that the ingestion rate and filtration rate of starved D. magna, comparing with satiated groups, on C. pyrenoidosa increased significantly, while, inverse trends was observed in C. sajao. Base on selectivity coefficient of D. magna, we observed that when D. magna was in satiation C. sajao will be preferred, while, C. pyrenoidosa will be selected when D. magna was in starvation, and moreover, these foraging behaviors were not influenced by the relative food abundance of each green alga. Therefore, a tradeoff between food palatability (physical makeup) and food nutritional content (chemical composition) can be hypothesized in the foraging behavior of D. magna, which is modified by the starvation of feeder. High valuable food is always selected by D. magna as predicted by optimal foraging theory. However, when D. magna is in satiation food diets with adequate size and easy digestibility will be preferred, while, those foods with relatively higher lipid or total carbon content will be selected when D. magna is in starvation. 相似文献
The Bay of Bengal (BoB) is a 2,600,000 km2 expanse in the Indian Ocean upon which many humans rely. However, the primary producers underpinning food chains here remain poorly characterized. We examined phytoplankton abundance and diversity along strong BoB latitudinal and vertical salinity gradients—which have low temperature variation (27–29°C) between the surface and subsurface chlorophyll maximum (SCM). In surface waters, Prochlorococcus averaged 11.7 ± 4.4 × 104 cells ml−1, predominantly HLII, whereas LLII and ‘rare’ ecotypes, HLVI and LLVII, dominated in the SCM. Synechococcus averaged 8.4 ± 2.3 × 104 cells ml−1 in the surface, declined rapidly with depth, and population structure of dominant Clade II differed between surface and SCM; Clade X was notable at both depths. Across all sites, Ostreococcus Clade OII dominated SCM eukaryotes whereas communities differentiated strongly moving from Arabian Sea-influenced high salinity (southerly; prasinophytes) to freshwater-influenced low salinity (northerly; stramenopiles, specifically, diatoms, pelagophytes, and dictyochophytes, plus the prasinophyte Micromonas) surface waters. Eukaryotic phytoplankton peaked in the south (1.9 × 104 cells ml−1, surface) where a novel Ostreococcus was revealed, named here Ostreococcus bengalensis. We expose dominance of a single picoeukaryote and hitherto ‘rare’ picocyanobacteria at depth in this complex ecosystem where studies suggest picoplankton are replacing larger phytoplankton due to climate change. 相似文献
Eighteen bacterial strains were isolated from soil samples and screened for alkaline, thermophilic lipase production. Pseudomonas fluorescens NS2W was selected and its production of lipase was optimized in shake flasks using a statistical experimental design. Cell
growth and lipase production were studied in shake flasks and in a 1-l fermenter in the optimized medium. Maximum lipase yields
were 69.7 and 68.7 U ml−1, respectively. The optimized medium resulted in about a five-fold increase in the enzyme production, compared to that obtained
in the basal medium. The lipase had an optimal activity at pH 9.0 and was stable over a wide pH range of 3–11 with more than
70% activity retention. The lipase had an optimal activity at 55°C and was stable up to 60°C with more than 70% activity retention
for at least 2 h. Journal of Industrial Microbiology & Biotechnology (2002) 28, 344–348 DOI: 10.1038/sj/jim/7000254
Received 06 September 2001/ Accepted in revised form 15 March 2002 相似文献
The influence of initial glucose concentrations on the production of biomass and lutein by Chlorella protothecoides CS-41 was investigated in batch cultures using both shake flasks and fermentors. The maximum biomass concentration increased from 4·9 to 31·2 g litre−1 dry cells with an increase in initial glucose concentration from 10 to 80 g litre−1. An even higher initial glucose concentration (100 g litre−1) resulted in a lower biomass concentration, a lower specific growth rate, a lower growth yield coefficient and a considerably longer lag phase, which might be due to substrate inhibition. The initial glucose level also had a significant effect on the production of lutein. In a 3·7-litre fermentor an increase in lutein production from 19·39 to 76·56 mg litre−1 was obtained with an increase in initial glucose concentration from 10 to 40 g litre−1, within which range, lutein yield coefficient was constant (YItn=1·90±0·02 mg g−1). A simple substrate inhibition model was developed, which fitted the experimental data better than the classical Haldane model. A group of time-dependent kinetic models for algal cultivation in fermentors were also constructed, which were in good agreement with the experimental results and could be employed to predict the production of biomass and lutein, and the consumption of glucose in fermentor cultures. 相似文献
Chroococcoid cyanobacteria, (mean size = 0.79 μm, likely Synchetocystis limnetica Popovsk) and total eubacteria (mean size = 0.33 μm), from Lake Baikal, USSR, were enumerated using epifluorescence microscopy and sized with image analysis. Bacterial densities ranged from 0.44 · 106 cells ml−1 at 250 m to 2.3 · 106 cells ml−1 at the surface. Mean eubacterial abundance was 1.3 · 106 cells ml−1. Cyanobacterial densities were more variable, ranging from 0.42 · 104 cells ml−1 at 250 m to 9.8 · 104 cells ml−1 at the surface, with a mean abundance of 2.7 · 104 cells ml−1. The cyanobacteria, in particular, occurred in clusters resembling “marine snow”. Our results indicate that Lake Baikal picoplankton size and density are similar to other large lakes but may have a more diverse community structure than in other large oligotrophic lakes. 相似文献
A gene expression system using recombinant Autographa californica nuclear polyhedrosis virus (baculovirus) and Sf-9 cells has been scaled up to the 10-L tank level and shown to be capable
of producing herpes simplex virus (HSV) protease in serum-free media. High densities of Spodoptera frugiperda (Sf-9) cells were achieved by modifying two 10-L Biolafitte fermenters specifically for insect cell growth. The existing
Rushton impellers were replaced by marine impellers to reduce shear and the aeration system was modified to allow external
addition of air/O2 mixtures at low flow rates through either the sparge line or into the head space of the fermenter. To inoculate the tanks,
Sf-9 cells were adapted to grow to high cell densities (6–10 × 106 cells ml−1) in shake flasks in serum-free media. With these procedures, cell densities of 5 × 106 cells ml−1 were routinely achieved in the 10-L tanks. These cells were readily infected with recombinant baculovirus expressing the
247-amino acid catalytic domain of the HSV-1 strain 17 protease UL26 gene as a glutathione-S-transferase (GST) fusion protein (GST-247). Three days after infection at a multiplicity of infection (MOI) of 3 pfu cell−1, the GST-247 fusion protein was purified from a cytoplasmic lysate by Glutathione Sepharose 4-B affinity chromatography
with reproducible yields of 11–38 mg L−1 of recombinant protein and ≥ 90% purity. Maximum production of this protein was observed at a cell density of 5.0 × 106 cells ml−1.
Received 09 December 1996/ Accepted in revised form 13 April 1997 相似文献
In 1992 we examined the morphological characteristics and space-time distribution of picoplankton cells in Lake Maggiore, a subalpine lake in which oligotrophication is in progress. We measured by image analyser the biovolume of autotrophic (APP), eukaryotic and prokaryotic. and heterotrophic (HPP) picoplankton. Among the APP < 2μm the yellow fluoresceing are the dominating cells in the euphoric zone. The red cells, mainly eukaryotic, on average are only 11% of the total abundance of cells < 5μm. The APP cell numbers range from 9.5 × 103 cells ml−1 to 1.3 × 105 cells ml−1 (average: 5 × 104 cells ml−1). Their mean biovolume shows a minimum value of 7.8mm3 m−3 in March and a maximum of 186.3mm3 m-3 in September. The contribution of biovolume of yellow cells to total phytoplankton biovolume varies between 0.3% and 27%. suggesting that picocyanobacteria, at this stage of lake recovery, are not yet a dominant component. The HPP cell density is two order of magnitude higher than the APP with a mean value of 2.6 × 106 cells ml−1. APP mean cell size fluctuates from a minimum of 0.5 μm to a maximum of 1.4urn (0.26–1.69μm3 volume), while HPP range from 0.4 to 0.7 um (0.07–0.57 μm3 volume), making it easier to distinguish them on a dimensional basis for most of the year. During the period of thermal stratification, a peak in abundance was noted in the central part of the metalimnion at depths receiving less than 10% of surface irradiance. The total picoplanktonic carbon fraction (APP and HPP) varied from 38 to 384 μgC 1−1 with a mean value of 133μgC 1−1 which represents 42% of POC collected on GF/C filters. Most of the picoplankton carbon is made up of HPP cells (34% of the total POC). 相似文献
A recombinant strain of Escherichia coli with CYP102A1 gene was developed for the demethylation of colchicine into their derivatives. The CYP102A1 gene responsible for demethylation was isolated from Bacillus megaterium ACBT03 and amplified using suitable primers. The amplified product was cloned into pET28a+ expression vector using host E. coli BL21(DE3) cells. The CYP3A4 (product of CYP102A1 gene) protein expression and other parameters like substrate toxicity, product toxicity and enzyme activity were optimized in shake flasks; and further scaled-up to 5 l bioreactor with 3 l working volume. In 5 l bioreactor, dissolved oxygen (DO) was optimized for maximum specific growth and enhanced 3-demethylated colchicine (3-DMC) production. The optimized conditions from shake flasks were scaled-up to 70 l bioreactor and resulted into ∼80% conversion of 20 mM colchicine in 48 h with a volumetric productivity of 6.62 mg l−1 h−1. Scale-up factors were measured as volumetric oxygen transfer coefficient (kLa) i.e., 56 h−1 and impeller tip velocity (Vtip) i.e., 7.065 m s−1, respectively. The kinetic parameters Km, kcat, and kcat/Km of the CYP3A4 enzyme using colchicine as the substrate were determined to be 271 ± 30 μM, 8533 ± 25 min−1, and 31.49 μM min−1, respectively, when IPTG induced recombinant E. coli culture was used. 相似文献
Pyrolysate obtained from the pyrolysis of waste cotton is a source of fermentable sugars that could be fermented into bioethanol fuel and other chemicals via microbial fermentation. However, pyrolysate is a complex mixture of fermentable and non-fermentable substrates causing inhibition of the microbial growth. The aim of this study was to detoxify the hydrolysate and then ferment it into bio-ethanol fuel in shake flasks and fermenter applying yeast strain Saccharomyces cerevisiae 2.399. Pyrolysate was hydrolyzed to glucose with 0.2 M sulfuric acid, neutralized with Ba(OH)2 followed by treatment with ethyl acetate and activated carbon to remove fermentation inhibitors. The effect of various fermentation parameters such as inoculum concentration, pH and hydrolysate glucose was evaluated in shake flasks for optimum ethanol fermentation. With respect to inoculum concentration, 20% v/v inoculum i.e. 8.0 × 108–1.2 × 109 cells/mL was the optimum level for producing 8.62 ± 0.33 g/L ethanol at 9 h of fermentation with a maximum yield of 0.46 g ethanol/g glucose. The optimum pH for hydrolysate glucose fermentation was found to be 6.0 that produced 8.57 ± 0.66 g/L ethanol. Maximum ethanol concentration, 14.78 g/L was obtained for 4% hydrolysate glucose concentration after 16 h of fermentation. Scale-up studies in stirred fermenter produced much higher productivity (1.32 g/L/h–1) compared to shake flask fermentation (0.92 g/L/h–1). The yield of ethanol reached a maximum of 91% and 89% of the theoretical yield of ethanol in shake flasks and fermenter, respectively. The complex of integrated models of development was applied, that has been successfully tested previously for the mathematical analysis of the fermentation processes. 相似文献
The batch production of gluconic acid in the presence of glucose, sucrose and molasses was investigated using free mycelia of Aspergillus foetidus NRRL 337 in shake flasks. Eight growth parameters were chosen as independent variables. The temperature, pH, substrate type and initial concentrations, inoculum percentage and shake rate directly affected the specific microorganism growth and gluconic acid production rates. The optimum temperature and initial pH values were found to be 33°C and five to six, respectively. The maximum specific growth and gluconic acid production rates were established as 57 g/dm3 of glucose, 75 g/dm3 of sucrose and 150 g/dm3 of molasses. The optimum values of the shake rate, inoculum percentage and initial ammonium nitrate concentration were determined as 100 1/min, 0.5% and 1.5 g/dm3, respectively. The maximum gluconic acid concentrations corresponding to these initial substrate concentrations were observed to be 8.3 g/dm3, 17.4 g/dm3 37.0 g/dm3, respectively. The optimum specific microbial growth and gluconic acid production rates were found as 0.0145 1/h and 0.0375 g/g × h, respectively, for the fermentation conditions of SGo = 57 g/dm3, T = 28°C, initial pH = 6.5, N = 84 1/min, A = 0.5 g/dm3 and I = 0.5%. 相似文献