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PubMed2003

Increase of the yields of eicosapentaenoic and docosahexaenoic acids by the microalga Pavlova lutheri following random mutagenesis.

The high commercial values of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids have driven a strain-improvement program, aimed at increasing the content of those fatty acids in the microalga Pavlova lutheri (SMBA 60) as parent strain. After a round of mutation using UV-light as mutagenic agent, an isolate strain (tentatively called II#2) was obtained, the EPA and DHA contents of which (in % dry biomass) were 32.8% and 32.9% higher than those of the control, native strain. The final EPA yields, when the cultures were maintained under appropriate conditions, were 17.4 and 23.1 mg. g(-1) dry biomass, for the wild-type and the II#2 strain, respectively, whereas the final DHA yields were 8.0 and 10.6 mg. g(-1) dry biomass, respectively. These results suggest that random mutagenesis can successfully be applied to increase the yield of n-3 fatty acids by microalgae.

Docosahexaenoic Acids↗
PubMed2001

Determination of 3-deoxy-D-arabino-heptulosonate 7-phosphate productivity and yield from glucose in Escherichia coli devoid of the glucose phosphotransferase transport system.

The effect of inactivation of the glucose phosphotransferase transport system (PTS) on 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) productivity and yield from glucose in Escherichia coli is reported. Strains used in this study were the PTS(+) PB103 and its PTS(-) glucose(+) derivative NF9. Their aroB(-) derivatives PB103B and NF9B were constructed to allow accurate measurement of total carbon flow into the aromatic pathway. The measured specific rates of DAHP synthesis were 0.55 and 0.94 mmol/g-dcw. h and the DAHP molar yields from glucose were 0.43 and 0.71 mol/mol for the PTS(+) aroB(-)and the PTS(-) glucose(+) aroB(-)strains, respectively. For the latter strain, this value represents 83% of the maximum theoretical yield for DAHP synthesis from glucose.

Biological Transport↗
PubMed2004

Metabolic engineering and protein directed evolution increase the yield of L-phenylalanine synthesized from glucose in Escherichia coli.

L-phenylalanine (L-Phe) is an aromatic amino acid with diverse commercial applications. Technologies for industrial microbial synthesis of L-Phe using glucose as a starting raw material currently achieve a relatively low conversion yield (Y(Phe/Glc)). The purpose of this work was to study the effect of PTS (phosphotransferase transport system) inactivation and overexpression of different versions of feedback inhibition resistant chorismate mutase-prephenate dehydratase (CM-PDT) on the yield (Y(Phe/Glc)) and productivity of L-Phe synthesized from glucose. The E. coli JM101 strain and its mutant derivative PB12 (PTS(-)Glc(+) phenotype) were used as hosts. PB12 has an inactive PTS, but is capable of transporting and phosphorylating glucose by using an alternative system constituted by galactose permease (GalP) and glucokinase activities (Glk). JM101 and PB12 were transformed with three plasmids, harboring genes that encode for a feedback inhibition resistant DAHP synthase (aroG(fbr)), transketolase (tktA) and either a truncated CM-PDT (pheA(fbr)) or its derived evolved genes (pheA(ev1) or pheA(ev2)). Resting-cells experiments with these engineered strains showed that JM101 and PB12 strains expressing either pheA(ev1) or pheA(ev2) genes produced l-Phe from glucose with Y(Phe/Glc) of 0.21 and 0.33 g/g, corresponding to 38 and 60% of the maximum theoretical yield (0.55 g/g), respectively. In addition, in both engineered strains the reached q(Phe) high levels of 40 mg/g-dcw.h. The metabolic engineering strategy followed in this work, including a strain with an inactive PTS, resulted in a positive impact over the Y(Phe/Glc), enhancing it nearly 57% compared with its PTS(+) counterpart. This is the first report wherein PTS inactivation was a successful strategy to improve the Y(Phe/Glc).

Directed Molecular Evolution↗
PubMed2006

Influence of substrate concentration on the stability and yield of continuous biohydrogen production.

The effect of substrate concentration (sucrose) on the stability and yield of a continuous fermentative process producing hydrogen was studied. High substrate concentrations are attractive from an energy standpoint as they would minimise the energy required for heating. The reactor was a CSTR; temperature was maintained at 35 degrees C; pH was controlled between 5.2 and 5.3, and the hydraulic retention time (HRT) was 12 h. Online measurements were taken for ORP, pH, temperature, %CO2, gas output and %H2, and data logged using a MatLAB data acquisition toolbox. Steady-state operation was obtained at 10, 20 and 40 g/L of sucrose in the influent, but a subsequent step change to 50 g/L was unsustainable. The hydrogen content ranged between 50% and 60%. The yield of hydrogen decreased as the substrate concentration increased from 1.7 +/- 0.2 mol/mol hexose added at 10 g/L, to 0.8 +/- 0.1 mol/mol at 50 g/L. Sparging with nitrogen improved the hydrogen yield by at least 35% at 40 g/L and at least 33% at 50 g/L sucrose. Sparging also enabled steady-state operation at 50 g/L sucrose. Addition of an extra 4 g/L of n-butyric acid to the reactor operating at 40 g/L sucrose increased the butyrate concentration from 9,830 to 18,900 mg/L, immediately stopping gas production and initiating the production of propionate, whilst the addition of 2 g/L taking the butyrate concentration to 12,200 mg/L did not do so. It was shown that operation at 50 g/L sucrose in a CSTR in butyrate fermentation is possible.

Acetic Acid↗
PubMed2006

Bioconversion of hybrid poplar to ethanol and co-products using an organosolv fractionation process: optimization of process yields.

An organosolv process involving extraction with hot aqueous ethanol has been evaluated for bioconversion of hybrid poplar to ethanol. The process resulted in fractionation of poplar chips into a cellulose-rich solids fraction, an ethanol organosolv lignin (EOL) fraction, and a water-soluble fraction containing hemicellulosic sugars, sugar breakdown products, degraded lignin, and other components. The influence of four independent process variables (temperature, time, catalyst dose, and ethanol concentration) on product yields was analyzed over a broad range using a small composite design and response surface methodology. Center point conditions for the composite design (180 degrees C, 60 min, 1.25% H(2)SO(4), and 60% ethanol), yielded a solids fraction containing approximately 88% of the cellulose present in the untreated poplar. Approximately 82% of the total cellulose in the untreated poplar was recovered as monomeric glucose after hydrolysis of the solids fraction for 24 h using a low enzyme loading (20 filter paper units of cellulase/g cellulose); approximately 85% was recovered after 48 h hydrolysis. Total recovery of xylose (soluble and insoluble) was equivalent to approximately 72% of the xylose present in untreated wood. Approximately 74% of the lignin in untreated wood was recovered as EOL. Other cooking conditions resulted in either similar or inferior product yields although the distribution of components between the various fractions differed markedly. Data analysis generated regression models that describe process responses for any combination of the four variables.

Biodegradation, Environmental↗
PubMed2007

Thermodynamic electron equivalents model for bacterial yield prediction: modifications and comparative evaluations.

Modifications are made to an earlier thermodynamic model (TEEM1) for prediction of maximum microbial yields from aerobic and anaerobic as well as heterotrophic and autotrophic growth. The revised model (TEEM2) corrects for lower yields found with aerobic oxidations of organic compounds where an oxygenase is involved and with growth on single-carbon (C1) compounds. TEEM1 and TEEM2 are based on energy release and consumption as determined from the reduction potential or Gibbs free energy of (1/2)-reaction reduction equations together with losses of energy during energy transfer. Energy transfer efficiency is a key parameter needed to make predictions with TEEM2, and was determined through evaluations with extensive data sets on aerobic heterotrophic yield available in the literature. For compounds following normal catabolic pathways, the best-fit value for energy transfer efficiency was 0.37, which permitted accurate predictions of growth with a precision of 15%-20% as determined by standard deviation. Using the same energy transfer efficiency, a similar precision, but somewhat less accuracy was found for organic compounds where oxidation involves an oxygenase (estimates 8% too high) and for C1 compounds (estimates 17% too high). In spite of the somewhat lower accuracy, the TEEM2 modifications resulted in improved predictions over TEEM1 and the comparison models.

Bacteria↗
PubMed1977

Theoretical conversion yields for penicillin synthesis.

The efficiency of conversion of the carbon-energy source to product is of primary importance in many fermentation processes. In order to assess the efficiency of a process, one must know how close the actual conversion yield is to the theoretical maximum. Theoretical conversion yields are useful, therefore, as guides in improving a process. This knowledge is particularly important today because the cost of raw materials is rapidly rising. In this study, the biochemical pathway of penicillin synthesis was used to estimate the theoretical yield of penicillin from glucose, ammonia, and sulfate. These values are compared with experimental data from the literature. An analysis of the role of glucose in the synthesis of cell mass and penicillin and in the maintenance of cells makes it possible to assess the efficiency of carbon-source utilization and to direct further advances in penicillin fermentations.

Ammonia↗
PubMed1976

Altered viscosity and yield stress in patients with abdominal malignancy: relationship to deep vein thrombosis.

Twenty-eight patients undergoing laparotomy were studied. Blood viscosity at both high and low rates of shear and a yield stress index were measured preoperatively and correlated with the incidence of post-operative deep vein thrombosis (DVT). There was a correlation between a raised index of yield stress and the incidence of DVT. Patients with cancer had a very high incidence of postoperative DVT and a high index of yield stress.

Abdominal Neoplasms↗
PubMed2004

Interpretive yields of screening Pap tests and diagnostic Pap tests.

Recently, our laboratory has introduced the Medicare classification "diagnostic Papanicolaou (Pap) test" (DPT) for both conventional and liquid-based Pap tests. Because little is known regarding the diagnostic yield of DPT, we review our experience with these tests in a community with a low incidence of squamous intraepithelial lesions and contrast this with our experience with screening Pap tests (SPTs). A search of our laboratory's computerized data system identified all Pap tests classified as SPT and DPT from December 3, 2002 to December 2, 2003. The interpretations were tabulated and statistical comparisons were made. Between December 3, 2002 and December 2, 2003 63,626 Pap tests were interpreted (57,922 SPTs and 5704 DPTs). DPTs were far more likely to yield abnormal results (atypical squamous cells or worse, P<0.001) and were especially more likely to yield malignant results. These differences have numerous possible implications regarding screening, rescreening, and the design of studies that investigate the screening value of the Pap test.

False Negative Reactions↗
PubMed1992

Cytology of transitional-cell carcinoma of the urinary bladder: diagnostic yield and histologic basis.

The diagnostic yield of cytology in histologically proven transitional-cell carcinoma (TCC) of the urinary bladder has been studied in 100 cases. Cytohistologic correlation rates were 20 percent, 61.7 percent, and 92.8 percent, respectively, for grade 1, 2, and 3 tumors. When further evaluated, G2 cases were segregated into 2 subcategories, G2a and G2b, based on histologic preservation of nuclear polarity, pleomorphism, and other cellular irregularities. Correlation rates were rather low for G2a cases (6/18, 33%) and high for G2b cases, (23/29; 79%). The prevalence of atypical cells was 2 (11.1%) cases in G2a and 16 (55.2%) cases in G2b. The results of this study confirm that cytology has an extremely varying diagnostic yield in urinary bladder TCC. Greater cell exfoliation, increased atypia, and a tendency to infiltration of G2b and G3 cases probably account for the higher diagnostic yield detected in these groups.

Carcinoma, Transitional Cell↗
PubMed2006

Viable human buccal mucosa cells do not yield typical nucleoids: impacts on the single-cell gel electrophoresis/Comet assay.

Buccal mucosa (BM) cells have been used in human biomonitoring studies for detecting DNA adducts and chromosomal damage in an epithelial cell population. In the present study, we have investigated if human BM cells are suitable for use in the single-cell gel electrophoresis (SCGE)/Comet assay as an approach for estimating the exposure of epithelial cells to DNA-damaging agents. Our results indicate that only a few cells from BM cell samples yield comets that can be analyzed by current methods, and that the yield of cells with comets is independent of the percentage of viable BM cells in the sample. Data generated after enzymatic enrichment of viable cells and immunomagnetic separation of epithelial cells suggest that most of the BM cells that do form comets are probably leukocytes. Moreover, by reevaluating specific cells after running the Comet assay, we found that viable epithelial BM cells give rise to atypical comets that are not included in the analysis. Comparing DNA migration patterns between small groups of smokers and nonsmokers indicated that long-term smoking had no effect on the subpopulation of cells that yield typical comets. Our results indicate that the SCGE assay, as it is commonly performed, may not be useful for genotoxicity monitoring in human epithelial BM cells.

Adult↗
PubMed1987

Enhancement of concurrent yield of spermatogonial, meiotic I, and meiotic II metaphase chromosomes from Chinese hamster testes.

A procedure to enhance the concurrent yield of large numbers of spermatogonial, meiotic I, and meiotic II metaphases from Chinese hamster testes has been developed. Animals were injected intraperitoneally with 5, 10, 20, or 40 mg of colchicine/kg and killed 3 hr after the treatment. Both testes from each animal were excised; one testis was minced, and germ cells were isolated by using 0.1% trypsin solution; the other testis was minced, and germ cells were isolated by using a conventional procedure. Chromosomal preparations were made with a standard air-drying technique. Examination of slides revealed that with the trypsin-isolation procedure the concurrent yields of spermatogonial, meiotic I, and meiotic II metaphases were significantly higher (P less than .05) than the yields obtained with the conventional procedure. Furthermore, 40 mg of colchicine/kg produced maximum numbers of all three types of germ cell metaphases. At this maximum concentration, metaphases were stable with no evidence of structural aberrations, and the frequency of hyperploidy was not significantly different (P greater than .05) from hypoploidy.

Animals↗
PubMed2003

High-yield isolation of murine microglia by mild trypsinization.

Microglia can be isolated with high purity but low yield by shaking off loosely adherent cells from mixed glial cultures. Here we describe a new technique for isolating microglia with an average yield close to 2,000,000 microglial cells/mouse pup, more than five times higher than that of the shaking method. Confluent mixed glial cultures are subjected to mild trypsinization (0.05-0.12%) in the presence of 0.2-0.5 mM EDTA and 0.5-0.8 mM Ca2+. This results in the detachment of an intact layer of cells containing virtually all the astrocytes, leaving undisturbed a population of firmly attached cells identified as >98% microglia. These almost pure microglial preparations can be kept in culture for weeks and show proliferation and phagocytosis. Treatment with macrophage colony-stimulating factor and lipopolysaccharide, alone or in the presence of interferon gamma, induces typical microglial responses in terms of proliferation, morphological changes, nuclear factor-kappaB translocation, NO, and tumor necrosis alpha release and phagocytosis. This method allows for the preparation of highly enriched mouse or rat microglial cultures with ease and reproducibility. Because of its high yield, it can be especially convenient when high amounts of microglial protein/mRNA are required or in cases in which the starting material is limited, such as microglial cultures from transgenic animals.

Animals↗
PubMed1986

Theoretically possible yields of microbial secondary products.

Balance equations for some representatives of the main biogenetic groups of secondary metabolites were formulated which provide the known and probable reactions within the biosynthetic routes. The calculated yields Yp/s max with respect to the usual substrate glucose range between 0.37 and 0.9 and, obviously, they agree well with the theoretical yields of biochemically related parts of cell dry mass. The sugar derivatives correspond to carbohydrates, the beta-lactams and shikimate derivatives to proteins, and finally, the polyketides and mevalonate derivatives to lipids. From this stoichiometric point of view the secondary metabolism thus seems to possess the same biosynthetic efficiency as adequate parts of primary metabolism under the conditions of retarded growth. The introduction of the Yp/s max values into an equation for substrate consumption regarding the growth, maintenance and product formation (van der Beek and Roels 1984) delivered yields which demonstrated that more than 70% of the glucose flowed into byproducts or waste metabolites, respectively.

Anti-Bacterial Agents↗
PubMed1996

Type-I collagen mutation compromises the post-yield behavior of Mov13 long bone.

Despite recent advances in our understanding of the molecular basis of skeletal fragility, little is known about how these molecular alterations lead to whole bone brittleness. In the current study, we investigated the relationship between a type-I collagen mutation and post-yield behavior of whole bone in Mov13 transgenic mice by considering tissue-level organizational issues known to be important for normal bone fracture. Mechanical assays revealed that the post-yield deflection of Mov13 femurs was reduced by 61% relative to littermate controls. Fractographic images revealed that lamellar interfaces which were important for dissipating energy during the failure process of control femurs, were not effective in Mov13 mice. Further investigation revealed that a 22% reduction in bone collagen content, a 2-fold increase in tissue porosity, and significant alterations in collagen organization interfered with normal energy dissipation mechanisms of Mov13 microstructure. Collectively, the results provided the first evidence that the reduced ductility associated with a type-I collagen mutation was mediated by alterations in intermediate structures that normally contribute to the post-yield behavior of cortical bone. The results suggest that, to better understand the pathogenesis of skeletal fragility, it is important to consider the effects of molecular alterations on higher-level structures, particularly those structures that contribute to the failure mechanisms in normal bone.

Animals↗
PubMed2002

Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality.

The aim of this study is to examine the effect of bovine oocyte maturation, fertilization or culture in vivo or in vitro on the proportion of oocytes reaching the blastocyst stage, and on blastocyst quality as measured by survival following vitrification. In Experiment 1, 4 groups of oocytes were used: (1) immature oocytes from 2-6 mm follicles; (2) immature oocytes from > 6 mm follicles; (3) immature oocytes recovered in vivo just before the LH surge; and (4) in vivo matured oocytes. Significantly more blastocysts developed from oocytes matured in vivo than those recovered just before the LH surge or than oocytes from 2-6 mm follicles. Results from > 6 mm follicles were intermediate. All blastocysts had low survival following vitrification. In Experiment 2, in vivo matured oocytes were either (1) fertilized in vitro or (2) fertilized in vivo by artificial insemination and the resulting presumptive zygotes recovered on day 1. Both groups were then cultured in vitro. In vivo fertilized oocytes had a significantly higher blastocyst yield than those fertilized in vitro. Blastocyst quality was similar between the groups. Both groups had low survival following vitrification. In Experiment 3a, presumptive zygotes produced by in vitro maturation (IVM)/fertilization (IVF) were cultured either in vitro in synthetic oviduct fluid, or in vivo in the ewe oviduct. In Experiment 3b, in vivo matured/in vivo fertilized zygotes were either surgically recovered on day 1 and cultured in vitro in synthetic oviduct fluid, or were nonsurgically recovered on day 7. There was no difference in blastocyst yields between groups of zygotes originating from the same source (in vivo or in vitro fertilization) irrespective of whether culture took place in vivo or in vitro. However, there was a dramatic effect on blastocyst quality with those blastocysts produced following in vivo culture surviving vitrification at significantly higher rates than their in vitro cultured counterparts. Collectively, these results indicate that the intrinsic quality of the oocyte is the main factor affecting blastocyst yields, while the conditions of embryo culture have a crucial role in determining blastocyst quality.

Animals↗
PubMed1993

Renaturation of lysozyme--temperature dependence of renaturation rate, renaturation yield, and aggregation: identification of hydrophobic folding intermediates.

Renaturation of denatured-reduced hen egg white lysozyme was analyzed at temperatures between 4 and 70 degrees C using the reduced/oxidized glutathione renaturation system. With an increase in temperature to 50 degrees C both renaturation rate constant and renaturation yield increased while formation of aggregates decreased. Denatured-reduced lysozyme and early folding intermediates were less stable against heat than native lysozyme at temperatures above 60 degrees C. Renaturation at 70 degrees C resulted in no reconstitution of lysozyme activity but the highest level of aggregation. Renaturation of denatured-reduced hen egg white lysozyme was further analyzed in the presence of the hydrophobicity-indicating fluorescence dye 1-anilinonaphalene-8-sulfonate at temperatures between 10 and 40 degrees C. The change in fluorescence intensity, the generation of enzyme activity, renaturation yield, and the formation of aggregates were studied. The results showed that early folding intermediates possess a strong hydrophobic nature. With an increase in temperature both the renaturation rate and the decay rate of hydrophobicity-mediated fluorescence increased. Consequently, with increasing temperature, accumulation of hydrophobic folding intermediates and formation of insoluble aggregates decreased, leading to an increase in the renaturation yield.

Animals↗
PubMed1999

Manganese and iron porphyrins catalyze peroxynitrite decomposition and simultaneously increase nitration and oxidant yield: implications for their use as peroxynitrite scavengers in vivo.

Twelve substituted metalloporphyrins (MPs), some of which have been previously characterized with respect to superoxide dismutase and peroxynitrite decomposing activities, were evaluated for their ability to scavenge peroxynitrite in vitro at 37 degrees C. Because the overall effectiveness of MPs as catalytic peroxynitrite scavengers is a function of (1) how fast they react with peroxynitrite, (2) how fast they cycle back to the starting compound, and (3) how well they contain or quench the reactive intermediates generated, all of these properties were evaluated and compared directly under the same conditions. Of the various MPs tested, only the iron and manganese porphyrins showed significant reactivity with peroxynitrite. The Mn(IV) intermediates resulting from oxidation by peroxynitrite were relatively stable and rereduction to the Mn(III) forms was rate-limiting to catalytic decomposition of peroxynitrite. However, in the presence of oxidizeable substrates like phenolics, rereduction of Mn(IV) forms occurred very rapidly and both the Mn- and Fe-porphyrins catalyzed nitration and oxidation by peroxynitrite. Mn- and Fe-porphyrins enhanced the yield of nitrated phenolics by peroxynitrite as much as 5-fold at pH 7.4 and up to 12-fold at pH 9. 1, while total oxidative yield was more than doubled. Nitration enhancement by MPs was effectively inhibited by ascorbate, glutathione, or serum, although much higher concentrations of ascorbate were required to inhibit nitration catalyzed by either Mn or Fe tetramethylpyridyl porphyrin. Catalysis of peroxynitrite nitration by MPs appears to proceed via a radical-mediated reaction mechanism whereby the phenolic substrate rapidly reduces Mn(IV) = O or Fe[IV] = O to the +3 state to yield phenoxyl radical which then combines with the other primary product, nitrogen dioxide. Based on the rate constants and the proposed reaction mechanism, it is reasonable to suggest that Mn and Fe porphyrins could detoxify peroxynitrite in vivo by efficiently trapping the relatively unreactive peroxynitrite anion and, in effect, channeling it into a single reaction pathway which could then be more effectively scavenged by cellular reductants like ascorbate.

Ascorbic Acid↗